First Genome Sequence of Leptospira interrogans Serovar Pomona, Isolated from a Bovine Abortion

PubMed Central

Varni, Vanina; Koval, Ariel; Nagel, Ariel; Ruybal, Paula

2016-01-01

Leptospirosis is a widespread zoonosis and a re-emergent disease of global distribution with major relevance in veterinary production. Here, we report the whole-genome sequence of Leptospira interrogans serovar Pomona strain AKRFB, isolated from a bovine abortion during a leptospirosis outbreak in Argentina. PMID:27198013

Expression and characterization of an iron-regulated hemin-binding protein, HbpA, from Leptospira interrogans serovar Lai.

PubMed

Asuthkar, Swapna; Velineni, Sridhar; Stadlmann, Johannes; Altmann, Friedrich; Sritharan, Manjula

2007-09-01

In an earlier study, based on the ferric enterobactin receptor FepA of Escherichia coli, we identified and modeled a TonB-dependent outer membrane receptor protein (LB191) from the genome of Leptospira interrogans serovar Lai. Based on in silico analysis, we hypothesized that this protein was an iron-dependent hemin-binding protein. In this study, we provide experimental evidence to prove that this protein, termed HbpA (hemin-binding protein A), is indeed an iron-regulated hemin-binding protein. We cloned and expressed the full-length 81-kDa recombinant rHbpA protein and a truncated 55-kDa protein from L. interrogans serovar Lai, both of which bind hemin-agarose. Assay of hemin-associated peroxidase activity and spectrofluorimetric analysis provided confirmatory evidence of hemin binding by HbpA. Immunofluorescence studies by confocal microscopy and the microscopic agglutination test demonstrated the surface localization and the iron-regulated expression of HbpA in L. interrogans. Southern blot analysis confirmed our earlier observation that the hbpA gene was present only in some of the pathogenic serovars and was absent in Leptospira biflexa. Hemin-agarose affinity studies showed another hemin-binding protein with a molecular mass of approximately 44 kDa, whose expression was independent of iron levels. This protein was seen in several serovars, including nonpathogenic L. biflexa. Sequence analysis and immunoreactivity with specific antibodies showed this protein to be LipL41.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection.

PubMed

Gómez, Ricardo M; Vieira, Monica L; Schattner, Mirta; Malaver, Elisa; Watanabe, Monica M; Barbosa, Angela S; Abreu, Patricia A E; de Morais, Zenaide M; Cifuente, Javier O; Atzingen, Marina V; Oliveira, Tatiane R; Vasconcellos, Silvio A; Nascimento, Ana L T O

2008-01-01

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L. interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L. interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira.

Management practices as risk factors for the presence of bulk milk antibodies to Salmonella, Neospora caninum and Leptospira interrogans serovar hardjo in Irish dairy herds.

PubMed

O' Doherty, E; Berry, D P; O' Grady, L; Sayers, R

2014-06-01

A survey of management practices in 309 Irish dairy herds was used to identify risk factors for the presence of antibodies to Salmonella, Neospora caninum and Leptospira interrogans serovar hardjo in extensively managed unvaccinated dairy herds. A previous study documented a herd-level seroprevalence in bulk milk of 49%, 19% and 86% for Salmonella, Neospora caninum and leptospira interrogans serovar hardjo, respectively in the unvaccinated proportion of these 309 herds in 2009. Association analyses in the present study were carried out using multiple logistic regression models. Herds where cattle were purchased or introduced had a greater likelihood of being positive to leptospira interrogans serovar hardjo (P<0.01) and Salmonella (P<0.01). Larger herds had a greater likelihood of recording a positive bulk milk antibody result to leptospira interrogans serovar hardjo (P<0.05). Herds that practiced year round calving were more likely to be positive to Neospora caninum (P<0.05) compared to herds with a spring-calving season, with no difference in risk between herds that practiced split calving compared to herds that practiced spring calving. No association was found between presence of dogs on farms and prevalence of Neospora caninum possibly due to limited access of dogs to infected materials including afterbirths. The information from this study will assist in the design of suitable control programmes for the diseases under investigation in pasture-based livestock systems.

SEROPREVALENCE OF NINE LEPTOSPIRA INTERROGANS SEROVARS IN WILD CARNIVORES, UNGULATES, AND PRIMATES FROM A ZOO POPULATION IN A METROPOLITAN REGION OF CHILE.

PubMed

Moreno-Beas, Eduardo; Abalos, Pedro; Hidalgo-Hermoso, Ezequiel

2015-12-01

Serum samples from 130 individuals representing 42 species of carnivores, ungulates, and primates from a population of captive mammals in Metropolitan Region in Chile were tested for antibodies against nine serovars of Leptospira interrogans using the microscopic agglutination test. Ten percent of the animals were seropositive to one or more serovars. Seroprevalence was significantly higher in ungulates (20.4%) compared to carnivores (3.8%) and primates (3.4%). There were no significant differences in seroprevalence among sex and age ranges. The most frequent serovar detected was Autumnalis, present in 53.4% of antibody-positive animals. Most positive animals had titers of ≤1 : 200, except for a maned wolf ( Chrysocyon brachyurus ) with titers of 1 : 400 against serovar Hardjo. To the authors' knowledge, this is the first report of Leptospira exposure detected in native endangered pudu ( Pudu puda ) and the first confirmation of exposure to L. interrogans in captive wild mammals in Chile. Leptospirosis should be considered as a differential diagnosis in future disease presentation for hepatitis or abortions in captive mammals in Chile.

Effects of temperature on gene expression patterns in Leptospira interrogans serovar Lai as assessed by whole-genome microarrays.

PubMed

Lo, Miranda; Bulach, Dieter M; Powell, David R; Haake, David A; Matsunaga, James; Paustian, Michael L; Zuerner, Richard L; Adler, Ben

2006-10-01

Leptospirosis is an important zoonosis of worldwide distribution. Humans become infected via exposure to pathogenic Leptospira spp. from infected animals or contaminated water or soil. The availability of genome sequences for Leptospira interrogans, serovars Lai and Copenhageni, has opened up opportunities to examine global transcription profiles using microarray technology. Temperature is a key environmental factor known to affect leptospiral protein expression. Leptospira spp. can grow in artificial media at a range of temperatures reflecting conditions found in the environment and the mammalian host. Therefore, transcriptional changes were compared between cultures grown at 20 degrees C, 30 degrees C, 37 degrees C, and 39 degrees C to represent ambient temperatures in the environment, growth under laboratory conditions, and temperatures in healthy and febrile hosts. Data from direct pairwise comparisons of the four temperatures were consolidated to examine transcriptional changes at two generalized biological conditions representing mammalian physiological temperatures (37 degrees C and 39 degrees C) versus environmental temperatures (20 degrees C and 30 degrees C). Additionally, cultures grown at 30 degrees C then shifted overnight to 37 degrees C were compared with those grown long-term at 30 degrees C and 37 degrees C to identify genes potentially expressed in the early stages of infection. Comparison of data sets from physiological versus environmental experiments with upshift experiments provided novel insights into possible transcriptional changes at different stages of infection. Changes included differential expression of chemotaxis and motility genes, signal transduction systems, and genes encoding proteins involved in alteration of the outer membrane. These findings indicate that temperature is an important factor regulating expression of proteins that facilitate invasion and establishment of disease.

Methylation and in vivo expression of the surface-exposed Leptospira interrogans outer-membrane protein OmpL32.

PubMed

Eshghi, Azad; Pinne, Marija; Haake, David A; Zuerner, Richard L; Frank, Ami; Cameron, Caroline E

2012-03-01

Recent studies have revealed that bacterial protein methylation is a widespread post-translational modification that is required for virulence in selected pathogenic bacteria. In particular, altered methylation of outer-membrane proteins has been shown to modulate the effectiveness of the host immune response. In this study, 2D gel electrophoresis combined with MALDI-TOF MS identified a Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 protein, corresponding to ORF LIC11848, which undergoes extensive and differential methylation of glutamic acid residues. Immunofluorescence microscopy implicated LIC11848 as a surface-exposed outer-membrane protein, prompting the designation OmpL32. Indirect immunofluorescence microscopy of golden Syrian hamster liver and kidney sections revealed expression of OmpL32 during colonization of these organs. Identification of methylated surface-exposed outer-membrane proteins, such as OmpL32, provides a foundation for delineating the role of this post-translational modification in leptospiral virulence.

Leptospira interrogans serovars Bratislava and Muenchen animal infections: Implications for epidemiology and control.

PubMed

Arent, Z; Frizzell, C; Gilmore, C; Allen, A; Ellis, W A

2016-07-15

Strains of Leptospira interrogans belonging to two very closely related serovars - Bratislava and Muenchen - have been associated with disease in domestic animals, in particular pigs, but also in horses and dogs. Similar strains have also been recovered from various wildlife species. Their epidemiology is poorly understood. Two hundred and forty seven such isolates, from UK domestic animal and wildlife species, were examined by restriction endonuclease analysis in an attempt to elucidate their epidemiology. A representative sub-sample of 65 of these isolates was further examined by multiple-locus variable-number tandem repeat analysis and 22 by secY sequencing. Ten restriction pattern types were identified. The majority of isolates fell into one of three restriction endonuclease analysis pattern types designated B2a, B2b and M2a. B2a was ubiquitous and was isolated from 10 species and represented the majority of the horse and all dog isolates. B2b was very different, being isolated only from pigs, indicating that this type was maintained by pigs. The pattern M2a was reported for the majority of isolates from pigs but also was common in small rodents isolates. Five restriction pattern types were found only in wildlife suggesting that they are unlikely to pose a disease threat to domestic animals. Multiple-locus variable-number tandem repeat analysis identified six clusters. The REA types B2a and B2b were all found in one MLVA cluster while the majority of the M2a strains examined occurred in another cluster. The secY sequencing detected only one sequence type, clustered with other serovars of Leptospira interrogans. Copyright © 2016 Elsevier B.V. All rights reserved.

Characterization of leptospiral proteins that afford partial protection in hamsters against lethal challenge with Leptospira interrogans.

PubMed

Atzingen, Marina V; Gonçales, Amane P; de Morais, Zenaide M; Araújo, Eduardo R; De Brito, Thales; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-09-01

Leptospirosis is a worldwide zoonosis caused by pathogenic Leptospira. The whole-genome sequence of Leptospira interrogans serovar Copenhageni together with bioinformatic tools allow us to search for novel antigen candidates suitable for improved vaccines against leptospirosis. This study focused on three genes encoding conserved hypothetical proteins predicted to be exported to the outer membrane. The genes were amplified by PCR from six predominant pathogenic serovars in Brazil. The genes were cloned and expressed in Escherichia coli strain BL21-SI using the expression vector pDEST17. The recombinant proteins tagged with N-terminal 6xHis were purified by metal-charged chromatography. The proteins were recognized by antibodies present in sera from hamsters that were experimentally infected. Immunization of hamsters followed by challenge with a lethal dose of a virulent strain of Leptospira showed that the recombinant protein rLIC12730 afforded statistically significant protection to animals (44 %), followed by rLIC10494 (40 %) and rLIC12922 (30 %). Immunization with these proteins produced an increase in antibody titres during subsequent boosters, suggesting the involvement of a T-helper 2 response. Although more studies are needed, these data suggest that rLIC12730 and rLIC10494 are promising candidates for a multivalent vaccine for the prevention of leptospirosis.

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis.

PubMed

Silva, Everton F; Medeiros, Marco A; McBride, Alan J A; Matsunaga, Jim; Esteves, Gabriela S; Ramos, João G R; Santos, Cleiton S; Croda, Júlio; Homma, Akira; Dellagostin, Odir A; Haake, David A; Reis, Mitermayer G; Ko, Albert I

2007-08-14

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund's adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P<0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis.

Molecular identification of the ompL1 gene within Leptospira interrogans standard serovars.

PubMed

Dezhbord, Mehrangiz; Esmaelizad, Majid; Khaki, Pejvak; Fotohi, Fariba; Zarehparvar Moghaddam, Athena

2014-06-11

Leptospirosis, caused by infection with pathogenic Leptospira species, is one of the most prevalent zoonotic diseases in the world. Current leptospiral vaccines are mainly multivalent dead whole-cell mixtures made of several local dominant serovars. Therefore, design and construction of an efficient recombinant vaccine for leptospirosis control is very important. OmpL1 is an immunogenic porin protein that could be of special significance in vaccination and serodiagnosis for leptospirosis. Three strains belonging to pathogenic L. interrogans were analyzed. The specific primers for proliferation of the ompL1 gene were designed. The amplified gene was cloned. In order to investigate the ompL1 nucleotide sequence and homological analysis of this gene, ompL1 genes cloned from standard vaccinal Leptospira serovars prevalent in Iran were sequenced and cloned. PCR amplification of the ompL1 gene using the designed primers resulted in a 963 bp ompL1 gene product. The PCR based on the ompL1 gene detected all pathogenic reference serovars of Leptospira spp. tested. Based on alignment and phylogenetic analysis, although the ompL1 nucleotide sequence was slightly different within three vaccinal serovars (100%-85% identity), amino acid alignment of the OmpL1 proteins revealed that there would be inconsiderable difference among them. The ompL1 gene of the three isolates was well conserved, differing only by a total of 6 bp and the proteins by 2 amino acids. The cloned gene could be further used for expression and recombinant OmpL1 as an efficient and conserved antigen, and may be a useful vaccine candidate against leptospirosis in our region.

Helix handedness of Leptospira interrogans as determined by scanning electron microscopy.

PubMed Central

Carleton, O; Charon, N W; Allender, P; O'Brien, S

1979-01-01

Representative serovars and strains of the seven genetic groups of Leptospira interrogans, and two previously studied serovars, were all found to form exclusively right-handed helices as determined by scanning electron microscopy. No change in handedness occurred in cells grown in a minimal medium (Tween-80 albumin) compared to cells grown in a rich medium (rabbit serum). The right-handedness of the organisms was related to the evolution, cell wall structure, and the mechanism of motility of L. interrogans. Images PMID:438122

A Model System for Studying the Transcriptomic and Physiological Changes Associated with Mammalian Host-Adaptation by Leptospira interrogans Serovar Copenhageni

PubMed Central

Caimano, Melissa J.; Sivasankaran, Sathesh K.; Allard, Anna; Hurley, Daniel; Hokamp, Karsten; Grassmann, André A.; Hinton, Jay C. D.; Nally, Jarlath E.

2014-01-01

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for

The terminal portion of leptospiral immunoglobulin-like protein LigA confers protective immunity against lethal infection in the hamster model of leptospirosis

PubMed Central

Silva, Éverton F.; Medeiros, Marco A.; McBride, Alan J. A.; Matsunaga, Jim; Esteves, Gabriela S.; Ramos, João G. R.; Santos, Cleiton S.; Croda, Júlio; Homma, Akira; Dellagostin, Odir A.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

2007-01-01

Subunit vaccines are a potential intervention strategy against leptospirosis, which is a major public health problem in developing countries and a veterinary disease in livestock and companion animals worldwide. Leptospiral immunoglobulin-like (Lig) proteins are a family of surface-exposed determinants that have Ig-like repeat domains found in virulence factors such as intimin and invasin. We expressed fragments of the repeat domain regions of LigA and LigB from Leptospira interrogans serovar Copenhageni. Immunization of Golden Syrian hamsters with Lig fragments in Freund’s adjuvant induced robust antibody responses against recombinant protein and native protein, as detected by ELISA and immunoblot, respectively. A single fragment, LigANI, which corresponds to the six carboxy-terminal Ig-like repeat domains of the LigA molecule, conferred immunoprotection against mortality (67-100%, P <0.05) in hamsters which received a lethal inoculum of L. interrogans serovar Copenhageni. However, immunization with this fragment did not confer sterilizing immunity. These findings indicate that the carboxy-terminal portion of LigA is an immunoprotective domain and may serve as a vaccine candidate for human and veterinary leptospirosis. PMID:17629368

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells

PubMed Central

Sato, Hiromi

2017-01-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways

Leptospira interrogans causes quantitative and morphological disturbances in adherens junctions and other biological groups of proteins in human endothelial cells.

PubMed

Sato, Hiromi; Coburn, Jenifer

2017-07-01

Pathogenic Leptospira transmits from animals to humans, causing the zoonotic life-threatening infection called leptospirosis. This infection is reported worldwide with higher risk in tropical regions. Symptoms of leptospirosis range from mild illness to severe illness such as liver damage, kidney failure, respiratory distress, meningitis, and fatal hemorrhagic disease. Invasive species of Leptospira rapidly disseminate to multiple tissues where this bacterium damages host endothelial cells, increasing vascular permeability. Despite the burden in humans and animals, the pathogenic mechanisms of Leptospira infection remain to be elucidated. The pathogenic leptospires adhere to endothelial cells and permeabilize endothelial barriers in vivo and in vitro. In this study, human endothelial cells were infected with the pathogenic L. interrogans serovar Copenhageni or the saprophyte L. biflexa serovar Patoc to investigate morphological changes and other distinctive phenotypes of host cell proteins by fluorescence microscopy. Among those analyzed, 17 proteins from five biological classes demonstrated distinctive phenotypes in morphology and/or signal intensity upon infection with Leptospira. The affected biological groups include: 1) extracellular matrix, 2) intercellular adhesion molecules and cell surface receptors, 3) intracellular proteins, 4) cell-cell junction proteins, and 5) a cytoskeletal protein. Infection with the pathogenic strain most profoundly disturbed the biological structures of adherens junctions (VE-cadherin and catenins) and actin filaments. Our data illuminate morphological disruptions and reduced signals of cell-cell junction proteins and filamentous actin in L. interrogans-infected endothelial cells. In addition, Leptospira infection, regardless of pathogenic status, influenced other host proteins belonging to multiple biological classes. Our data suggest that this zoonotic agent may damage endothelial cells via multiple cascades or pathways

Expression and characterization of recombinant leptospiral outer membrane protein LipL32 from Leptospira interrogans serovar autumnalis.

PubMed

Boonsathorn, Naphatsawan; Konghom, Ganokrot; Mongkolsiri, Kaveewan; Jirapongwattana, Chanin; Balachandra, Kruavon; Naigowit, Pimjai; Sawanpanyalert, Pathom

2009-01-01

Leptospira interrogans serovar autumnalis, a causative agent of leptospirosis in Thailand, was isolated from a patient for DNA extraction and amplification of LipL32 gene by polymerase chain reaction (PCR). The 782 bp PCR product was obtained, which was inserted into pAE plasmid with polyhistidine (His6 tag) to construct pAE-LipL32. This recombinant plasmid was transfected into E. coli BL21 (DE3). His6-LipL32 was purified by Ni-NTA affinity chromatography. The recombinant protein was used as antigen for testing with sera from leptospirosis and syphilis patients by dot-ELISA technique. It reacted positively with leptospirosis patient sera and negatively with syphilis and healthy sera.

Role of sph2 Gene Regulation in Hemolytic and Sphingomyelinase Activities Produced by Leptospira interrogans.

PubMed

Narayanavari, Suneel A; Lourdault, Kristel; Sritharan, Manjula; Haake, David A; Matsunaga, James

2015-01-01

Pathogenic members of the genus Leptospira are the causative agents of leptospirosis, a neglected disease of public and veterinary health concern. Leptospirosis is a systemic disease that in its severest forms leads to renal insufficiency, hepatic dysfunction, and pulmonary failure. Many strains of Leptospira produce hemolytic and sphingomyelinase activities, and a number of candidate leptospiral hemolysins have been identified based on sequence similarity to well-characterized bacterial hemolysins. Five of the putative hemolysins are sphingomyelinase paralogs. Although recombinant forms of the sphingomyelinase Sph2 and other hemolysins lyse erythrocytes, none have been demonstrated to contribute to the hemolytic activity secreted by leptospiral cells. In this study, we examined the regulation of sph2 and its relationship to hemolytic and sphingomyelinase activities produced by several L. interrogans strains cultivated under the osmotic conditions found in the mammalian host. The sph2 gene was poorly expressed when the Fiocruz L1-130 (serovar Copenhageni), 56601 (sv. Lai), and L495 (sv. Manilae) strains were cultivated in the standard culture medium EMJH. Raising EMJH osmolarity to physiological levels with sodium chloride enhanced Sph2 production in all three strains. In addition, the Pomona subtype kennewicki strain LC82-25 produced substantially greater amounts of Sph2 during standard EMJH growth than the other strains, and sph2 expression increased further by addition of salt. When 10% rat serum was present in EMJH along with the sodium chloride supplement, Sph2 production increased further in all strains. Osmotic regulation and differences in basal Sph2 production in the Manilae L495 and Pomona strains correlated with the levels of secreted hemolysin and sphingomyelinase activities. Finally, a transposon insertion in sph2 dramatically reduced hemolytic and sphingomyelinase activities during incubation of L. interrogans at physiologic osmolarity

[Sequences and expression pattern of mce gene in Leptospira interrogans of different serogroups].

PubMed

Zhang, Lei; Xue, Feng; Yan, Jie; Mao, Ya-fei; Li, Li-wei

2008-11-01

To determine the frequency of mce gene in Leptospira interrogans, and to investigate the gene transcription levels of L. interrogans before and after infecting cells. The segments of entire mce genes from 13 L.interrogans strains and 1 L.biflexa strain were amplified by PCR and then sequenced after T-A cloning. A prokaryotic expression system of mce gene was constructed; the expression and output of the target recombinant protein rMce were examined by SDS-PAGE and Western Blot assay. Rabbits were intradermally immunized with rMce to prepare the antiserum, the titer of antiserum was measured by immunodiffusion test. The transcription levels of mce gene in L.interrogans serogroup Icterohaemorrhagiae serovar lai strain 56601 before and after infecting J774A.1 cells were monitored by real-time fluorescence quantitative RT-PCR. mce gene was carried in all tested L.interrogans strains, but not in L.biflexa serogroup Semaranga serovar patoc strain Patoc I. The similarities of nucleotide and putative amino acid sequences of the cloned mce genes to the reported sequences (GenBank accession No: NP712236) were 99.02%-100% and 97.91%-100%, respectively. The constructed prokaryotic expression system of mce gene expressed rMce and the output of rMce was about 5% of the total bacterial proteins. The antiserum against whole cell of L.interrogans strain 56601 efficiently recognized rMce. After infecting J774A.1 cells, transcription levels of the mce gene in L.interrogans strain 56601 were remarkably up-regulated. The constructed prokaryotic expression system of mce gene and the prepared antiserum against rMce provide useful tools for further study of the gene function.

Post-translational Modification of LipL32 during Leptospira interrogans Infection

PubMed Central

Witchell, Timothy D.; Eshghi, Azad; Nally, Jarlath E.; Hof, Rebecca; Boulanger, Martin J.; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Cameron, Caroline E.

2014-01-01

Background Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. Methodology/Principal Findings Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. Conclusions/Significance The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although

[Construction and expression of recombinant Mycobacterium bovis BCG with the ompA-like membrane protein gene Loa22 of Leptospira interrogans serovar].

PubMed

Li, Dao-kun; Bao, Lang; Zhang, Ying; Sun, Zhan

2010-03-01

To study the immunity of Loa22 from Leptospira interrogans serovar Lai strain 56601 by expressing its protein in BCG. Amplified the mature peptide of Loa22 gene from the genome of of Leptospira interrogans serovar Lai strain 56601 and constructed recombinant plasmid rpMV36l-1oa22 with the E. coli-BCG integrating shuttle plasmid pMV361 and the Loa22 mature peptide gene. The rpMV36l-1oa22 plasmid was transformed into BCG by electroporation. The rBCG bearing rpMV36l-1oa22 was induced by high temperature of 45 degrees C and expressed protein was identified by SDS-PAGE and Western Blotting. Fifth 6-week-old BALB/c mice were randomly divided into five groups, which were inoculated intraperitoneally two times at 0-day and 21-day with BCG, rBCG-pMV361, rI3CG-1oa22, Loa22 and killed whole-leptospires respectively. All animals were dislocated from cervical vertebra on the 14Ih day after the last immunization. The proliferative reaction of splenic lymphocyte in tuitro were tested by XTT. The rpMV36l-1oa22 plasmid was constructed successfully and transformed into BCG. The rBCG expressed a 19 X io specifical protein identified by SDS-PAGE and Western Blotting. The splenic lymphocyte proliferate activity (SI) in rBCG-ioa22 group in intro was significantly higher than those in BCG group and rBCG-pMV361 group. We explored the expressing feasibility of Loa22 in Mycobacterium bovis BCG. may therefore make further researches on the induction of protective immunity against human and animal leptospirosis.

Cloning, expression, and homology modeling of GroEL protein from Leptospira interrogans serovar autumnalis strain N2.

PubMed

Natarajaseenivasan, Kalimuthusamy; Shanmughapriya, Santhanam; Velineni, Sridhar; Artiushin, Sergey C; Timoney, John F

2011-10-01

Leptospirosis is an infectious bacterial disease caused by Leptospira species. In this study, we cloned and sequenced the gene encoding the immunodominant protein GroEL from L. interrogans serovar Autumnalis strain N2, which was isolated from the urine of a patient during an outbreak of leptospirosis in Chennai, India. This groEL gene encodes a protein of 60 kDa with a high degree of homology (99% similarity) to those of other leptospiral serovars. Recombinant GroEL was overexpressed in Escherichia coli. Immunoblot analysis indicated that the sera from confirmed leptospirosis patients showed strong reactivity with the recombinant GroEL while no reactivity was observed with the sera from seronegative control patient. In addition, the 3D structure of GroEL was constructed using chaperonin complex cpn60 from Thermus thermophilus as template and validated. The results indicated a Z-score of -8.35, which is in good agreement with the expected value for a protein. The superposition of the Ca traces of cpn60 structure and predicted structure of leptospiral GroEL indicates good agreement of secondary structure elements with an RMSD value of 1.5 Å. Further study is necessary to evaluate GroEL for serological diagnosis of leptospirosis and for its potential as a vaccine component. Copyright © 2011 Beijing Genomics Institute. Published by Elsevier Ltd. All rights reserved.

Studies on equine recurrent uveitis. II: The role of infection with Leptospira interrogans serovar pomona.

PubMed

Halliwell, R E; Brim, T A; Hines, M T; Wolf, D; White, F H

1985-10-01

An enzyme linked immunosorbent assay was developed for the detection of immunoglobulin class specific antibodies to Leptospira interrogans serovar pomona in the serum and aqueous humor of horses. Serum antibody was also assayed by microscopic agglutination tests. Although higher levels of antibody were found in sera from horses with signs of uveitis, the association was not statistically significant. Antibodies to pomona were detected in the aqueous of 12 eyes from the 101 horses sampled at a slaughterhouse, and in most instances, a comparison of the aqueous/serum antibody level with that of the total aqueous/serum IgG level indicated intraocular antibody synthesis. Antibodies were also found in 4 aqueous (or vitreous) samples out of 9 obtained from horses with clinically documented uveitis and the above comparison again indicated intraocular antibody synthesis. The data point to an important role for pomona as an etiology of equine recurrent uveitis but also emphasize that the initiating cause for this disease is often obscure in that association with leptospirosis cannot be shown in many instances.

Identification of Cell-Binding Adhesins of Leptospira interrogans

PubMed Central

Evangelista, Karen V.; Hahn, Beth; Wunder, Elsio A.; Ko, Albert I.; Haake, David A.; Coburn, Jenifer

2014-01-01

Leptospirosis is a globally distributed bacterial infectious disease caused by pathogenic members of the genus Leptospira. Infection can lead to illness ranging from mild and non-specific to severe, with jaundice, kidney and liver dysfunction, and widespread endothelial damage. The adhesion of pathogenic Leptospira species (spp.), the causative agent of leptospirosis, to host tissue components is necessary for infection and pathogenesis. While it is well-established that extracellular matrix (ECM) components play a role in the interaction of the pathogen with host molecules, we have shown that pathogenic Leptospira interrogans binds to host cells more efficiently than to ECM components. Using in vitro phage display to select for phage clones that bind to EA.hy926 endothelial cells, we identified the putative lipoproteins LIC10508 and LIC13411, and the conserved hypothetical proteins LIC12341 and LIC11574, as candidate L. interrogans sv. Copenhageni st. Fiocruz L1–130 adhesins. Recombinant LIC11574, but not its L. biflexa homologue LBF1629, exhibited dose-dependent binding to both endothelial and epithelial cells. In addition, LIC11574 and LIC13411 bind to VE-cadherin, an endothelial cell receptor for L. interrogans. Extraction of bacteria with the non-ionic detergent Triton X-114 resulted in partitioning of the candidate adhesins to the detergent fraction, a likely indication that these proteins are outer membrane localized. All candidate adhesins were recognized by sera obtained from leptospirosis patients but not by sera from healthy individuals as assessed by western blot. This work has identified bacterial adhesins that are potentially involved in L. interrogans infection of the mammalian host, and through cadherin binding, may contribute to dissemination and vascular damage. Our findings may be of value in leptospirosis control and prevention, with the bacterial adhesins potentially serving as targets for development of diagnostics, therapeutics, and

Mouse model for sublethal Leptospira interrogans infection.

PubMed

Richer, Luciana; Potula, Hari-Hara; Melo, Rita; Vieira, Ana; Gomes-Solecki, Maria

2015-12-01

Although Leptospira can infect a wide range of mammalian species, most studies have been conducted in golden Syrian hamsters, a species particularly sensitive to acute disease. Chronic disease has been well characterized in the rat, one of the natural reservoir hosts. Studies in another asymptomatic reservoir host, the mouse, have occasionally been done and have limited infection to mice younger than 6 weeks of age. We analyzed the outcome of sublethal infection of C3H/HeJ mice older than age 10 weeks with Leptospira interrogans serovar Copenhageni. Infection led to bloodstream dissemination of Leptospira, which was followed by urinary shedding, body weight loss, hypothermia, and colonization of the kidney by live spirochetes 2 weeks after infection. In addition, Leptospira dissemination triggered inflammation in the kidney but not in the liver or lung, as determined by increased levels of mRNA transcripts for the keratinocyte-derived chemokine, RANTES, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-1β, inducible nitric oxide synthase, interleukin-6, and gamma interferon in kidney tissue. The acquired humoral response to Leptospira infection led to the production of IgG mainly of the IgG1 subtype. Flow cytometric analysis of splenocytes from infected mice revealed that cellular expansion was primarily due to an increase in the levels of CD4(+) and double-negative T cells (not CD8(+) cells) and that CD4(+) T cells acquired a CD44(high) CD62L(low) effector phenotype not accompanied by increases in memory T cells. A mouse model for sublethal Leptospira infection allows understanding of the bacterial and host factors that lead to immune evasion, which can result in acute or chronic disease or resistance to infection (protection). Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Six new leptospiral serovars isolated from wild animals in Peru.

PubMed Central

Liceras de Hidalgo, J L; Sulzer, K R

1984-01-01

Six new serovars of Leptospira interrogans were isolated from opossums (Didelphis marsupialis and Philander opossum) trapped in the Peruvian jungle. The proposed names, type strain designation, and serogroup of the serovars, respectively, were: huallaga, strain M-7, Djasiman serogroup; luis, strain M-6, Tarassovi serogroup; machiguenga, strain MMD-3, Icterohaemorrhagiae serogroup; rioja, strain MR-12, Bataviae serogroup; rupa rupa, strain M-3, Sejroe serogroup; and tingomaria, strain M-13, Cynopteri serogroup. PMID:6470106

Male-specific pulmonary hemorrhage and cytokine gene expression in golden hamster in early-phase Leptospira interrogans serovar Hebdomadis infection.

PubMed

Tomizawa, Rina; Sugiyama, Hiromu; Sato, Ryoichi; Ohnishi, Makoto; Koizumi, Nobuo

2017-10-01

Leptospirosis causes severe clinical signs more frequently in men than in women, but the mechanism underlying the gender differences in leptospirosis remains unclear. In this study, petechial hemorrhage was observed in male but not in female hamster lung tissues infected with Leptospira interrogans serovar Hebdomadis at 120 h pi, demonstrating that male hamsters were more susceptible to the development of a severe disease upon Leptospira infection. No leptospiral DNA was detected in the lung tissues at 120 h pi when pulmonary hemorrhage was observed, indicating that pulmonary hemorrhage is attributable to the immune reactions of the host rather than from the direct effect of leptospires. The upregulation of nitric oxide synthase genes in the hamsters without pulmonary hemorrhage, inos and enos in female hamsters at 96 h pi and enos in male animals without hemorrhage at 120 h pi, may suggest that nitric oxide has a suppressive effect on leptospirosis-associated pulmonary hemorrhage. Copyright © 2017 Elsevier Ltd. All rights reserved.

Infection rate of Leptospira interrogans in the field rodent, Apodemus agrarius, in Korea.

PubMed Central

Cho, M. K.; Kee, S. H.; Song, H. J.; Kim, K. H.; Song, K. J.; Baek, L. J.; Kim, H. H.; Oh, H. B.; Kim, Y. W.; Chang, W. H.

1998-01-01

Leptospirosis has significantly decreased in Korea since 1988, following the leptospiral vaccination programme initiated in 1988. Whether this wholly explains the decreased incidence is uncertain. As an initial step to answer this question, infection rates of Leptospira interrogans in field rodents, Apodemis agrarius, were examined and compared with previous data. Two hundred and twenty-two A. agrarius were captured during October-December 1996. Spirochaetes were isolated from 22 (9.9%) and leptospiral DNA was detected in an additional 6 rodents (12.6%). Subsequent microscopic agglutination tests (MAT) classified all these isolates as L. interrogans serogroup Icterohaemorrhagiae serovar lai. The above data did not significantly differ from previous surveys in 1984-7. There was no significant change of L. interrogans infection in field rodents following the introduction of the vaccination programme in Korea. Further studies are needed to determine the role of human vaccination in reducing incidence. PMID:10030719

Refolding of the recombinant protein OmpA70 from Leptospira interrogans from inclusion bodies using high hydrostatic pressure and partial characterization of its immunological properties.

PubMed

Fraga, Tatiana R; Chura-Chambi, Rosa M; Gonçales, Amane P; Morais, Zenaide M; Vasconcellos, Sílvio A; Morganti, Ligia; Martins, Elizabeth A L

2010-07-20

Leptospira is the etiological agent of leptospirosis, a life-threatening disease that affects human populations worldwide. Available vaccines have demonstrated limited effectiveness, and therapeutic interventions are complicated by the difficulty of establishing an early diagnosis. The genome of Leptospira strains was sequenced, and bioinformatic analyses revealed potential vaccine and serodiagnosis candidates. The present work studied OmpA70, a putative outer membrane protein from Leptospira interrogans serovar Copenhageni that combines structural features of Loa22, the first genetically defined virulence factor in Leptospira, and Lp49, a protein that reacts with sera from early and convalescent patients. Recombinant OmpA was produced in Escherichia coli in an insoluble form. Considering the importance of the structural integrity of a protein to confer immune protection, high hydrostatic pressure (HHP) was used to refold OmpA70 aggregated as inclusion bodies. HHP was applied in association with redox-shuffling reagents (oxidized and reduced glutathione) and guanidine hydrochloride or l-arginine. About 40% of the protein was refolded by applying 200MPa for 16h in concentrations of l-arginine above 0.4M. Circular dichroism revealed the presence of secondary structure. OmpA70 has immunogenic and antigenic properties as high antibody titers were seen after immunization with this protein, and sera from infected hamsters reacted with soluble OmpA70. Copyright (c) 2010 Elsevier B.V. All rights reserved.

Isolation and Molecular Characterization of Leptospira interrogans and Leptospira borgpetersenii Isolates from the Urban Rat Populations of Kuala Lumpur, Malaysia

PubMed Central

Benacer, Douadi; Zain, Siti Nursheena Mohd; Amran, Fairuz; Galloway, Renee L.; Thong, Kwai Lin

2013-01-01

Rats are considered the principal maintenance hosts of Leptospira. The objectives of this study were isolation and identification of Leptospira serovars circulating among urban rat populations in Kuala Lumpur. Three hundred urban rats (73% Rattus rattus and 27% R. norvegicus) from three different sites were trapped. Twenty cultures were positive for Leptospira using dark-field microscopy. R. rattus was the dominant carrier (70%). Polymerase chain reaction (PCR) confirmed that all isolates were pathogenic Leptospira species. Two Leptospira serogroups, Javanica and Bataviae, were identified using microscopic agglutination test (MAT). Pulsed-field gel electrophoresis (PFGE) identified two serovars in the urban rat populations: L. borgpetersenii serovar Javanica (85%) and L. interrogans serovar Bataviae (15%). We conclude that these two serovars are the major serovars circulating among the urban rat populations in Kuala Lumpur. Despite the low infection rate reported, the high pathogenicity of these serovars raises concern of public health risks caused by rodent transmission of leptospirosis. PMID:23358635

Serum and vitreous humor antibody titers in and isolation of Leptospira interrogans from horses with recurrent uveitis.

PubMed

Wollanke, B; Rohrbach, B W; Gerhards, H

2001-09-15

To measure antibody titers against Leptospira interrogans in serum and vitreous humor and determine the prevalence of L interrogans in vitreous humor of horses with recurrent uveitis. Cross-sectional study. 242 horses (270 eyes) with recurrent uveitis undergoing vitrectomy and 39 control horses (54 eyes) without any history or clinical signs of recurrent uveitis undergoing euthanasia or enucleation for unrelated reasons. Serum and vitreous humor were tested for antibodies against 13 serovars of L interrogans. Vitreous humor was submitted for leptospiral culture; isolates were typed to the serogroup level. Leptospira interrogans was isolated from vitreous humor from 120/229 (52%) horses (126/252 [50%] eyes) with recurrent uveitis but was not isolated from vitreous humor from 36 eyes of 21 control horses. Duration of recurrent uveitis was > or = 1 year for 45 of the 120 (38%) horses from which the organism was isolated. Geometric mean antibody titers against L interrogans in the vitreous humor and serum of horses with recurrent uveitis were 1:1,332 and 1:186, respectively. Only 91 of 120 (76%) horses from which the organism was isolated had a 4-fold or greater difference between serum and vitreous humor antibody titers. Results suggest that persistent ocular infection with L interrogans is common in horses with recurrent uveitis. A 4-fold increase in vitreous humor versus serum antibody titers may not be a sensitive test for the diagnosis of L interrogans-induced recurrent uveitis. We hypothesize that the immune component of recurrent uveitis can be directly induced and maintained by persistent infection of the eye with L interrogans.

A conserved region of leptospiral immunoglobulin-like A and B proteins as a DNA vaccine elicits a prophylactic immune response against leptospirosis.

PubMed

Forster, Karine M; Hartwig, Daiane D; Seixas, Fabiana K; Bacelo, Kátia L; Amaral, Marta; Hartleben, Cláudia P; Dellagostin, Odir A

2013-05-01

The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine.

A Conserved Region of Leptospiral Immunoglobulin-Like A and B Proteins as a DNA Vaccine Elicits a Prophylactic Immune Response against Leptospirosis

PubMed Central

Forster, Karine M.; Hartwig, Daiane D.; Seixas, Fabiana K.; Bacelo, Kátia L.; Amaral, Marta; Hartleben, Cláudia P.

2013-01-01

The leptospiral immunoglobulin-like (Lig) proteins LigA and LigB possess immunoglobulin-like domains with 90-amino-acid repeats and are adhesion molecules involved in pathogenicity. They are conserved in pathogenic Leptospira spp. and thus are of interest for use as serodiagnostic antigens and in recombinant vaccine formulations. The N-terminal amino acid sequences of the LigA and LigB proteins are identical, but the C-terminal sequences vary. In this study, we evaluated the protective potential of five truncated forms of LigA and LigB proteins from Leptospira interrogans serovar Canicola as DNA vaccines using the pTARGET mammalian expression vector. Hamsters immunized with the DNA vaccines were subjected to a heterologous challenge with L. interrogans serovar Copenhageni strain Spool via the intraperitoneal route. Immunization with a DNA vaccine encoding LigBrep resulted in the survival of 5/8 (62.5%) hamsters against lethal infection (P < 0.05). None of the control hamsters or animals immunized with the other vaccine preparations survived. The vaccine induced an IgG antibody response and, additionally, conferred sterilizing immunity in 80% of the surviving animals. Our results indicate that the LigBrep DNA vaccine is a promising candidate for inclusion in a protective leptospiral vaccine. PMID:23486420

[Prokaryotic expression of Leptospira interrogans groEL gene and immunoprotection of its products in hamsters].

PubMed

Li, Xiaoyu; Wang, Yinhuan; Yan, Jie; Cheng, Dongqing

2013-03-01

To construct a prokaryotic expression system of groEL gene of Leptospira interrogans serogroup Icterohaemorrhagia serovar Lai strain Lai, and to determine the immunoprotective effect of recombinant GroEL protein (rGroEL) in LVG hamsters. The groEL gene was amplified by high fidelity PCR and the amplification products were then sequenced. A prokaryotic expression system of groEL gene was constructed using routine genetic engineering technique. SDS-PAGE plus Bio-Rad Gel Image Analyzer was applied to examine the expression and dissolubility of rGroEL protein while Ni-NTA affinity chromatography was used to extract the expressed rGroEL. The immunoprotective rate in rGroEL-immunized LVG hamsters was determined after challenge with L.interrogans strain Lai. The cross agglutination titers of sera from immunized hamsters with different L.interrogans serogroups were detected using MAT. The nucleotide and amino acid sequences of the cloned groEL gene were the same as those reported in GenBank. The constructed prokaryotic expression system of groEL gene expressed soluble rGroEL. The immunoprotective rates of 100 and 200 μg rGroEL in LVG hamsters were 50.0 % and 75.0%, respectively. The sera from the rGroEL-immunized LVG hamsters agglutinated all the L.interrogans serogroups tested with different levels. The GroEL protein is a genus-specific immunoprotective antigen of L.interrogans and can be used to develop an universal genetically engineering vaccine of Leptospira.

A DNA fragment of Leptospira interrogans encodes a protein which shares epitopes with equine cornea.

PubMed

Lucchesi, P M; Parma, A E

1999-11-30

Horses infected with Leptospira interrogans present several clinical disorders, one of them being recurrent uveitis. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. With the aim to make progress on defining the molecular basis and pathogenesis of equine recurrent uveitis, here we describe the cloning of one DNA fragment from a Leptospira interrogans serovar pomona genomic lambda gt11 library. Although there are references of transcription of leptospiral genes in E. coli from their own leptospiral promoters, in this recombinant construction the leptospiral DNA was located under the control of lacZ promoter since no expression could be detected in the absence of IPTG. This clone, isolated by expression screening with polyclonal serum raised against equine corneal proteins, encodes a 90 kDa protein of L. interrogans which crossreacts with equine cornea as proved Western-blotting. Antibodies directed against this leptospiral protein strongly recognised a 66 kDa equine corneal protein, one of those recognised by an anti-equine cornea serum. Our findings suggest that an immune response to 90 kDa protein participates in pathogenesis of equine uveitis.

In Vivo-Expressed Proteins of Virulent Leptospira interrogans Serovar Autumnalis N2 Elicit Strong IgM Responses of Value in Conclusive Diagnosis.

PubMed

Raja, Veerapandian; Shanmughapriya, Santhanam; Kanagavel, Murugesan; Artiushin, Sergey C; Velineni, Sridhar; Timoney, John F; Natarajaseenivasan, Kalimuthusamy

2016-01-01

Leptospirosis is a serious zoonosis that is underdiagnosed because of limited access to laboratory facilities in Southeast Asia, Central and South America, and Oceania. Timely diagnosis of locally distributed serovars of high virulence is crucial for successful care and outbreak management. Using pooled patient sera, an expression gene library of a virulent Leptospira interrogans serovar Autumnalis strain N2 isolated in South India was screened. The identified genes were characterized, and the purified recombinant proteins were used as antigens in IgM enzyme-linked immunosorbent assay (ELISA) either singly or in combination. Sera (n = 118) from cases of acute leptospirosis along with sera (n = 58) from healthy subjects were tested for reactivity with the identified proteins in an ELISA designed to detect specific IgM responses. We have identified nine immunoreactive proteins, ArgC, RecA, GlpF, FliD, TrmD, RplS, RnhB, Lp28.6, and Lrr44.9, which were found to be highly conserved among pathogenic leptospires. Apparently, the proteins ArgC, RecA, GlpF, FliD, TrmD, and Lrr44.9 are expressed during natural infection of the host and undetectable in in vitro cultures. Among all the recombinant proteins used as antigens in IgM ELISA, ArgC had the highest sensitivity and specificity, 89.8% and 95.5%, respectively, for the conclusive diagnosis of leptospirosis. The use of ArgC and RecA in combination for IgM ELISA increased the sensitivity and specificity to 95.7% and 94.9%, respectively. ArgC and RecA thus elicited specific IgM responses and were therefore effective in laboratory confirmation of Leptospira infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Immunological and molecular characterization of Leptospira interrogans isolated from a bovine foetus.

PubMed

Monte, Leonardo Garcia; Ridieri, Karine Forster; Jorge, Sérgio; Oliveira, Natasha Rodrigues; Hartwig, Daiane Drawanz; Amaral, Marta Gonçalves; Hartleben, Cláudia Pinho; Dellagostin, Odir Antonio

2015-06-01

Cattle are commonly infected with pathogenic leptospires, and similarly to rodents, they excrete the bacteria in their urine and can transmit the pathogen from animal to animal or animal to human. Thus, surveillance and monitoring systems for detection of new Leptospira serovars are important for the control of leptospirosis. Here, we report the isolation of a spirochete from a stillborn bovine foetus and its characterization by immunological and molecular techniques. A variable number tandem repeat profile using seven discriminatory primers identified the spirochete as belonging to species Leptospira interrogans serogroup Australis serovar Muenchen. A phenotypic analysis using monoclonal antibodies (mAbs) against leptospiral membrane-associated proteins confirmed the expression of important virulence and pathogenicity factors (LipL32 and LigBrep). Out of 120 reference sera tested, 22 positive (36.66%) and 9 negative (15%) also reacted with the new isolate. Furthermore, the serovar Muenchen isolate was virulent in hamster model. The animal inoculated developed acute lethal infection characterized by hepatic, pulmonary and renal lesions. Local isolates exhibited unique characteristics that differed from those of reference strains; therefore, isolation of leptospires is useful in the surveillance of local pathogenic serovars. In conclusion, the data obtained from this study can contribute to the epidemiological understanding and control of leptospirosis in southern Brazil. Copyright © 2015 Elsevier Ltd. All rights reserved.

Evaluation of a recombinant LigB protein of Leptospira interrogans serovar Canicola in an enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

PubMed

Sankar, Surya; Harshan, Hiron M; Somarajan, S R; Srivastava, S K

2010-06-01

A recombinant leptospiral lipoprotein, LigB, was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLigB IgG ELISA). The standard reference test (Microscopic agglutination test, MAT) of 200 serum samples from cattle suspected of leptospirosis showed that 95 (47.5%) samples had positive agglutination titres, which ranged from 100 to 1600. In rLigB IgG ELISA, 49% of the samples were positive. Sensitivity of IgG ELISA for 95 bovine sera, which had MAT titres of greater than or equal to 100, were 100%. ELISA showed a specificity of 97.1% with 105 bovine sera, which were negative at a 1:50 dilution in MAT for Leptospira interrogans serovars. The results of ELISA and MAT correspond very good. When analytical specificity of IgG ELISA was evaluated using bovine serum samples from animals showing the serum antibodies to other pathogens, no cross-reaction was observed. Thus the recombinant LigB IgG ELISA can be used instead of the MAT as an aid to the diagnosis of bovine leptospirosis. Copyright 2009. Published by Elsevier India Pvt Ltd.

Health assessment of wild lowland tapir (Tapirus terrestris) populations in the Atlantic Forest and Pantanal biomes, Brazil (1996-2012).

PubMed

Medici, Emília Patrícia; Mangini, Paulo Rogerio; Fernandes-Santos, Renata Carolina

2014-10-01

Abstract The lowland tapir (Tapirus terrestris) is found in South America and is listed as Vulnerable to Extinction by the International Union for Conservation of Nature, Red List of Threatened Species. Health issues, particularly infectious diseases, are potential threats for the species. Health information from 65 wild tapirs from two Brazilian biomes, Atlantic Forest (AF) and Pantanal (PA), were collected during a long-term study (1996-2012). The study included physic, hematologic and biochemical evaluations, microbiologic cultures, urinalysis, and serologic analyses for antibodies against 13 infectious agents (viral and bacterial). The AF and PA tapirs were significantly different for several hematologic and biochemical parameters. Ten bacteria taxa were identified in the AF and 26 in the PA. Antibodies against five viruses were detected: Bluetongue virus, eastern equine encephalitis virus, western equine encephalitis virus, infectious bovine rhinotracheitis virus, and porcine parvovirus. A high prevalence of exposure to Leptospira interrogans (10 serovars: Autumnalis, Bratislava, Canicola, Copenhageni, Grippotyphosa, Hardjo, Hebdomadis, Icterohaemorrhagiae, Pomona, and Pyrogenes) was detected in both the AF and PA sites. A greater diversity of serovars and higher antibody titers were found in the PA. Statistically significant differences between sites were found for L. interrogans, equine encephalitis virus, and porcine parvovirus. Based on physical evaluations, both AF and PA populations were healthy. The differences in the overall health profile of the AF and PA tapir populations appear to be associated with environmental factors and infectious diseases ecology. The extensive datasets on hematology, biochemistry, urinalysis, and microbiology results from this paper can be used as reference values for wild tapirs.

Presence of antigen and antibodies in serum and genital discharges of cows from dairy herds naturally infected with Leptospira interrogans serovar hardjo.

PubMed

Dhaliwal, G S; Murray, R D; Dobson, H; Montgomery, J; Ellis, W A

1996-03-01

Samples of cervico-vaginal mucus from 163 bulling cows (group 1) and post calving discharges from 59 newly calved cows (group 2) in five dairy herds naturally infected with Leptospira interrogans serovar hardjo were examined for the presence of antigen and IgG and IgA antibodies by using two ELISA systems which were protein or carbohydrate based. Corresponding serum samples were examined for systemic immune responses by using a microscopic agglutination test (MAT) and IgG-ELISA tests. Antigen was detected by direct immunofluorescence in six of the 163 samples of cervico-vaginal mucus. Both IgG and IgA antibodies were detected by ELISA in the genital discharges with a prevalence much higher than that obtained by the MAT but lower than that observed with the serum IgG-ELISA. Combining both groups, none of the MAT-positive cattle was negative by serum-ELISA. By using the protein or carbohydrate fraction serum IgG-ELISA assays, respectively, 29 or 41 per cent of the MAT-negative cows were positive at a titre of at least 1:40. Similarly, eight or 23 samples (10 or 27 per cent) had titres of at least 1:20 in the genital discharge ELISA for IgG and IgA antibodies, respectively. The serum IgG-ELISA was the most efficient in detecting hardjo antibodies, but in group 2 the IgG- and IgA-ELISA of the post calving discharge proved to be equally effective.

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment.

PubMed

Hu, Wei-Lin; Pappas, Christopher J; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie; Picardeau, Mathieu; Yang, X Frank

2017-02-01

Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans In this pathway, EbpA, a σ 54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ 54 ) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans IMPORTANCE: Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen achieves its

The EbpA-RpoN Regulatory Pathway of the Pathogen Leptospira interrogans Is Essential for Survival in the Environment

PubMed Central

Hu, Wei-Lin; Pappas, Christopher J.; Zhang, Jun-Jie; Yang, You-Yun; Yan, Jie

2016-01-01

ABSTRACT Leptospira interrogans is the agent of leptospirosis, a reemerging zoonotic disease. It is transmitted to humans through environmental surface waters contaminated by the urine of mammals chronically infected by pathogenic strains able to survive in water for long periods. Little is known about the regulatory pathways underlying environmental sensing and host adaptation of L. interrogans during its enzootic cycle. This study identifies the EbpA-RpoN regulatory pathway in L. interrogans. In this pathway, EbpA, a σ54 activator and putative prokaryotic enhancer-binding protein (EBP), and the alternative sigma factor RpoN (σ54) control expression of at least three genes, encoding AmtB (an ammonium transport protein) and two proteins of unknown function. Electrophoresis mobility shift assay demonstrated that recombinant RpoN and EbpA bind to the promoter region and upstream of these three identified genes, respectively. Genetic disruption of ebpA in L. interrogans serovar Manilae virtually abolished expression of the three genes, including amtB in two independent ebpA mutants. Complementation of the ebpA mutant restored expression of these genes. Intraperitoneal inoculation of gerbils with the ebpA mutant did not affect mortality. However, the ebpA mutant had decreased cell length in vitro and had a significantly lowered cell density at stationary phase when grown with l-alanine as the sole nitrogen source. Furthermore, the ebpA mutant has dramatically reduced long-term survival ability in water. Together, these studies identify a regulatory pathway, the EbpA-RpoN pathway, that plays an important role in the zoonotic cycle of L. interrogans. IMPORTANCE Leptospirosis is a reemerging disease with global importance. However, our understanding of gene regulation of the spirochetal pathogen Leptospira interrogans is still in its infancy, largely due to the lack of robust tools for genetic manipulation of this spirochete. Little is known about how the pathogen

Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans.

PubMed

Fujita, Rie; Koizumi, Nobuo; Sugiyama, Hiromu; Tomizawa, Rina; Sato, Ryoichi; Ohnishi, Makoto

2015-01-01

Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis

Comparison of Bacterial Burden and Cytokine Gene Expression in Golden Hamsters in Early Phase of Infection with Two Different Strains of Leptospira interrogans

PubMed Central

Fujita, Rie; Koizumi, Nobuo; Sugiyama, Hiromu; Tomizawa, Rina; Sato, Ryoichi; Ohnishi, Makoto

2015-01-01

Leptospirosis, a zoonotic infection with worldwide prevalence, is caused by pathogenic spirochaetes of Leptospira spp., and exhibits an extremely broad clinical spectrum in human patients. Although previous studies indicated that specific serovars or genotypes of Leptospira spp. were associated with severe leptospirosis or its outbreak, the mechanism underlying the difference in virulence of the various Leptospira serotypes or genotypes remains unclear. The present study addresses this question by measuring and comparing bacterial burden and cytokine gene expression in hamsters infected with strains of two L. interrogans serovars Manilae (highly virulent) and Hebdomadis (less virulent). The histopathology of kidney, liver, and lung tissues was also investigated in infected hamsters. A significantly higher bacterial burden was observed in liver tissues of hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.01). The average copy number of the leptospiral genome was 1,302 and 20,559 in blood and liver, respectively, of hamsters infected with serovar Manilae and 1,340 and 4,896, respectively, in hamsters infected with serovar Hebdomadis. The expression levels of mip1alpha in blood; tgfbeta, il1beta, mip1alpha, il10, tnfalpha and cox2 in liver; and tgfbeta, il6, tnfalpha and cox2 in lung tissue were significantly higher in hamsters infected with serovar Manilae than those infected with serovar Hebdomadis (p < 0.05). In addition, infection with serovar Manilae resulted in a significantly larger number of hamsters with tnfalpha upregulation (p = 0.04). Severe distortion of tubular cell arrangement and disruption of renal tubules in kidney tissues and hemorrhage in lung tissues were observed in Manilae-infected hamsters. These results demonstrate that serovar Manilae multiplied more efficiently in liver tissues and induced significantly higher expression of genes encoding pro- and anti-inflammatory cytokines than serovar Hebdomadis

Leptospira interrogans Binds to Cadherins

PubMed Central

Evangelista, Karen; Franco, Ricardo; Schwab, Andrew; Coburn, Jenifer

2014-01-01

Leptospirosis, caused by pathogenic species of Leptospira, is the most widespread zoonosis and has emerged as a major public health problem worldwide. The adhesion of pathogenic Leptospira to host cells, and to extracellular matrix (ECM) components, is likely to be necessary for the ability of leptospires to penetrate, disseminate and persist in mammalian host tissues. Previous work demonstrated that pathogenic L. interrogans binds to host cells more efficiently than to ECM. Using two independent screening methods, mass spectrometry and protein arrays, members of the cadherin family were identified as potential L. interrogans receptors on mammalian host surfaces. We focused our investigation on vascular endothelial (VE)-cadherin, which is widely expressed on endothelia and is primarily responsible for endothelial cell-cell adhesion. Monolayers of EA.hy926 and HMEC-1 endothelial cells produce VE-cadherin, bind L. interrogans in vitro, and are disrupted upon incubation with the bacteria, which may reflect the endothelial damage seen in vivo. Dose-dependent and saturable binding of L. interrogans to the purified VE-cadherin receptor was demonstrated and pretreatment of purified receptor or endothelial cells with function-blocking antibody against VE-cadherin significantly inhibited bacterial attachment. The contribution of VE-cadherin to leptospiral adherence to host endothelial cell surfaces is biologically significant because VE-cadherin plays an important role in maintaining the barrier properties of the vasculature. Attachment of L. interrogans to the vasculature via VE-cadherin may result in vascular damage, facilitating the escape of the pathogen from the bloodstream into different tissues during disseminated infection, and may contribute to the hemorrhagic manifestations of leptospirosis. This work is first to describe a mammalian cell surface protein as a receptor for L. interrogans. PMID:24498454

Identification of a novel prophage-like gene cluster actively expressed in both virulent and avirulent strains of Leptospira interrogans serovar Lai.

PubMed

Qin, Jin-Hong; Zhang, Qing; Zhang, Zhi-Ming; Zhong, Yi; Yang, Yang; Hu, Bao-Yu; Zhao, Guo-Ping; Guo, Xiao-Kui

2008-06-01

DNA microarray analysis was used to compare the differential gene expression profiles between Leptospira interrogans serovar Lai type strain 56601 and its corresponding attenuated strain IPAV. A 22-kb genomic island covering a cluster of 34 genes (i.e., genes LA0186 to LA0219) was actively expressed in both strains but concomitantly upregulated in strain 56601 in contrast to that of IPAV. Reverse transcription-PCR assays proved that the gene cluster comprised five transcripts. Gene annotation of this cluster revealed characteristics of a putative prophage-like remnant with at least 8 of 34 sequences encoding prophage-like proteins, of which the LA0195 protein is probably a putative prophage CI-like regulator. The transcription initiation activities of putative promoter-regulatory sequences of transcripts I, II, and III, all proximal to the LA0195 gene, were further analyzed in the Escherichia coli promoter probe vector pKK232-8 by assaying the reporter chloramphenicol acetyltransferase (CAT) activities. The strong promoter activities of both transcripts I and II indicated by the E. coli CAT assay were well correlated with the in vitro sequence-specific binding of the recombinant LA0195 protein to the corresponding promoter probes detected by the electrophoresis mobility shift assay. On the other hand, the promoter activity of transcript III was very low in E. coli and failed to show active binding to the LA0195 protein in vitro. These results suggested that the LA0195 protein is likely involved in the transcription of transcripts I and II. However, the identical complete DNA sequences of this prophage remnant from these two strains strongly suggests that possible regulatory factors or signal transduction systems residing outside of this region within the genome may be responsible for the differential expression profiling in these two strains.

Serovar prevalence of Leptospira in semirural India and the development of an IgM-based indirect ELISA.

PubMed

Chandy, Sara; Kirubanandhan, Lokeshwaran; Hemavathy, Priya; Khadeeja, Anees Mohammad; Kurian, Siby Jacob; Venkataraman, Krishnan; Mørch, Kristine; Mathai, Dilip; Manoharan, Anand

2017-03-31

Leptospirosis is a major public health problem in India. However, it has been underreported and under-diagnosed due to a lack of awareness of the disease, a functional surveillance system, and appropriate laboratory diagnostic facilities. This multicenter study aimed to understand the Leptospira serovars causing leptospirosis in seven secondary-level hospitals in six states in India. Since early and accurate diagnosis of leptospirosis is one of the challenges faced by clinicians in India due to the poor specificity and sensitivity of commercially available diagnostic systems, an in-house indirect enzyme-linked immunosorbent assay (ELISA) was developed. Genomic DNA from L. interrogans serovar Canicola was used for polymerase chain reaction amplification, cloning, and expression of the lipL32 gene in E. coli to amplify, clone, and express the lipL32 gene. Australis was the common serovar seen at all the study centers. Serovar Icterohaemorrhagiae was seen in samples from Tamil Nadu and Assam. In-house ELISA was standardized using the purified recombinant LipL32 polypeptide and was used to evaluate serum. Subsequently, acute serum samples from leptospirosis patients (n = 60) were screened. Compared to the gold standard, the microscopic agglutination test, sensitivity and specificity of the in-house ELISA was 95% and 90%, respectively. Understanding Leptospira serovars circulating in leptospirosis-endemic areas will help to formulate better vaccines. LipL32-based ELISA may serve as a valuable tool for early diagnosis of leptospirosis.

MHC class II DRB diversity predicts antigen recognition and is associated with disease severity in California sea lions naturally infected with Leptospira interrogans

USGS Publications Warehouse

Acevedo-Whitehouse, Karina; Gulland, Frances; Bowen, Lizabeth

2018-01-01

We examined the associations between California sea lion MHC class II DRB (Zaca-DRB) configuration and diversity, and leptospirosis. As Zaca-DRB gene sequences are involved with antigen presentation of bacteria and other extracellular pathogens, we predicted that they would play a role in determining responses to these pathogenic spirochaetes. Specifically, we investigated whether Zaca-DRB diversity (number of genes) and configuration (presence of specific genes) explained differences in disease severity, and whether higher levels of Zaca-DRB diversity predicted the number of specific Leptospira interrogans serovars that a sea lion's serum would react against. We found that serum from diseased sea lions with more Zaca-DRB loci reacted against a wider array of serovars. Specific Zaca-DRB loci were linked to reactions with particular serovars. Interestingly, sea lions with clinical manifestation of leptospirosis that had higher numbers of Zaca-DRB loci were less likely to recover from disease than those with lower diversity, and those that harboured Zaca-DRB.C or –G were 4.5 to 5.3 times more likely to die from leptospirosis, regardless of the infective serovars. We propose that for leptospirosis, a disadvantage of having a wider range of antigen presentation might be increased disease severity due to immunopathology. Ours is the first study to examine the importance of Zaca-DRB diversity for antigen detection and disease severity following natural exposure to infective leptospires.

[Detection of leptospira by culture of vitreous humor and detection of antibodies against leptospira in vitreous humor and serum of 225 horses with equine recurrent uveitis].

PubMed

Dorrego-Keiter, Elisa; Tóth, József; Dikker, Lieke; Sielhorst, Jutta; Schusser, Gerald Fritz

2016-01-01

In the ongoing discussion regarding the aetiopathogenesis of equine recurrent uveitis (ERU) it was the aim of the present study to elucidate the relationship of leptospira infection and ERU. In a population of 225 horses leptospira were examined in vitreous humor by culture and leptospira antibody were detected in vitreous humor and serum samples. Preoperative serum samples were collected from 221/225 ERU patients of different age, gender and breed. Undiluted vitreous humor was aseptically taken from 198/225 patients that underwent pars plana vitrectomy at the beginning of surgery and from 27/225 patients' eyeball after enucleation: Serum and vitreous humor were tested for specific leptospiral antibodies by microscopic agglutination test (MAT). Furthermore, vitreous humor was examined by culture. 20 patients which were euthanized due to a live-threatening disease other than ERU served as a control group. A total of 127/221 (57.5%) horses had serum antibodies (≥ 1:100). Most frequently antibodies against L. interrogans serovar Grippotyphosa were detected (79/127), followed by L. interrogans serovar lcterohaemorrhagiae (34/127) and L. interrogans serovar Bratislava (29/127). Only 79/225 horses (35.1%) had leptospiral antibodies in vitreous humor, in which L. interrogans serovar Grippotyphosa (67/79) was identified most frequently followed by L. interrogans serovar Pomona (18/79) and L. interrogans serovar lcterohaemorrhagiae (8/79) which was identified as single or multiple reaction. Isolation of leptospira from vitreous humor was positive in 34/212 horses (16%). 10/20 control horses had a positive antibody titer against leptospira in serum and 2/20 horses in vitreous humor, whereas there was no leptospira detected in culture. The result of 84% negative cultures from vitreous humor of 212 ERU patients is decisive for the diagnosis and therapy of ERU.

[Expression changes of major outer membrane protein antigens in Leptospira interrogans during infection and its mechanism].

PubMed

Zheng, Linli; Ge, Yumei; Hu, Weilin; Yan, Jie

2013-03-01

To determine expression changes of major outer membrane protein(OMP) antigens of Leptospira interrogans serogroup Icterohaemorrhagiae serovar Lai strain Lai during infection of human macrophages and its mechanism. OmpR encoding genes and OmpR-related histidine kinase (HK) encoding gene of L.interrogans strain Lai and their functional domains were predicted using bioinformatics technique. mRNA level changes of the leptospiral major OMP-encoding genes before and after infection of human THP-1 macrophages were detected by real-time fluorescence quantitative RT-PCR. Effects of the OmpR-encoding genes and HK-encoding gene on the expression of leptospiral OMPs during infection were determined by HK-peptide antiserum block assay and closantel inhibitive assays. The bioinformatics analysis indicated that LB015 and LB333 were referred to OmpR-encoding genes of the spirochete, while LB014 might act as a OmpR-related HK-encoding gene. After the spirochete infecting THP-1 cells, mRNA levels of leptospiral lipL21, lipL32 and lipL41 genes were rapidly and persistently down-regulated (P <0.01), whereas mRNA levels of leptospiral groEL, mce, loa22 and ligB genes were rapidly but transiently up-regulated (P<0.01). The treatment with closantel and HK-peptide antiserum partly reversed the infection-based down-regulated mRNA levels of lipL21 and lipL48 genes (P <0.01). Moreover, closantel caused a decrease of the infection-based up-regulated mRNA levels of groEL, mce, loa22 and ligB genes (P <0.01). Expression levels of L.interrogans strain Lai major OMP antigens present notable changes during infection of human macrophages. There is a group of OmpR-and HK-encoding genes which may play a major role in down-regulation of expression levels of partial OMP antigens during infection.

Isolation and molecular characterization of Leptospira borgpetersenii serovar Hardjo strain Hardjobovis in the urine of naturally infected cattle in Brazil.

PubMed

Chideroli, R T; Pereira, U P; Gonçalves, D D; Nakamura, A Y; Alfieri, A A; Alfieri, A F; Freitas, J C

2016-02-19

Most epidemiologic studies on bovine leptospirosis are based on serological tests that use antibodies against several serotypes, including the serovar Hardjo, which is widespread and considered to be the most adapted to bovine hosts. However, using only serological studies is not sufficient to identify and distinguish species of leptospires. The aim of this study was report the first isolation in Brazil of two strains serovar Hardjo obtained in urine samples from naturally infected cows in a small Brazilian dairy herd and find the genetic species and consequently the type strain Hardjobovis by molecular characterization. Fifteen dairy cows with a history of reproductive failure, such as abortion and infertility, were selected. Urine samples obtained from each animal were immediately seeded in tubes containing Ellinghausen-McCullough-Johnson-Harris culture medium. The identification of the isolates was performed by Multilocus variable-number tandem-repeat analysis (MLVA) technique and phylogenetic analysis of partial sequence of gene sec Y. From the 15 urine samples evaluated, two Leptospira were found and identified as the Londrina 49 and Londrina 54 strains. The MLVA profiles and sequencing of gene sec Y characterized the isolates as L. borgpetersenii serovar Hardjo strain Hadjobovis because it has different genetic pattern of Leptospira interrogans serovar Hardjo strain Hardjoprajitno. Therefore, more studies are needed including isolation and molecular characterization from regional strains to obtain a better knowledge about epidemiology of serovar Hardjo in bovine which may assist in future strategies of prevention and control of bovine leptospirosis.

Safety and efficacy of a new octavalent combined Erysipelas, Parvo and Leptospira vaccine in gilts against Leptospira interrogans serovar Pomona associated disease and foetal death.

PubMed

Jacobs, A A C; Harks, F; Hoeijmakers, M; Collell, M; Segers, R P A M

2015-07-31

The safety and protective efficacy of a new octavalent combination vaccine containing inactivated Erysipelothrix rhusiopathiae, Parvovirus, and Leptospira interrogans (sensu lato) serogroups Canicola, Icterohaemorrhagiae, Australis (Bratislava), Grippotyphosa, Pomona and Tarassovi - Porcilis(®) Ery+Parvo+Lepto - was evaluated in laboratory studies and under field conditions. The safety (2× overdose and repeated dose) was tested in 26 gilts. In this study, neither vaccine related temperature increase nor other systemic reactions were observed after intramuscular vaccination. No local reactions were observed except for one animal that had a small local reaction (2cm diameter) that lasted for 5 days after the third vaccination. Efficacy was tested in 40 gilts. A group of 20 gilts was vaccinated at 20 and 24 weeks of age with Porcilis(®) Ery+Parvo+Lepto and a group of 20 age- and source-matched animals served as the control group. The gilts were inseminated at 41 weeks or 66 weeks of age and were challenged with serovar Pomona 10 weeks after insemination, corresponding to 6 months (n=2×10) and 12 months (n=2×10) after the last vaccination. After both the 6- and 12-month challenges the control animals developed clinical signs (fever, lethargy and anorexia) and leptospiraemia as determined by positive blood culture. In addition, both the 6- and 12-month challenges resulted in death of 21% and 27% of the total number of foetuses in the control groups, respectively. Clinical signs and leptospiraemia were statistically significantly lower in vaccinated gilts after both the 6- and 12-month challenges. In addition, foetal death was statistically significantly lower (3% and 2%, respectively) in vaccinated gilts after both the 6- and 12 month challenges. The vaccine was tested further under field conditions on a Portuguese farm with a history of an increasing abortion rate associated with a Leptospira serovar Pomona infection (confirmed by PCR and serology). This study was

Pathogenomic Inference of Virulence-Associated Genes in Leptospira interrogans

PubMed Central

Lehmann, Jason S.; Fouts, Derrick E.; Haft, Daniel H.; Cannella, Anthony P.; Ricaldi, Jessica N.; Brinkac, Lauren; Harkins, Derek; Durkin, Scott; Sanka, Ravi; Sutton, Granger; Moreno, Angelo; Vinetz, Joseph M.; Matthias, Michael A.

2013-01-01

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens. PMID:24098822

Pathogenomic inference of virulence-associated genes in Leptospira interrogans.

PubMed

Lehmann, Jason S; Fouts, Derrick E; Haft, Daniel H; Cannella, Anthony P; Ricaldi, Jessica N; Brinkac, Lauren; Harkins, Derek; Durkin, Scott; Sanka, Ravi; Sutton, Granger; Moreno, Angelo; Vinetz, Joseph M; Matthias, Michael A

2013-01-01

Leptospirosis is a globally important, neglected zoonotic infection caused by spirochetes of the genus Leptospira. Since genetic transformation remains technically limited for pathogenic Leptospira, a systems biology pathogenomic approach was used to infer leptospiral virulence genes by whole genome comparison of culture-attenuated Leptospira interrogans serovar Lai with its virulent, isogenic parent. Among the 11 pathogen-specific protein-coding genes in which non-synonymous mutations were found, a putative soluble adenylate cyclase with host cell cAMP-elevating activity, and two members of a previously unstudied ∼15 member paralogous gene family of unknown function were identified. This gene family was also uniquely found in the alpha-proteobacteria Bartonella bacilliformis and Bartonella australis that are geographically restricted to the Andes and Australia, respectively. How the pathogenic Leptospira and these two Bartonella species came to share this expanded gene family remains an evolutionary mystery. In vivo expression analyses demonstrated up-regulation of 10/11 Leptospira genes identified in the attenuation screen, and profound in vivo, tissue-specific up-regulation by members of the paralogous gene family, suggesting a direct role in virulence and host-pathogen interactions. The pathogenomic experimental design here is generalizable as a functional systems biology approach to studying bacterial pathogenesis and virulence and should encourage similar experimental studies of other pathogens.

Prevalence of leptospirosis in dairy cattle from small rural production units in Toluca Valley, State of Mexico.

PubMed

Leon, L L; Garcia, R C; Diaz, C O; Valdez, R B; Carmona, G C A; Velazquez, B L G

2008-12-01

In order to know the seroprevalence of Leptospira spp. in stabled dairy cattle, a study was conducted from 2004 to 2006 in which 416 sera were tested using a microscopic agglutination test conducted on microplates. A collection of culture reference antigens, each representing a serogroup, was used for these tests. Results showed that 10.33% (43) of the animals had antibody titers ranging from 1:100 to 1:1600. The main serovars detected in these tests were L. interrogans serovar hardjo and L. interrogans serovar canicola. It is important to note that these serovars represent a high risk for transmission to other susceptible animal species, between individuals, and to human health. This serological survey provides useful information establishing the presence or absence of these serovars in this type of herd. The range of antigens used in this study included serovars representative of all common serogroups.

Isolation and characterization of Leptospira interrogans from pigs slaughtered in São Paulo State, Brazil

PubMed Central

Miraglia, Fabiana; Moreno, Andréa Mike; Gomes, Cleise Ribeiro; Paixão, Renata; Liuson, Esequiel; Morais, Zenaide Maria; Maiorka, Paulo; Seixas, Fabiana Kömmling; Dellagostin, Odir Antonio; Vasconcellos, Silvio Arruda

2008-01-01

With the aim of isolating Leptospira spp., blood serum, kidney, liver and genital tract of 137 female swine (40 sows and 97 gilts) and also urine samples from 22 sows were collected in a slaughterhouse in the State of São Paulo, from April 2003 to August 2004. Four isolates were obtained from animals that presented microagglutination test (MAT) titers ≥ 100 for the serovar Pomona and one was obtained from an animal negative by MAT in which Leptospira was isolated from the liver and reproductive tract. The presence of leptospiral DNA was investigated by PCR, and positive results were found in kidneys of 11 females, liver of two, genital tract of two and urine of one of them. Nephrosis, interstitial multifocal nephritis, moderate to severe changing, hyalines cylinders and hemorrhagic focuses, hepatic and uterine horns congestion were histological lesions observed in higher frequency in animals positive for leptospira. The silver impregnation (Warthin Starry) confirmed the presence of spirochetes in renal tubules of four females with positive leptospira cultures from kidneys. The serogroup of the five isolates was identified as Pomona by cross agglutination with reference polyclonal antibodies. Molecular characterization of the isolates was carried out by variable-number tandem-repeats analysis. All the isolates revealed a pattern distinct from the L. interrogans Pomona type strain, but identical to a previously identified pattern from strains isolated in Argentina belonging to serovar Pomona. PMID:24031254

Investigation of infectious reproductive pathogens of large ruminants: Are neosporosis, brucellosis, leptospirosis and BVDV of relevance in Lao PDR?

PubMed

Olmo, L; Dye, M T; Reichel, M P; Young, J R; Nampanya, S; Khounsy, S; Thomson, P C; Windsor, P A; Bush, R D

2018-01-01

N. caninum, bovine viral diarrhoea virus, Brucella abortus and Leptospira interrogans serovar Hardjo are globally significant reproductive pathogens that cause abortion and reproductive loss in large ruminants. Prevalence information is lacking in Lao People's Democratic Republic (Laos) despite the poor reproductive performance of cattle and buffalo. Serological examination of frozen cattle (n=90) and buffalo (n=61) sera by commercially available enzyme-linked immunosorbent assays provided the first reported screening of some of these pathogens in Laos. Seroprevalence differed amongst these large ruminant species, with N. caninum, BVDV and L. interrogans serovar Hardjo antibodies found in 68.9% (95% CI±11.6), 4.9% (95% CI±5.4) and 3.3% (95% CI±4.5) of buffalo sera, respectively, and in 7.8% (95% CI±5.5), 10.0% (95% CI±6.2) and 22.2% (95% CI±8.6) of cattle sera, respectively. Buffalo sera had a significantly higher seroprevalence of N. caninum compared to cattle (p<0.001) and cattle sera had a significantly higher seroprevalence of L. interrogans serovar Hardjo compared to buffalo (p=0.003). Variability was also observed across provinces for N. caninum in buffalo (p=0.007) and for L. interrogans serovar Hardjo in cattle (p=0.071), suggesting provincial risk factors conducive to pathogen transmission. BVDV and N. caninum seropositivity were negatively associated in buffalo (p=0.018) and cattle (p=0.003). In buffalo, L. interrogans serovar Hardjo and BVDV seropositivity were associated (p=0.035, p=0.039). The identification of antibodies against three major abortifacient pathogens in Laos prompts further research to determine if infection is associated with low reproductive efficiency and the risk factors for infection. This is needed for the development of evidence based prevention strategies for improved large ruminant reproductive management among smallholders in Laos. Copyright © 2017 Elsevier B.V. All rights reserved.

Lsa63, a newly identified surface protein of Leptospira interrogans binds laminin and collagen IV.

PubMed

Vieira, Monica L; de Morais, Zenaide M; Gonçales, Amane P; Romero, Eliete C; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease that affects populations worldwide. We have identified in proteomic studies a protein that is encoded by the gene LIC10314 and expressed in virulent strain of L. interrogans serovar Pomona. This protein was predicted to be surface exposed by PSORT program and contains a p83/100 domain identified by BLAST analysis that is conserved in protein antigens of several strains of Borrelia and Treponema spp. The proteins containing this domain have been claimed antigen candidates for serodiagnosis of Lyme borreliosis. Thus, we have cloned the LIC10314 and expressed the protein in Escherichia coli BL21-SI strain by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. This protein is conserved among several species of pathogenic Leptospira and absent in the saprophytic strain L. biflexa. We confirm by liquid-phase immunofluorescence assays with living organisms that this protein is most likely a new surface leptospiral protein. The ability of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC10314, named Lsa63 (Leptospiral surface adhesin of 63kDa), binds strongly to laminin and collagen IV in a dose-dependent and saturable fashion. In addition, Lsa63 is probably expressed during infection since it was recognized by antibodies of serum samples of confirmed-leptospirosis patients in convalescent phase of the disease. Altogether, the data suggests that this novel identified surface protein may be involved in leptospiral pathogenesis. 2009 The British Infection Society. Published by Elsevier Ltd. All rights reserved.

Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans.

PubMed Central

Ellis, W A; Montgomery, J M; Thiermann, A B

1991-01-01

Restriction endonuclease analysis was performed on DNAs from the type strains of the Australis serogroup of Leptospira interrogans by using 20 restriction enzymes, and the electrophoretic patterns obtained were compared with patterns obtained from 162 Australis serogroup isolates from pigs. It proved to be a quick and reliable method for typing such strains. All of the pig isolates were identified as either serovar bratislava or muenchen. It also showed differences at the subserovar level which may be important in (i) understanding the epidemiology of the Australis serogroup, (ii) the development of suitable vaccines, and (iii) pathogenesis and pathogenicity studies. Two genotypes (B2b and M2) accounted for 92% of isolates from aborted or stillborn piglets, while a third genotype (B2a) was the only one recovered from the brains of piglets with meningitis. Images PMID:1647408

Restriction endonuclease analysis as a taxonomic tool in the study of pig isolates belonging to the Australis serogroup of Leptospira interrogans.

PubMed

Ellis, W A; Montgomery, J M; Thiermann, A B

1991-05-01

Restriction endonuclease analysis was performed on DNAs from the type strains of the Australis serogroup of Leptospira interrogans by using 20 restriction enzymes, and the electrophoretic patterns obtained were compared with patterns obtained from 162 Australis serogroup isolates from pigs. It proved to be a quick and reliable method for typing such strains. All of the pig isolates were identified as either serovar bratislava or muenchen. It also showed differences at the subserovar level which may be important in (i) understanding the epidemiology of the Australis serogroup, (ii) the development of suitable vaccines, and (iii) pathogenesis and pathogenicity studies. Two genotypes (B2b and M2) accounted for 92% of isolates from aborted or stillborn piglets, while a third genotype (B2a) was the only one recovered from the brains of piglets with meningitis.

Leptospira Immunoglobulin-Like Proteins as a Serodiagnostic Marker for Acute Leptospirosis▿

PubMed Central

Croda, Julio; Ramos, João G. R.; Matsunaga, James; Queiroz, Adriano; Homma, Akira; Riley, Lee W.; Haake, David A.; Reis, Mitermayer G.; Ko, Albert I.

2007-01-01

There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease. PMID:17360842

Leptospira immunoglobulin-like proteins as a serodiagnostic marker for acute leptospirosis.

PubMed

Croda, Julio; Ramos, João G R; Matsunaga, James; Queiroz, Adriano; Homma, Akira; Riley, Lee W; Haake, David A; Reis, Mitermayer G; Ko, Albert I

2007-05-01

There is an urgent need for improved diagnosis of leptospirosis, an emerging infectious disease which imparts a large disease burden in developing countries. We evaluated the use of Leptospira immunoglobulin (Ig)-like (Lig) proteins as a serodiagnostic marker for leptospirosis. Lig proteins have bacterial immunoglobulin-like (Big) tandem repeat domains, a moiety found in virulence factors in other pathogens. Sera from patients identified during urban outbreaks in Brazil reacted strongly with immunoblots of a recombinant fragment comprised of the second to sixth Big domains of LigB from L. interrogans serovar Copenhageni, the principal agent for transmission in this setting. Furthermore, the sera recognized an analogous LigB fragment derived from L. kirschneri serovar Grippotyphosa, a pathogenic serovar which is not endemic to the study area. The immunoblot assay detected anti-LigB IgM antibodies in sera from 92% (95% confidence interval, 85 to 96%) of patients during acute-phase leptospirosis. The assay had a sensitivity of 81% for sera from patients with less than 7 days of illness. Anti-LigB antibodies were found in sera from 57% of the patients who did not have detectable anti-whole-Leptospira responses as detected by IgM enzyme-linked immunosorbent assay and microagglutination test. The specificities of the assay were 93 to 100% and 90 to 97% among sera from healthy individuals and patients with diseases that have clinical presentations that overlap with those of leptospirosis, respectively. These findings indicate that the antibody response to this putative virulence determinant is a sensitive and specific marker for acute infection. The use of this marker may aid the prompt and timely diagnosis required to reduce the high mortality associated with severe forms of the disease.

Leptospira interrogans induces uterine inflammatory responses and abnormal expression of extracellular matrix proteins in dogs.

PubMed

Wang, Wei; Gao, Xuejiao; Guo, Mengyao; Zhang, Wenlong; Song, Xiaojing; Wang, Tiancheng; Zhang, Zecai; Jiang, Haichao; Cao, Yongguo; Zhang, Naisheng

2014-10-01

Leptospira interrogans (L. interrogans), a worldwide zoonosis, infect humans and animals. In dogs, four syndromes caused by leptospirosis have been identified: icteric, hemorrhagic, uremic (Stuttgart disease) and reproductive (abortion and premature or weak pups), and also it caused inflammation. Extracellular matrix (ECM) is a complex mixture of matrix molecules that is crucial to the reproduction. Both inflammatory response and ECM are closed relative to reproductive. The aim of this study was to clarify how L. interrogans affected the uterus of dogs, by focusing on the inflammatory responses, and ECM expression in dogs uterine tissue infected by L. interrogans. In the present study, 27 dogs were divided into 3 groups, intrauterine infusion with L. interrogans, to make uterine infection, sterile EMJH, and normal saline as a control, respectively. The uteruses were removed by surgical operation in 10, 20, and 30 days, respectively. The methods of histopathological analysis, ELISA, Western blot and qPCR were used. The results showed that L. interrogans induced significantly inflammatory responses, which were characterized by inflammatory cellular infiltration and high expression levels of tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6) in uterine tissue of these dogs. Furthermore, L. interrogans strongly down-regulated the expression of ECM (collagens (CL) IV, fibronectins (FN) and laminins (LN)) in mRNA and protein levels. These data indicated that strongly inflammatory responses, and abnormal regulation of ECM might contribute to the proliferation of dogs infected by L. interrogans. Copyright © 2014 Elsevier Ltd. All rights reserved.

Ecology of Leptospira interrogans in Norway rats (Rattus norvegicus) in an inner-city neighborhood of Vancouver, Canada.

PubMed

Himsworth, Chelsea G; Bidulka, Julie; Parsons, Kirbee L; Feng, Alice Y T; Tang, Patrick; Jardine, Claire M; Kerr, Thomas; Mak, Sunny; Robinson, John; Patrick, David M

2013-01-01

Leptospira interrogans is a bacterial zoonosis with a worldwide distribution for which rats (Rattus spp.) are the primary reservoir in urban settings. In order to assess, monitor, and mitigate the risk to humans, it is important to understand the ecology of this pathogen in rats. The objective of this study was to characterize the ecology of L. interrogans in Norway rats (Rattus norvegicus) in an impoverished inner-city neighborhood of Vancouver, Canada. Trapping was performed in 43 city blocks, and one location within the adjacent port, over a 12 month period. Kidney samples were tested for the presence of L. interrogans using PCR and sequencing. A multivariable model was built to predict L. interrogans infection status in individual rats using season and morphometric data (e.g., weight, sex, maturity, condition, etc.) as independent variables. Spatial analysis was undertaken to identify clusters of high and low L. interrogans prevalence. The prevalence of L. interrogans varied remarkably among blocks (0-66.7%), and spatial clusters of both high and low L. interrogans prevalence were identified. In the final cluster-controlled model, characteristics associated with L. interrogans-infection in rats included weight (OR = 1.14, 95% CI = 1.07-1.20), increased internal fat (OR = 2.12, 95% CI = 1.06-4.25), and number of bite wounds (OR = 1.20, 95% CI = 0.96-1.49). Because L. interrogans prevalence varied with weight, body fat, and bite wounds, this study suggests that social structure and interactions among rats may influence transmission. The prevalence and distribution of L. interrogans in rats was also highly variable even over a short geographic distance. These factors should be considered in future risk management efforts.

Effect of Leptospira interrogans outer membrane proteins LipL32 on HUVEC.

PubMed

Sun, Zhan; Bao, Lang; Li, DaoKun; Huang, Bi; Wu, Bingting

2010-09-01

Leptospira cause disease through a toxin-mediated process by inducing vascular injury, particularly a small-vessel vasculitis. Breakdown of vessel endothelial cell integrity may increase vessel permeability which is correlated with the changes of tight junction and/or apoptosis in vessel endothelial cells. The specific toxin responsible remains unidentified. In this study, we amplified outer membrane protein LipL32 from the genome of Leptospira interrogans serovar Lai, and it was subcloned in pET32a(+) vector to express thioredoxin(Trx)-LipL32 fusion protein in Escherichia coli BL21(DE3). The protein was expressed and purified, and Trx-LipL32 was administered to culture with human umbilical vein endothelial cells (HUVEC) to elucidate the role of leptospiral outer membrane proteins in vessel endothelial cell. The purified recombinant protein was capable to increase the permeability of HUVECs. And the protein was able to decrease the expression of ZO-1 and induce F-actin in HUVECs display thickening and clustering. Moreover, apoptosis of HUVEC was significantly accelerated. But the fusion partner had no effect in these regards. It is possible that LipL32 is involved in the vessel lesions. Copyright 2010 Elsevier Ltd. All rights reserved.

Prevalence of antibodies against Leptospira sp. in snakes, lizards and turtles in Slovenia

PubMed Central

2013-01-01

Background Leptospiral infections in poikilothermic (cold blooded) animals have received very little attention and the literature concerning natural infections of these animals is limited. The aim of this study was to determine the prevalence of leptospiral antibodies in reptiles, imported into Slovenia and intended to be pets in close contact with humans. A total of 297 reptiles (22 snakes, 210 lizards and 65 turtles) were tested for specific antibodies against serovars of Leptospira interrogans sensu stricto using the microscopic agglutination test (MAT). Live cultures of different serovars were used as antigens. MAT was performed according to standard procedures and the degree of reaction was interpreted by estimating the percentage of agglutinated leptospires. Samples showing titres of ≥ 50 against one or more serovars were considered as positive. Results Antibodies against seven pathogenic serovars of L. interrogans sensu stricto were detected in 46 of 297 reptiles. Among 22 snakes, specific antibodies against pathogenic serovars of three Leptospira species (L. interrogans, L. kirschneri and L. borgpetersenii) at titre levels from 1:50 to 1:400 were detected in 6 snakes. In 31 of 210 lizards, specific antibodies were found in titres from 1:50 to 1:1000 and, finally, among 65 turtles (terrapins and tortoises), 9 had specific antibodies at titre levels between 1:50 and 1:1600. Animals imported from non-EU countries showed significantly higher prevalence (25.0%; 95 confidence interval: 16.7–33.3%) than animals from EU member states (10.4%; confidence interval: 6.1–14.7%). Conclusions Reptiles may be considered as potential reservoirs of L. interrogans sensu stricto. Origin of the animals is a risk factor for presence of leptospiral antibodies, especially in lizards. Special attention should be focused on animals from non-EU member states. PMID:24020619

Ecology of Leptospira interrogans in Norway Rats (Rattus norvegicus) in an Inner-City Neighborhood of Vancouver, Canada

PubMed Central

Himsworth, Chelsea G.; Bidulka, Julie; Parsons, Kirbee L.; Feng, Alice Y. T.; Tang, Patrick; Jardine, Claire M.; Kerr, Thomas; Mak, Sunny; Robinson, John; Patrick, David M.

2013-01-01

Background Leptospira interrogans is a bacterial zoonosis with a worldwide distribution for which rats (Rattus spp.) are the primary reservoir in urban settings. In order to assess, monitor, and mitigate the risk to humans, it is important to understand the ecology of this pathogen in rats. The objective of this study was to characterize the ecology of L. interrogans in Norway rats (Rattus norvegicus) in an impoverished inner-city neighborhood of Vancouver, Canada. Methodology/Principal Findings Trapping was performed in 43 city blocks, and one location within the adjacent port, over a 12 month period. Kidney samples were tested for the presence of L. interrogans using PCR and sequencing. A multivariable model was built to predict L. interrogans infection status in individual rats using season and morphometric data (e.g., weight, sex, maturity, condition, etc.) as independent variables. Spatial analysis was undertaken to identify clusters of high and low L. interrogans prevalence. The prevalence of L. interrogans varied remarkably among blocks (0–66.7%), and spatial clusters of both high and low L. interrogans prevalence were identified. In the final cluster-controlled model, characteristics associated with L. interrogans-infection in rats included weight (OR = 1.14, 95% CI = 1.07–1.20), increased internal fat (OR = 2.12, 95% CI = 1.06–4.25), and number of bite wounds (OR = 1.20, 95% CI = 0.96–1.49). Conclusions/Significance Because L. interrogans prevalence varied with weight, body fat, and bite wounds, this study suggests that social structure and interactions among rats may influence transmission. The prevalence and distribution of L. interrogans in rats was also highly variable even over a short geographic distance. These factors should be considered in future risk management efforts. PMID:23818996

Molecular modeling and in-silico engineering of Cardamom mosaic virus coat protein for the presentation of immunogenic epitopes of Leptospira LipL32.

PubMed

Kumar, Vikram; Damodharan, S; Pandaranayaka, Eswari P J; Madathiparambil, Madanan G; Tennyson, Jebasingh

2016-01-01

Expression of Cardamom mosaic virus (CdMV) coat protein (CP) in E. coli forms virus-like particles. In this study, the structure of CdMV CP was predicted and used as a platform to display epitopes of the most abundant surface-associated protein, LipL32 of Leptospira at C, N, and both the termini of CdMV CP. In silico, we have mapped sequential and conformational B-cell epitopes from the crystal structure of LipL32 of Leptospira interrogans serovar Copenhageni str. Fiocruz L1-130 using IEDB Elipro, ABCpred, BCPRED, and VaxiJen servers. Our results show that the epitopes displayed at the N-terminus of CdMV CP are promising vaccine candidates as compared to those displayed at the C-terminus or at both the termini. LipL32 epitopes, EP2, EP3, EP4, and EP6 are found to be promising B-cell epitopes for vaccine development. Based on the type of amino acids, length, surface accessibility, and docking energy with CdMV CP model, the order of antigenicity of the LipL32 epitopes was found to be EP4 > EP3 > EP2 > EP6.

Cross-Species Surveillance of Leptospira in Domestic and Peri-Domestic Animals in Mahalla City, Gharbeya Governorate, Egypt

PubMed Central

Felt, Stephen A.; Wasfy, Momtaz O.; El-Tras, Wael F.; Samir, Ahmed; Rahaman, Bassem Abdel; Boshra, Marie; Parker, Tina M.; Hatem, Mahmoud Essam; El-Bassiouny, Ahmed Ahmed; Murray, Clinton K.; Pimentel, Guillermo

2011-01-01

A survey of 179 animals (black rats, dogs, sheep, buffaloes, cattle, donkeys, weasels, and cats) for Leptospira infection was conducted in Mahalla City (Lower Egypt). Blood, urine, and kidney were collected and tested by culture, microscopic agglutination test (MAT), and/or polymerase chain reaction (PCR). Among rats, 26% were positive by PCR, including 7% that were also positive by culture for L. interrogans serovars Grippotyphosa, Pyrogenes, and Icterohaemorrhagiae. L. borpetersenii serovar Polonica was isolated for the first time in Egypt in three rats. MAT titers ≥ 1:800 were observed in 11% of rats and 12% of dogs. L. interrogans serovar Grippotyphosa was detected in one cat. Sheep and donkeys were negative for leptospirosis by all methods. Buffaloes and cattle were seropositive in 20% and 44% of animals, respectively. Data indicate that several pathogenic serovars are circulating in the animals, which may pose exposure risks and account for high rates of acute febrile illness. PMID:21363980

Changes in the prevalence of Salmonella serovars associated swine production and correlations of avian, bovine and swine-associated serovars with human-associated serovars in the United States (1997-2015).

PubMed

Yuan, C; Krull, A; Wang, C; Erdman, M; Fedorka-Cray, P J; Logue, C M; O'Connor, A M

2018-04-23

As Salmonella enterica is an important pathogen of food animals, surveillance programmes for S. enterica serovars have existed for many years in the United States. Surveillance programmes serve many purposes, one of which is to evaluate alterations in the prevalence of serovars that may signal changes in the ecology of the target organism. The primary aim of this study was to evaluate changes in the proportion of S. enterica serovars isolated from swine over a near 20-year observation period (1997-2015) using four longitudinal data sets from different food animal species. The secondary aim was to evaluate correlations between changes in S. enterica serovars frequently recovered from food animals and changes in S. enterica serovars associated with disease in humans. We found decreasing proportions of S. enterica serovar Typhimurium, serovar Derby and serovar Heidelberg and increasing proportions of S. enterica serovar 4,[5],12:i:-, serovar Infantis and serovar Johannesburg in swine over time. We also found positive correlations for the yearly changes in S. enterica serovar 4,[5],12:i:-, serovar Anatum and serovar Johannesburg between swine and human data; in S. enterica Worthington between avian and human data; and in S. enterica serovar 4,[5],12:i:- between bovine and human data. We found negative correlations for the yearly changes in S. enterica serovar 4,[5],12:i:- and serovar Johannesburg between avian and human data. © 2018 Blackwell Verlag GmbH.

Molecular characterization of the pL40 protein in Leptospira interrogans.

PubMed

Zhao, Wei; Chen, Chun-Yan; Zhang, Xiang-Yan; Lai, Wei-Qiang; Hu, Bao-Yu; Zhao, Guo-Ping; Qin, Jin-Hong; Guo, Xiao-Kui

2009-06-01

Leptospirosis is a widespread zoonotic disease caused by pathogenic leptospires. The identification of outer membrane proteins (OMPs) conserved among pathogenic leptospires, which are exposed on the leptospiral surface and expressed during mammalian infection, has become a major focus of leptospirosis research. pL40, a 40 kDa protein coded by the LA3744 gene in Leptospira interrogans, was found to be unique to Leptospira. Triton X-114 fractionation and flow cytometry analyses indicate that pL40 is a component of the leptospiral outer membrane. The conservation of pL40 among Leptospira strains prevalent in China was confirmed by both Western blotting and PCR screening. Furthermore, the pL40 antigen could be recognized by sera from guinea pigs and mice infected with low-passage L. interrogans. These findings indicate that pL40 may serve as a useful serodiagnostic antigen and vaccine candidate for L. interrogans.

Leptospira interrogans in Rodents from Cape Verde.

PubMed

Plata-Luis, Josué; Foronda, Pilar; Martín-Alonso, Aaron; Feliu, Carlos; Alves, Joana; Gil, Horacio; Valladares, Basilio

2016-11-01

Leptospirosis is an important worldwide zoonotic disease that can infect both animals and humans. In most cases, leptospirosis is a nonspecific self-limiting illness, but some patients can develop a severe form with a high mortality. This study was carried out in Santiago Island, Cape Verde, in 2012-2013. A total of 62 wild rodents (Rattus rattus and Mus domesticus) were analyzed. The lipL32 gene, present only in pathogenic Leptospira spp., was amplified by PCR, and 16 samples were positive (25.8%). In both rodent species, Leptospira interrogans was identified. The results show the presence of pathogenic Leptospira in the three localities analyzed in Santiago. The presence of L. interrogans demonstrates a serious health risk for the population, since this species has been associated with the most severe form of leptospirosis, the Weil's disease in humans, a severe infection with jaundice, renal failure, and hemorrhage.

Whole Genome Analysis of Leptospira licerasiae Provides Insight into Leptospiral Evolution and Pathogenicity

PubMed Central

Selengut, Jeremy D.; Harkins, Derek M.; Patra, Kailash P.; Moreno, Angelo; Lehmann, Jason S.; Purushe, Janaki; Sanka, Ravi; Torres, Michael; Webster, Nicholas J.; Vinetz, Joseph M.; Matthias, Michael A.

2012-01-01

The whole genome analysis of two strains of the first intermediately pathogenic leptospiral species to be sequenced (Leptospira licerasiae strains VAR010 and MMD0835) provides insight into their pathogenic potential and deepens our understanding of leptospiral evolution. Comparative analysis of eight leptospiral genomes shows the existence of a core leptospiral genome comprising 1547 genes and 452 conserved genes restricted to infectious species (including L. licerasiae) that are likely to be pathogenicity-related. Comparisons of the functional content of the genomes suggests that L. licerasiae retains several proteins related to nitrogen, amino acid and carbohydrate metabolism which might help to explain why these Leptospira grow well in artificial media compared with pathogenic species. L. licerasiae strains VAR010T and MMD0835 possess two prophage elements. While one element is circular and shares homology with LE1 of L. biflexa, the second is cryptic and homologous to a previously identified but unnamed region in L. interrogans serovars Copenhageni and Lai. We also report a unique O-antigen locus in L. licerasiae comprised of a 6-gene cluster that is unexpectedly short compared with L. interrogans in which analogous regions may include >90 such genes. Sequence homology searches suggest that these genes were acquired by lateral gene transfer (LGT). Furthermore, seven putative genomic islands ranging in size from 5 to 36 kb are present also suggestive of antecedent LGT. How Leptospira become naturally competent remains to be determined, but considering the phylogenetic origins of the genes comprising the O-antigen cluster and other putative laterally transferred genes, L. licerasiae must be able to exchange genetic material with non-invasive environmental bacteria. The data presented here demonstrate that L. licerasiae is genetically more closely related to pathogenic than to saprophytic Leptospira and provide insight into the genomic bases for its infectiousness

NEW WILDLIFE HOSTS OF Leptospira interrogans IN CAMPECHE, MEXICO

PubMed Central

ESPINOSA-MARTÍNEZ, Deborah V.; SÁNCHEZ-MONTES, Daniel Sokani; LEÓN-PANIAGUA, Livia; RÍOS-MUÑOZ, César A.; BERZUNZA-CRUZ, Miriam; BECKER, Ingeborg

2015-01-01

Leptospira interrogans has been identified to cause leptospirosis, a widespread zoonotic disease that has been identified in domestic and wild animals. This work analyzed kidneys from two species of wild rodents from the state of Campeche, Mexico. Analyses were made by PCR using specific primers for detection of Leptospira interrogans DNA. The rodent species that tested positive were Heteromys gaumeri and Ototylomys phyllotis, both of which are new hosts for the bacteria in Southeastern Mexico. These records provide new insights into the disease’s transmission that should be studied carefully in order to identify other potential host species, including humans, which are at risk of becoming infected if they are in contact with infected wildlife. PMID:25923901

Development of Transcriptional Fusions to Assess Leptospira interrogans Promoter Activity

PubMed Central

Cerqueira, Gustavo M.; Souza, Natalie M.; Araújo, Eduardo R.; Barros, Aline T.; Morais, Zenaide M.; Vasconcellos, Sílvio A.; Nascimento, Ana L. T. O.

2011-01-01

Background Leptospirosis is a zoonotic infectious disease that affects both humans and animals. The existing genetic tools for Leptospira spp. have improved our understanding of the biology of this spirochete as well as the interaction of pathogenic leptospires with the mammalian host. However, new tools are necessary to provide novel and useful information to the field. Methodology and Principal Findings A series of promoter-probe vectors carrying a reporter gene encoding green fluorescent protein (GFP) were constructed for use in L. biflexa. They were tested by constructing transcriptional fusions between the lipL41, Leptospiral Immunoglobulin-like A (ligA) and Sphingomielynase 2 (sph2) promoters from L. interrogans and the reporter gene. ligA and sph2 promoters were the most active, in comparison to the lipL41 promoter and the non-induced controls. The results obtained are in agreement with LigA expression from the L. interrogans Fiocruz L1-130 strain. Conclusions The novel vectors facilitated the in vitro evaluation of L. interrogans promoter activity under defined growth conditions which simulate the mammalian host environment. The fluorescence and rt-PCR data obtained closely reflected transcriptional regulation of the promoters, thus demonstrating the suitability of these vectors for assessing promoter activity in L. biflexa. PMID:21445252

Occurrence of antibodies anti -Toxoplasma gondii, Neospora caninum and Leptospira interrogans in a captive deer herd in Southern Brazil.

PubMed

Zimpel, Cristina Kraemer; Grazziotin, Ana Laura; de Barros Filho, Ivan Roque; Guimaraes, Ana Marcia de Sa; dos Santos, Leonilda Correia; de Moraes, Wanderlei; Cubas, Zalmir Silvino; de Oliveira, Marcos Jose; Pituco, Edviges Maristela; Lara, Maria do Carmo Custódio de Souza Hunold; Villalobos, Eliana Monteforte Cassaro; Silva, Lília Marcia Paulin; Cunha, Elenice Maria Sequetin; Castro, Vanessa; Biondo, Alexander Welker

2015-01-01

A large number of Brazilian zoos keep many endangered species of deer, however, very few disease surveillance studies have been conducted among captive cervids. Blood samples from 32 Brazilian deer (Blastocerus dichotomus, Mazama nana and Mazama americana) kept in captivity at Bela Vista Biological Sanctuary (Foz do Iguaçu, Brazil) were investigated for 10 ruminant pathogens, with the aims of monitoring deer health status and evaluating any potential zoonotic risk. Deer serum samples were tested for Brucella abortus, Leptospira (23 serovars), Toxoplasma gondii, Neospora caninum, bovine viral diarrhea virus, infectious bovine rhinotracheitis virus, foot-and-mouth disease virus, western equine encephalitis virus, eastern equine encephalitis virus and Venezuelan equine encephalitis virus. Antibodies against T. gondii (15.6%), N. caninum (6.2%) and L. interrogans serogroup Serjoe (3.1%) were detected. The serological results for all other infectious agents were negative. The deer were considered to be clinically healthy and asymptomatic regarding any disease. Compared with studies on free-ranging deer, the prevalences of the same agents tested among the captive deer kept at the Sanctuary were lower, thus indicating good sanitary conditions and high-quality management practices at the zoo.

Serologic Survey for Selected Viral and Bacterial Swine Pathogens in Colombian Collared Peccaries ( Pecari tajacu) and Feral Pigs ( Sus scrofa).

PubMed

Montenegro, Olga L; Roncancio, Nestor; Soler-Tovar, Diego; Cortés-Duque, Jimena; Contreras-Herrera, Jorge; Sabogal, Sandra; Acevedo, Luz Dary; Navas-Suárez, Pedro Enrique

2018-06-14

In South America, wild populations of peccaries coexist with domestic and feral pigs, with poorly understood consequences. We captured 58 collared peccaries ( Pecari tajacu) and 15 feral pigs ( Sus scrofa) in locations of Colombia where coexistence of these species is known. Blood samples were tested for antibodies against four viral agents, classical swine fever virus (CSFV), Aujeszky's disease virus (ADV), porcine circovirus (PCV-2), and vesicular stomatitis virus (New Jersey and Indiana subtypes) and two bacterial agents, Brucella spp. and six serovars of Leptospira interrogans. The prevalence of CSFV was 5% (3/58) in collared peccaries and 7% (1/15) in feral pigs. The prevalence of PCV-2 was 7% (1/15) in collared peccaries and 67% (2/3) in feral pigs. Vesicular stomatitis prevalence was 33% (8/24) in collared peccaries and 67% (4/6) in feral pigs. Leptospira prevalence was 78% (39/50) in collared peccary and 100% (8/8) in feral pigs; bratislava, grippotyphosa, icterohaemorrhagiae, and pomona were the most frequent serovars. Also, the only white-lipped peccary ( Tayassu pecari) sampled was positive for L. interrogans serovar bratislava and for vesicular stomatitis virus, New Jersey strain. No samples were positive for ADV or Brucella. The seroprevalence of antibodies against L. interrogans was similar to that observed in other studies. Icterohaemorrhagiae appears to be a common serovar among in situ and ex situ peccary populations. Positive antibodies against PVC-2 represent a novel report of exposure to this pathogen in Colombian peccaries. Our results indicate the possible transmission of various pathogens, important for pig farms, in the studied pig and peccaries.

Leptospira Interrogans Induces Fibrosis in the Mouse Kidney through Inos-Dependent, TLR- and NLR-Independent Signaling Pathways

PubMed Central

Fanton d'Andon, Martine; Quellard, Nathalie; Fernandez, Béatrice; Ratet, Gwenn; Lacroix-Lamandé, Sonia; Vandewalle, Alain; Boneca, Ivo G.; Goujon, Jean-Michel; Werts, Catherine

2014-01-01

Background Leptospira (L.) interrogans are bacteria responsible for a worldwide reemerging zoonosis. Rodents carry L. interrogans asymptomatically in their kidneys and excrete bacteria in the urine, contaminating the environment. Humans get infected through skin contact and develop a mild or severe leptospirosis that may lead to renal failure and fibrosis. L. interrogans provoke an interstitial nephritis, but the induction of fibrosis caused by L. interrogans has not been studied in murine models. Innate immune receptors from the TLR and NLR families have recently been shown to play a role in the development and progression of tissue fibrosis in the lung, liver and kidneys under different pathophysiological situations. We recently showed that TLR2, TLR4, and NLRP3 receptors were crucial in the defense against leptospirosis. Moreover, infection of a human cell line with L. interrogans was shown to induce TLR2-dependent production of fibronectin, a component of the extracellular matrix. Therefore, we thought to assess the presence of renal fibrosis in L. interrogans infected mice and to analyze the contribution of some innate immune pathways in this process. Methodology/principal findings Here, we characterized by immunohistochemical studies and quantitative real-time PCR, a model of Leptospira-infected C57BL/6J mice, with chronic carriage of L. interrogans inducing mild renal fibrosis. Using various strains of transgenic mice, we determined that the renal infiltrates of T cells and, unexpectedly, TLR and NLR receptors, are not required to generate Leptospira-induced renal fibrosis. We also show that the iNOS enzyme, known to play a role in Leptospira-induced interstitial nephritis, also plays a role in the induction of renal fibrosis. Conclusion/significance To our knowledge, this work provides the first experimental murine model of sustained renal fibrosis induced by a chronic bacterial infection that may be peculiar, since it does not rely on TLR or NLR receptors

Longitudinal serological survey and herd-level risk factors for Leptospira spp. serovars Hardjo-bovis and Pomona on deer farms with sheep and/or beef cattle.

PubMed

Subharat, S; Wilson, Pr; Heuer, C; Collins-Emerson, Jm

2012-07-01

To investigate the seroprevalence of Leptospira spp. serovars Hardjo-bovis and Pomona on deer and mixed deer, sheep and/or beef cattle farms in the lower North Island of New Zealand and to examine associations between putative risk factors for seropositive deer herds. Serological screening was conducted on 19 commercial deer farms, 16 with sheep and/or beef cattle, between August and October each year between 2006 and 2008. No leptospiral vaccination had been conducted on the farms. On each farm every year, serum samples were collected from a random sample of 20 or more rising 2-year-old replacement animals from each species. The microscopic agglutination test (MAT) was used to detect leptospiral antibodies against Leptospira borgpetersenii serovar Hardjo-bovis and Leptospira interrogans serovar Pomona. For both serovars, a titre of ≥1:48 was considered positive and a herd was considered seropositive if >3 of 20 serum samples were positive. Information on potential herd-level risk factors for deer herds being seropositive was obtained from a questionnaire completed by the farm owner or manager. The mean percentage of deer, cattle and sheep herds seropositive for Hardjo-bovis alone over 3 years was 42%, 53% and 54%, respectively, and for serovar Pomona was 7%, 5% and 0%, respectively. Antibodies to both serovars were found in 23%, 16% and 10% of deer, cattle and sheep herds, respectively. At the individual animal level, 228/1,107 (21%) deer, 308/767 (40%) cattle and 369/1,244 (30%) sheep were seropositive for Hardjo-bovis, 102 (9%) deer, 51 (7%) cattle and 23 (2%) sheep were seropositive for Pomona, and 49 (4%) deer, 28 (4%) cattle and 18 (1%) sheep were seropositive for both serovars. Deer herds were more likely to be seropositive for Hardjo-bovis in 2006 than 2008 (p=0.008), when seropositive in the preceding year (p=0.016) and on hilly compared with flat topography (p<0.001). Deer herds were more likely to be seropositive for Pomona when seropositive in the

Effects of Culling on Leptospira interrogans Carriage by Rats

PubMed Central

Byers, Kaylee A.; Donovan, Christina M.; Bidulka, Julie J.; Stephen, Craig; Patrick, David M.; Himsworth, Chelsea G.

2018-01-01

We found that lethal, urban rat control is associated with a significant increase in the odds that surviving rats carry Leptospira interrogans. Our results suggest that human interventions have the potential to affect and even increase the prevalence of zoonotic pathogens within rat populations. PMID:29350160

Leptospira interrogans at the human-wildlife interface in northern Botswana: a newly identified public health threat.

PubMed

Jobbins, S E; Sanderson, C E; Alexander, K A

2014-03-01

Leptospirosis is the most widespread zoonosis in the world. In northern Botswana, humans live in close proximity to a diversity of wildlife and peridomestic rodents and may be exposed to a variety of zoonotic pathogens. Little is known regarding the occurrence and epidemiology of L. interrogans in Africa despite the recognized global importance of this zoonotic disease and the threat it poses to public health. In Botswana, banded mongooses (Mungos mungo) live in close proximity to humans across protected and unprotected landscapes and may be a useful sentinel species for assessing the occurrence of zoonotic organisms, such as L. interrogans. We utilized PCR to screen banded mongoose kidneys for leptospiral DNA and identified 41.5% prevalence of renal carriage of L. interrogans (exact binomial 95% CI 27.7-56.7%, n = 41). Renal carriage was also detected in one Selous' mongoose (Paracynictis selousi). This is the first published confirmation of carriage of L. interrogans in either species. This is also the first report of L. interrogans occurrence in northern Botswana and the only report of this organism in a wildlife host in the country. Pathogenic Leptospira are usually transmitted indirectly to humans through soil or water contaminated with infected urine. Other avenues, such as direct contact between humans and wildlife, as well as consumption of mongooses and other wildlife as bushmeat, may pose additional exposure risk and must be considered in public health management of this newly identified zoonotic disease threat. There is a critical need to characterize host species involvement and pathogen transmission dynamics, including human-wildlife interactions that may increase human exposure potential and infection risk. We recommend that public health strategy be modified to include sensitization of medical practitioners to the presence of L. interrogans in the region, the potential for human infection, and implementation of clinical screening. This study

Cloning and Molecular Characterization of an Immunogenic LigA Protein of Leptospira interrogans

PubMed Central

Palaniappan, Raghavan U. M.; Chang, Yung-Fu; Jusuf, S. S. D.; Artiushin, S.; Timoney, John F.; McDonough, Sean P.; Barr, Steve C.; Divers, Thomas J.; Simpson, Kenneth W.; McDonough, Patrick L.; Mohammed, Hussni O.

2002-01-01

A clone expressing a novel immunoreactive leptospiral immunoglobulin-like protein A of 130 kDa (LigA) from Leptospira interrogans serovar pomona type kennewicki was isolated by screening a genomic DNA library with serum from a mare that had recently aborted due to leptospiral infection. LigA is encoded by an open reading frame of 3,675 bp, and the deduced amino acid sequence consists of a series of 90-amino-acid tandem repeats. A search of the NCBI database found that homology of the LigA repeat region was limited to an immunoglobulin-like domain of the bacterial intimin binding protein of Escherichia coli, the cell adhesion domain of Clostridium acetobutylicum, and the invasin of Yersinia pestis. Secondary structure prediction analysis indicates that LigA consists mostly of beta sheets with a few alpha-helical regions. No LigA was detectable by immunoblot analysis of lysates of the leptospires grown in vitro at 30°C or when cultures were shifted to 37°C. Strikingly, immunohistochemistry on kidney from leptospira-infected hamsters demonstrated LigA expression. These findings suggest that LigA is specifically induced only in vivo. Sera from horses, which aborted as a result of natural Leptospira infection, strongly recognize LigA. LigA is the first leptospiral protein described to have 12 tandem repeats and is also the first to be expressed only during infection. Thus, LigA may have value in serodiagnosis or as a protective immunogen in novel vaccines. PMID:12379666

Pathogenic Leptospira interrogans Exoproteins Are Primarily Involved in Heterotrophic Processes

PubMed Central

Eshghi, Azad; Pappalardo, Elisa; Hester, Svenja; Thomas, Benjamin; Pretre, Gabriela

2015-01-01

Leptospirosis is a life-threatening and emerging zoonotic disease with a worldwide annual occurrence of more than 1 million cases. Leptospirosis is caused by spirochetes belonging to the genus Leptospira. The mechanisms of disease manifestation in the host remain elusive, and the roles of leptospiral exoproteins in these processes have yet to be determined. Our aim in this study was to assess the composition and quantity of exoproteins of pathogenic Leptospira interrogans and to construe how these proteins contribute to disease pathogenesis. Label-free quantitative mass spectrometry of proteins obtained from Leptospira spirochetes cultured in vitro under conditions mimicking infection identified 325 exoproteins. The majority of these proteins are conserved in the nonpathogenic species Leptospira biflexa, and proteins involved in metabolism and energy-generating functions were overrepresented and displayed the highest relative abundance in culture supernatants. Conversely, proteins of unknown function, which represent the majority of pathogen-specific proteins (presumably involved in virulence mechanisms), were underrepresented. Characterization of various L. interrogans exoprotein mutants in the animal infection model revealed host mortality rates similar to those of hosts infected with wild-type L. interrogans. Collectively, these results indicate that pathogenic Leptospira exoproteins primarily function in heterotrophic processes (the processes by which organisms utilize organic substances as nutrient sources) to maintain the saprophytic lifestyle rather than the virulence of the bacteria. The underrepresentation of proteins homologous to known virulence factors, such as toxins and effectors in the exoproteome, also suggests that disease manifesting from Leptospira infection is likely caused by a combination of the primary and potentially moonlight functioning of exoproteins. PMID:25987703

Positive regulation of Leptospira interrogans kdp expression by KdpE as Demonstrated with a novel β-galactosidase reporter in Leptospira biflexa.

PubMed

Matsunaga, James; Coutinho, Mariana L

2012-08-01

Leptospirosis is a potentially deadly zoonotic disease that afflicts humans and animals. Leptospira interrogans, the predominant agent of leptospirosis, encounters diverse conditions as it proceeds through its life cycle, which includes stages inside and outside the host. Unfortunately, the number of genetic tools available for examining the regulation of gene expression in L. interrogans is limited. Consequently, little is known about the genetic circuits that control gene expression in Leptospira. To better understand the regulation of leptospiral gene expression, the L. interrogans kdp locus, encoding homologs of the P-type ATPase KdpABC potassium transporter with their KdpD sensors and KdpE response regulators, was selected for analysis. We showed that a kdpE mutation in L. interrogans prevented the increase in kdpABC mRNA levels observed in the wild-type L. interrogans strain when external potassium levels were low. To confirm that KdpE was a positive regulator of kdpABC transcription, we developed a novel approach for constructing chromosomal genetic fusions to the endogenous bgaL (β-galactosidase) gene of the nonpathogen Leptospira biflexa. We demonstrated positive regulation of a kdpA'-bgaL fusion in L. biflexa by the L. interrogans KdpE response regulator. A control lipL32'-bgaL fusion was not regulated by KdpE. These results demonstrate the utility of genetic fusions to the bgaL gene of L. biflexa for examining leptospiral gene regulation.

Evaluation of a recombinant LipL41 antigen of Leptospira interrogans serovar canicola in ELISA for serodiagnosis of bovine leptospirosis.

PubMed

Mariya, R; Chaudhary, Pallab; Kumar, A A; Thangapandian, E; Amutha, R; Srivastava, S K

2006-11-01

The efficacy of a recombinant leptospiral lipoprotein LipL41 as an antigen for conducting enzyme-linked immunosorbent assay (ELISA) for diagnosis of bovine leptospirosis was evaluated. Using known positive and known negative cattle sera the recombinant antigen was found to be highly reactive in the concentration of 100 ng/well. Using a total of 321 field cattle sera the sensitivity of ELISA as compared to microscopic agglutination test (MAT) was calculated to be 100% whereas the specificity was 85.3%. The seropositivity of leptospirosis among bovine population was found to be 21.18% having the predominance of serovars Sejroe and Pomona. It was concluded that rLipL41 protein could be a putative diagnostic candidate for serodiagnosis of bovine leptospirosis.

Isolation of Salmonella enterica serovar Enteritidis from houseflies (Musca domestica) found in rooms containing Salmonella serovar Enteritidis-challenged hens.

PubMed

Holt, Peter S; Geden, Christopher J; Moore, Randle W; Gast, Richard K

2007-10-01

Houseflies (Musca domestica) released into rooms containing hens challenged with Salmonella enterica serovar Enteritidis (Salmonella serovar Enteritidis) rapidly became contaminated with Salmonella serovar Enteritidis. Forty to 50% of the flies were contaminated at 48 h, and the percentage increased to 50 to 70% at 4 and 7 days postexposure and then decreased to 30% at day 15. Initial attempts at recovering surface organisms for culture using an aqueous rinse were largely unsuccessful, while cultures of internal contents readily recovered Salmonella serovar Enteritidis. However, when 0.5% detergent was incorporated into the rinse, high recovery levels of bacteria were observed from both external and internal culture regimens, indicating equal distribution of the organism on and in the fly and a tighter interaction of the organism with the host than previously thought. Salmonella serovar Enteritidis was isolated routinely from the fly gut, on rare occasions from the crop, and never from the salivary gland. Feeding contaminated flies to hens resulted in gut colonization of a third of the birds, but release of contaminated flies in a room containing previously unchallenged hens failed to result in colonization of any of the subject birds. These results indicate that flies exposed to an environment containing Salmonella serovar Enteritidis can become colonized with the organism and might serve as a source for transmission of Salmonella serovar Enteritidis within a flock situation.

Isolation of Salmonella enterica Serovar Enteritidis from Houseflies (Musca domestica) Found in Rooms Containing Salmonella Serovar Enteritidis-Challenged Hens▿

PubMed Central

Holt, Peter S.; Geden, Christopher J.; Moore, Randle W.; Gast, Richard K.

2007-01-01

Houseflies (Musca domestica) released into rooms containing hens challenged with Salmonella enterica serovar Enteritidis (Salmonella serovar Enteritidis) rapidly became contaminated with Salmonella serovar Enteritidis. Forty to 50% of the flies were contaminated at 48 h, and the percentage increased to 50 to 70% at 4 and 7 days postexposure and then decreased to 30% at day 15. Initial attempts at recovering surface organisms for culture using an aqueous rinse were largely unsuccessful, while cultures of internal contents readily recovered Salmonella serovar Enteritidis. However, when 0.5% detergent was incorporated into the rinse, high recovery levels of bacteria were observed from both external and internal culture regimens, indicating equal distribution of the organism on and in the fly and a tighter interaction of the organism with the host than previously thought. Salmonella serovar Enteritidis was isolated routinely from the fly gut, on rare occasions from the crop, and never from the salivary gland. Feeding contaminated flies to hens resulted in gut colonization of a third of the birds, but release of contaminated flies in a room containing previously unchallenged hens failed to result in colonization of any of the subject birds. These results indicate that flies exposed to an environment containing Salmonella serovar Enteritidis can become colonized with the organism and might serve as a source for transmission of Salmonella serovar Enteritidis within a flock situation. PMID:17675422

Salmonella enterica: Survival, Colonization, and Virulence Differences among Serovars

PubMed Central

Andino, A.; Hanning, I.

2015-01-01

Data indicate that prevalence of specific serovars of Salmonella enterica in human foodborne illness is not correlated with their prevalence in feed. Given that feed is a suboptimal environment for S. enterica, it appears that survival in poultry feed may be an independent factor unrelated to virulence of specific serovars of Salmonella. Additionally, S. enterica serovars appear to have different host specificity and the ability to cause disease in those hosts is also serovar dependent. These differences among the serovars may be related to gene presence or absence and expression levels of those genes. With a better understanding of serovar specificity, mitigation methods can be implemented to control Salmonella at preharvest and postharvest levels. PMID:25664339

Immunoprotective properties of recombinant LigA and LigB in a hamster model of acute leptospirosis

PubMed Central

Lourdault, Kristel; Matsunaga, James; Haake, David A.

2017-01-01

Leptospirosis is the most widespread zoonosis and is considered a major public health problem worldwide. Currently, there is no widely available vaccine against leptospirosis for use in humans. A purified, recombinant subunit vaccine that includes the last six immunoglobulin-like (Ig-like) domains of the leptospiral protein LigA (LigA7’-13) protects against lethal infection but not renal colonization after challenge by Leptospira interrogans. In this study, we examined whether the addition of the first seven Ig-like domains of LigB (LigB0-7) to LigA7’-13, can enhance immune protection and confer sterilizing immunity in the Golden Syrian hamster model of acute leptospirosis. Hamsters were subcutaneously immunized with soluble, recombinant LigA7’-13, LigB0-7, or a combination of LigA7’-13 and LigB0-7 in Freund’s adjuvant. Immunization with Lig proteins generated a strong humoral immune response with high titers of IgG that recognized homologous protein, and cross-reacted with the heterologous protein as assessed by ELISA. LigA7’-13 alone, or in combination with LigB0-7, protected all hamsters from intraperitoneal challenge with a lethal dose of L. interrogans serovar Copenhageni strain Fiocruz L1-130. However, bacteria were recovered from the kidneys of all animals. Of eight animals immunized with LigB0-7, only three survived Leptospira challenge, one of which lacked renal colonization and had antibodies to native LigB by immunoblot. In addition, sera from two of the three LigB0-7 immunized survivors cross-reacted with LigA11-13, a region of LigA that is sufficient for protection. In summary, we confirmed that LigA7’-13 protects hamsters from death but not infection, and immunization with LigB0-7, either alone or in combination with LigA7’-13, did not confer sterilizing immunity. PMID:28704385

Environmental Factors Influencing White-Tailed Deer (Odocoileus virginianus) Exposure to Livestock Pathogens in Wisconsin

PubMed Central

Kern, Bryant; Mahoney, Kathleen; Norton, Andrew; Patnayak, Devi; Van Deelen, Timothy

2015-01-01

White-tailed deer (Odocoileus virginianus) are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland) in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo), bovine viral diarrhea virus (BVDV-1 and BVDV-2), infectious bovine rhinotracheitis virus (IBR), and parainfluenza 3 virus (PI3). We used logistic regression to model potential intrinsic (e.g., age, sex) and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens) variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010–2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%), IBR (7.9%), Leptospira interrogans serovar pomona (11.7%), L. i. bratislava (1.0%), L. i. grippotyphosa (2.5%) and L. i. hardjo (0.3%). Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119) were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens. PMID:26030150

Environmental Factors Influencing White-Tailed Deer (Odocoileus virginianus) Exposure to Livestock Pathogens in Wisconsin.

PubMed

Dubay, Shelli; Jacques, Christopher; Golden, Nigel; Kern, Bryant; Mahoney, Kathleen; Norton, Andrew; Patnayak, Devi; Van Deelen, Timothy

2015-01-01

White-tailed deer (Odocoileus virginianus) are commonly exposed to disease agents that affect livestock but environmental factors that predispose deer to exposure are unknown for many pathogens. We trapped deer during winter months on two study areas (Northern Forest and Eastern Farmland) in Wisconsin from 2010 to 2013. Deer were tested for exposure to six serovars of Leptospira interrogans (grippotyphosa, icterohaemorrhagiae, canicola, bratislava, pomona, and hardjo), bovine viral diarrhea virus (BVDV-1 and BVDV-2), infectious bovine rhinotracheitis virus (IBR), and parainfluenza 3 virus (PI3). We used logistic regression to model potential intrinsic (e.g., age, sex) and extrinsic (e.g., land type, study site, year, exposure to multiple pathogens) variables we considered biologically meaningful to exposure of deer to livestock pathogens. Deer sampled in 2010-2011 did not demonstrate exposure to BVDV, so we did not test for BVDV in subsequent years. Deer had evidence of exposure to PI3 (24.7%), IBR (7.9%), Leptospira interrogans serovar pomona (11.7%), L. i. bratislava (1.0%), L. i. grippotyphosa (2.5%) and L. i. hardjo (0.3%). Deer did not demonstrate exposure to L. interrogans serovars canicola and icterohaemorrhagiae. For PI3, we found that capture site and year influenced exposure. Fawns (n = 119) were not exposed to L. i. pomona, but land type was an important predictor of exposure to L. i. pomona for older deer. Our results serve as baseline exposure levels of Wisconsin white-tailed deer to livestock pathogens, and helped to identify important factors that explain deer exposure to livestock pathogens.

Interaction of bovine peripheral blood polymorphonuclear cells and Leptospira species; innate responses in the natural bovine reservoir host.

USDA-ARS?s Scientific Manuscript database

Cattle are the reservoir hosts of Leptospira borgpetersenii serovar Hardjo, and also be reservoir hosts of other Leptospira species such as L. kirschneri, and L. interrogans. As a reservoir host, cattle shed Leptospira, infecting other animals, including humans. Previous studies with human and murin...

Salmonella enterica serovar-specific transcriptional reprogramming of infected cells.

PubMed

Hannemann, Sebastian; Galán, Jorge E

2017-07-01

Despite their high degree of genomic similarity, different Salmonella enterica serovars are often associated with very different clinical presentations. In humans, for example, the typhoidal S. enterica serovar Typhi causes typhoid fever, a life-threatening systemic disease. In contrast, the non-typhoidal S. enterica serovar Typhimurium causes self-limiting gastroenteritis. The molecular bases for these different clinical presentations are incompletely understood. The ability to re-program gene expression in host cells is an essential virulence factor for typhoidal and non-typhoidal S. enterica serovars. Here, we have compared the transcriptional profile of cultured epithelial cells infected with S. Typhimurium or S. Typhi. We found that both serovars stimulated distinct transcriptional responses in infected cells that are associated with the stimulation of specific signal transduction pathways. These specific responses were associated with the presence of a distinct repertoire of type III secretion effector proteins. These observations provide major insight into the molecular bases for potential differences in the pathogenic mechanisms of typhoidal and non-typhoidal S. enterica serovars.

Molecular differentiation between Salmonella enterica subsp enterica serovar Pullorum and Salmonella enterica subsp enterica serovar Gallinarum

PubMed Central

Ribeiro, Simone Alves Mendes; de Paiva, Jaqueline Boldrin; Zotesso, Fábio; Lemos, Manoel Victor Franco; Berchieri Jánior, Ângelo

2009-01-01

S. Pullorum (SP) and S. Gallinarum (SG) are very similar. They are the agents of pullorum disease and fowl typhoid, respectively, and the two diseases are responsible for economic losses in poultry production. Although SP and SG are difficult to be differentiated in routine laboratory procedures, the ability to metabolize ornithine is a biochemical test that may be used to achieve this aim. While SP is able to decarboxylate this amino acid, SG is not. However, the isolation of strains showing atypical biochemical behavior has made this differentiation difficult. One of the genes associated with the metabolization of the amino acid ornithine is called speC, and is found in both serovars. The analysis of 21 SP and 15 SG strains by means of PCR did not enable the differentiation of the two serovars, because fragments produced were identical. However, after enzymatic treatment with restriction enzyme Eco RI, the band pattern of each serovar showed to be different, even in samples of atypical biochemical behavior. This fact enabled the standardization of the technique for a quick and safe differentiation of serovars Pullorum and Gallinarum. PMID:24031341

Development of two real-time polymerase chain reaction assays to detect Actinobacillus pleuropneumoniae serovars 1-9-11 and serovar 2.

PubMed

Marois-Créhan, Corinne; Lacouture, Sonia; Jacques, Mario; Fittipaldi, Nahuel; Kobisch, Marylène; Gottschalk, Marcelo

2014-01-01

Two real-time, or quantitative, polymerase chain reaction (qPCR) assays were developed to detect Actinobacillus pleuropneumoniae serovars 1-9-11 (highly related serovars with similar virulence potential) and serovar 2, respectively. The specificity of these assays was verified on a collection of 294 strains, which included all 16 reference A. pleuropneumoniae strains (including serovars 5a and 5b), 263 A. pleuropneumoniae field strains isolated between 1992 and 2009 in different countries, and 15 bacterial strains other than A. pleuropneumoniae. The detection levels of both qPCR tests were evaluated using 10-fold dilutions of chromosomal DNA from reference strains of A. pleuropneumoniae serovars 1 and 2, and the detection limit for both assays was 50 fg per assay. The analytical sensitivities of the qPCR tests were also estimated by using pure cultures and tonsils experimentally spiked with A. pleuropneumoniae. The detection threshold was 2.5 × 10(4) colony forming units (CFU)/ml and 2.9 × 10(5) CFU/0.1 g of tonsil, respectively, for both assays. These specific and sensitive tests can be used for the serotyping of A. pleuropneumoniae in diagnostic laboratories to control porcine pleuropneumonia.

Isoleucine Biosynthesis in Leptospira interrogans Serotype lai Strain 56601 Proceeds via a Threonine-Independent Pathway† ‡

PubMed Central

Xu, Hai; Zhang, Yuzhen; Guo, Xiaokui; Ren, Shuangxi; Staempfli, Andreas A.; Chiao, Juishen; Jiang, Weihong; Zhao, Guoping

2004-01-01

Three leuA-like protein-coding sequences were identified in Leptospira interrogans. One of these, the cimA gene, was shown to encode citramalate synthase (EC 4.1.3.-). The other two encoded α-isopropylmalate synthase (EC 4.1.3.12). Expressed in Escherichia coli, the citramalate synthase was purified and characterized. Although its activity was relatively low, it was strictly specific for pyruvate as the keto acid substrate. Unlike the citramalate synthase of the thermophile Methanococcus jannaschii, the L. interrogans enzyme is temperature sensitive but exhibits a much lower Km (0.04 mM) for pyruvate. The reaction product was characterized as (R)-citramalate, and the proposed β-methyl-d-malate pathway was further confirmed by demonstrating that citraconate was the substrate for the following reaction. This alternative pathway for isoleucine biosynthesis from pyruvate was analyzed both in vitro by assays of leptospiral isopropylmalate isomerase (EC 4.2.1.33) and β-isopropylmalate dehydrogenase (EC 1.1.1.85) in E. coli extracts bearing the corresponding clones and in vivo by complementation of E. coli ilvA, leuC/D, and leuB mutants. Thus, the existence of a leucine-like pathway for isoleucine biosynthesis in L. interrogans under physiological conditions was unequivocally proven. Significant variations in either the enzymatic activities or mRNA levels of the cimA and leuA genes were detected in L. interrogans grown on minimal medium supplemented with different levels of the corresponding amino acids or in cells grown on serum-containing rich medium. The similarity of this metabolic pathway in leptospires and archaea is consistent with the evolutionarily primitive status of the eubacterial spirochetes. PMID:15292141

Neutrophil Extracellular Traps are Involved in the Innate Immune Response to Infection with Leptospira

PubMed Central

Scharrig, Emilia; Carestia, Agostina; Ferrer, María F.; Cédola, Maia; Pretre, Gabriela; Drut, Ricardo; Picardeau, Mathieu; Schattner, Mirta; Gómez, Ricardo M.

2015-01-01

NETosis is a process by which neutrophils extrude their DNA together with bactericidal proteins that trap and/or kill pathogens. In the present study, we evaluated the ability of Leptospira spp. to induce NETosis using human ex vivo and murine in vivo models. Microscopy and fluorometric studies showed that incubation of human neutrophils with Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 (LIC) resulted in the release of DNA extracellular traps (NETs). The bacteria number, pathogenicity and viability were relevant factors for induction of NETs, but bacteria motility was not. Entrapment of LIC in the NETs resulted in LIC death; however, pathogenic but not saprophytic Leptospira sp. exerted nuclease activity and degraded DNA. Mice infected with LIC showed circulating NETs after 2 days post-infection (dpi). Depletion of neutrophils with mAb1A8 significantly reduced the amount of intravascular NETs in LIC-infected mice, increasing bacteremia at 3 dpi. Although there was a low bacterial burden, scarce neutrophils and an absence of inflammation in the early stages of infection in the kidney and liver, at the beginning of the leptospiruric phase, the bacterial burden was significantly higher in kidneys of neutrophil-depleted-mice compared to non-depleted and infected mice. Surprisingly, interstitial nephritis was of similar intensity in both groups of infected mice. Taken together, these data suggest that LIC triggers NETs, and that the intravascular formation of these DNA traps appears to be critical not only to prevent early leptospiral dissemination but also to preclude further bacterial burden. PMID:26161745

LruA and LruB, novel lipoproteins of pathogenic Leptospira interrogans associated with equine recurrent uveitis.

PubMed

Verma, Ashutosh; Artiushin, Sergey; Matsunaga, James; Haake, David A; Timoney, John F

2005-11-01

Recurrent uveitis as a sequela to Leptospira infection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library of Leptospira interrogans DNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation of L. interrogans in medium containing [14C]palmitic acid, confirming that it is a lipoprotein. lruA and lruB were detected by Southern blotting in infectious Leptospira interrogans but not in nonpathogenic Leptospira biflexa. Fractionation data from cultured Leptospira indicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis.

LruA and LruB, Novel Lipoproteins of Pathogenic Leptospira interrogans Associated with Equine Recurrent Uveitis

PubMed Central

Verma, Ashutosh; Artiushin, Sergey; Matsunaga, James; Haake, David A.; Timoney, John F.

2005-01-01

Recurrent uveitis as a sequela to Leptospira infection is the most common infectious cause of blindness and impaired vision of horses worldwide. Leptospiral proteins expressed during prolonged survival in the eyes of horses with lesions of chronic uveitis were identified by screening a phage library of Leptospira interrogans DNA fragments with eye fluids from uveitic horses. Inserts of reactive phages encoded several known leptospiral proteins and two novel putative lipoproteins, LruA and LruB. LruA was intrinsically labeled during incubation of L. interrogans in medium containing [14C]palmitic acid, confirming that it is a lipoprotein. lruA and lruB were detected by Southern blotting in infectious Leptospira interrogans but not in nonpathogenic Leptospira biflexa. Fractionation data from cultured Leptospira indicate that LruA and LruB are localized in the inner membrane. Uveitic eye fluids contained significantly higher levels of immunoglobulin A (IgA) and IgG specific for each protein than did companion sera, indicating strong local antibody responses. Moreover, LruA- and LruB-specific antisera reacted with equine ocular components, suggesting an immunopathogenic role in leptospiral uveitis. PMID:16239521

Camel as a transboundary vector for emerging exotic Salmonella serovars.

PubMed

Ghoneim, Nahed H; Abdel-Moein, Khaled A; Zaher, Hala

2017-05-01

The current study was conducted to shed light on the role of imported camels as a transboundary vector for emerging exotic Salmonella serovars. Fecal samples were collected from 206 camels directly after slaughtering including 25 local camels and 181 imported ones as well as stool specimens were obtained from 50 slaughterhouse workers at the same abattoir. The obtained samples were cultured while Salmonella serovars were identified through Gram's stain films, biochemical tests and serotyping with antisera kit. Moreover, the obtained Salmonella serovars were examined by PCR for the presence of invA and stn genes. The overall prevalence of Salmonella serovars among the examined camels was 8.3%. Stn gene was detected in the vast majority of exotic strains (11/14) 78.6% including emerging serovars such as Salmonella Saintpaul, S. Chester, S. Typhimurium whereas only one isolate from local camels carried stn gene (1/3) 33.3%. On the other hand, none of the examined humans yielded positive result. Our findings highlight the potential role of imported camels as a transboundary vector for exotic emerging Salomenella serovars.

Multidrug-resistant Salmonella enterica serovar Infantis, Israel.

PubMed

Gal-Mor, Ohad; Valinsky, Lea; Weinberger, Miriam; Guy, Sara; Jaffe, Joseph; Schorr, Yosef Ilan; Raisfeld, Abraham; Agmon, Vered; Nissan, Israel

2010-11-01

To determine whether rapid emergence of Salmonella enterica serovar Infantis in Israel resulted from an increase in different biotypes or spread of 1 clone, we characterized 87 serovar Infantis isolates on the genotypic and phenotypic levels. The emerging strain comprised 1 genetic clone with a distinct pulsed-field gel electrophoresis profile and a common antimicrobial drug resistance pattern.

Multidrug-Resistant Salmonella enterica Serovar Infantis, Israel

PubMed Central

Valinsky, Lea; Weinberger, Miriam; Guy, Sara; Jaffe, Joseph; Schorr, Yosef Ilan; Raisfeld, Abraham; Agmon, Vered; Nissan, Israel

2010-01-01

To determine whether rapid emergence of Salmonella enterica serovar Infantis in Israel resulted from an increase in different biotypes or spread of 1 clone, we characterized 87 serovar Infantis isolates on the genotypic and phenotypic levels. The emerging strain comprised 1 genetic clone with a distinct pulsed-field gel electrophoresis profile and a common antimicrobial drug resistance pattern. PMID:21029536

Diagnosis and seroprevalence of leptospirosis in California sea lions from coastal California.

PubMed

Colagross-Schouten, Angela M; Mazet, Jonna A K; Gulland, Frances M D; Miller, Melissa A; Hietala, Sharon

2002-01-01

The sensitivity and specificity of the microscopic agglutination test (MAT) as a method for detection of exposure to Leptospira spp. in California sea lions (Zalophus californianus) were determined. Sera came from individuals that demonstrated clinical signs of renal disease, had lesions suggestive of leptospirosis at necropsy, and had visible leptospires in silver stained kidney sections as positive controls. Sera from unexposed captive individuals were used as negative controls. The test was 100% sensitive at 1:3,200 for confirming renal infection and 100% specific at negative < 1:100 for detection of Leptospira interrogans scrovar pomona antibodies by MAT in California sea lions. Leptospira interrogans serovar pomona was used as a screening serovar because it has been isolated previously from the kidneys and placentas of California sea lions, and there appears to be cross-reactivity between serovar pomona and other serovars. Sera from 225 free-ranging California sea lions presented to one of three participating California (USA) coastal marine mammal rehabilitation centers in 1996 were then evaluated for antibodies to serovar pomona using the MAT. The overall seroprevalence was 38.2% (86/225), although the prevalence varied among locations from 100% (38/38) in animals at the Marine Mammal Care Center (Fort MacArthur, California, USA) to 0% (0/14) at SeaWorld California (San Diego, California). At The Marine Mammal Center (Sausalito, California) [prevalence 27.8% (48/173)], the majority of seropositive animals were subadults and adults, and males were 4.7 times more likely to be seropositive to serovar pomona than females. When combining results from all three centers, subadult and adult animals were more likely to be seropositive than pups and juvenile sea lions, and the highest proportion of seropositive animals presented during the autumn months. Serum elevations of blood urea nitrogen, creatinine, phosphorus, and/or calcium were associated with seropositivity

Seroepidemiology of leptospirosis in livestock in Trinidad.

PubMed

Suepaul, Sharianne M; Carrington, Christine V; Campbell, Mervyn; Borde, Gustave; Adesiyun, Abiodun Adewale

2011-02-01

A study was conducted to determine the seroprevalence of leptospirosis and infecting serovars across livestock (cattle, sheep, goats, and pigs) in Trinidad using the microscopic agglutination test with an international panel of 23 serovars. Of a total of 590 cattle tested, 21.5% were seropositive with agglutinations to 13 of the 23 antigens used in the panel. Icterohaemorrhagiae (9.3%), Sejroe (4.1%), Ballum (4.1%), and Autumnalis (1.9%) were the predominant serogroups detected in the cattle sampled (n = 590). Of 222 sheep tested, 5.0% were seropositive with agglutinations to five serovars belonging to two serogroups. These serogroups were Autumnalis at 2.7%, and Icterohaemorrhagiae at 2.3% of all sheep tested (n = 222). Of a total of 180 goats tested, 3.3% were seropositive, all agglutinating to the Icterohaemorrhagiae serogroup, 1.7% to serovar Copenhageni, 1.1% to serovar Mankarso, and 0.6% to serovar Icterohaemorrhagiae. Among pigs (n = 200), 5.0% were seropositive for five serovars belonging to three serogroups. These serogroups were Icterohaemorrhagiae at 2.5%, Australis at 2%, and Ballum at 0.5%. Overall, age and sex of animals were not significantly associated with leptospirosis with the exception of cattle where age was a significant factor for seropositivity. It was concluded that for livestock, leptospirosis may be an important zoonotic and economic disease, particularly in the case of cattle. It is imperative that the impact of leptospirosis on abortion, stillbirths, and decreased milk production in livestock in the country be assessed.

Serologic survey for leptospirae in European brown bears (Ursus arctos) in Croatia.

PubMed

Modrić, Z; Huber, D

1993-10-01

From 1981 to 1991, sera of 42 European brown bears (Ursus arctos) from three areas in Croatia were tested for antibodies against 12 Leptospira interrogans serovars: grippotyphosa, sejroe, australis, pomona, canicola, icterohaemorrhagiae, tarassovi, saxkoebing, ballum, bataviae, poi, and hardjo. Diagnostic levels of antibody were found in 17 (40%) of 42 sera. Evidence of exposure to at least one of the serovars was found in seven of 14 free-ranging bears from the Lika region, four of 12 free-ranging bears from the Gorski Kotar region, zero of six orphaned cubs from the Gorski Kotar region, and six of 10 captive bears from the Zagreb Zoo. Based on the antibody titers, we implicated the following serovars: australis in five bears, sejroe in two bears, canicola in one bear, and icterohaemorrhagiae in one bear. There was a strong correlation between serovars implicated by this survey and serovars previously isolated from small mammals in Croatia.

A Single Multilocus Sequence Typing (MLST) Scheme for Seven Pathogenic Leptospira Species

PubMed Central

Amornchai, Premjit; Wuthiekanun, Vanaporn; Bailey, Mark S.; Holden, Matthew T. G.; Zhang, Cuicai; Jiang, Xiugao; Koizumi, Nobuo; Taylor, Kyle; Galloway, Renee; Hoffmaster, Alex R.; Craig, Scott; Smythe, Lee D.; Hartskeerl, Rudy A.; Day, Nicholas P.; Chantratita, Narisara; Feil, Edward J.; Aanensen, David M.; Spratt, Brian G.; Peacock, Sharon J.

2013-01-01

Background The available Leptospira multilocus sequence typing (MLST) scheme supported by a MLST website is limited to L. interrogans and L. kirschneri. Our aim was to broaden the utility of this scheme to incorporate a total of seven pathogenic species. Methodology and Findings We modified the existing scheme by replacing one of the seven MLST loci (fadD was changed to caiB), as the former gene did not appear to be present in some pathogenic species. Comparison of the original and modified schemes using data for L. interrogans and L. kirschneri demonstrated that the discriminatory power of the two schemes was not significantly different. The modified scheme was used to further characterize 325 isolates (L. alexanderi [n = 5], L. borgpetersenii [n = 34], L. interrogans [n = 222], L. kirschneri [n = 29], L. noguchii [n = 9], L. santarosai [n = 10], and L. weilii [n = 16]). Phylogenetic analysis using concatenated sequences of the 7 loci demonstrated that each species corresponded to a discrete clade, and that no strains were misclassified at the species level. Comparison between genotype and serovar was possible for 254 isolates. Of the 31 sequence types (STs) represented by at least two isolates, 18 STs included isolates assigned to two or three different serovars. Conversely, 14 serovars were identified that contained between 2 to 10 different STs. New observations were made on the global phylogeography of Leptospira spp., and the utility of MLST in making associations between human disease and specific maintenance hosts was demonstrated. Conclusion The new MLST scheme, supported by an updated MLST website, allows the characterization and species assignment of isolates of the seven major pathogenic species associated with leptospirosis. PMID:23359622

Identification of genes associated with survival of salmonellaenterica serovar enteridis in chicken egg albumen

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clavijo, Raul I.; Loui, Cindy; Andersen, Gary L.

Salmonella enterica consists of over 2,000 serovars that aremajor causes of morbidity and mortality associated with contaminatedfood. Despite similarities among serovars of Salmonella enterica, manydemonstrate unique host specificities, epidemiological characteristics,and clinical manifestations. One of the unique epidemiologicalcharacteristics of the serovar Enteritidis is that it is the onlybacterium routinely transmitted to humans through intact chicken eggs.Therefore, Salmonella enterica serovar Enteritidis must be able topersist inside chicken eggs to be transmitted to humans, and its survivalin egg is important for its transmission to the human population. Theability of Salmonella enterica serovar Enteritidis to survive in andtransmit through eggs may have contributed tomore » its drastically increasedprevalence in the 1980s and 1990s. In the present study, usingtransposon-mediated mutagenesis, we have identified genes important forthe association of Salmonella enterica serovar Enteritidis with chickeneggs. Our results indicate that genes involved in cell wall structuraland functional integrity, and nucleic acid and amino acid metabolism areimportant for Salmonella enterica serovar Enteritidis to persist in eggalbumen. Two regions unique toSalmonella enterica serovar Enteritidiswere also identified, one of which enhanced the survival of a Salmonellaenterica serovar Typhimurium isolate in egg albumen. The implication ofour results to the serovar specificity of Salmonella enterica is alsoexplored in the present study.« less

Comparative Genomic Analyses of Transport Proteins Encoded Within the Genomes of Leptospira Species

PubMed Central

Buyuktimkin, Bora; Saier, Milton H.

2015-01-01

Select species of the bacterial genus Leptospira are causative agents of leptospirosis, an emerging global zoonosis affecting nearly one million people worldwide annually. We examined two Leptospira pathogens, L. interrogans serovar Lai str. 56601 and L. borgpetersenii serovar Hardjo-bovis str. L550, as well as the free-living leptospiral saprophyte, L. biflexa serovar Patoc str. ‘Patoc 1 (Ames)’. The transport proteins of these leptospires were identified and compared using bioinformatics to gain an appreciation for which proteins may be related to pathogenesis and saprophytism. L. biflexa possesses a disproportionately high number of secondary carriers for metabolite uptake and environmental adaptability as well as an increased number of inorganic cation transporters providing ionic homeostasis and effective osmoregulation in a rapidly changing environment. L. interrogans and L. borgpetersenii possess far fewer transporters, but those that they have are remarkably similar, with near-equivalent representation in most transporter families. These two Leptospira pathogens also possess intact sphingomyelinases, holins, and virulence-related outer membrane porins. These virulence-related factors, in conjunction with decreased transporter substrate versatility, indicate that pathogenicity was accompanied by progressively narrowing ecological niches and the emergence of a limited set of proteins responsible for host invasion. The variability of host tropism and mortality rates by infectious leptospires suggests that small differences in individual sets of proteins play important physiological and pathological roles. PMID:26247102

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) for the characterisation of pathogenic leptospires: intra-serovar divergence, inter-serovar convergence, and evidence of attenuation in Leptospira reference collections.

PubMed

Tulsiani, S M; Craig, S B; Graham, G C; Cobbold, R C; Dohnt, M F; Burns, M-A; Jansen, C C; Leung, L K-P; Field, H E; Smythe, L D

2010-07-01

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Isolation and genetic characterization of an Actinobacillus pleuropneumoniae serovar K12:O3 strain.

PubMed

Ito, Hiroya; Matsumoto, Atsuko

2015-01-01

An atypical Actinobacillus pleuropneumoniae serovar 12 strain, termed QAS106, was isolated from a clinical case of porcine pleuropneumonia in Japan. An immunodiffusion (ID) test identified the strain as serovar 12. However, the ID test also demonstrated that strain QAS106 shared antigenic determinants with both the serovar 3 and 15 reference strains. Strain QAS106 was positive in the capsular serovar 12-specific polymerase chain reaction (PCR) assay, while the PCR toxin gene profiling and omlA PCR typing assays indicated that strain QAS106 was similar to serovar 3. The nucleotide sequence of the 16S ribosomal DNA (rDNA) of strain QAS106 was identical with that of serovars 3 and 12, but it showed 99.7% identity with that of serovar 15. Nucleotide sequence analysis revealed that genes involved in biosynthesis of the capsular polysaccharide (CPS) of strain QAS106 were identical to those of serovar 12 at the amino acid level. On the other hand, strain QAS106 would express putative proteins involved in the biosynthesis of lipopolysaccharide (LPS) O-polysaccharide (O-PS), the amino acid sequences of which were identical or nearly identical to those of serovars 3 and 15. In conclusion, strain QAS106 should be recognized as K12:O3, even though typical serovar 12 strains are K12:O12. The emergence of an atypical A. pleuropneumoniae serovar 12 strain expressing a rare combination of CPS and O-PS antigens would hamper precise serodiagnosis by the use of either CPS- or LPS-based serodiagnostic methodology alone. © 2014 The Author(s).

The emergence of Leptospira borgpetersenii serovar Arborea in Queensland, Australia, 2001 to 2013.

PubMed

Lau, Colleen L; Skelly, Chris; Dohnt, Michael; Smythe, Lee D

2015-06-14

Leptospirosis is an emerging infectious disease, with increasing frequency and severity of outbreaks, changing epidemiology of populations at risk, and the emergence of new serovars. Environmental drivers of disease transmission include flooding, urbanisation, poor sanitation, changes in land use and agricultural practices, and socioeconomic factors. In Queensland, human infection with Leptosira borgpetersenii serovar Arborea was first reported in 2001. This study aims to report the emergence of serovar Arborea in Queensland from 2001 to 2013, and investigate potential risk factors for infection and drivers of emergence. Data on laboratory-confirmed cases of human leptospirosis in Queensland were obtained from the enhanced surveillance system at the WHO/FAO/OIE Collaborating Centre for Reference and Research on Leptospirosis in Brisbane, Australia. The changing epidemiology of serovar Arborea from 2001 to 2003 was described with respect to case numbers, proportion of leptospirosis cases attributed to the serovar, and geographic distribution. Differences in risk factors for the most common serovars were compared. During this period, 1289 cases of leptospirosis were reported, including 233 cases attributed to serovar Arborea. Risk factors for infection include male gender (91 % of cases), occupation, and recreational exposure. Most common occupations recorded were banana workers (28.4 %), meat workers (7.2 %), dairy farmers (5.8 %), graziers/stockmen (5.5 %), 'other agricultural/rural workers' (16.4 %), and tourists or tourism operators (4.6 %). Time trend analysis showed that while non-Arborea cases decreased over the study period, Arborea cases increased by 3.4 cases per year. The proportion of annual cases attributed to Arborea peaked at 49 % in 2011 after unprecedented flooding in Queensland. Mapping of cases by residential location showed expansion of the geographic range of serovar Arborea, concentrating mostly around Brisbane, Cairns and Innisfail. Serovars

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog

USDA-ARS?s Scientific Manuscript database

Leptospira interrogans is the causative agent of leptospirosis, a zoonosis of global significance. Iron is essential for growth of most bacterial species. Since availability of iron is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In ...

Salmonella Serovars from Humans and Other Sources in Thailand, 1993–2002

PubMed Central

Bangtrakulnonth, Aroon; Pornreongwong, Srirat; Pulsrikarn, Chaiwat; Sawanpanyalert, Pathom; Hendriksen, Rene S.; Wong, Danilo M. A. Lo Fo

2004-01-01

We serotyped 44,087 Salmonella isolates from humans and 26,148 from other sources from 1993 through 2002. The most common serovar causing human salmonellosis in Thailand was Salmonella enterica Weltevreden. Serovars causing human infections in Thailand differ from those in other countries and seem to be related to Salmonella serovars in different food products and reservoirs. PMID:15078609

Transcriptional response of turkeys to MDR Salmonella enterica serovar heidelberg

USDA-ARS?s Scientific Manuscript database

Food-producing animals such as swine, cattle and poultry are a major reservoir of the human foodborne pathogen Salmonella. While some Salmonella serovars can cause disease in food-producing animals, most serovars colonize these animals asymptomatically, resulting in the hosts becoming carriers and ...

Haemophilus parasuis serovars isolated from pathological samples in Northern Italy.

PubMed

Luppi, A; Bonilauri, P; Dottori, M; Iodice, G; Gherpelli, Y; Merialdi, G; Maioli, G; Martelli, P

2013-04-01

From January 2007 to December 2011, a total of 106 Haemophilus parasuis strains isolated from pigs were serotyped by agar gel diffusion test (DG). Serovar 4 was the most prevalent (24.5%), followed by serovar 13 (19.8%) and serovar 5 (11.3%). Twenty-nine strains were non-typeable (27.3%). The strains were divided into two groups, depending on whether they were isolated from specific pathological lesions of systemic disease such as polyserositis, arthritis or meningitis (73 cases of 106) or from the lower respiratory tract of pigs suffering from bronchopneumonia (33 cases of 106). Serovars 4 and 13 had a higher prevalence in systemic infection (polyserositis) than in respiratory disease only. Pasteurella multocida (14/106), Streptococcus suis (7/106), Actinobacillus pleuropneumoniae (4/106), Bordetella bronchiseptica (3/106) and Arcanobacterium pyogenes (3/106) were isolated in association with H. parasuis. © 2012 Blackwell Verlag GmbH.

Characterization of Isolates of Salmonella enterica Serovar Stanley, a Serovar Endemic to Asia and Associated with Travel

PubMed Central

Le Hello, Simon; Bortolaia, Valeria; Pulsrikarn, Chaiwat; Nielsen, Eva Møller; Pornruangmong, Srirat; Chaichana, Phattharaporn; Svendsen, Christina Aaby; Weill, François-Xavier; Aarestrup, Frank M.

2012-01-01

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding blaCMY-2 gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad. PMID:22205822

Characterization of isolates of Salmonella enterica serovar Stanley, a serovar endemic to Asia and associated with travel.

PubMed

Hendriksen, Rene S; Le Hello, Simon; Bortolaia, Valeria; Pulsrikarn, Chaiwat; Nielsen, Eva Møller; Pornruangmong, Srirat; Chaichana, Phattharaporn; Svendsen, Christina Aaby; Weill, François-Xavier; Aarestrup, Frank M

2012-03-01

Salmonella enterica serovar Stanley (S. Stanley) is a common serovar in Southeast Asia and was the second most common serovar implicated in human salmonellosis in Thailand in the years 2002 to 2007. In contrast, this serovar is relatively uncommon in Europe. The objective of this study was to characterize a collection of S. Stanley strains isolated from Thai (n = 62), Danish (n = 39), and French (n = 24) patients to gain a broader understanding of the genetic diversity, population dynamics, and susceptibility to antimicrobials. All isolates were characterized by pulsed-field gel electrophoresis and antimicrobial susceptibility testing. The molecular mechanisms of resistance to extended-spectrum cephalosporins and plasmid-mediated resistance to quinolones were characterized by PCR and sequencing. Plasmid profiling, replicon typing, and microarray analysis were used to characterize the genetic mechanisms of antimicrobial resistance in 10 extended-spectrum cephalosporinase-producing isolates. Considerable genetic diversity was observed among the isolates characterized with 91 unique XbaI pulsed-field gel electrophoresis (PFGE) patterns, including 17 distinct clusters consisting of two to seven indistinguishable isolates. We found some of the S. Stanley isolates isolated from patients in Europe were acquired during travel to Southeast Asia, including Thailand. The presence of multiple plasmid lineages carrying the extended-spectrum cephalosporinase-encoding bla(CMY-2) gene in S. Stanley isolates from the central part of Thailand was confirmed. Our results emphasize that Thai authorities, as well as authorities in other countries lacking prudent use of antimicrobials, should improve the ongoing efforts to regulate antimicrobial use in agriculture and in clinical settings to limit the spread of multidrug-resistant Salmonella isolates and plasmids among humans and pigs in Thailand and abroad.

A putative regulatory genetic locus modulates virulence in the pathogen Leptospira interrogans.

PubMed

Eshghi, Azad; Becam, Jérôme; Lambert, Ambroise; Sismeiro, Odile; Dillies, Marie-Agnès; Jagla, Bernd; Wunder, Elsio A; Ko, Albert I; Coppee, Jean-Yves; Goarant, Cyrille; Picardeau, Mathieu

2014-06-01

Limited research has been conducted on the role of transcriptional regulators in relation to virulence in Leptospira interrogans, the etiological agent of leptospirosis. Here, we identify an L. interrogans locus that encodes a sensor protein, an anti-sigma factor antagonist, and two genes encoding proteins of unknown function. Transposon insertion into the gene encoding the sensor protein led to dampened transcription of the other 3 genes in this locus. This lb139 insertion mutant (the lb139(-) mutant) displayed attenuated virulence in the hamster model of infection and reduced motility in vitro. Whole-transcriptome analyses using RNA sequencing revealed the downregulation of 115 genes and the upregulation of 28 genes, with an overrepresentation of gene products functioning in motility and signal transduction and numerous gene products with unknown functions, predicted to be localized to the extracellular space. Another significant finding encompassed suppressed expression of the majority of the genes previously demonstrated to be upregulated at physiological osmolarity, including the sphingomyelinase C precursor Sph2 and LigB. We provide insight into a possible requirement for transcriptional regulation as it relates to leptospiral virulence and suggest various biological processes that are affected due to the loss of native expression of this genetic locus.

Characterization of two new putative adhesins of Leptospira interrogans.

PubMed

Figueredo, Jupciana M; Siqueira, Gabriela H; de Souza, Gisele O; Heinemann, Marcos B; Vasconcellos, Silvio A; Chapola, Erica G B; Nascimento, Ana L T O

2017-01-01

We here report the characterization of two novel proteins encoded by the genes LIC11122 and LIC12287, identified in the genome sequences of Leptospira interrogans, annotated, respectively, as a putative sigma factor and a hypothetical protein. The CDSs LIC11122 and LIC12287 have signal peptide SPII and SPI and are predicted to be located mainly at the cytoplasmic membrane of the bacteria. The genes were cloned and the proteins expressed using Escherichia coli. Proteinase K digestion showed that both proteins are surface exposed. Evaluation of interaction of recombinant proteins with extracellular matrix components revealed that they are laminin binding and they were called Lsa19 (LIC11122) and Lsa14 (LIC12287), for Leptospiral-surface adhesin of 19 and 14 kDa, respectively. The bindings were dose-dependent on protein concentration, reaching saturation, fulfilling the ligand-binding criteria. Reactivity of the recombinant proteins with leptospirosis human sera has shown that Lsa19 and, to a lesser extent, Lsa14, are recognized by antibodies, suggesting that, most probably, Lsa19 is expressed during infection. The proteins interact with plasminogen and generate plasmin in the presence of urokinase-type plasminogen activator. Plasmin generation in Leptospira has been associated with tissue penetration and immune evasion strategies. The presence of a sigma factor on the cell surface playing a secondary role, probably mediating host -pathogen interaction, suggests that LIC11122 is a moonlighting protein candidate. Although the biological significance of these putative adhesins will require the generation of mutants, our data suggest that Lsa19 is a potential candidate for future evaluation of its role in adhesion/colonization activities during L. interrogans infection.

Genome-Wide Transcriptional Start Site Mapping and sRNA Identification in the Pathogen Leptospira interrogans

PubMed Central

Zhukova, Anna; Fernandes, Luis Guilherme; Hugon, Perrine; Pappas, Christopher J.; Sismeiro, Odile; Coppée, Jean-Yves; Becavin, Christophe; Malabat, Christophe; Eshghi, Azad; Zhang, Jun-Jie; Yang, Frank X.; Picardeau, Mathieu

2017-01-01

Leptospira are emerging zoonotic pathogens transmitted from animals to humans typically through contaminated environmental sources of water and soil. Regulatory pathways of pathogenic Leptospira spp. underlying the adaptive response to different hosts and environmental conditions remains elusive. In this study, we provide the first global Transcriptional Start Site (TSS) map of a Leptospira species. RNA was obtained from the pathogen Leptospira interrogans grown at 30°C (optimal in vitro temperature) and 37°C (host temperature) and selectively enriched for 5′ ends of native transcripts. A total of 2865 and 2866 primary TSS (pTSS) were predicted in the genome of L. interrogans at 30 and 37°C, respectively. The majority of the pTSSs were located between 0 and 10 nucleotides from the translational start site, suggesting that leaderless transcripts are a common feature of the leptospiral translational landscape. Comparative differential RNA-sequencing (dRNA-seq) analysis revealed conservation of most pTSS at 30 and 37°C. Promoter prediction algorithms allow the identification of the binding sites of the alternative sigma factor sigma 54. However, other motifs were not identified indicating that Leptospira consensus promoter sequences are inherently different from the Escherichia coli model. RNA sequencing also identified 277 and 226 putative small regulatory RNAs (sRNAs) at 30 and 37°C, respectively, including eight validated sRNAs by Northern blots. These results provide the first global view of TSS and the repertoire of sRNAs in L. interrogans. These data will establish a foundation for future experimental work on gene regulation under various environmental conditions including those in the host. PMID:28154810

[Leptospirosis outbreak in calves from Corrientes Province, Argentina].

PubMed

Draghi, María G; Brihuega, Bibiana; Benítez, Daniel; Sala, Juan M; Biotti, Graciela M; Pereyra, Matilde; Homse, Alberto; Guariniello, Luciano

2011-01-01

Leptospirosis is an infectious disease resulting in significant economic losses in livestock production. This disease causes abortion, embryo death, death of calves within the first few days of life and mastitis. We report a leptospirosis outbreak in calf growing and fattening. Histopathological and hemoparasite studies, immunofluorescence, and bacterial cultures were performed. A strain of Leptospira interrogans serovar Pomona was isolated from samples collected from dead calves.

Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups.

PubMed

Jiang, Yanfen; Kulkarni, Raveendra R; Parreira, Valeria R; Poppe, Cornelius; Roland, Kenneth L; Prescott, John F

2010-10-01

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 10⁸ colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 10⁶ CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine's value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars.

Assessment of 2 Salmonella enterica serovar Typhimurium-based vaccines against necrotic enteritis in reducing colonization of chickens by Salmonella serovars of different serogroups

PubMed Central

Jiang, Yanfen; Kulkarni, Raveendra R.; Parreira, Valeria R.; Poppe, Cornelius; Roland, Kenneth L.; Prescott, John F.

2010-01-01

This study assessed the protective efficacy of oral vaccination with 2 experimental attenuated Salmonella Typhimurium-vectored vaccines for necrotic enteritis in protecting chickens against intestinal colonization by common serovars of Salmonella belonging to the 4 major serogroups affecting chickens. Birds were vaccinated orally with 1 × 108 colony-forming units (CFU) of 1 of the vaccine strains χ9241 and χ9352, which express a plasmid-encoded partial recombinant hypothetical protein gene (tHP) of Clostridium perfringens, at days 1 and 7 of age, and then were challenged at 14 d of age with 106 CFU of Salmonella serovars Anatum, Enteritidis, Heidelberg, Kentucky, or Typhimurium (representative serovars of serogroups B, C, D, and E). Birds were necropsied at 4 wk of age, and samples were collected to determine reduction in tissue and intestinal colonization. The chickens vaccinated with χ9241-tHP showed reduced colonization by Salmonella Enteritidis (serogroup D) and by Salmonella Heidelberg and Salmonella Typhimurium (serogroup B) compared with the control birds. No reduction in colonization was observed in the chickens vaccinated with χ9352-tHP. There was an association between the efficacy of these vaccine strains in protecting against necrotic enteritis, assessed on an earlier occasion, and their efficacy in protecting against Salmonella colonization. Thus, the choice of an attenuated Salmonella Typhimurium vaccine vector for delivery of heterologous antigens to chickens should be based partly on the vaccine’s value in protecting against colonization by serovars within serogroups B and D. Such vectors would have the additional benefit of reducing colonization of important Salmonella serovars. PMID:21197226

Il-10 deficient mice express IFN-γ mRNA and clear Leptospira interrogans from their kidneys more rapidly than normal C57BL/6 mice.

PubMed

Devlin, Amy A; Halvorsen, Priya J; Miller, Jennifer C; Laster, Scott M

2017-05-01

Leptospira interrogans (L. interrogans), the causative agent of leptospirosis, is a widespread zoonotic spirochete that lives a dual lifestyle. L. interrogans infects mice, rats, and wildlife in a persistent and asymptomatic fashion, while also causing productive and acute infections in other mammals such as humans and hamsters. Infections in humans can be fatal, accompanied by a cytokine storm and shock-like symptoms. Production of IL-10 has been noted in both rodent and human infections which has led a number of investigators to hypothesize that IL-10 plays a role in the pathogenesis of this disease. To test this hypothesis we have compared bacteremia and the cytokine response of normal and IL-10 deficient C57Bl/6 mice following ip infection with L. interrogans. In normal mice bacterial 16s mRNA was detected in both lung and kidney tissues within a day after infection. Levels of 16s mRNA then dropped in both organs with complete elimination from the lung by day 3 but persistence in the kidney for 7days after infection. In contrast, in IL-10 deficient mice, the organism was eliminated more rapidly from the kidney. We found that infection of both control and IL-10 deficient mice produced similar levels of a number of pro-inflammatory cytokine mRNAs. On the other hand, IFN-γ mRNA was only induced in IL-10 deficient mice. These results support the hypothesis that L. interrogans ability to induce IL-10, which in turn prevents production of IFN-γ and inhibits T cell immunity, may contribute to the persistent growth of this microorganism in the murine kidney. Copyright © 2017 Elsevier GmbH. All rights reserved.

Immunoreactivity of the AAA+ chaperone ClpB from Leptospira interrogans with sera from Leptospira-infected animals.

PubMed

Krajewska, Joanna; Arent, Zbigniew; Więckowski, Daniel; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2016-07-16

Leptospira interrogans is a spirochaete responsible for leptospirosis in mammals. The molecular mechanisms of the Leptospira virulence remain mostly unknown. Recently, it has been demonstrated that L. interrogans ClpB (ClpBLi) is essential for bacterial survival under stressful conditions and also during infection. The aim of this study was to provide further insight into the role of ClpB in L. interrogans and answer the question whether ClpBLi as a potential virulence factor may be a target of the humoral immune response during leptospiral infections in mammals. ClpBLi consists of 860 amino acid residues with a predicted molecular mass of 96.3 kDa and shows multi-domain organization similar to that of the well-characterized ClpB from Escherichia coli. The amino acid sequence identity between ClpBLi and E. coli ClpB is 52 %. The coding sequence of the clpB Li gene was cloned and expressed in E. coli BL21(DE3) strain. Immunoreactivity of the recombinant ClpBLi protein was assessed with the sera collected from Leptospira-infected animals and uninfected healthy controls. Western blotting and ELISA analysis demonstrated that ClpBLi activates the host immune system, as evidenced by an increased level of antibodies against ClpBLi in the sera from infected animals, as compared to the control group. Additionally, ClpBLi was found in kidney tissues of Leptospira-infected hamsters. ClpBLi is both synthesized and immunogenic during the infectious process, further supporting its involvement in the pathogenicity of Leptospira. In addition, the immunological properties of ClpBLi point to its potential value as a diagnostic antigen for the detection of leptospirosis.

Generation of mammalian host-adapted Leptospira interrogans by cultivation in peritoneal dialysis membrane chamber implantation in rats

USDA-ARS?s Scientific Manuscript database

Leptospira interrogans can infect a myriad of mammalian hosts, including humans (Bharti, Nally et al. 2003, Ko, Goarant et al. 2009). Following acquisition by a suitable host, leptospires disseminate via the bloodstream to multiple tissues, including the kidneys, where they adhere to and colonize th...

Leptospiral outer membrane protein microarray, a novel approach to identification of host ligand-binding proteins.

PubMed

Pinne, Marija; Matsunaga, James; Haake, David A

2012-11-01

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes belonging to the genus Leptospira. The leptospiral life cycle involves transmission via freshwater and colonization of the renal tubules of their reservoir hosts. Infection requires adherence to cell surfaces and extracellular matrix components of host tissues. These host-pathogen interactions involve outer membrane proteins (OMPs) expressed on the bacterial surface. In this study, we developed an Leptospira interrogans serovar Copenhageni strain Fiocruz L1-130 OMP microarray containing all predicted lipoproteins and transmembrane OMPs. A total of 401 leptospiral genes or their fragments were transcribed and translated in vitro and printed on nitrocellulose-coated glass slides. We investigated the potential of this protein microarray to screen for interactions between leptospiral OMPs and fibronectin (Fn). This approach resulted in the identification of the recently described fibronectin-binding protein, LIC10258 (MFn8, Lsa66), and 14 novel Fn-binding proteins, denoted Microarray Fn-binding proteins (MFns). We confirmed Fn binding of purified recombinant LIC11612 (MFn1), LIC10714 (MFn2), LIC11051 (MFn6), LIC11436 (MFn7), LIC10258 (MFn8, Lsa66), and LIC10537 (MFn9) by far-Western blot assays. Moreover, we obtained specific antibodies to MFn1, MFn7, MFn8 (Lsa66), and MFn9 and demonstrated that MFn1, MFn7, and MFn9 are expressed and surface exposed under in vitro growth conditions. Further, we demonstrated that MFn1, MFn4 (LIC12631, Sph2), and MFn7 enable leptospires to bind fibronectin when expressed in the saprophyte, Leptospira biflexa. Protein microarrays are valuable tools for high-throughput identification of novel host ligand-binding proteins that have the potential to play key roles in the virulence mechanisms of pathogens.

Distinct antibody responses of patients with mild and severe leptospirosis determined by whole proteome microarray analysis

PubMed Central

Lessa-Aquino, Carolina; Lindow, Janet C.; Randall, Arlo; Wunder, Elsio; Pablo, Jozelyn; Nakajima, Rie; Jasinskas, Algis; Cruz, Jaqueline S.; Damião, Alcineia O.; Nery, Nívison; Ribeiro, Guilherme S.; Costa, Federico; Hagan, José E.; Reis, Mitermayer Galvão; Ko, Albert I.; Medeiros, Marco Alberto; Felgner, Philip L.

2017-01-01

Background Leptospirosis is an important zoonotic disease worldwide. Humans usually present a mild non-specific febrile illness, but a proportion of them develop more severe outcomes, such as multi-organ failure, lung hemorrhage and death. Such complications are thought to depend on several factors, including the host immunity. Protective immunity is associated with humoral immune response, but little is known about the immune response mounted during naturally-acquired Leptospira infection. Methods and principal findings Here, we used protein microarray chip to profile the antibody responses of patients with severe and mild leptospirosis against the complete Leptospira interrogans serovar Copenhageni predicted ORFeome. We discovered a limited number of immunodominant antigens, with 36 antigens specific to patients, of which 11 were potential serodiagnostic antigens, identified at acute phase, and 33 were potential subunit vaccine targets, detected after recovery. Moreover, we found distinct antibody profiles in patients with different clinical outcomes: in the severe group, overall IgM responses do not change and IgG responses increase over time, while both IgM and IgG responses remain stable in the mild patient group. Analyses of individual patients’ responses showed that >74% of patients in the severe group had significant IgG increases over time compared to 29% of patients in the mild group. Additionally, 90% of IgM responses did not change over time in the mild group, compared to ~51% in the severe group. Conclusions In the present study, we detected antibody profiles associated with disease severity and speculate that patients with mild disease were protected from severe outcomes due to pre-existing antibodies, while patients with severe leptospirosis demonstrated an antibody profile typical of first exposure. Our findings represent a significant advance in the understanding of the humoral immune response to Leptospira infection, and we have identified new

Complete genome sequence of Leptospira alstonii serovar room 22, strain GWTS#1

USDA-ARS?s Scientific Manuscript database

We report the complete genome sequence of Leptospira alstonii serovar room 22 strain GWTS#1. This is the first isolate of L. alstonii to be cultured from a mammal, in Western Europe, and represents a new serovar of pathogenic leptospires....

Serovar distribution of a DNA sequence involved in the antigenic relationship between Leptospira and equine cornea.

PubMed

Lucchesi, Paula M A; Parma, Alberto E; Arroyo, Guillermo H

2002-01-01

Horses infected with Leptospira present several clinical disorders, one of them being recurrent uveitis. A common endpoint of equine recurrent uveitis is blindness. Serovar pomona has often been incriminated, although others have also been reported. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. A leptospiral DNA fragment that encodes cross-reacting epitopes was previously cloned and expressed in Escherichia coli. A region of that DNA fragment was subcloned and sequenced. Samples of leptospiral DNA from several sources were analysed by PCR with two primer pairs designed to amplify that region. Reference strains from serovars canicola, icterohaemorrhagiae, pomona, pyrogenes, wolffi, bataviae, sentot, hebdomadis and hardjo rendered products of the expected sizes with both pairs of primers. The specific DNA region was also amplified from isolates from Argentina belonging to serogroups Canicola and Pomona. Both L. biflexa serovar patoc and L. borgpetersenii serovar tarassovi rendered a negative result. The DNA sequence related to the antigen mimicry with equine cornea was not exclusively found in serovar pomona as it was also detected in several strains of Leptospira belonging to different serovars. The results obtained with L. biflexa serovar patoc strain Patoc I and L. borgpetersenii serovar tarassovi strain Perepelicin suggest that this sequence is not present in these strains, which belong to different genomospecies than those which gave positive results. This is an interesting finding since L. biflexa comprises nonpathogenic strains and serovar tarassovi has not been associated clinically with equine uveitis.

Leptospirosis in Sheep in Western Canada

PubMed Central

Kingscote, B.

1985-01-01

A survey of 930 ovine sera and kidneys from 33 sheep was conducted to assess the rate of leptospiral infection in sheep slaughtered in Alberta. Sera were tested for the presence of agglutinins to indigenous serovars of Leptospira interrogans. Kidneys with gross lesions were examined for the presence of leptospires by means of an indirect fluorescent antibody test (FAT) and by culture. Antibodies to serovars pomona and hardjo were present at rates of 1.0% and 0.4%, respectively, in sheep from Saskatchewan, Alberta and British Columbia. Sera from 120 feedlot lambs shipped from Oregon reacted to serovars pomona, hardjo and grippotyphosa at rates of 1.7%, 61.7% and 59.1%, respectively. Fluorescent antibody test detected serovars (presumptively) hardjo in 52% of Oregon feedlot lambs and grippotyphosa in 32% of the same group, a finding supported by the isolation of both these serovars from a pool of two fluorescent antibody test-positive kidneys. The grippotyphosa strain was highly virulent for hamsters, producing intense icterus and death. Leptospires, presumptively serovar grippotyphosa were demonstrated by fluorescent antibody test in one Alberta lamb kidney. The possibility of spreading leptospirosis by movement of breeding stock through public facilities and by assembling lambs in feedlots is discussed. PMID:17422531

Isolation and Identification of Putative Protein Substrates of the AAA+ Molecular Chaperone ClpB from the Pathogenic Spirochaete Leptospira interrogans.

PubMed

Krajewska, Joanna; Arent, Zbigniew; Zolkiewski, Michal; Kędzierska-Mieszkowska, Sabina

2018-04-18

Bacterial ClpB is an ATP-dependent Hsp100 chaperone that reactivates aggregated proteins in cooperation with the DnaK chaperone system and promotes survival of bacteria under stress conditions. A large number of publications also indicate that ClpB supports the virulence of bacteria, including a pathogenic spirochaete Leptospira interrogans responsible for leptospirosis in both animals and humans. However, the exact role of ClpB in bacterial pathogenicity remains poorly characterized. It can be assumed that ClpB, due to its role as the molecular chaperone, mediates refolding of essential bacterial proteins, including the known virulence factors, which may become prone to aggregation under infection-induced stresses. In this study, we identified putative substrates of ClpB from L. interrogans (ClpB Li ). For this purpose, we used a proteomic approach combining the ClpB-Trap affinity pull-down assays, Liquid chromatography-tandem mass spectrometry (LC-MS-MS/MS), and bioinformatics analyses. Most of the identified proteins were enzymes predominantly associated with major metabolic pathways like the tricarboxylic acid (TCA) cycle, glycolysis–gluconeogenesis and amino acid and fatty acid metabolism. Based on our proteomic study, we suggest that ClpB can support the virulence of L. interrogans by protecting the conformational integrity and catalytic activity of multiple metabolic enzymes, thus maintaining energy homeostasis in pathogen cells.

Molecular Characterization of Multiresistant d-Tartrate-Positive Salmonella enterica Serovar Paratyphi B Isolates

PubMed Central

Miko, Angelika; Guerra, Beatriz; Schroeter, Andreas; Dorn, Christina; Helmuth, Reiner

2002-01-01

Since 1996, the National Salmonella Reference Laboratory of Germany has received an increasing number of Salmonella enterica subsp. enterica serovar Paratyphi B isolates. Nearly all of these belonged to the dextrorotatory tartrate-positive variant (S. enterica subsp. enterica serovar Paratyphi B dT+), formerly called S. enterica subsp. enterica serovar Java. A total of 55 selected contemporary and older S. enterica subsp. enterica serovar Paratyphi B dT+ isolates were analyzed by plasmid profiling, antimicrobial resistance testing, pulsed-field gel electrophoresis, IS200 profiling, and PCR-based detection of integrons. The results showed a high genetic heterogeneity among 10 old strains obtained from 1960 to 1993. In the following years, however, new distinct multiresistant S. enterica subsp. enterica serovar Paratyphi B dT+ clones emerged, and one clonal lineage successfully displaced the older ones. Since 1994, 88% of the isolates investigated were multiple drug resistant. Today, a particular clone predominates in some German poultry production lines, poultry products, and various other sources. It was also detected in contemporary isolates from two neighboring countries as well. PMID:12202551

C3 fixed in vivo to cornea from horses inoculated with Leptospira interrogans.

PubMed

Parma, A E; Cerone, S I; Sansinanea, S A; Ghezzi, M

1992-10-01

C3 was detected bound in vivo to the opaque cornea of horses inoculated with killed Leptospira interrogans. Employing epithelial corneal cells isolated from a monolayer in tissue culture, we proved that C3 is fixed in vitro to the intact cell surface after incubation with a fresh equine anti-Leptospira serum. These findings, in addition to the infiltration of cornea with neutrophils and lymphocytes, may explain the mechanisms of tissue damage in recurrent uveitis of horses with leptospirosis.

Electrocardiographic changes in hospitalized patients with leptospirosis over a 10-year period.

PubMed

Škerk, Vedrana; Markotić, Alemka; Puljiz, Ivan; Kuzman, Ilija; Čeljuska Tošev, Elvira; Habuš, Josipa; Turk, Nenad; Begovac, Josip

2011-07-01

The aim of this study was to investigate the incidence and type of ECG changes in patients with leptospirosis regardless of clinical evidence of cardiac involvement. A total of 97 patients with serologically confirmed leptospirosis treated at the University Hospital for Infectious Diseases "Dr. Fran Mihaljević" in Zagreb, Croatia, were included in this retrospective study. A 12-lead resting ECG was routinely performed in the first 2 days after hospital admission. Thorough past and current medical history was obtained, and careful physical examination and laboratory tests were performed. Abnormal ECG findings were found in 56 of 97 (58%) patients. Patients with abnormal ECG had significantly elevated values of bilirubin and alanine aminotransferase, lower values of potassium and lower number of platelets, as well as more frequently recorded abnormal chest x-ray. Non-specific ventricular repolarization disturbances were the most common abnormal ECG finding. Other recorded ECG abnormalities were sinus tachycardia, right branch conduction disturbances, low voltage of the QRS complex in standard limb leads, supraventricular and ventricular extrasystoles, intraventricular conduction disturbances, atrioventricular block first-degree and atrial fibrillation. Myopericarditis was identified in 4 patients. Regardless of ECG changes, the most commonly detected infection was with Leptospira interrogans serovar Australis, Leptospira interrogans serovar Saxkoebing and Leptospira kirschneri serovar Grippotyphosa. The ECG abnormalities are common at the beginning of disease and are possibly caused by the direct effect of leptospires or are the non-specific result of a febrile infection and metabolic and electrolyte abnormalities. New studies are required for better understanding of the mechanism of ECG alterations in leptospirosis.

Ureaplasma serovars & their antimicrobial susceptibility in patients of infertility & genital tract infections.

PubMed

Dhawan, Benu; Malhotra, Neena; Sreenivas, Vishnubhatla; Rawre, Jyoti; Khanna, Neena; Chaudhry, Rama; Mittal, Suneeta

2012-12-01

Ureaplasmas have been implicated in a variety of clinical conditions. However, only certain serovars of ureaplasmas are disease associated. Only a few classes of antimicrobial agents are available for the treatment of mycoplasmal infections in humans. Increase of resistance of genital mycoplasmas to antimicrobials has been reported worldwide. The aim of the present study was to determine the occurrence of Ureaplasma serovars in patients with infertility and genital tract infections with polymerase chain reaction (PCR)-based serotyping. The antimicrobial susceptibilities of Ureaplasma spp. and Mycoplasma hominis were also assessed to determine the most suitable treatment strategy. Sexually active adults (n=147) with symptoms of genital tract infections and 115 infertile women were enrolled. Endocervical swabs from women and urethral swabs from men were subjected to culture and multiplex PCR for detection of genital mycoplasmas. Serotyping of Ureaplasma was done by PCR and antimicrobial susceptibility to doxycycline, azithromycin, josamycin and ofloxacin was done by microbroth dilution method. Ureaplasma was detected in 25.8 per cent patients with genital tract infections and 20.8 per cent in infertile women. Serovar 3/14 was the most frequent isolate followed by serovar 1 and serovar 6. The majority of Ureaplasma isolates were susceptible to doxycycline (91%) and josamycin (86%) followed by ofloxacin (77%) and azithromycin (71%). All the isolates of M. hominis were uniformly susceptible to doxycycline, josamycin and ofloxacin. The predominance of Ureaplasma serovar 3/14 suggests their possible pathogenic role in genital tract infections and infertility. For empirical treatment, doxycycline could be the drug of choice for genital mycoplasmas.

Genotypes of pathogenic Leptospira spp isolated from rodents in Argentina

PubMed Central

Loffler, Sylvia Grune; Pavan, Maria Elisa; Vanasco, Bibiana; Samartino, Luis; Suarez, Olga; Auteri, Carmelo; Romero, Graciela; Brihuega, Bibiana

2014-01-01

Leptospirosis is the most widespread zoonosis in the world and significant efforts have been made to determine and classify pathogenic Leptospira strains. This zoonosis is maintained in nature through chronic renal infections of carrier animals, with rodents and other small mammals serving as the most important reservoirs. Additionally, domestic animals, such as livestock and dogs, are significant sources of human infection. In this study, a multiple-locus variable-number tandem repeat analysis (MLVA) was applied to genotype 22 pathogenic Leptospira strains isolated from urban and periurban rodent populations from different regions of Argentina. Three MLVA profiles were identified in strains belonging to the species Leptospira interrogans (serovars Icterohaemorrhagiae and Canicola); one profile was observed in serovar Icterohaemorrhagiae and two MLVA profiles were observed in isolates of serovars Canicola and Portlandvere. All strains belonging to Leptospira borgpetersenii serovar Castellonis exhibited the same MLVA profile. Four different genotypes were isolated from urban populations of rodents, including both mice and rats and two different genotypes were isolated from periurban populations. PMID:24676656

Serovar distribution of a DNA sequence involved in the antigenic relationship between Leptospira and equine cornea

PubMed Central

Lucchesi, Paula MA; Parma, Alberto E; Arroyo, Guillermo H

2002-01-01

Background Horses infected with Leptospira present several clinical disorders, one of them being recurrent uveitis. A common endpoint of equine recurrent uveitis is blindness. Serovar pomona has often been incriminated, although others have also been reported. An antigenic relationship between this bacterium and equine cornea has been described in previous studies. A leptospiral DNA fragment that encodes cross-reacting epitopes was previously cloned and expressed in Escherichia coli. Results A region of that DNA fragment was subcloned and sequenced. Samples of leptospiral DNA from several sources were analysed by PCR with two primer pairs designed to amplify that region. Reference strains from serovars canicola, icterohaemorrhagiae, pomona, pyrogenes, wolffi, bataviae, sentot, hebdomadis and hardjo rendered products of the expected sizes with both pairs of primers. The specific DNA region was also amplified from isolates from Argentina belonging to serogroups Canicola and Pomona. Both L. biflexa serovar patoc and L. borgpetersenii serovar tarassovi rendered a negative result. Conclusions The DNA sequence related to the antigen mimicry with equine cornea was not exclusively found in serovar pomona as it was also detected in several strains of Leptospira belonging to different serovars. The results obtained with L. biflexa serovar patoc strain Patoc I and L. borgpetersenii serovar tarassovi strain Perepelicin suggest that this sequence is not present in these strains, which belong to different genomospecies than those which gave positive results. This is an interesting finding since L. biflexa comprises nonpathogenic strains and serovar tarassovi has not been associated clinically with equine uveitis. PMID:11869455

A Unique Capsule Locus in the Newly Designated Actinobacillus pleuropneumoniae Serovar 16 and Development of a Diagnostic PCR Assay.

PubMed

Bossé, Janine T; Li, Yanwen; Sárközi, Rita; Gottschalk, Marcelo; Angen, Øystein; Nedbalcova, Katerina; Rycroft, Andrew N; Fodor, László; Langford, Paul R

2017-03-01

Actinobacillus pleuropneumoniae causes pleuropneumonia, an economically significant lung disease of pigs. Recently, isolates of A. pleuropneumoniae that were serologically distinct from the previously characterized 15 serovars were described, and a proposal was put forward that they comprised a new serovar, serovar 16. Here we used whole-genome sequencing of the proposed serovar 16 reference strain A-85/14 to confirm the presence of a unique capsular polysaccharide biosynthetic locus. For molecular diagnostics, primers were designed from the capsule locus of strain A-85/14, and a PCR was formulated that differentiated serovar 16 isolates from all 15 known serovars and other common respiratory pathogenic/commensal bacteria of pigs. Analysis of the capsule locus of strain A-85/14 combined with the previous serological data show the existence of a sixteenth serovar-designated serovar 16-of A. pleuropneumoniae . Copyright © 2017 Bossé et al.

A temporal study of Salmonella serovars in animals in Alberta between 1990 and 2001

PubMed Central

2005-01-01

Abstract Passive laboratory-based surveillance data from Alberta Agriculture Food and Rural Development were analyzed for common Salmonella serovars, prevalences, trends, and for the presence of temporal clusters. There were 1767 isolates between October 1990 and December 2001 comprising 63 different serovars, including 961 isolates from chickens, 418 from cattle, 108 from pigs, 102 from turkeys, and 178 from all other species combined. Salmonella Typhimurium, Heidelberg, Hadar, Kentucky, and Thompson were the 5 most frequently isolated serovars. Approximately 60% of the S. Typhimurium were isolated from cattle, whereas over 90% of the S. Heidelberg, Hadar, Kentucky, and Thompson were isolated from chickens. Salmonella Enteritidis was rarely isolated. There was an increasing trend in isolates from chickens, cattle, and pigs, and a decreasing trend in isolates from turkeys. Temporal clusters were observed in 11 of 15 serovars examined in chickens (S. Anatum, Heidelberg, Infantis, Kentucky, Mbandaka, Montevideo, Nienstedten, Oranienburg, Thompson, Typhimurium, and Typhimurium var. Copenhagen), 5 of 5 serovars in cattle (S. Dublin, Montevideo, Muenster, Typhimurium, and Typhimurium var. Copenhagen), and 1 of 3 serovars in pigs (S. Typhimurium). Short-duration clusters may imply point source infections, whereas long-duration clusters may indicate an increase in the prevalence of the serovar, farm-to-farm transmission, or a wide-spread common source. A higher concentration of clusters in the winter months may reflect greater confinement, reduced ventilation, stressors, or increased exposure to wildlife vectors that are sharing housing during the winter. Detection of large clusters of Salmonella may have public health implications in addition to animal health concerns. PMID:15971672

Genomic comparison of the closely-related Salmonella enterica serovars enteritidis, dublin and gallinarum

DOE Office of Scientific and Technical Information (OSTI.GOV)

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content betweenmore » strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. As a result, the loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.« less

Genomic comparison of the closely-related Salmonella enterica serovars enteritidis, dublin and gallinarum

DOE PAGES

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.; ...

2015-06-03

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content betweenmore » strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. As a result, the loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars.« less

Genomic Comparison of the Closely-Related Salmonella enterica Serovars Enteritidis, Dublin and Gallinarum

PubMed Central

Matthews, T. David; Schmieder, Robert; Silva, Genivaldo G. Z.; Busch, Julia; Cassman, Noriko; Dutilh, Bas E.; Green, Dawn; Matlock, Brian; Heffernan, Brian; Olsen, Gary J.; Farris Hanna, Leigh; Schifferli, Dieter M.; Maloy, Stanley; Dinsdale, Elizabeth A.; Edwards, Robert A.

2015-01-01

The Salmonella enterica serovars Enteritidis, Dublin, and Gallinarum are closely related but differ in virulence and host range. To identify the genetic elements responsible for these differences and to better understand how these serovars are evolving, we sequenced the genomes of Enteritidis strain LK5 and Dublin strain SARB12 and compared these genomes to the publicly available Enteritidis P125109, Dublin CT 02021853 and Dublin SD3246 genome sequences. We also compared the publicly available Gallinarum genome sequences from biotype Gallinarum 287/91 and Pullorum RKS5078. Using bioinformatic approaches, we identified single nucleotide polymorphisms, insertions, deletions, and differences in prophage and pseudogene content between strains belonging to the same serovar. Through our analysis we also identified several prophage cargo genes and pseudogenes that affect virulence and may contribute to a host-specific, systemic lifestyle. These results strongly argue that the Enteritidis, Dublin and Gallinarum serovars of Salmonella enterica evolve by acquiring new genes through horizontal gene transfer, followed by the formation of pseudogenes. The loss of genes necessary for a gastrointestinal lifestyle ultimately leads to a systemic lifestyle and niche exclusion in the host-specific serovars. PMID:26039056

Porcine response to a multidrug-resistant Salmonella enterica serovar I 4,[5],12:i:- outbreak isolate

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar I 4,[5],12:i:- has emerged as a common nontyphoidal Salmonella serovar to cause human foodborne illness. An interesting trait of serovar I 4,[5],12:i:- is it only expresses the fliC gene for bacterial motility (i.e. monophasic), while most Salmonella strains alternately e...

Survival and Filamentation of Salmonella enterica Serovar Enteritidis PT4 and Salmonella enterica Serovar Typhimurium DT104 at Low Water Activity

PubMed Central

Mattick, K. L.; Jørgensen, F.; Legan, J. D.; Cole, M. B.; Porter, J.; Lappin-Scott, H. M.; Humphrey, T. J.

2000-01-01

In this study we investigated the long-term survival of and morphological changes in Salmonella strains at low water activity (aw). Salmonella enterica serovar Enteritidis PT4 and Salmonella enterica serovar Typhimurium DT104 survived at low aw for long periods, but minimum humectant concentrations of 8% NaCl (aw, 0.95), 96% sucrose (aw, 0.94), and 32% glycerol (aw, 0.92) were bactericidal under most conditions. Salmonella rpoS mutants were usually more sensitive to bactericidal levels of NaCl, sucrose, and glycerol. At a lethal aw, incubation at 37°C resulted in more rapid loss of viability than incubation at 21°C. At aw values of 0.93 to 0.98, strains of S. enterica serovar Enteritidis and S. enterica serovar Typhimurium formed filaments, some of which were at least 200 μm long. Filamentation was independent of rpoS expression. When the preparations were returned to high-aw conditions, the filaments formed septa, and division was complete within approximately 2 to 3 h. The variable survival of Salmonella strains at low aw highlights the importance of strain choice when researchers produce modelling data to simulate worst-case scenarios or conduct risk assessments based on laboratory data. The continued increase in Salmonella biomass at low aw (without a concomitant increase in microbial count) would not have been detected by traditional microbiological enumeration tests if the tests had been performed immediately after low-aw storage. If Salmonella strains form filaments in food products that have low aw values (0.92 to 0.98), there are significant implications for public health and for designing methods for microbiological monitoring. PMID:10742199

Streptomycin Induced Stress Response in Salmonella enterica Serovar Typhimurium Shows Distinct Colony Scatter Signature

PubMed Central

Singh, Atul K.; Drolia, Rishi; Bai, Xingjian; Bhunia, Arun K.

2015-01-01

We investigated the streptomycin-induced stress response in Salmonella enterica serovars with a laser optical sensor, BARDOT (bacterial rapid detection using optical scattering technology). Initially, the top 20 S. enterica serovars were screened for their response to streptomycin at 100 μg/mL. All, but four S. enterica serovars were resistant to streptomycin. The MIC of streptomycin-sensitive serovars (Enteritidis, Muenchen, Mississippi, and Schwarzengrund) varied from 12.5 to 50 μg/mL, while streptomycin-resistant serovar (Typhimurium) from 125–250 μg/mL. Two streptomycin-sensitive serovars (Enteritidis and Mississippi) were grown on brain heart infusion (BHI) agar plates containing sub-inhibitory concentration of streptomycin (1.25–5 μg/mL) and a streptomycin-resistant serovar (Typhimurium) was grown on BHI containing 25–50 μg/mL of streptomycin and the colonies (1.2 ± 0.1 mm diameter) were scanned using BARDOT. Data show substantial qualitative and quantitative differences in the colony scatter patterns of Salmonella grown in the presence of streptomycin than the colonies grown in absence of antibiotic. Mass-spectrometry identified overexpression of chaperonin GroEL, which possibly contributed to the observed differences in the colony scatter patterns. Quantitative RT-PCR and immunoassay confirmed streptomycin-induced GroEL expression while, aminoglycoside adenylyltransferase (aadA), aminoglycoside efflux pump (aep), multidrug resistance subunit acrA, and ribosomal protein S12 (rpsL), involved in streptomycin resistance, were unaltered. The study highlights suitability of the BARDOT as a non-invasive, label-free tool for investigating stress response in Salmonella in conjunction with the molecular and immunoassay methods. PMID:26252374

Prevalence of Salmonella spp., and serovars isolated from captive exotic reptiles in New Zealand.

PubMed

Kikillus, K H; Gartrell, B D; Motion, E

2011-07-01

To investigate the prevalence of Salmonella spp. in captive exotic reptile species in New Zealand, and identify the serovars isolated from this population. Cloacal swabs were obtained from 378 captive exotic reptiles, representing 24 species, residing in 25 collections throughout New Zealand between 2008 and 2009. Samples were cultured for Salmonella spp., and suspected colonies were serotyped by the Institute of Environmental Science and Research (ESR). Forty-three of the 378 (11.4%) reptiles sampled tested positive for Salmonella spp., with 95% CI for the estimated true prevalence being 12-25% in exotic reptiles in this study population. Lizards tested positive for Salmonella spp. more often than chelonians. Agamid lizards tested positive more often than any other family group, with 95% CI for the estimated true prevalence being 56-100%.. Six Salmonella serovars from subspecies I and two from subspecies II were isolated. The serovar most commonly isolated was S. Onderstepoort (30.2%), followed by S. Thompson (20.9%), S. Potsdam (14%), S. Wangata (14%), S. Infantis (11.6%) and S. Eastbourne (2.3%). All of the subspecies I serovars have been previously reported in both reptiles and humans in New Zealand, and include serovars previously associated with disease in humans. This study showed that Salmonella spp. were commonly carried by exotic reptiles in the study population in New Zealand. Several serovars of Salmonella spp. with known pathogenicity to humans were isolated, including S. Infantis, which is one of the most common serovars isolated from both humans and non-human sources in New Zealand. The limitations of this study included the bias engendered by the need for voluntary involvement in the study, and the non-random sampling design. Based on the serovars identified in this and previous studies, it is recommended native and exotic reptiles be segregated within collections, especially when native reptiles may be used for biodiversity restoration

Differential antibacterial response of chicken granulosa cells to invasion by Salmonella serovars.

PubMed

Babu, Uma S; Harrison, Lisa M; Patel, Isha R; Ramirez, Gerardo A; Williams, Kristina M; Pereira, Marion; Balan, Kannan V

2016-06-01

In the United States, Salmonella enterica ser. Enteritidis (SE) is among the leading bacterial cause of foodborne illness via consumption of raw or undercooked eggs. The top Salmonella serovars implicated in U.S. foodborne outbreaks associated with chicken consumption include SE, Typhimurium (ST), Heidelberg (SH), Montevideo, Mbandka, Braenderup, and Newport. While enforcement actions target the eradication of SE from layer hens, there is a growing concern that other serovars could occupy this niche and be a cause of egg-transmitted human salmonellosis. Therefore, we tested the invasion and survival of SE, SH, ST, and Salmonella enterica ser. Hadar (S. Hadar) at 4 and 20 h post infection (hpi) in chicken ovarian granulosa cells (cGC); a cellular layer which surrounds the previtelline layer and central yolk in egg-forming follicles. We also evaluated cGC transcriptional changes, using an antibacterial response PCR array, to assess host response to intracellular SalmonellaWe observed that invasion of cGC by SE, SH, and ST was significantly higher than invasion by S. Hadar, with ST showing the highest level of invasion. The Bacterial Survival Index, defined as the ratio of intracellular bacteria at 20 and 4 h, were 18.94, 7.35, and 15.27 for SE, SH, and ST, respectively, with no significant difference in survival between SE or ST compared to SH. Evaluation of cGC anti-Salmonella gene responses indicated that at 4 hpi there was a significant decrease in Toll-like receptor (TLR)-4 mRNA in cGC infected with SE, whereas TLR5 and myeloid differentiation primary response gene 88 were significantly down regulated across all serovars. At 4 hpi, invasion by Salmonella serovars resulted in significant upregulation of several antimicrobial genes, and proinflammatory cytokines and chemokines (PICs). At 20 hpi, all the serovars induced PICs with SH being the strongest inducer. Additionally, SE, SH and ST differentially induced signal transduction pathways. Although only a single

Influence of Environmental Factors and Human Activity on the Presence of Salmonella Serovars in a Marine Environment

PubMed Central

Martinez-Urtaza, Jaime; Saco, Montserrat; de Novoa, Jacobo; Perez-Piñeiro, Pelayo; Peiteado, Jesus; Lozano-Leon, Antonio; Garcia-Martin, Oscar

2004-01-01

The temporal and spatial distribution of Salmonella contamination in the coastal waters of Galicia (northwestern Spain) relative to contamination events with different environmental factors (temperature, wind, hours of sunlight, rainfall, and river flow) were investigated over a 4-year period. Salmonellae were isolated from 127 of 5,384 samples of molluscs and seawater (2.4%), and no significant differences (P < 0.05) between isolates obtained in different years were observed. The incidence of salmonellae was significantly higher in water column samples (2.9%) than in those taken from the marine benthos (0.7%). Of the 127 strains of Salmonella isolated, 20 different serovars were identified. Salmonella enterica serovar Senftenberg was the predominant serovar, being represented by 54 isolates (42.5%), followed by serovar Typhimurium (19 isolates [15%]) and serovar Agona (12 isolates [9.4%]). Serovar Senftenberg was detected at specific points on the coast and could not be related to any of the environmental parameters analyzed. All serovars except Salmonella serovar Senftenberg were found principally in the southern coastal areas close to the mouths of rivers, and their incidence was associated with high southwestern wind and rainfall. Using multiple logistic regression analysis models, the prevalence of salmonellae was best explained by environmental parameters on the day prior to sampling. Understanding this relationship may be useful for the control of molluscan shellfish harvests, with wind and rainfall serving as triggers for closure. PMID:15066800

Serological survey for diseases in free-ranging coyotes (Canis latrans) in Yellowstone National Park, Wyoming.

PubMed

Gese, E M; Schultz, R D; Johnson, M R; Williams, E S; Crabtree, R L; Ruff, R L

1997-01-01

From October 1989 to June 1993, we captured and sampled 110 coyotes (Canis latrans) for various diseases in Yellowstone National Park, Wyoming (USA). Prevalence of antibodies against canine parvovirus (CPV) was 100% for adults (> 24 months old), 100% for yearlings (12 to 24 months old), and 100% for old pups (4 to 12 months old); 0% of the young pups (< 3 months old) had antibodies against CPV. Presence of antibodies against canine distemper virus (CDV) was associated with the age of the coyote, with 88%, 54%, 23%, and 0% prevalence among adults, yearlings, old pups, and young pups, respectively. Prevalence of CDV antibodies declined over time from 100% in 1989 to 33% in 1992. The prevalence of canine infectious hepatitis (ICH) virus antibodies was 97%, 82%, 54%, and 33%, for adults, yearlings, old pups, and young pups, respectively. The percentage of coyotes with ICH virus antibodies also declined over time from a high of 100% in 1989 to 31% in 1992, and 42% in 1993. Prevalence of antibodies against Yersinia pestis was 86%, 33%, 80%, and 7%, for adults, yearlings, old pups, and young pups, respectively, and changed over time from 57% in 1991 to 0% in 1993. The prevalence of antibodies against Francisella tularensis was 21%, 17%, 10%, and 20%, for adults, yearlings, old pups, and young pups, respectively. No coyotes had serologic evidence of exposure to brucellosis, either Brucella abortus or Brucella canis. No coyotes were seropositive to Leptospira interrogans (serovars canicola, hardjo, and icterohemorrhagiae). Prevalence of antibodies against L. interrogans serovar pomona was 7%, 0%, 0%, and 9%, for adults, yearlings, old pups, and young pups, respectively. Antibodies against L. interrogans serovar grippotyphosa were present in 17% of adults and 0% of yearlings, old pups, and young pups. Many infectious canine pathogens (CPV, CDV, ICH virus) are prevalent in coyotes in Yellowstone National Park, with CPV influencing coyote pup survival during the first 3 months

Electrocardiographic changes in hospitalized patients with leptospirosis over a 10-year period

PubMed Central

Škerk, Vedrana; Markotić, Alemka; Puljiz, Ivan; Kuzman, Ilija; Tošev, Elvira Čeljuska; Habuš, Josipa; Turk, Nenad; Begovac, Josip

2011-01-01

Summary Background The aim of this study was to investigate the incidence and type of ECG changes in patients with leptospirosis regardless of clinical evidence of cardiac involvement. Material/Methods A total of 97 patients with serologically confirmed leptospirosis treated at the University Hospital for Infectious Diseases „Dr. Fran Mihaljević‟ in Zagreb, Croatia, were included in this retrospective study. A 12-lead resting ECG was routinely performed in the first 2 days after hospital admission. Thorough past and current medical history was obtained, and careful physical examination and laboratory tests were performed. Results Abnormal ECG findings were found in 56 of 97 (58%) patients. Patients with abnormal ECG had significantly elevated values of bilirubin and alanine aminotransferase, lower values of potassium and lower number of platelets, as well as more frequently recorded abnormal chest x-ray. Non-specific ventricular repolarization disturbances were the most common abnormal ECG finding. Other recorded ECG abnormalities were sinus tachycardia, right branch conduction disturbances, low voltage of the QRS complex in standard limb leads, supraventricular and ventricular extrasystoles, intraventricular conduction disturbances, atrioventricular block first-degree and atrial fibrillation. Myopericarditis was identified in 4 patients. Regardless of ECG changes, the most commonly detected infection was with Leptospira interrogans serovar Australis, Leptospira interrogans serovar Saxkoebing and Leptospira kirschneri serovar Grippotyphosa. Conclusions The ECG abnormalities are common at the beginning of disease and are possibly caused by the direct effect of leptospires or are the non-specific result of a febrile infection and metabolic and electrolyte abnormalities. New studies are required for better understanding of the mechanism of ECG alterations in leptospirosis. PMID:21709630

Virulence of Serovar C-1 Strains of Avibacterium paragallinarum.

PubMed

Trujillo-Ruíz, H H; Shivaprasad, H L; Morales-Erasto, V; Talavera-Rojas, M; Salgado-Miranda, C; Salazar-García, F; Blackall, P J; Soriano-Vargas, E

2016-12-01

The bacterium Avibacterium paragallinarum is the etiologic agent of infectious coryza of chickens. There are nine serovars of A. paragallinarum , and serovar C-1 has emerged in outbreaks of infectious coryza in layer hens in the Americas, with all isolates having been obtained from infectious coryza-vaccinated chickens. In the current study, the clinical and histopathologic outcomes of experimental infections in chickens with A. paragallinarum of serovar C-1 were investigated. The Japanese serovar reference strain, H-18, and a Mexican isolate, ESV-135, were included in the study. No differences in clinical sign scores or morbidity were observed between the two strains. The two bacterial strains caused microscopic lesions of lymphoplasmacytic inflammation in the mucosa of the nasal cavity, infraorbital sinus, and trachea. Similar severe lesions were observed in birds inoculated with both H-18 and ESV-135 strains. The lesions were present 48 hr after inoculation and persisted until day 10 after inoculation. Slight to severe, extensive hemorrhages were observed in the lumen, mucous membranes, and lamina propria of the nasal cavity and infraorbital sinus in most of the chickens inoculated with either the reference strain H-18 or the ESV-135 isolate. Hemorrhages in the upper respiratory tract of chickens experimentally infected with A. paragallinarum are reported here for the first time. The results have confirmed the high virulence of the reference strain H-18 as previously reported and have shown that the Mexican isolate was as virulent as the reference strain. The virulence of A. paragallinarum isolates may play a role in explaining why severe infectious coryza outbreaks are being seen in both vaccinated and nonvaccinated chicken flocks.

Salmonella Pathogenicity and Host Adaptation in Chicken-Associated Serovars

PubMed Central

Johnson, Timothy J.; Ricke, Steven C.; Nayak, Rajesh; Danzeisen, Jessica

2013-01-01

SUMMARY Enteric pathogens such as Salmonella enterica cause significant morbidity and mortality. S. enterica serovars are a diverse group of pathogens that have evolved to survive in a wide range of environments and across multiple hosts. S. enterica serovars such as S. Typhi, S. Dublin, and S. Gallinarum have a restricted host range, in which they are typically associated with one or a few host species, while S. Enteritidis and S. Typhimurium have broad host ranges. This review examines how S. enterica has evolved through adaptation to different host environments, especially as related to the chicken host, and continues to be an important human pathogen. Several factors impact host range, and these include the acquisition of genes via horizontal gene transfer with plasmids, transposons, and phages, which can potentially expand host range, and the loss of genes or their function, which would reduce the range of hosts that the organism can infect. S. Gallinarum, with a limited host range, has a large number of pseudogenes in its genome compared to broader-host-range serovars. S. enterica serovars such as S. Kentucky and S. Heidelberg also often have plasmids that may help them colonize poultry more efficiently. The ability to colonize different hosts also involves interactions with the host's immune system and commensal organisms that are present. Thus, the factors that impact the ability of Salmonella to colonize a particular host species, such as chickens, are complex and multifactorial, involving the host, the pathogen, and extrinsic pressures. It is the interplay of these factors which leads to the differences in host ranges that we observe today. PMID:24296573

Evaluation of Molecular Methods for Identification of Salmonella Serovars

PubMed Central

Gurnik, Simone; Ahmad, Aaminah; Blimkie, Travis; Murphy, Stephanie A.; Kropinski, Andrew M.; Nash, John H. E.

2016-01-01

Classification by serotyping is the essential first step in the characterization of Salmonella isolates and is important for surveillance, source tracking, and outbreak detection. To improve detection and reduce the burden of salmonellosis, several rapid and high-throughput molecular Salmonella serotyping methods have been developed. The aim of this study was to compare three commercial kits, Salm SeroGen (Salm Sero-Genotyping AS-1 kit), Check&Trace (Check-Points), and xMAP (xMAP Salmonella serotyping assay), to the Salmonella genoserotyping array (SGSA) developed by our laboratory. They were assessed using a panel of 321 isolates that represent commonly reported serovars from human and nonhuman sources globally. The four methods correctly identified 73.8% to 94.7% of the isolates tested. The methods correctly identified 85% and 98% of the clinically important Salmonella serovars Enteritidis and Typhimurium, respectively. The methods correctly identified 75% to 100% of the nontyphoidal, broad host range Salmonella serovars, including Heidelberg, Hadar, Infantis, Kentucky, Montevideo, Newport, and Virchow. The sensitivity and specificity of Salmonella serovars Typhimurium and Enteritidis ranged from 85% to 100% and 99% to 100%, respectively. It is anticipated that whole-genome sequencing will replace serotyping in public health laboratories in the future. However, at present, it is approximately three times more expensive than molecular methods. Until consistent standards and methodologies are deployed for whole-genome sequencing, data analysis and interlaboratory comparability remain a challenge. The use of molecular serotyping will provide a valuable high-throughput alternative to traditional serotyping. This comprehensive analysis provides a detailed comparison of commercial kits available for the molecular serotyping of Salmonella. PMID:27194688

Identification of three extra-chromosomal replicons in Leptospira pathogenic strain and development of new shuttle vectors.

PubMed

Zhu, Weinan; Wang, Jin; Zhu, Yongzhang; Tang, Biao; Zhang, Yunyi; He, Ping; Zhang, Yan; Liu, Boyu; Guo, Xiaokui; Zhao, Guoping; Qin, Jinhong

2015-02-15

The genome of pathogenic Leptospira interrogans contains two chromosomes. Plasmids and prophages are known to play specific roles in gene transfer in bacteria and can potentially serve as efficient genetic tools in these organisms. Although plasmids and prophage remnants have recently been reported in Leptospira species, their characteristics and potential applications in leptospiral genetic transformation systems have not been fully evaluated. Three extrachromosomal replicons designated lcp1 (65,732 bp), lcp2 (56,757 bp), and lcp3 (54,986 bp) in the L. interrogans serovar Linhai strain 56609 were identified through whole genome sequencing. All three replicons were stable outside of the bacterial chromosomes. Phage particles were observed in the culture supernatant of 56609 after mitomycin C induction, and lcp3, which contained phage-related genes, was considered to be an inducible prophage. L. interrogans-Escherichia coli shuttle vectors, constructed with the predicted replication elements of single rep or rep combined with parAB loci from the three plasmids were shown to successfully transform into both saprophytic and pathogenic Leptospira species, suggesting an essential function for rep genes in supporting auto-replication of the plasmids. Additionally, a wide distribution of homologs of the three rep genes was identified in L. interrogans isolates, and correlation tests showed that the transformability of the shuttle vectors in L. interrogans isolates depended, to certain extent, on genetic compatibility between the rep sequences of both plasmid and host. Three extrachromosomal replicons co-exist in L. interrogans, one of which we consider to be an inducible prophage. The vectors constructed with the rep genes of the three replicons successfully transformed into saprophytic and pathogenic Leptospira species alike, but this was partly dependent on genetic compatibility between the rep sequences of both plasmid and host.

Lymphogranuloma Venereum-Serovar L2b Presenting With Painful Genital Ulceration: An Emerging Clinical Presentation?

PubMed

Haber, Roger; Maatouk, Ismaël; de Barbeyrac, Bertille; Bagot, Martine; Janier, Michel; Fouéré, Sébastien

2017-05-01

These 5 cases of atypical inflammatory lymphogranula venereum (LGV) serovar L2b presenting initially with edema and persistent painful ulceration illustrate that clinical manifestations of LGV in the current outbreak in men who have sex with men reflect the influence of both the serovars virulence and the host immune system and are not confined to proctitis. L2b serovar could have a particular high virulence profile, and the need for awareness of LGV as a cause of genital ulceration is crucial.

Passive surveillance of Leptospira infection in swine in Germany.

PubMed

Strutzberg-Minder, Katrin; Tschentscher, Astrid; Beyerbach, Martin; Homuth, Matthias; Kreienbrock, Lothar

2018-01-01

As no current data are available on the prevalence of leptospiral infection in swine in Germany, we analysed laboratory data from diagnostic examinations carried out on samples from swine all over Germany from January 2011 to September 2016. A total of 29,829 swine sera were tested by microscopic agglutination test (MAT) for antibodies against strains of eleven Leptospira serovars. Overall, 20.2% (6025) of the total sample collection tested positive for leptospiral infection. Seropositivity ranged between 16.3% (964) in 2011 and 30.9% (941) in 2016 (January to September only). Of all samples, 11.6% (57.3% of the positives) reacted with only one Leptospira serovar, and only 8.6% (42.7% of the positives) reacted simultaneously with two or more serovars. The most frequently detected serovar was Bratislava, which was found in 11.6% (3448) of all samples, followed by the serovars Australis in 7.3% (2185), Icterohaemorrhagiae in 4.0% (1191), Copenhageni in 4.0% (1182), Autumnalis in 3.7% (1054), Canicola in 2.0% (585), and Pomona in 1.2% (368). Modelling shows that both the year and the reason for testing at the laboratory had statistically strong effects on the test results; however, no interactions were determined between those factors. The results support the suggestion that the seropositivities found may be considered to indicate the state of leptospiral infections in the German swine population. Although data from passive surveillance are prone to selection bias, stratified analysis by initial reason for examination and analyses by model approaches may correct for biases. A prevalence of about 20% for a leptospiral infection is most probable for sows with reproductive problems in Germany, with an increasing trend. Swine in Germany are probably a reservoir host for serovar Bratislava, but in contrast to other studies not for Pomona and Tarassovi.

Molecular analysis of Leptospira spp. isolated from humans by restriction fragment length polymorphism, real-time PCR and pulsed-field gel electrophoresis.

PubMed

Turk, Nenad; Milas, Zoran; Mojcec, Vesna; Ruzic-Sabljic, Eva; Staresina, Vilim; Stritof, Zrinka; Habus, Josipa; Postic, Daniele

2009-11-01

A total of 17 Leptospira clinical strains isolated from humans in Croatia were serologically and genetically analysed. For serovar identification, the microscopic agglutination test (MAT) and pulsed-field gel electrophoresis (PFGE) were used. To identify isolates on genomic species level, PCR-based restriction fragment length polymorphism (RFLP) and real-time PCR were performed. MAT revealed the following serogroup affinities: Grippotyphosa (seven isolates), Icterohaemorrhagiae (eight isolates) and Javanica (two isolates). RFLP of PCR products from a 331-bp-long fragment of rrs (16S rRNA gene) digested with endonucleases MnlI and DdeI and real-time PCR revealed three Leptospira genomic species. Grippotyphosa isolates belonged to Leptospira kirschneri, Icterohaemorrhagiae isolates to Leptospira interrogans and Javanica isolates to Leptospira borgpetersenii. Genomic DNA from 17 leptospiral isolates was digested with NotI and SgrAI restriction enzymes and analysed by PFGE. Results showed that seven isolates have the same binding pattern to serovar Grippotyphosa, eight isolates to serovar Icterohaemorrhagiae and two isolates to serovar Poi. Results demonstrate the diversity of leptospires circulating in Croatia. We point out the usefulness of a combination of PFGE, RFLP and real-time PCR as appropriate molecular methods in molecular analysis of leptospires.

Refined Live Attenuated Salmonella enterica Serovar Typhimurium and Enteritidis Vaccines Mediate Homologous and Heterologous Serogroup Protection in Mice

PubMed Central

Schmidlein, Patrick; Simon, Raphael; Pasetti, Marcela F.; Galen, James E.; Levine, Myron M.

2015-01-01

Invasive nontyphoidal Salmonella (NTS) infections constitute a major health problem among infants and toddlers in sub-Saharan Africa; these infections also occur in infants and the elderly in developed countries. We genetically engineered a Salmonella enterica serovar Typhimurium strain of multilocus sequence type 313, the predominant genotype circulating in sub-Saharan Africa. We evaluated the capacities of S. Typhimurium and Salmonella enterica serovar Enteritidis ΔguaBA ΔclpX live oral vaccines to protect mice against a highly lethal challenge dose of the homologous serovar and determined protection against other group B and D serovars circulating in sub-Saharan Africa. The vaccines S. Typhimurium CVD 1931 and S. Enteritidis CVD 1944 were immunogenic and protected BALB/c mice against 10,000 50% lethal doses (LD50) of S. Typhimurium or S. Enteritidis, respectively. S. Typhimurium CVD 1931 protected mice against the group B serovar Salmonella enterica serovar Stanleyville (91% vaccine efficacy), and S. Enteritidis CVD 1944 protected mice against the group D serovar Salmonella enterica serovar Dublin (85% vaccine efficacy). High rates of survival were observed when mice were infected 12 weeks postimmunization, indicating that the vaccines elicited long-lived protective immunity. Whereas CVD 1931 did not protect against S. Enteritidis R11, CVD 1944 did mediate protection against S. Typhimurium D65 (81% efficacy). These findings suggest that a bivalent (S. Typhimurium and S. Enteritidis) vaccine would provide broad protection against the majority of invasive NTS infections in sub-Saharan Africa. PMID:26351285

Chemogenomics profiling of drug targets of peptidoglycan biosynthesis pathway in Leptospira interrogans by virtual screening approaches.

PubMed

Bhattacharjee, Biplab; Simon, Rose Mary; Gangadharaiah, Chaithra; Karunakar, Prashantha

2013-06-28

Leptospirosis is a worldwide zoonosis of global concern caused by Leptospira interrogans. The availability of ligand libraries has facilitated the search for novel drug targets using chemogenomics approaches, compared with the traditional method of drug discovery, which is time consuming and yields few leads with little intracellular information for guiding target selection. Recent subtractive genomics studies have revealed the putative drug targets in peptidoglycan biosynthesis pathways in Leptospira interrogans. Aligand library for the murD ligase enzyme in the peptidoglycan pathway has also been identified. Our approach in this research involves screening of the pre-existing ligand library of murD with related protein family members in the putative drug target assembly in the peptidoglycan biosynthesis pathway. A chemogenomics approach has been implemented here, which involves screening of known ligands of a protein family having analogous domain architecture for identification of leads for existing druggable protein family members. By means of this approach, one murC and one murF inhibitor were identified, providing a platform for developing an antileptospirosis drug targeting the peptidoglycan biosynthesis pathway. Given that the peptidoglycan biosynthesis pathway is exclusive to bacteria, the in silico identified mur ligase inhibitors are expected to be broad-spectrum Gram-negative inhibitors if synthesized and tested in in vitro and in vivo assays.

Circulating serovars of Leptospira in cart horses of central and southern Ethiopia and associated risk factors.

PubMed

Tsegay, K; Potts, A D; Aklilu, N; Lötter, C; Gummow, B

2016-03-01

Little work has been done on diseases of horses in Ethiopia or tropical regions of the world. Yet, Ethiopia has the largest horse population in Africa and their horses play a pivotal role in their economy as traction animals. A serological and questionnaire survey was therefore conducted to determine the circulating serovars of Leptospira and their association with potential risk factors in the cart horse population of Central and Southern Ethiopia. A total of 184 out of 418 cart horses from 13 districts had antibody titres of 1:100 or greater to at least one of 16 serovars of Leptospira species in Central and Southern Ethiopian horses. A significantly higher seropositivity (62.1%) was noted in horses from the highland agroecology followed by midland (44.4%) and lowland (39.8%). Serovar Bratislava (34.5%) was the predominant serovar followed by serovars Djasiman (9.8%), Topaz (5.98%) and Pomona (5.3%). Age and location proved to be associated with seropositive horses with older horses being more commonly affected and the districts of Ziway (Batu) (Apparent Prevalence (AP)=65.5%), Shashemene (AP=48.3%) and Sebeta (AP=41.4%) having the highest prevalence. Multivariable logistic regression found risk factors significantly associated with Leptospira seropositive horses were drinking river water (OR=2.8) and horses 7-12 years old (OR=5) and risk factors specifically associated with serovar Bratislava seropositive horses were drinking river water (OR=2.5), horses ≥13 years (OR=3.5) and the presence of dogs in adjacent neighbouring properties (OR=0.3). Dogs had a protective effect against seropositivity to serovars Bratislava and Djasiman, which may be due to their ability to control rodents. The high seroprevalence confirm that leptospirosis is endemic among horses of Central and Southern Ethiopia. The predominance of serovar Bratislava supports the idea that serovar Bratislava may be adapted to and maintained by the horse population of Central and Southern Ethiopia

Haemophilus parasuis serovar 5 Nagasaki strain adheres and invades PK-15 cells.

PubMed

Frandoloso, Rafael; Martínez-Martínez, Sonia; Gutiérrez-Martín, César B; Rodríguez-Ferri, Elías F

2012-01-27

Haemophilus parasuis is the agent responsible for causing Glässer's disease, which is characterized by fibrinous polyserositis, polyarthritis and meningitis in pigs. The purpose of this study was to investigate the in vitro ability of two H. parasuis serovars of different virulence (serovar 5, Nagasaki strain, highly virulent, belonging to serovar 5, and SW114 strain, nonvirulent, belonging to serovar 3) to adhere to and invade porcine kidney epithelial cells (PK-15 line). Nagasaki strain was able to attach at high levels from 60 to 180 min of incubation irrespective of the concentrations compared (10(7)-10(10)CFU), and a substantial increase of surface projections could be seen in PK-15 cells by scanning electron microscopy. This virulent strain was also able to invade effectively these epithelial cells, and the highest invasion capacity was reached at 180 min of infection. On the contrary, nonvirulent SW114 strain hardly adhered to PK-15 cells, and it did not invade these cells, thus suggesting that adherence and invasion of porcine kidney epithelial cells could be a virulence mechanism involved in the lesions caused by H. parasuis Nagasaki strain in this organ. Copyright © 2011 Elsevier B.V. All rights reserved.

Evaluation of a multiplex PCR to identify and serotype Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15.

PubMed

Turni, C; Singh, R; Schembri, M A; Blackall, P J

2014-10-01

The aim of this study was to validate a multiplex PCR for the species identification and serotyping of Actinobacillus pleuropneumoniae serovars 1, 5, 7, 12 and 15. All 15 reference strains and 411 field isolates (394 from Australia, 11 from Indonesia, five from Mexico and one from New Zealand) of A. pleuropneumoniae were tested with the multiplex PCR. The specificity of this multiplex PCR was validated on 26 non-A. pleuropneumoniae species. The multiplex PCR gave the expected results with all 15 serovar reference strains and agreed with conventional serotyping for all field isolates from serovars 1 (n = 46), 5 (n = 81), 7 (n = 80), 12 (n = 16) and serovar 15 (n = 117). In addition, a species-specific product was amplified in the multiplex PCR with all 411 A. pleuropneumoniae field isolates. Of 25 nontypeable field isolates only two did not yield a serovar-specific band in the multiplex PCR. This multiplex PCR for serovars 1, 5, 7, 12 and 15 is species specific and capable of serotyping isolates from diverse locations. Significance and impact of the study: A multiplex PCR that can recognize serovars 1, 5, 7, 12 and 15 of A. pleuropneumoniae was developed and validated. This novel diagnostic tool will enable frontline laboratories to provide key information (the serovar) to guide targeted prevention and control programmes for porcine pleuropneumonia, a serious economic disease of pigs. The previous technology, traditional serotyping, is typically provided by specialized reference laboratories, limiting the capacity to respond to this key disease. © 2014 The Society for Applied Microbiology.

Refined live attenuated Salmonella enterica serovar Typhimurium and Enteritidis vaccines mediate homologous and heterologous serogroup protection in mice.

PubMed

Tennant, Sharon M; Schmidlein, Patrick; Simon, Raphael; Pasetti, Marcela F; Galen, James E; Levine, Myron M

2015-12-01

Invasive nontyphoidal Salmonella (NTS) infections constitute a major health problem among infants and toddlers in sub-Saharan Africa; these infections also occur in infants and the elderly in developed countries. We genetically engineered a Salmonella enterica serovar Typhimurium strain of multilocus sequence type 313, the predominant genotype circulating in sub-Saharan Africa. We evaluated the capacities of S. Typhimurium and Salmonella enterica serovar Enteritidis ΔguaBA ΔclpX live oral vaccines to protect mice against a highly lethal challenge dose of the homologous serovar and determined protection against other group B and D serovars circulating in sub-Saharan Africa. The vaccines S. Typhimurium CVD 1931 and S. Enteritidis CVD 1944 were immunogenic and protected BALB/c mice against 10,000 50% lethal doses (LD50) of S. Typhimurium or S. Enteritidis, respectively. S. Typhimurium CVD 1931 protected mice against the group B serovar Salmonella enterica serovar Stanleyville (91% vaccine efficacy), and S. Enteritidis CVD 1944 protected mice against the group D serovar Salmonella enterica serovar Dublin (85% vaccine efficacy). High rates of survival were observed when mice were infected 12 weeks postimmunization, indicating that the vaccines elicited long-lived protective immunity. Whereas CVD 1931 did not protect against S. Enteritidis R11, CVD 1944 did mediate protection against S. Typhimurium D65 (81% efficacy). These findings suggest that a bivalent (S. Typhimurium and S. Enteritidis) vaccine would provide broad protection against the majority of invasive NTS infections in sub-Saharan Africa. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Construction and application of prokaryotic expression system of Leptospira interrogans lipL32/1-lipL41/1 fusion gene].

PubMed

Luo, Dong-jiao; Yan, Jie; Mao, Ya-fei; Li, Shu-ping; Luo, Yi-hui; Li, Li-wei

2005-01-01

To construct lipL32/1-lipL41/1 fusion gene and its prokaryotic expression system and to determine frequencies of carrying and expression of lipL32 and lipL41 genes in L.interrogans wild strains and specific antibody levels in sera from leptospirosis patients. lipL32/1-lipL41/1 fusion gene was constructed using linking primer PCR method and the prokaryotic expression system of the fusion gene done with routine techniques. SDS-PAGE was used to examine expression of the target recombinant protein rLipL32/1-rLipL41/1. Immunogenicity of rLipL32/1-rLipL41/1 was identified by Western blot. PCR and MAT were performed to detect carrying and expression of lipL32 and lipL41 genes in 97 wild L.interrogans strains. Antibodies against products of lipL32 and lipL41 genes in serum samples from 228 leptospirosis patients were detected by ELISA method. The homogeneity of nucleotide and putative amino acid sequence of lipL32/1-lipL41/1 fusion gene were 99.9 % and 99.8 % in comparison with the reported sequences. Expression output of the target recombinant protein rLipL32/1-rLipL41/1, mainly present in inclusion body, accounted for 10 % of the total bacterial proteins. Both the rabbit antisera against rLipL32/1 and rLipL41/1 could combine to rLipL32/1-rLipL41/1. 97.9 % and 87.6 % of the L.interrogans wild strains had lipL32 and lipL41 genes, respectively. 95.9 % and 84.5 % of the wild strains were positive for MAT with titers of 1:4 - 1:128 using rabbit anti-rLipL32s or anti-rLipL41s sera, respectively. 94.7 % - 97.4 % of the patients'serum samples were positive for rLipL32s antibodies, while 78.5 % - 84.6 % of them were rLipL41s antibodies detectable. lipL32/1-jlipL41/1 fusion gene and its prokaryotic expression system were successfully constructed. The expressed fusion protein had qualified immunogenicity. Both the lipL32 and lipL41 genes are extensively carried and frequently expressed by different serogroups of L.interrogans, and their expression products exhibit cross-antigenicity.

Investigation on predominant Leptospira serovars and its distribution in humans and livestock in Thailand, 2010-2015.

PubMed

Chadsuthi, Sudarat; Bicout, Dominique J; Wiratsudakul, Anuwat; Suwancharoen, Duangjai; Petkanchanapong, Wimol; Modchang, Charin; Triampo, Wannapong; Ratanakorn, Parntep; Chalvet-Monfray, Karine

2017-02-01

Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand's regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand.

Characterization of a T7-like lytic bacteriophage (phiSG-JL2) of Salmonella enterica serovar gallinarum biovar gallinarum.

PubMed

Kwon, Hyuk-Joon; Cho, Sun-Hee; Kim, Tae-Eun; Won, Yong-Jin; Jeong, Jihye; Park, Se Chang; Kim, Jae-Hong; Yoo, Han-Sang; Park, Yong-Ho; Kim, Sun-Joong

2008-11-01

PhiSG-JL2 is a newly discovered lytic bacteriophage infecting Salmonella enterica serovar Gallinarum biovar Gallinarum but is nonlytic to a rough vaccine strain of serovar Gallinarum biovar Gallinarum (SG-9R), S. enterica serovar Enteritidis, S. enterica serovar Typhimurium, and S. enterica serovar Gallinarum biovar Pullorum. The phiSG-JL2 genome is 38,815 bp in length (GC content, 50.9%; 230-bp-long direct terminal repeats), and 55 putative genes may be transcribed from the same strand. Functions were assigned to 30 genes based on high amino acid similarity to known proteins. Most of the expected proteins except tail fiber (31.9%) and the overall organization of the genomes were similar to those of yersiniophage phiYeO3-12. phiSG-JL2 could be classified as a new T7-like virus and represents the first serovar Gallinarum biovar Gallinarum phage genome to be sequenced. On the basis of intraspecific ratios of nonsynonymous to synonymous nucleotide changes (Pi[a]/Pi[s]), gene 2 encoding the host RNA polymerase inhibitor displayed Darwinian positive selection. Pretreatment of chickens with phiSG-JL2 before intratracheal challenge with wild-type serovar Gallinarum biovar Gallinarum protected most birds from fowl typhoid. Therefore, phiSG-JL2 may be useful for the differentiation of serovar Gallinarum biovar Gallinarum from other Salmonella serotypes, prophylactic application in fowl typhoid control, and understanding of the vertical evolution of T7-like viruses.

Assessment of exposure to Leptospira serovars in veterinary staff and dog owners in contact with infected dogs.

PubMed

Barmettler, Reto; Schweighauser, Ariane; Bigler, Susanne; Grooters, Amy M; Francey, Thierry

2011-01-15

To assess patterns of seroreactivity to Leptospira serovars in veterinary professional staff and dog owners exposed to dogs with acute leptospirosis and to contrast these patterns in people with those observed in dogs. Cross-sectional study. Human subjects consisted of 91 people (50 veterinarians, 19 technical staff, 9 administrative personnel, and 13 dog owners) exposed to dogs with leptospirosis. Canine subjects consisted of 52 dogs with naturally occurring leptospirosis admitted to the University of Bern Vetsuisse Faculty Small Animal Clinic in 2007 and 2008. People were tested for seroreactivity to regionally prevalent Leptospira serovars by use of a complement fixation test. A questionnaire designed to identify risk factors associated with seropositivity was used to collect demographic information from each study participant. Dogs were tested for seroreactivity to Leptospira serovars by use of a microscopic agglutination test. On the basis of microscopic agglutination test results, infected dogs were seropositive for antibodies against Leptospira serovars as follows (in descending order): Bratislava (43/52 [83%]), Australis (43/52 [83%]), Grippotyphosa (18/52 [35%]), Pomona (12/52 [23%]), Autumnalis (6/52 [12%]), Icterohemorrhagiae (4/52 [8%]), Tarassovi (2/52 [4%]), and Canicola (1/52 [2%]). All 91 people were seronegative for antibodies against Leptospira serovars. Therefore, statistical evaluation of risk factors and comparison of patterns of seroreactivity to Leptospira serovars between human and canine subjects were limited to theoretical risks. Seroreactivity to Leptospira serovars among veterinary staff adhering to standard hygiene protocols and pet owners exposed to dogs with acute leptospirosis was uncommon.

Ocular sensitization of mice by live (but not irradiated) Chlamydia trachomatis serovar A

DOE Office of Scientific and Technical Information (OSTI.GOV)

Colley, D.G.; Goodman, T.G.; Barsoum, I.S.

1986-10-01

Ocular exposure of mice to live elementary bodies of Chlamydia trachomatis serovar A results in immunological sensitization of the mice. This reactivity is manifested by the development of early (5 h) and delayed-type (24 h) dermal reactivity and serovar-specific antibody formation against either live or irradiated (100 kilorads) elementary bodies. Parallel ocular exposure of mice to irradiated elementary bodies does not result in this sensitization. The early and late dermal immune responses induced by ocular exposure to live organisms can be transferred to unexposed mice by serum and lymphoid cell transfers, respectively. It appears that successful murine ocular sensitization bymore » human C. trachomatis serovar A elementary bodies is an ability manifested by live organisms and not by inactivated but antigenic organisms.« less

Investigation on predominant Leptospira serovars and its distribution in humans and livestock in Thailand, 2010-2015

PubMed Central

Chadsuthi, Sudarat; Bicout, Dominique J.; Wiratsudakul, Anuwat; Suwancharoen, Duangjai; Petkanchanapong, Wimol; Modchang, Charin; Triampo, Wannapong; Ratanakorn, Parntep; Chalvet-Monfray, Karine

2017-01-01

Background Leptospirosis is a worldwide zoonotic bacterial disease caused by infection with leptospires. Leptospirosis in humans and livestock is an endemic and epidemic disease in Thailand. Livestock may act as reservoirs for leptospires and source for human infection. Methodology/Principal findings Data on leptospirosis infection in humans and livestock (Buffaloes, Cattle, and Pigs) species during 2010 to 2015 were analyzed. Serum samples were examined using Microscopic Agglutination Test (MAT) to identify antibodies against Leptospira serovars using a cut-off titer ≥ 1:100. The seroprevalence was 23.7% in humans, 24.8% in buffaloes, 28.1% in cattle, and 11.3% in pigs. Region specific prevalence among humans and livestock was found in a wide range. The most predominant serovars were Shermani, followed by Bratislava, Panama, and Sejroe in human, Shermani, Ranarum, and Tarassovi in buffaloes, and Shermani and Ranarum in cattle and pigs. Equally highest MAT titers against multiple serovars per one sample were found mainly in buffaloes and cattle showing equally titers against Ranarum and Shermani. The correlations of distribution of serovars across Thailand’s regions were found to be similar in pattern for cattle but not for buffaloes. In humans, the serovar distribution in the south differed from other regions. By logistic regression, the results indicated that livestock is more susceptible to infection by serovar Shermani when compared to humans. Conclusions/Significance This study gives a detailed picture of the predominance of Leptospira serovars in relation to region, humans and typical livestock. The broad spatial distribution of seroprevalence was analyzed across and within species as well as regions in Thailand. Our finding may guide public health policy makers to implement appropriate control measures and help to reduce the impact of leptospirosis in Thailand. PMID:28182662

Expression of Toll-like receptors, interleukin 8, macrophage migration inhibitory factor, and osteopontin in tissues from pigs challenged with Salmonella enterica serovar Typhimurium or serovar Choleraesuis.

PubMed

Burkey, T E; Skjolaas, K A; Dritz, S S; Minton, J E

2007-02-15

Two serovars of Salmonella enterica, namely serovar Typhimurium (ST) and serovar Choleraesuis (SC) account for the vast majority of clinical cases of swine salmonellosis worldwide. These serovars are thought to be transmitted among pigs in production settings mainly through fecal-oral routes. Yet, few studies have evaluated effects of these serovars on expression of innate immune targets when presented to pigs via repeated oral dosing in an attempt to model transmission in production settings. Thus, a primary objective of the current experiments was to evaluate expression of Toll-like receptors (TLR) and selected chemoattractive mediators (interleukin 8, IL8; macrophage migration inhibitory factor, MIF; osteopontin, OPN) in tissues from pigs exposed to ST or SC that had been transformed with kanamycin resistance and green (STG) or red (SCR) fluorescent protein to facilitate isolation from pen fecal samples. In vitro studies confirmed that STG and SCR largely (though not completely) retained their ability to upregulate IL8 and CC chemokine ligand 20 (CCL20) in cultured swine jejunal epithelial cells. Transformed bacteria were then fed to pigs in an in vivo study to determine tissue specific effects on mRNA relative expression. Pigs were fed cookie dough inoculated with bacteria on days 0, 3, 7, and 10 with 10(8)CFU STG (n=8) or SCR (n=8), while control (CTL) pigs (n=8) received dough without bacteria. Animals were sacrificed 14 days from the initial bacterial challenge and samples of tonsil, jejunum, ileum, colon, mesenteric lymph node (MLN), spleen, and liver were removed for subsequent RNA isolation. Expression of mRNA in tissues was determined using real-time quantitative PCR and expressed relative to 18S rRNA. Within CTL pigs, when expressed relative to the content in liver, mRNA for all targets demonstrated substantial tissue effects (P<0.001 for all TLR; MIF, and OPN; P<0.05 for IL8). Feeding STG and SCR resulted in significant (P

Physiological and molecular responses of Lactuca sativa to colonization by Salmonella enterica serovar Dublin.

PubMed

Klerks, M M; van Gent-Pelzer, M; Franz, E; Zijlstra, C; van Bruggen, A H C

2007-08-01

This paper describes the physiological and molecular interactions between the human-pathogenic organism Salmonella enterica serovar Dublin and the commercially available mini Roman lettuce cv. Tamburo. The association of S. enterica serovar Dublin with lettuce plants was first determined, which indicated the presence of significant populations outside and inside the plants. The latter was evidenced from significant residual concentrations after highly efficient surface disinfection (99.81%) and fluorescence microscopy of S. enterica serovar Dublin in cross sections of lettuce at the root-shoot transition region. The plant biomass was reduced significantly compared to that of noncolonized plants upon colonization with S. enterica serovar Dublin. In addition to the physiological response, transcriptome analysis by cDNA amplified fragment length polymorphism analysis also provided clear differential gene expression profiles between noncolonized and colonized lettuce plants. From these, generally and differentially expressed genes were selected and identified by sequence analysis, followed by reverse transcription-PCR displaying the specific gene expression profiles in time. Functional grouping of the expressed genes indicated a correlation between colonization of the plants and an increase in expressed pathogenicity-related genes. This study indicates that lettuce plants respond to the presence of S. enterica serovar Dublin at physiological and molecular levels, as shown by the reduction in growth and the concurrent expression of pathogenicity-related genes. In addition, it was confirmed that Salmonella spp. can colonize the interior of lettuce plants, thus potentially imposing a human health risk when processed and consumed.

Genetic and antigenic characteristics of ApxIIA and ApxIIIA from Actinobacillus pleuropneumoniae serovars 2, 3, 4, 6, 8 and 15.

PubMed

To, Ho; Nagai, Shinya; Iwata, Akira; Koyama, Tomohiro; Oshima, Atsushi; Tsutsumi, Nobuyuki

2016-07-01

Apx toxins produced by Actinobacillus pleuropneumoniae are essential components of new generation vaccines. In this study, apxIIA and apxIIIA genes of serovars 2, 3, 4, 6, 8 and 15 were cloned and sequenced. Amino acid sequences of ApxIIA proteins of serovars 2, 3, 4, 6, 8 and 15 were almost identical to those of serovars 1, 5, 7, 9 and 11-13. Immunoblot analysis showed that rApxIIA from serovars 2 and 15 reacts strongly with sera from animals infected with various serovars. Sequence analysis revealed that ApxIIIA proteins has two variants, one in strains of serovar 2 and the other in strains of serovars 3, 4, 6, 8 and 15. A mouse cross-protection study showed that mice actively immunized with rApxIIIA/2 or rApxIIIA/15 are protected against challenge with A. pleuropneumoniae strains of serovars 3, 4, 6, 8, 15, and 2 expressing ApxIII/15 and ApxIII/2, respectively. Similarly, mice passively immunized with rabbit anti-rApxIIIA/2 or anti-rApxIIIA/15 sera were found to be protected against challenge with strains of serovars 2 and 15. Our study revealed antigenic and sequence similarities within ApxIIA and ApxIIIA proteins, which may help in the development of effective vaccines against disease caused by A. pleuropneumoniae. © 2016 The Societies and John Wiley & Sons Australia, Ltd.

LipL53, a temperature regulated protein from Leptospira interrogans that binds to extracellular matrix molecules.

PubMed

Oliveira, Tatiane R; Longhi, Mariana T; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2010-03-01

The regulation of gene expression by environmental signals, such as temperature and osmolarity, has been correlated with virulence. In this study, we characterize the protein LipL53 from Leptospira interrogans, previously shown to react with serum sample of individual diagnosed with leptospirosis and to be up-regulated by shift to physiological osmolarity. The recombinant protein was expressed in Escherichia coli system, in insoluble form, recovered by urea solubilization and further refolded by decreasing the denaturing agent concentration during the purification procedure. The secondary structure content of the recombinant LipL53, as assessed by circular dichroism, showed a mixture of beta-strands and alpha-helix. The presence of LipL53 transcript at 28 degrees C was only detected within the virulent strains. However, upon shifted of attenuated cultures of pathogenic strains from 28 degrees C to 37 degrees C and to 39 degrees C, this transcript could also be observed. LipL53 binds laminin, collagen IV, cellular and plasma fibronectin in dose-dependent and saturable manner. Animal challenge studies showed that LipL53, although immunogenic, elicited only partial protection in hamsters. LipL53 is probably surface exposed as seen through immunofluorescence confocal microscopy. Our results suggest that LipL53 is a novel temperature regulated adhesin of L. interrogans that may be relevant in the leptospiral pathogenesis. Copyright 2009 Elsevier Masson SAS. All rights reserved.

Salmonella enterica serovar Kentucky flagella are required for broiler skin adhesion and Caco-2 cell invasion

USDA-ARS?s Scientific Manuscript database

Nontyphoidal Salmonella strains are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry-processing plants. Previous studies showed that flagella are one...

Salmonella enterica Serovar Kentucky Flagella are Required for Broiler Skin Adhesion and Caco-2 Cell Invasion

USDA-ARS?s Scientific Manuscript database

Non-typhoidal Salmonella are the main source of pathogenic bacterial contamination in the poultry industry. Recently, Salmonella enterica serovar Kentucky has been recognized as the most prominent serovar on carcasses in poultry processing plants. Previous studies showed that flagella are one of the...

Infection of Mice by Salmonella enterica Serovar Enteritidis Involves Additional Genes That Are Absent in the Genome of Serovar Typhimurium

PubMed Central

Silva, Cecilia A.; Blondel, Carlos J.; Quezada, Carolina P.; Porwollik, Steffen; Andrews-Polymenis, Helene L.; Toro, Cecilia S.; Zaldívar, Mercedes; Contreras, Inés

2012-01-01

Salmonella enterica serovar Enteritidis causes a systemic, typhoid-like infection in newly hatched poultry and mice. In the present study, a library of 54,000 transposon mutants of S. Enteritidis phage type 4 (PT4) strain P125109 was screened for mutants deficient in the in vivo colonization of the BALB/c mouse model using a microarray-based negative-selection screening. Mutants in genes known to contribute to systemic infection (e.g., Salmonella pathogenicity island 2 [SPI-2], aro, rfa, rfb, phoP, and phoQ) and enteric infection (e.g., SPI-1 and SPI-5) in this and other Salmonella serovars displayed colonization defects in our assay. In addition, a strong attenuation was observed for mutants in genes and genomic islands that are not present in S. Typhimurium or in most other Salmonella serovars. These genes include a type I restriction/modification system (SEN4290 to SEN4292), the peg fimbrial operon (SEN2144A to SEN2145B), a putative pathogenicity island (SEN1970 to SEN1999), and a type VI secretion system remnant SEN1001, encoding a hypothetical protein containing a lysin motif (LysM) domain associated with peptidoglycan binding. Proliferation defects for mutants in these individual genes and in exemplar genes for each of these clusters were confirmed in competitive infections with wild-type S. Enteritidis. A ΔSEN1001 mutant was defective for survival within RAW264.7 murine macrophages in vitro. Complementation assays directly linked the SEN1001 gene to phenotypes observed in vivo and in vitro. The genes identified here may perform novel virulence functions not characterized in previous Salmonella models. PMID:22083712

Human Leptospira Isolates Circulating in Mayotte (Indian Ocean) Have Unique Serological and Molecular Features

PubMed Central

Bourhy, P.; Collet, L.; Lernout, T.; Zinini, F.; Hartskeerl, R. A.; van der Linden, Hans; Thiberge, J. M.; Diancourt, L.; Brisse, S.; Giry, C.; Pettinelli, F.

2012-01-01

Leptospirosis is one of the most widespread zoonoses in the world. However, there is a lack of information on circulating Leptospira strains in remote parts of the world. We describe the serological and molecular features of leptospires isolated from 94 leptospirosis patients in Mayotte, a French department located in the Comoros archipelago, between 2007 and 2010. Multilocus sequence typing identified these isolates as Leptospira interrogans, L. kirschneri, L. borgpetersenii, and members of a previously undefined phylogenetic group. This group, consisting of 15 strains, could represent a novel species. Serological typing revealed that 70% of the isolates belonged to the serogroup complex Mini/Sejroe/Hebdomadis, followed by the serogroups Pyrogenes, Grippotyphosa, and Pomona. However, unambiguous typing at the serovar level was not possible for most of the strains because the isolate could belong to more than one serovar or because serovar and species did not match the original classification. Our results indicate that the serovar and genotype distribution in Mayotte differs from what is observed in other regions, thus suggesting a high degree of diversity of circulating isolates worldwide. These results are essential for the improvement of current diagnostic tools and provide a starting point for a better understanding of the epidemiology of leptospirosis in this area of endemicity. PMID:22162544

Complete Genome Sequence of a Multidrug-Resistant Salmonella enterica Serovar Typhimurium var. 5- Strain Isolated from Chicken Breast.

PubMed

Hoffmann, Maria; Muruvanda, Tim; Allard, Marc W; Korlach, Jonas; Roberts, Richard J; Timme, Ruth; Payne, Justin; McDermott, Patrick F; Evans, Peter; Meng, Jianghong; Brown, Eric W; Zhao, Shaohua

2013-12-19

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5- CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar.

Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts.

PubMed

Mgode, Georgies F; Machang'u, Robert S; Mhamphi, Ginethon G; Katakweba, Abdul; Mulungu, Loth S; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A; Belmain, Steven R

2015-12-01

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries.

Leptospira Serovars for Diagnosis of Leptospirosis in Humans and Animals in Africa: Common Leptospira Isolates and Reservoir Hosts

PubMed Central

Mgode, Georgies F.; Machang’u, Robert S.; Mhamphi, Ginethon G.; Katakweba, Abdul; Mulungu, Loth S.; Durnez, Lies; Leirs, Herwig; Hartskeerl, Rudy A.; Belmain, Steven R.

2015-01-01

The burden of leptospirosis in humans and animals in Africa is higher than that reported from other parts of the world. However, the disease is not routinely diagnosed in the continent. One of major factors limiting diagnosis is the poor availability of live isolates of locally circulating Leptospira serovars for inclusion in the antigen panel of the gold standard microscopic agglutination test (MAT) for detecting antibodies against leptospirosis. To gain insight in Leptospira serovars and their natural hosts occurring in Tanzania, concomitantly enabling the improvement of the MAT by inclusion of fresh local isolates, a total of 52 Leptospira isolates were obtained from fresh urine and kidney homogenates, collected between 1996 and 2006 from small mammals, cattle and pigs. Isolates were identified by serogrouping, cross agglutination absorption test (CAAT), and molecular typing. Common Leptospira serovars with their respective animal hosts were: Sokoine (cattle and rodents); Kenya (rodents and shrews); Mwogolo (rodents); Lora (rodents); Qunjian (rodent); serogroup Grippotyphosa (cattle); and an unknown serogroup from pigs. Inclusion of local serovars particularly serovar Sokoine in MAT revealed a 10-fold increase in leptospirosis prevalence in Tanzania from 1.9% to 16.9% in rodents and 0.26% to 10.75% in humans. This indicates that local serovars are useful for diagnosis of human and animal leptospirosis in Tanzania and other African countries. PMID:26624890

Expression of hilA in response to mild acid stress in Salmonella enterica is serovar and strain dependent.

PubMed

González-Gil, Francisco; Le Bolloch, Alexandre; Pendleton, Sean; Zhang, Nan; Wallis, Audra; Hanning, Irene

2012-05-01

Salmonella enterica is the leading cause of foodborne illness with poultry and poultry products being primary sources of infection. The 2 most common S. enterica serovars associated with human infection are Typhimurium and Enteritidis. However, Kentucky and Heidelburg and the 2 most prevalent serovars isolated from poultry environments. Given the prevalence of other serovars in poultry products and environments, research is needed to understand virulence modulation in response to stress in serovars other than Typhimurium and Enteritidis. Thus, the objective of this research was to compare hilA gene expression (a master regulator of the virulence pathogenicity island) in response to acid stress among different strains and serovars of Salmonella. A total of 11 serovars consisting of 15 strains of S. enterica were utilized for these experiments. Cultures were suspended in tryptic soy broth (TSB) adjusted to pH 7.2, 6.2, or 5.5 with HCl or acetic acid. Total RNA was extracted from cultures at specific time points (0, 2, 4, and 24 h). Gene expression of hilA was measured with quantitative reverse transcriptase real time PCR (qRT-PCR). Growth and pH were measured throughout the 24 h time frame. Regulation of hilA in response to acid stress varied by serovar and strain and type of acid. The results of these experiments indicate that hilA regulation may have some impact on virulence and colonization of S. enterica. However, these results warrant further research to more fully understand the significance of hilA regulation in response to mild acid stress in S. enterica. © 2012 Institute of Food Technologists®

Arginine-Dependent Acid Resistance in Salmonella enterica Serovar Typhimurium

PubMed Central

Kieboom, Jasper; Abee, Tjakko

2006-01-01

Salmonella enterica serovar Typhimurium does not survive a pH 2.5 acid challenge under conditions similar to those used for Escherichia coli (J. W. Foster, Nat. Rev. Microbiol. 2:898-907, 2004). Here, we provide evidence that S. enterica serovar Typhimurium can display arginine-dependent acid resistance (AR) provided the cells are grown under anoxic conditions and not under the microaerobic conditions used for assessment of AR in E. coli. The role of the arginine decarboxylase pathway in Salmonella AR was shown by the loss of AR in mutants lacking adiA, which encodes arginine decarboxylase; adiC, which encodes the arginine-agmatine antiporter; or adiY, which encodes an AraC-like regulator. Transcription of adiA and adiC was found to be dependent on AdiY, anaerobiosis, and acidic pH. PMID:16855258

Induction of TNF-alfa and CXCL-2 mRNAs in different organs of mice infected with pathogenic Leptospira.

PubMed

da Silva, Josefa B; Carvalho, Enéas; Covarrubias, Ambart E; Ching, Ana Tung C; Mattaraia, Vania G M; Paiva, Delhi; de Franco, Marcelo; Fávaro, Regiane Degan; Pereira, Martha M; Vasconcellos, Silvio; Zorn, Telma T M; Ho, Paulo Lee; Martins, Elizabeth A L

2012-04-01

The role of innate immune response in protection against leptospirosis is poorly understood. We examined the expression of the chemokine CXCL2/MIP-2 and the cytokine TNF-α in experimental resistant and susceptible mice models, C3H/HeJ, C3H/HePas and BALB/c strains, using a virulent strain of Leptospira interrogans serovar Copenhageni. Animals were infected intraperitoneally with 10(7) cells and the development of the disease was followed. Mortality of C3H/HeJ mice was observed whereas C3H/HePas presented jaundice and BALB/c mice remained asymptomatic. The infection was confirmed by the presence of leptospiral DNA in the organs of the animals, demonstrated by PCR. Sections of the organs were analyzed, after H&E stain. The relative expression of mRNA of chemokine CXCL2/MIP-2 and cytokine TNF-α was measured in lung, kidney and liver of the mice by qPCR. The concentrations of these proteins were measured in extracts of tissues and in serum of the animals, by ELISA. Increasing levels of transcripts and protein CXCL2/MIP-2 were detected since the first day of infection. The highest expression was observed at third day of infection in kidney, liver and lung of BALB/c mice. In C3H/HeJ the expression of CXCL2/MIP-2 was delayed, showing highest protein concentration in lung and kidney at the 5th day. Increasing in TNF-α transcripts were detected after infection, in kidney and liver of animals from the three mice strains. The expression of TNF-α protein in C3H/HeJ was also delayed, being detected in kidney and lung. Our data demonstrated that Leptospira infection stimulates early expression of CXCL2/MIP-2 and TNF-α in the resistant strain of mice. Histological analysis suggests that the expression of those molecules may be related to the influx of distinct immune cells and plays a role in the naturally acquired protective immunity. Copyright © 2012 Elsevier Ltd. All rights reserved.

Human migration is important in the international spread of exotic Salmonella serovars in animal and human populations.

PubMed

Iveson, J B; Bradshaw, S D; How, R A; Smith, D W

2014-11-01

The exposure of indigenous humans and native fauna in Australia and the Wallacea zoogeographical region of Indonesia to exotic Salmonella serovars commenced during the colonial period and has accelerated with urbanization and international travel. In this study, the distribution and prevalence of exotic Salmonella serovars are mapped to assess the extent to which introduced infections are invading native wildlife in areas of high natural biodiversity under threat from expanding human activity. The major exotic Salmonella serovars, Bovismorbificans, Derby, Javiana, Newport, Panama, Saintpaul and Typhimurium, isolated from wildlife on populated coastal islands in southern temperate areas of Western Australia, were mostly absent from reptiles and native mammals in less populated tropical areas of the state. They were also not recorded on the uninhabited Mitchell Plateau or islands of the Bonaparte Archipelago, adjacent to south-eastern Indonesia. Exotic serovars were, however, isolated in wildlife on 14/17 islands sampled in the Wallacea region of Indonesia and several islands off the west coast of Perth. Increases in international tourism, involving islands such as Bali, have resulted in the isolation of a high proportion of exotic serovar infections suggesting that densely populated island resorts in the Asian region are acting as staging posts for the interchange of Salmonella infections between tropical and temperate regions.

Rapid Detection of Chlamydia trachomatis and Typing of the Lymphogranuloma venereum associated L-Serovars by TaqMan PCR

PubMed Central

Schaeffer, Anke; Henrich, Birgit

2008-01-01

Background Infection due to Chlamydia trachomatis is the most common sexually transmitted bacterial disease of global health significance, and especially the L-serovars causing lymphogranuloma venereum are increasingly being found in Europe in men who have sex with men. Results The design and evaluation of a rapid, multiplex, real-time PCR targeting the major outer membrane protein (omp-1) -gene and a L-serovar-specific region of the polymorphic protein H (pmp-H) -gene for the detection of Chlamydia trachomatis is reported here. The PCR takes place as a single reaction with an internal control. For L1-, L2- and L3-serovar differentiation a second set of real-time PCRs was evaluated based on the amplification of serovar-specific omp-1-regions. The detection limit of each real-time PCR, multiplexed or not, was 50 genome copies per reaction with an efficiency ranging from 90,5–95,2%. In a retrospective analysis of 50 ocular, rectal and urogenital specimens formerly tested to be positive for C. trachomatis we identified six L2-serovars in rectal specimens of HIV-positive men, one in a double-infection with L3, and one L2 in a urethral specimen of an HIV-negative male. Conclusion This unique real-time PCR is specific and convenient for the rapid routine-diagnostic detection of lymphogranuloma venereum-associated L-serovars and enables the subsequent differentiation of L1, L2 and L3 for epidemiologic studies. PMID:18447917

Rapid Determination of Lymphogranuloma Venereum Serovars of Chlamydia trachomatis by Quantitative High-Resolution Melt Analysis (HRMA)

PubMed Central

Stevens, Matthew P.; Garland, Suzanne M.; Zaia, Angelo M.; Tabrizi, Sepehr N.

2012-01-01

A quantitative high-resolution melt analysis assay was developed to differentiate lymphogranuloma venereum-causing serovars of Chlamydia trachomatis (L1 to L3) from other C. trachomatis serovars (D to K). The detection limit of this assay is approximately 10 copies per reaction, comparable to the limits of other quantitative-PCR-based methods. PMID:22933594

Complete Genome Sequence of a Multidrug-Resistant Salmonella enterica Serovar Typhimurium var. 5− Strain Isolated from Chicken Breast

PubMed Central

Muruvanda, Tim; Allard, Marc W.; Korlach, Jonas; Roberts, Richard J.; Timme, Ruth; Payne, Justin; McDermott, Patrick F.; Evans, Peter; Meng, Jianghong; Brown, Eric W.; Zhao, Shaohua

2013-01-01

Salmonella enterica subsp. enterica serovar Typhimurium is a leading cause of salmonellosis. Here, we report a closed genome sequence, including sequences of 3 plasmids, of Salmonella serovar Typhimurium var. 5− CFSAN001921 (National Antimicrobial Resistance Monitoring System [NARMS] strain ID N30688), which was isolated from chicken breast meat and shows resistance to 10 different antimicrobials. Whole-genome and plasmid sequence analyses of this isolate will help enhance our understanding of this pathogenic multidrug-resistant serovar. PMID:24356834

Identification and characterization of haemagglutinin epitopes of Avibacterium paragallinarum serovar C.

PubMed

Noro, Taichi; Oishi, Eiji; Kaneshige, Takahiro; Yaguchi, Kazuhiko; Amimoto, Katsuhiko; Shimizu, Mitsugu

2008-10-15

The objectives of this study were to identify haemagglutinin (HA) epitopes of Avibacterium paragallinarum serovar C that are capable of eliciting haemagglutination inhibition (HI) antibody, and to investigate their immunogenic role. Three conformational epitopes were detected on HA by blocking ELISA and immuno-dot blot analysis using a panel of five monoclonal antibodies (MAbs) with HI activity, designated 8C1C, 4G8B, 24E4D, 11E11B, and 10D1A. The minimum DNA regions coding these three epitopes were 3195, 2862, and 807bp in size, and mapped within a gene with 6117bp. Nine DNA fragments of various lengths were prepared, and their recombinant proteins were generated in E. coli. One recombinant protein, designated HPC5.5, was recognized by MAb 8C1C, and had strong ability to adsorb HI antibody to Av. paragallinarum serovar C. Other recombinant proteins designated HPC5.1, HPC4.8, and HPC2.5 did not react with MAb 8C1C and only slightly adsorbed HI antibody. All chickens immunized once with HPC5.5 did not show any typical clinical signs such as nasal discharge or facial edema against challenge inoculation with Av. paragallinarum serovar C. However, HPC5.1, which was recognized by four MAbs (not including MAb 8C1C), showed only partial protective immunity in five of eight immunized chickens. The results suggest that the HA epitope recognized by MAb 8C1C is the major epitope responsible for eliciting HI antibody, and HPC5.5 is a practical candidate protein to develop a new vaccine against avian infectious coryza caused by Av. paragallinarum serovar C.

Proteolytic Inhibition of Salmonella enterica Serovar Typhimurium-Induced Activation of the Mitogen-Activated Protein Kinases ERK and JNK in Cultured Human Intestinal Cells

PubMed Central

Mynott, Tracey L.; Crossett, Ben; Prathalingam, S. Radhika

2002-01-01

Bromelain, a mixture of cysteine proteases from pineapple stems, blocks signaling by the mitogen-activated protein (MAP) kinases extracellular regulated kinase 1 (ERK-1) and ERK-2, inhibits inflammation, and protects against enterotoxigenic Escherichia coli infection. In this study, we examined the effect of bromelain on Salmonella enterica serovar Typhimurium infection, since an important feature of its pathogenesis is its ability to induce activation of ERK-1 and ERK-2, which leads to internalization of bacteria and induction of inflammatory responses. Our results show that bromelain dose dependently blocks serovar Typhimurium-induced ERK-1, ERK-2, and c-Jun NH2-terminal kinase (JNK) activation in Caco-2 cells. Bromelain also blocked signaling induced by carbachol and anisomycin, pharmacological MAP kinase agonists. Despite bromelain inhibition of serovar Typhimurium-induced MAP kinase signaling, it did not prevent subsequent invasion of the Caco-2 cells by serovar Typhimurium or alter serovar Typhimurium -induced decreases in resistance across Caco-2 monolayers. Surprisingly, bromelain also did not block serovar Typhimurium-induced interleukin-8 (IL-8) secretion but synergized with serovar Typhimurium to enhance IL-8 production. We also found that serovar Typhimurium does not induce ERK phosphorylation in Caco-2 cells in the absence of serum but that serovar Typhimurium-induced invasion and decreases in monolayer resistance are unaffected. Collectively, these data indicate that serovar Typhimurium-induced invasion of Caco-2 cells, changes in the resistance of epithelial cell monolayers, and IL-8 production can occur independently of the ERK and JNK signaling pathways. Data also confirm that bromelain is a novel inhibitor of MAP kinase signaling pathways and suggest a novel role for proteases as inhibitors of signal transduction pathways in intestinal epithelial cells. PMID:11748167

Genome comparison of three serovar 5 pathogenic strains of Haemophilus parasuis: insights into an evolving swine pathogen.

PubMed

Bello-Ortí, Bernardo; Aragon, Virginia; Pina-Pedrero, Sonia; Bensaid, Albert

2014-09-01

Haemophilus parasuis is the causative agent of Glässer's disease, a systemic disorder characterized by polyarthritis, polyserositis and meningitis in pigs. Although it is well known that H. parasuis serovar 5 is the most prevalent serovar associated with the disease, the genetic differences among strains are only now being discovered. Genomes from two serovar 5 strains, SH0165 and 29755, are already available. Here, we present the draft genome of a third H. parasuis serovar 5 strain, the formal serovar 5 reference strain Nagasaki. An in silico genome subtractive analysis with full-length predicted genes of the three H. parasuis serovar 5 strains detected 95, 127 and 95 strain-specific genes (SSGs) for Nagasaki, SH0165 and 29755, respectively. We found that the genomic diversity within these three strains was high, in part because of a high number of mobile elements. Furthermore, a detailed analysis of large sequence polymorphisms (LSPs), encompassing regions ranging from 2 to 16 kb, revealed LSPs in virulence-related elements, such as a Toll-IL receptor, the AcrA multidrug efflux protein, an ATP-binding cassette (ABC) transporter, lipopolysaccharide-synthetizing enzymes and a tripartite ATP-independent periplasmic (TRAP) transporter. The whole-genome codon adaptation index (CAI) was also calculated and revealed values similar to other well-known bacterial pathogens. In addition, whole-genome SNP analysis indicated that nucleotide changes tended to be increased in membrane-related genes. This analysis provides further evidence that the genome of H. parasuis has been subjected to multiple lateral gene transfers (LGTs) and to fine-tuning of virulence factors, and has the potential for accelerated genome evolution. © 2014 The Authors.

Detection of Salmonella enterica Serovar Typhimurium by Using a Rapid, Array-Based Immunosensor

PubMed Central

Taitt, Chris Rowe; Shubin, Yura S.; Angel, Roselina; Ligler, Frances S.

2004-01-01

The multianalyte array biosensor (MAAB) is a rapid analysis instrument capable of detecting multiple analytes simultaneously. Rapid (15-min), single-analyte sandwich immunoassays were developed for the detection of Salmonella enterica serovar Typhimurium, with a detection limit of 8 × 104 CFU/ml; the limit of detection was improved 10-fold by lengthening the assay protocol to 1 h. S. enterica serovar Typhimurium was also detected in the following spiked foodstuffs, with minimal sample preparation: sausage, cantaloupe, whole liquid egg, alfalfa sprouts, and chicken carcass rinse. Cross-reactivity tests were performed with Escherichia coli and Campylobacter jejuni. To determine whether the MAAB has potential as a screening tool for the diagnosis of asymptomatic Salmonella infection of poultry, chicken excretal samples from a private, noncommercial farm and from university poultry facilities were tested. While the private farm excreta gave rise to signals significantly above the buffer blanks, none of the university samples tested positive for S. enterica serovar Typhimurium without spiking; dose-response curves of spiked excretal samples from university-raised poultry gave limits of detection of 8 × 103 CFU/g. PMID:14711637

Molecular typing of Salmonella enterica serovar typhi isolates from various countries in Asia by a multiplex PCR assay on variable-number tandem repeats.

PubMed

Liu, Yichun; Lee, May-Ann; Ooi, Eng-Eong; Mavis, Yeo; Tan, Ai-Ling; Quek, Hung-Hiang

2003-09-01

A multiplex PCR method incorporating primers flanking three variable-number tandem repeat (VNTR) loci (arbitrarily labeled TR1, TR2, and TR3) in the CT18 strain of Salmonella enterica serovar Typhi has been developed for molecular typing of S. enterica serovar Typhi clinical isolates from several Asian countries, including Singapore, Indonesia, India, Bangladesh, Malaysia, and Nepal. We have demonstrated that the multiplex PCR could be performed on crude cell lysates and that the VNTR banding profiles produced could be easily analyzed by visual inspection after conventional agarose gel electrophoresis. The assay was highly discriminative in identifying 49 distinct VNTR profiles among 59 individual isolates. A high level of VNTR profile heterogeneity was observed in isolates from within the same country and among countries. These VNTR profiles remained stable after the strains were passaged extensively under routine laboratory culture conditions. In contrast to the S. enterica serovar Typhi isolates, an absence of TR3 amplicons and a lack of length polymorphisms in TR1 and TR2 amplicons were observed for other S. enterica serovars, such as Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Enteritidis, and Salmonella enterica serovar Paratyphi A, B, and C. DNA sequencing of the amplified VNTR regions substantiated these results, suggesting the high stability of the multiplex PCR assay. The multiplex-PCR-based VNTR profiling developed in this study provides a simple, rapid, reproducible, and high-resolution molecular tool for the epidemiological analysis of S. enterica serovar Typhi strains.

Pre-treatment with Lactobacillus plantarum prevents severe pathogenesis in mice infected with Leptospira interrogans and may be associated with recruitment of myeloid cells

PubMed Central

Potula, Hari-Hara; Richer, Luciana; Werts, Catherine

2017-01-01

Recent estimates on global morbidity and mortality caused by Leptospirosis point to one million cases and almost 60,000 deaths a year worldwide, especially in resource poor countries. We analyzed how a commensal probiotic immunomodulator, Lactobacillus plantarum, affects Leptospira interrogans pathogenesis in a murine model of sub-lethal leptospirosis. We found that repeated oral pre-treatment of mice with live L. plantarum restored body weight to normal levels in mice infected with L. interrogans. Pre-treatment did not prevent L. interrogans access to the kidney but it affected the inflammatory response and it reduced histopathological signs of disease. Analysis of the immune cell profiles in lymphoid tissues of mice pre-treated with L. plantarum showed increased numbers of B cells as well as naïve and memory CD4+ helper T cell populations in uninfected mice that shifted towards increased numbers of effector CD4+ helper T in infected mice. CD8+ cytotoxic T cell profiles in pre-treated uninfected and infected mice mirrored the switch observed for CD4+ except that CD8+ memory T cells were not affected. In addition, pre-treatment led to increased populations of monocytes in lymphoid tissues of uninfected mice and to increased populations of macrophages in the same tissues of infected mice. Immunohistochemistry of kidney sections of pre-treated infected mice showed an enrichment of neutrophils and macrophages and a reduction of total leucocytes and T cells. Our results suggest that complex myeloid and T cell responses orchestrate the deployment of monocytes and other cells from lymphoid tissue and the recruitment of neutrophils and macrophages to the kidney, and that, the presence of these cells in the target organ may be associated with reductions in pathogenesis observed in infected mice treated with L. plantarum. PMID:28841659

Isolation and characterization of polyvalent bacteriophages infecting multi drug resistant Salmonella serovars isolated from broilers in Egypt.

PubMed

Mahmoud, Mayada; Askora, Ahmed; Barakat, Ahmed Barakat; Rabie, Omar El-Farouk; Hassan, Sayed Emam

2018-02-02

In this study, we isolated and characterized three phages named as Salmacey1, Salmacey2 and Salmacey3, infecting multi drug resistant Salmonella serovars isolated from broilers in Egypt. The most prevalent Salmonella serovars were S. typhimurium, S. enteritidis, and S. kentucky. All these Salmonella serovars were found to be resistant to more than two of the ten antimicrobial agents tested. Only S. kentucky was found to be resistant to seven antimicrobial agents. Examination of these phage particles by transmission electron microscopy (TEM), demonstrated that two phages (Salmacey1, Salmacey2) were found to belong to family Siphoviridae, and Salmacey3 was assigned to the family Myoviridae. The results of host range assay revealed that these bacteriophages were polyvalent and thus capable of infecting four strains of Salmonella serovars and Citrobacter freundii. Moreover, the two phages (Salmacey1, Salmacey2) had a lytic effect on Enterobacter cloacae and Salmacey3 was able to infect E. coli. All phages could not infect S. para Typhi, Staphylococus aureus and Bacillus cereus. One-step growth curves of bacteriophages revealed that siphovirus phages (Salmacey1, Salmacey2) have burst size (80 and 90pfu per infected cell with latent period 35min and 40min respectively), and for the myovirus Salmacey3 had a burst size 110pfu per infected cell with latent period 60min. Molecular analyses indicated that these phages contained double-stranded DNA genomes. The lytic activity of the phages against the most multidrug resistant serovars S. kentucky as host strain was evaluated. The result showed that these bacteriophages were able to completely stop the growth of S. kentucky in vitro. These results suggest that phages have a high potential for phage application to control Salmonella serovars isolated from broilers in Egypt. Copyright © 2017. Published by Elsevier B.V.

Salmonella enterica serovar Kentucky isolates from dairy cows and poultry demonstrate different evolutionary histories and host-specific polymorphisms

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subsp. enterica serovar Kentucky is commonly isolated from dairy cows and poultry in the United States. Although it is not among the most frequently isolated serovars from cases of human salmonellosis, its high prevalence in livestock and poultry indicate it is a potential public...

Global monitoring of Salmonella serovar distribution from the World Health Organization Global Foodborne Infections Network Country Data Bank: results of quality assured laboratories from 2001 to 2007.

PubMed

Hendriksen, Rene S; Vieira, Antonio R; Karlsmose, Susanne; Lo Fo Wong, Danilo M A; Jensen, Arne B; Wegener, Henrik C; Aarestrup, Frank M

2011-08-01

Salmonella enterica is commonly acquired from contaminated food and is an important cause of illness worldwide. Interventions are needed to control Salmonella; subtyping Salmonella by serotyping is useful for targeting such interventions. We, therefore, analyzed the global distribution of the 15 most frequently identified serovars of Salmonella isolated from humans from 2001 to 2007 in laboratories from 37 countries that participated in World Health Organization Global Foodborne Infections Network and demonstrated serotyping proficiency in the Global Foodborne Infections Network External Quality Assurance System. In all regions throughout the study period, with the exception of the Oceania and North American regions, Salmonella serovars Enteritidis and Typhimurium ranked as the most common and second most common serovar, respectively. In the North American and Oceania (Australia and New Zealand) regions, Salmonella serovar Typhimurium was the most common serovar reported, and Salmonella serovar Enteritidis was the second most common serovar. During the study period, the proportion of Salmonella isolates reported from humans that were Salmonella serovar Enteritidis was 43.5% (range: 40.6% [2007] to 44.9% [2003]), and Salmonella serovar Typhimurium was 17.1% (range: 15% [2007] to 18.9% [2001]). Salmonella serovars Newport (mainly observed in Latin and North American and European countries), Infantis (dominating in all regions), Virchow (mainly observed in Asian, European, and Oceanic countries), Hadar (profound in European countries), and Agona (intense in Latin and North American and European countries) were also frequently isolated with an overall proportion of 3.5%, 1.8%, 1.5%, 1.5%, and 0.8%, respectively. There were large differences in the most commonly isolated serovars between regions, but lesser differences between countries within the same region. The results also highlight the complexity of the global epidemiology of Salmonella and the need and importance

Salmonella enterica serovar Typhimurium and Escherichia coli contamination of root and leaf vegetables grown in soils with incorporated bovine manure.

PubMed

Natvig, Erin E; Ingham, Steven C; Ingham, Barbara H; Cooperband, Leslie R; Roper, Teryl R

2002-06-01

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P >or= 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10 degrees C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average

Salmonella enterica Serovar Typhimurium and Escherichia coli Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure

PubMed Central

Natvig, Erin E.; Ingham, Steven C.; Ingham, Barbara H.; Cooperband, Leslie R.; Roper, Teryl R.

2002-01-01

Bovine manure, with or without added Salmonella enterica serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The S. enterica serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for S. enterica serovar Typhimurium and Escherichia coli (indigenous in manure). After the 1 March manure application, S. enterica serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application, S. enterica serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil, S. enterica serovar Typhimurium died at a similar rate (P ≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with S. enterica serovar Typhimurium. Manure application under warmer (daily average maximum

Rapid multiplex PCR and Real-Time TaqMan PCR assays for detection of Salmonella enterica and the highly virulent serovars Choleraesuis and Paratyphi C

USDA-ARS?s Scientific Manuscript database

Salmonella enterica is a human pathogen with over 2,500 serovars characterized. S. enterica serovars Choleraesuis (Cs) and Paratyphi C (Pc) are two globally distributed serovars. We have developed a rapid molecular typing method to detect Cs and Pc in food samples by using a comparative genomics ap...

Genomic Comparison of Non-Typhoidal Salmonella enterica Serovars Typhimurium, Enteritidis, Heidelberg, Hadar and Kentucky Isolates from Broiler Chickens

PubMed Central

Dhanani, Akhilesh S.; Block, Glenn; Dewar, Ken; Forgetta, Vincenzo; Topp, Edward; Beiko, Robert G.; Diarra, Moussa S.

2015-01-01

Background Non-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. Methodology/Principal Finding The genomes of 25 S. enterica isolates covering five serovars (ten Typhimurium including three monophasic 4,[5],12:i:, four Enteritidis, three Hadar, four Heidelberg and four Kentucky) were sequenced. Most serovars were clustered in strongly supported phylogenetic clades, except for isolates of serovar Enteritidis that were scattered throughout the tree. Plasmids of varying sizes were detected in several isolates independently of serovars. Genes associated with the IncF plasmid and the IncI1 plasmid were identified in twelve and four isolates, respectively, while genes associated with the IncQ plasmid were found in one isolate. The presence of numerous genes associated with Salmonella pathogenicity islands (SPIs) was also confirmed. Components of the type III and IV secretion systems (T3SS and T4SS) varied in different isolates, which could explain in part, differences of their pathogenicity in humans and/or persistence in broilers. Conserved clusters of genes in the T3SS were detected that could be used in designing effective strategies (diagnostic, vaccination or treatments) to combat Salmonella. Antibiotic resistance genes (CMY, aadA, ampC, florR, sul1, sulI, tetAB, and srtA) and class I integrons were detected in resistant isolates while all isolates carried multidrug efflux pump systems regardless of their antibiotic susceptibility profile. Conclusions/Significance This study showed that the predominant

Genomic Comparison of Non-Typhoidal Salmonella enterica Serovars Typhimurium, Enteritidis, Heidelberg, Hadar and Kentucky Isolates from Broiler Chickens.

PubMed

Dhanani, Akhilesh S; Block, Glenn; Dewar, Ken; Forgetta, Vincenzo; Topp, Edward; Beiko, Robert G; Diarra, Moussa S

2015-01-01

Non-typhoidal Salmonella enterica serovars, associated with different foods including poultry products, are important causes of bacterial gastroenteritis worldwide. The colonization of the chicken gut by S. enterica could result in the contamination of the environment and food chain. The aim of this study was to compare the genomes of 25 S. enterica serovars isolated from broiler chicken farms to assess their intra- and inter-genetic variability, with a focus on virulence and antibiotic resistance characteristics. The genomes of 25 S. enterica isolates covering five serovars (ten Typhimurium including three monophasic 4,[5],12:i:, four Enteritidis, three Hadar, four Heidelberg and four Kentucky) were sequenced. Most serovars were clustered in strongly supported phylogenetic clades, except for isolates of serovar Enteritidis that were scattered throughout the tree. Plasmids of varying sizes were detected in several isolates independently of serovars. Genes associated with the IncF plasmid and the IncI1 plasmid were identified in twelve and four isolates, respectively, while genes associated with the IncQ plasmid were found in one isolate. The presence of numerous genes associated with Salmonella pathogenicity islands (SPIs) was also confirmed. Components of the type III and IV secretion systems (T3SS and T4SS) varied in different isolates, which could explain in part, differences of their pathogenicity in humans and/or persistence in broilers. Conserved clusters of genes in the T3SS were detected that could be used in designing effective strategies (diagnostic, vaccination or treatments) to combat Salmonella. Antibiotic resistance genes (CMY, aadA, ampC, florR, sul1, sulI, tetAB, and srtA) and class I integrons were detected in resistant isolates while all isolates carried multidrug efflux pump systems regardless of their antibiotic susceptibility profile. This study showed that the predominant Salmonella serovars in broiler chickens harbor genes encoding adhesins

Repeated isolation of Salmonella enterica Goverdhan, a very rare serovar, from Danish poultry surveillance samples.

PubMed

Pedersen, Karl; Sørensen, Gitte; Szabo, Istvan; Hächler, Herbert; Le Hello, Simon

2014-12-05

We report here the appearance of a very rare serovar of Salmonella, S. enterica subsp. enterica serovar Goverdhan, in routine Salmonella surveillance samples from Danish poultry production. S. Goverdhan was found on nine occasions: in one broiler breeder farm in October 2010, four broiler farms and one broiler breeder farm in June-September 2012, two broiler breeder flocks simultaneously in June 2013, and one layer flock in July 2013. The five isolates from 2012 and the three isolates from 2013 had identical pulsed-field gel electrophoresis profiles, whereas the profile of the isolate from 2010 deviated in a single band. It is the first time this serovar has been described in samples from poultry. The origin of the bacterium is still unknown, but it is suggested that it may have been a pseudo-outbreak caused by contaminated sampling material. Copyright © 2014 Elsevier B.V. All rights reserved.

Clinical and veterinary isolates of Salmonella enterica serovar Enteritidis defective in lipopolysaccharide O-chain polymerization

DOE Office of Scientific and Technical Information (OSTI.GOV)

Guard-Petter, J.; Parker, C.T.; Asokan, K.

1999-05-01

Twelve human and chicken isolates of Salmonella enterica serovar Enteritidis belonging to phage types 4, 8, 13a, and 23 were characterized for variability in lipopolysaccharide (LPS) composition. Isolates were differentiated into two groups, i.e., those that lacked immunoreactive O-chain, termed rough isolates, and those that had immunoreactive O-chain, termed smooth isolates. Isolates within these groups could be further differentiated by LPS compositional differences as detected by gel electrophoresis and gas liquid chromatography of samples extracted with water, which yielded significantly more LPS in comparison to phenol-chloroform extraction. The rough isolates were of two types, the O-antigen synthesis mutants and themore » O-antigen polymerization (wzy) mutants. Smooth isolates were also of two types, one producing low-molecular-weight (LMW) LPS and the other producing high-molecular-weight (HMW) LPS. To determine the genetic basis for the O-chain variability of the smooth isolates, the authors analyzed the effects of a null mutation in the O-chain length determinant gene, wzz (cld) of serovar Typhimurium. This mutation results in a loss of HMW LPS; however, the LMW LPS of this mutant was longer and more glucosylated than that from clinical isolates of serovar Enteritidis. Cluster analysis of these data and of those from two previously characterized isogenic strains of serovar Enteritidis that had different virulence attributes indicated that glucosylation of HMW LPS (via oafR function) is variable and results in two types of HMW structures, one that is highly glucosylated and one that is minimally glucosylated. These results strongly indicate that naturally occurring variability in wzy, wzz, and oafR function can be used to subtype isolates of serovar Enteritidis during epidemiological investigations.« less

Protective Immunity and Reduced Renal Colonization Induced by Vaccines Containing Recombinant Leptospira interrogans Outer Membrane Proteins and Flagellin Adjuvant

PubMed Central

Monaris, D.; Sbrogio-Almeida, M. E.; Dib, C. C.; Canhamero, T. A.; Souza, G. O.; Vasconcellos, S. A.; Ferreira, L. C. S.

2015-01-01

Leptospirosis is a global zoonotic disease caused by different Leptospira species, such as Leptospira interrogans, that colonize the renal tubules of wild and domestic animals. Thus far, attempts to develop effective leptospirosis vaccines, both for humans and animals, have failed to induce immune responses capable of conferring protection and simultaneously preventing renal colonization. In this study, we evaluated the protective immunity induced by subunit vaccines containing seven different recombinant Leptospira interrogans outer membrane proteins, including the carboxy-terminal portion of the immunoglobulinlike protein A (LigAC) and six novel antigens, combined with aluminum hydroxide (alum) or Salmonella flagellin (FliC) as adjuvants. Hamsters vaccinated with the different formulations elicited high antigen-specific antibody titers. Immunization with LigAC, either with alum or flagellin, conferred protective immunity but did not prevent renal colonization. Similarly, animals immunized with LigAC or LigAC coadministered with six leptospiral proteins with alum adjuvant conferred protection but did not reduce renal colonization. In contrast, immunizing animals with the pool of seven antigens in combination with flagellin conferred protection and significantly reduced renal colonization by the pathogen. The present study emphasizes the relevance of antigen composition and added adjuvant in the efficacy of antileptospirosis subunit vaccines and shows the complex relationship between immune responses and renal colonization by the pathogen. PMID:26108285

Development of Real Time PCR Using Novel Genomic Target for Detection of Multiple Salmonella Serovars from Milk and Chickens

USDA-ARS?s Scientific Manuscript database

Background: A highly sensitive and specific novel genomic and plasmid target-based PCR platform was developed to detect multiple Salmonella serovars (S. Heidelberg, S. Dublin, S. Hadar, S. Kentucky and S. Enteritidis). Through extensive genome mining of protein databases of these serovars and compar...

Clonality and antimicrobial resistance gene profiles of multidrug- resistant Salmonella enterica serovar infantis isolates from four public hospitals in Rio de Janeiro, Brazil.

PubMed

Fonseca, E L; Mykytczuk, O L; Asensi, M D; Reis, E M F; Ferraz, L R; Paula, F L; Ng, L K; Rodrigues, D P

2006-08-01

In Brazil, Salmonella enterica serovar Infantis resistant to various antimicrobials, including cephalosporins, has been identified as an etiological agent of severe gastroenteritis in hospitalized children since 1994. In this study, 35 serovar Infantis strains, isolated from children admitted to four different Rio de Janeiro, Brazil, hospitals between 1996 and 2001, were characterized by pulsed-field gel electrophoresis (PFGE) and antimicrobial susceptibility testing in order to determine their genetic relatedness and antimicrobial resistance profiles. Thirty-four serovar Infantis strains were resistant to at least two antibiotic classes, and all 35 strains were susceptible to fluoroquinolones, cephamycin, and carbapenem. Extended-spectrum beta-lactamase (ESBL) screening by double-disk diffusion indicated that 32 serovar Infantis strains (91.4%) produced beta-lactamases that were inhibited by clavulanic acid. Antimicrobial resistance gene profiles were determined by PCR for a subset of 11 multidrug-resistant serovar Infantis strains, and putative ESBLs were detected by isoelectric focusing. Ten serovar Infantis strains carried bla(TEM), catI, ant(3")Ia and/or ant(3")Ib, sulI and/or sulII, and tet(D) genes as well as an integron-associated aac(6')-Iq cassette. Eight strains possessed at least four different beta-lactamases with pI profiles that confirmed the presence of both ESBLs and non-ESBLs. Our PFGE profiles indicated that 33 serovar Infantis strains isolated from Rio de Janeiro hospitals came from the same genetic lineage.

Leptospira santorosai Serovar Shermani detergent extract induces an increase in fibronectin production through a Toll-like receptor 2-mediated pathway.

PubMed

Tian, Ya-Chung; Hung, Cheng-Chieh; Li, Yi-Jung; Chen, Yung-Chang; Chang, Ming-Yang; Yen, Tzung-Hai; Hsu, Hsiang-Hao; Wu, Mai-Szu; Phillips, Aled; Yang, Chih-Wei

2011-03-01

Leptospirosis can activate inflammatory responses through Toll-like receptors (TLRs) and may cause renal tubulointerstitial fibrosis characterized by the accumulation of extracellular matrix (ECM). We have previously demonstrated that Leptospira santorosai serovar Shermani detergent extract stimulates ECM accumulation in vitro. The aim of this study was to examine the mechanistic basis of these previous observations and, in particular, to examine the potential involvement of TLRs. The addition of serovar Shermani detergent extract led to an increase in fibronectin gene expression and production. Inhibition of TLR2 but not TLR4 expression abrogated serovar Shermani detergent extract-mediated increases in fibronectin production. This response was also blocked by the knockdown of the gene expression of the TLR2 downstream transducers myeloid differentiation factor 88 (MyD88) and tumor necrosis factor receptor-associated factor 6 (TRAF6). Serovar Shermani detergent extract also activated nuclear factor-κB, and its inhibition by curcumin-attenuated serovar Shermani detergent extract induced increases in fibronectin production. These effects were also mimicked by the specific TLR2 agonist, Pam(3)CsK(4), a response that was also abrogated by the knockdown of MyD88 and TRAF6. Similarly, the administration of live leptospires to cells also induced fibronectin production that was blocked by inhibition of TLR2 and MyD88 expression. In conclusion, serovar Shermani detergent extract can induce fibronectin production through the TLR2-associated cascade, providing evidence of an association between TLRs and leptospirosis-mediated ECM deposition.

SEROVARS AND ANTIMICROBIAL RESISTANCE OF Salmonella spp. ISOLATED FROM TURKEY AND BROILER CARCASSES IN SOUTHERN BRAZIL BETWEEN 2004 AND 2006

PubMed Central

PALMEIRA, Andre; dos SANTOS, Luciana Ruschel; BORSOI, Anderlise; RODRIGUES, Laura Beatriz; CALASANS, Max; do NASCIMENTO, Vladimir Pinheiro

2016-01-01

Salmonella spp. causes diseases in fowls, when species-specific serovars (Salmonella Pullorum and S.Gallinarum) are present in flocks, and public health problems, when non-typhoid serovars are isolated, as well as possible bacterial resistance induced by the preventive and therapeutic use of antimicrobials in animal production. This study describes the serovars and bacterial resistance of 280Salmonella spp. strains isolated from turkey and broiler carcasses in Southern Brazil between 2004 and 2006. SalmonellaEnteritidis was the most prevalent serovar (55.7%), followed by Heidelberg (5.0%), Agona (4.3%), Bredeney (3.9%), Hadar (3.2%), and Typhimurium (2.9%). Tennessee and S. Enterica subspecies enterica(O: 4.5) were isolated only in turkeys, and Hadar (18.6%) was the most prevalent serovar in this species. Antimicrobial susceptibility tests were performed in 178 isolates (43 from turkeys and 135 from broilers). All isolates were sensitive to amoxicillin + clavulanic acid, polymyxin B, ciprofloxacin, and norfloxacin, and were resistant to bacitracin and penicillin. Broiler carcass isolates showed resistance to nalidixic acid (48.9%), nitrofurantoin (34.3%), neomycin (9.6%), tetracycline (5.2%), and kanamycin (8.9%); and turkey carcass isolates were resistant to nalidixic acid (62.8%), tetracycline (34.9%), and neomycin (30.2%), with a significant difference in turkeys when compared to broiler carcass isolates. These results indicate the need for judicious use of antimicrobials in livestock production, given that the serovars identified are potential causes of food poisoning. PMID:27007562

Research note: Molecular subtyping of Salmonella enterica serovar Tshiongwe recently isolated in Malaysia during 2001-2002.

PubMed

Thong, Kwai Lin; Bakeri, Shamsilawani Ahmad; Lai, Kin Seng; Koh, Yin Tee; Taib, Mohd Zainuldin; Lim, V K E; Yasin, Rohani Md

2004-03-01

Pulsed field gel electrophoresis (PFGE) and antimicrobial susceptibility analysis were undertaken on twenty-three strains of Salmonella enterica serovar Tshiongwe, an unusual serovar, which recently emerged in Malaysia. Antimicrobial susceptibility analysis showed that all the strains were sensitive to ampicilin, chloramphenicol, cotrimoxazole, and kanamycin. Twenty (87%) and 8 (3.5%) strains had resistance to tetracycline and streptomycin respectively. PFGE analysis subtyped 23 strains into 10 profiles (Dice coefficient of similarity, F = 0.7-1.0). The predominant profile, X1 was found in both clinical and environmental isolates and was widely distributed in different parts of Malaysia during the study period. In addition, isolates recovered from food, a hand-towel, apron and the surface of a table-top in one particular location had unique, indistinguishable profiles (X4/4a) and identical antibiograms. Similarly, isolates from cooked meat and a chopping board had PFGE profiles similar to some human isolates. These probably indicated cross-contamination and poor hygiene in food practices, hence contributing to Salmonellosis. Factors causing the emergence of this rare Salmonella serovar being responsible for food poisoning episodes during the study period remained unclear. The study reiterated the usefulness and versatility of PFGE in the molecular subtyping of this rare Salmonella serovar in Malaysia.

Increased efficacy of inactivated vaccine candidates prepared with Salmonella enterica serovar Typhimurium strains of predominant genotypes in ducks.

PubMed

Youn, S Y; Kwon, Y K; Song, C S; Lee, H J; Jeong, O M; Choi, B K; Jung, S C; Kang, M S

2016-08-01

Salmonella enterica serovar Typhimurium has been a major causative agent of food-borne human disease, mainly due to consumption of contaminated food animal products. In particular, ducks serve as a reservoir of serovar Typhimurium, and are one of the common sources of human infection. To prevent infection of ducks, and therefore minimize human infection, it is critical to control the persistent epidemic strains in ducks. Here, we analyzed the genetic diversity and virulence of serovar Typhimurium isolates from ducks in Korea to identify the predominant strains that might be used as efficient vaccine candidates for ducks. Among the isolates, 2 representative isolates (ST26 and ST76) of predominant genotypes were selected as vaccine strains on the basis of genotypic analysis by pulsed-field gel electrophoresis and DNA microarrays. Two-week-old ducks were then injected intramuscularly with inactivated vaccine candidates prepared using ST26 or ST76 (10(8) cfu/0.5 mL/duck or 10(9) cfu/0.5 mL/duck), and oral challenge with a highly virulent serovar Typhimurium strain (10(9) cfu/0.5 mL/duck) was carried out 2 wk later. Shedding of the challenge strain was significantly decreased in group 2 after vaccination. The antibody levels by enzyme-linked immunosorbent assay in all vaccinated groups were enhanced significantly (P < 0.05) compared to the unvaccinated control group. Overall, vaccination with ST26 or ST76 reduced bacterial shedding and colonization in internal organs, and induced elevated antibody response. In particular, serovar Typhimurium ST26 (10(8) cfu/0.5 mL/duck) was the most effective vaccine candidate, which can provide efficient protection against serovar Typhimurium in ducks with higher effectiveness compared to a commercial vaccine currently used worldwide. © 2016 Poultry Science Association Inc.

Method for the detection of Salmonella enterica serovar Enteritidis

DOEpatents

Agron, Peter G.; Andersen, Gary L.; Walker, Richard L.

2008-10-28

Described herein is the identification of a novel Salmonella enterica serovar Enteritidis locus that serves as a marker for DNA-based identification of this bacterium. In addition, three primer pairs derived from this locus that may be used in a nucleotide detection method to detect the presence of the bacterium are also disclosed herein.

Bovine leptospirosis in urban and peri-urban dairy farming in low-income countries: a "One Health" issue?

PubMed

Rajala, Elisabeth Lindahl; Sattorov, Nosirjon; Boqvist, Sofia; Magnusson, Ulf

2017-12-12

Global trends in urbanization are increasing the spread of neglected zoonotic infections such as leptospirosis, and reducing the number of human cases of leptospirosis is best accomplished by controlling the infection in the animal reservoir. The aim of this study was to determine the seroprevalence of Leptospira borgpetersenii serovar Hardjo and L. interrogans serovar Hardjo (L. Hardjo) exposure and to assess the associated risk factors for infection in small-scale dairy farming in the urban and peri-urban area of Dushanbe, Tajikistan. The true individual seroprevalence among the dairy cows was 13%, and the level of seroprevalence was positively associated with older cows and with communal grazing practices. The study shows that dairy cows are commonly exposed to L. Hardjo in the study region, and this constitutes a public health risk and demonstrates the importance of including urban and peri-urban areas, where large numbers of humans and animals coexist, when investigating zoonotic infections and when planning and implementing control measures for cattle-associated leptospirosis.

Endemic, Epidemic Clone of Salmonella enterica Serovar Typhi Harboring a Single Multidrug-Resistant Plasmid in Vietnam between 1995 and 2002

PubMed Central

Le, Thi Anh Hong; Lejay-Collin, Monique; Grimont, Patrick A. D.; Hoang, Thuy Long; Nguyen, Thi Vinh; Grimont, Francine; Scavizzi, Maurice R.

2004-01-01

Salmonella enterica serovar Typhi strains resistant to ampicillin, chloramphenicol, tetracyclines, streptomycin, and cotrimoxazole, isolated from sporadic cases and minor outbreaks in Vietnam between 1995 and 2002, were typed and compared. Plasmid fingerprinting, Vi bacteriophage typing, XbaI pulsed-field gel electrophoresis, and PstI ribotyping showed that endemic, epidemic multidrug-resistant typhoid fever was due, for at least 74.1% of the isolates, to one or two clones of serovar Typhi harboring a single resistance plasmid. PstI ribotyping was used as a basic technique to ensure that a serovar Typhi expansion was clonal. PMID:15243066

Horizontal gene transfer of a ColV plasmid has resulted in a dominant avian clonal type of Salmonella enterica serovar Kentucky.

PubMed

Johnson, Timothy J; Thorsness, Jessica L; Anderson, Cole P; Lynne, Aaron M; Foley, Steven L; Han, Jing; Fricke, W Florian; McDermott, Patrick F; White, David G; Khatri, Mahesh; Stell, Adam L; Flores, Cristian; Singer, Randall S

2010-12-22

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard.

Horizontal Gene Transfer of a ColV Plasmid Has Resulted in a Dominant Avian Clonal Type of Salmonella enterica Serovar Kentucky

PubMed Central

Johnson, Timothy J.; Thorsness, Jessica L.; Anderson, Cole P.; Lynne, Aaron M.; Foley, Steven L.; Han, Jing; Fricke, W. Florian; McDermott, Patrick F.; White, David G.; Khatri, Mahesh; Stell, Adam L.; Flores, Cristian; Singer, Randall S.

2010-01-01

Salmonella enterica continues to be a significant cause of foodborne gastrointestinal illness in humans. A wide variety of Salmonella serovars have been isolated from production birds and from retail poultry meat. Recently, though, S. enterica subsp. enterica serovar Kentucky has emerged as one of the prominent Salmonella serovars isolated from broiler chickens. Recent work suggests that its emergence apparently coincides with its acquisition of a ColV virulence plasmid. In the present study, we examined 902 Salmonella isolates belonging to 59 different serovars for the presence of this plasmid. Of the serovars examined, the ColV plasmid was found only among isolates belonging to the serovars Kentucky (72.9%), Typhimurium (15.0%) and Heidelberg (1.7%). We demonstrated that a single PFGE clonal type of S. Kentucky harbors this plasmid, and acquisition of this plasmid by S. Kentucky significantly increased its ability to colonize the chicken cecum and cause extraintestinal disease. Comparison of the completed sequences of three ColV plasmids from S. Kentucky isolated from different geographical locales, timepoints and sources revealed a nearly identical genetic structure with few single nucleotide changes or insertions/deletions. Overall, it appears that the ColV plasmid was recently acquired by a single clonal type S. Kentucky and confers to its host enhanced colonization and fitness capabilities. Thus, the potential for horizontal gene transfer of virulence and fitness factors to Salmonella from other enteric bacteria exists in poultry, representing a potential human health hazard. PMID:21203520

Draft Genome Sequences of 20 Salmonella enterica subsp. enterica Serovar Typhimurium Strains Isolated from Swine in Santa Catarina, Brazil.

PubMed

Seribelli, Amanda Aparecida; Frazão, Miliane Rodrigues; Gonzales, Júlia Cunha; Cao, Guojie; Leon, Maria Sanchez; Kich, Jalusa Deon; Allard, Marc William; Falcão, Juliana Pfrimer

2018-04-19

Salmonellosis is a disease with a high incidence worldwide, and Salmonella enterica subsp. enterica serovar Typhimurium is one of the most clinically important serovars. We report here the draft genome sequences of 20 S. Typhimurium strains isolated from swine in Santa Catarina, Brazil. These draft genomes will improve our understanding of S. Typhimurium in Brazil.

Outer membrane protein a of Salmonella enterica serovar Typhimurium activates dendritic cells and enhances Th1 polarization

PubMed Central

2010-01-01

Background Typhoid, which is caused by Salmonella enterica serovar Typhimurium, remains a major health concern worldwide. Multidrug-resistant strains of Salmonella have emerged which exhibit increased survivability and virulence, thus leading to increased morbidity. However, little is known about the protective immune response against this microorganism. The outer membrane protein (Omp)A of bacteria plays an important role in pathogenesis. Results We purified OmpA from S. enterica serovar Typhimurium (OmpA-sal) and characterized the role of OmpA-sal in promoting adaptive and innate immune responses. OmpA-sal functionally activated bone marrow-derived dendritic cells by augmenting expression of CD80, CD86, and major histocompatibility complex classes I and II. Interestingly, OmpA-sal induced production of interferon-γ from T cells in mixed lymphocyte reactions, thus indicating Th1-polarizing capacity. The expression of surface markers and cytokine production in dendritic cells was mediated by the TLR4 signaling pathway in a TLR4 Knock-out system. Conclusions Our findings suggest that OmpA-sal modulates the adaptive immune responses to S. enterica serovar Typhimurium by activating dendritic cells and driving Th1 polarization, which are important properties to consider in the development of effective S. enterica serovar Typhimurium vaccines and immunotherapy adjuvant. PMID:20950448

Comparison of genome degradation in Paratyphi A and Typhi, human-restricted serovars of Salmonella enterica that cause typhoid.

PubMed

McClelland, Michael; Sanderson, Kenneth E; Clifton, Sandra W; Latreille, Phil; Porwollik, Steffen; Sabo, Aniko; Meyer, Rekha; Bieri, Tamberlyn; Ozersky, Phil; McLellan, Michael; Harkins, C Richard; Wang, Chunyan; Nguyen, Christine; Berghoff, Amy; Elliott, Glendoria; Kohlberg, Sara; Strong, Cindy; Du, Feiyu; Carter, Jason; Kremizki, Colin; Layman, Dan; Leonard, Shawn; Sun, Hui; Fulton, Lucinda; Nash, William; Miner, Tracie; Minx, Patrick; Delehaunty, Kim; Fronick, Catrina; Magrini, Vincent; Nhan, Michael; Warren, Wesley; Florea, Liliana; Spieth, John; Wilson, Richard K

2004-12-01

Salmonella enterica serovars often have a broad host range, and some cause both gastrointestinal and systemic disease. But the serovars Paratyphi A and Typhi are restricted to humans and cause only systemic disease. It has been estimated that Typhi arose in the last few thousand years. The sequence and microarray analysis of the Paratyphi A genome indicates that it is similar to the Typhi genome but suggests that it has a more recent evolutionary origin. Both genomes have independently accumulated many pseudogenes among their approximately 4,400 protein coding sequences: 173 in Paratyphi A and approximately 210 in Typhi. The recent convergence of these two similar genomes on a similar phenotype is subtly reflected in their genotypes: only 30 genes are degraded in both serovars. Nevertheless, these 30 genes include three known to be important in gastroenteritis, which does not occur in these serovars, and four for Salmonella-translocated effectors, which are normally secreted into host cells to subvert host functions. Loss of function also occurs by mutation in different genes in the same pathway (e.g., in chemotaxis and in the production of fimbriae).

Host-Nonspecific Iron Acquisition Systems and Virulence in the Zoonotic Serovar of Vibrio vulnificus

PubMed Central

Pajuelo, David; Lee, Chung-Te; Roig, Francisco J.; Lemos, Manuel L.; Hor, Lien-I

2014-01-01

The zoonotic serovar of Vibrio vulnificus (known as biotype 2 serovar E) is the etiological agent of human and fish vibriosis. The aim of the present work was to discover the role of the vulnibactin- and hemin-dependent iron acquisition systems in the pathogenicity of this zoonotic serovar under the hypothesis that both are host-nonspecific virulence factors. To this end, we selected three genes for three outer membrane receptors (vuuA, a receptor for ferric vulnibactin, and hupA and hutR, two hemin receptors), obtained single and multiple mutants as well as complemented strains, and tested them in a series of in vitro and in vivo assays, using eels and mice as animal models. The overall results confirm that hupA and vuuA, but not hutR, are host-nonspecific virulence genes and suggest that a third undescribed host-specific plasmid-encoded system could also be used by the zoonotic serovar in fish. hupA and vuuA were expressed in the internal organs of the animals in the first 24 h of infection, suggesting that they may be needed to achieve the population size required to trigger fatal septicemia. vuuA and hupA were sequenced in strains representative of the genetic diversity of this species, and their phylogenies were reconstructed by multilocus sequence analysis of selected housekeeping and virulence genes as a reference. Given the overall results, we suggest that both genes might form part of the core genes essential not only for disease development but also for the survival of this species in its natural reservoir, the aquatic environment. PMID:24478087

Large-scale purification and in vitro characterization of the assembly of MreB from Leptospira interrogans.

PubMed

Barkó, Szilvia; Szatmári, Dávid; Bódis, Emőke; Türmer, Katalin; Ujfalusi, Zoltán; Popp, David; Robinson, Robert C; Nyitrai, Miklós

2016-09-01

Weil's syndrome is caused by Leptospira interrogans infections, a Gram negative bacterium with a distinct thin corkscrew cell shape. The molecular basis for this unusual morphology is unknown. In many bacteria, cell wall synthesis is orchestrated by the actin homolog, MreB. Here we have identified the MreB within the L. interrogans genome and expressed the His-tagged protein product of the synthesized gene (Li-MreB) in Escherichia coli. Li-MreB did not purify under standard nucleotide-free conditions used for MreBs from other species, requiring the continual presence of ATP to remain soluble. Covalent modification of Li-MreB free thiols with Alexa488 produced a fluorescent version of Li-MreB. We developed native and denaturing/refolding purification schemes for Li-MreB. The purified product was shown to assemble and disassemble in MgCl2 and KCl dependent manners, as monitored by light scattering and sedimentation studies. The fluorescence spectrum of labeled Li-MreB-Alexa488 showed cation-induced changes in line with an activation process followed by a polymerization phase. The resulting filaments appeared as bundles and sheets under the fluorescence microscope. Finally, since the Li-MreB polymerization was cation dependent, we developed a simple method to measure monovalent cation concentrations within a test case prokaryote, E. coli. We have identified and initially characterized the cation-dependent polymerization properties of a novel MreB from a non-rod shaped bacterium and developed a method to measure cation concentrations within prokaryotes. This initial characterization of Li-MreB will enable future structural determination of the MreB filament from this corkscrew-shaped bacterium. Copyright © 2016 Elsevier B.V. All rights reserved.

High-Throughput Parallel Sequencing to Measure Fitness of Leptospira interrogans Transposon Insertion Mutants during Acute Infection

PubMed Central

Matsunaga, James; Haake, David A.

2016-01-01

Pathogenic species of Leptospira are the causative agents of leptospirosis, a zoonotic disease that causes mortality and morbidity worldwide. The understanding of the virulence mechanisms of Leptospira spp is still at an early stage due to the limited number of genetic tools available for this microorganism. The development of random transposon mutagenesis in pathogenic strains a decade ago has contributed to the identification of several virulence factors. In this study, we used the transposon sequencing (Tn-Seq) technique, which combines transposon mutagenesis with massive parallel sequencing, to study the in vivo fitness of a pool of Leptospira interrogans mutants. We infected hamsters with a pool of 42 mutants (input pool), which included control mutants with insertions in four genes previously analyzed by virulence testing (loa22, ligB, flaA1, and lic20111) and 23 mutants with disrupted signal transduction genes. We quantified the mutants in different tissues (blood, kidney and liver) at 4 days post-challenge by high-throughput sequencing and compared the frequencies of mutants recovered from tissues to their frequencies in the input pool. Control mutants that were less fit in the Tn-Seq experiment were attenuated for virulence when tested separately in the hamster model of lethal leptospirosis. Control mutants with unaltered fitness were as virulent as the wild-type strain. We identified two mutants with the transposon inserted in the same putative adenylate/guanylate cyclase gene (lic12327) that had reduced in vivo fitness in blood, kidney and liver. Both lic12327 mutants were attenuated for virulence when tested individually in hamsters. Growth of the control mutants and lic12327 mutants in culture medium were similar to that of the wild-type strain. These results demonstrate the feasibility of screening large pools of L. interrogans transposon mutants for those with altered fitness, and potentially attenuated virulence, by transposon sequencing. PMID

Human leptospirosis caused by a new, antigenically unique Leptospira associated with a Rattus species reservoir in the Peruvian Amazon.

PubMed

Matthias, Michael A; Ricaldi, Jessica N; Cespedes, Manuel; Diaz, M Monica; Galloway, Renee L; Saito, Mayuko; Steigerwalt, Arnold G; Patra, Kailash P; Ore, Carlos Vidal; Gotuzzo, Eduardo; Gilman, Robert H; Levett, Paul N; Vinetz, Joseph M

2008-04-02

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species "Leptospira licerasiae" serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010(T), which has been deposited into internationally accessible culture collections. By microscopic agglutination test, "Leptospira licerasiae" serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti-L. fainei serovar Hurstbridge at a titer of 1:100. LipL32, although not detectable by PCR, was detectable in "Leptospira licerasiae" serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against "Leptospira licerasiae" serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon.

Human Leptospirosis Caused by a New, Antigenically Unique Leptospira Associated with a Rattus Species Reservoir in the Peruvian Amazon

PubMed Central

Cespedes, Manuel; Diaz, M. Monica; Galloway, Renee L.; Saito, Mayuko; Steigerwalt, Arnold G.; Patra, Kailash P.; Ore, Carlos Vidal; Gotuzzo, Eduardo; Gilman, Robert H.; Levett, Paul N.; Vinetz, Joseph M.

2008-01-01

As part of a prospective study of leptospirosis and biodiversity of Leptospira in the Peruvian Amazon, a new Leptospira species was isolated from humans with acute febrile illness. Field trapping identified this leptospire in peridomestic rats (Rattus norvegicus, six isolates; R. rattus, two isolates) obtained in urban, peri-urban, and rural areas of the Iquitos region. Novelty of this species was proven by serological typing, 16S ribosomal RNA gene sequencing, pulsed-field gel electrophoresis, and DNA-DNA hybridization analysis. We have named this species “Leptospira licerasiae” serovar Varillal, and have determined that it is phylogenetically related to, but genetically distinct from, other intermediate Leptospira such as L. fainei and L. inadai. The type strain is serovar Varillal strain VAR 010T, which has been deposited into internationally accessible culture collections. By microscopic agglutination test, “Leptospira licerasiae” serovar Varillal was antigenically distinct from all known serogroups of Leptospira except for low level cross-reaction with rabbit anti–L. fainei serovar Hurstbridge at a titer of 1∶100. LipL32, although not detectable by PCR, was detectable in “Leptospira licerasiae” serovar Varillal by both Southern blot hybridization and Western immunoblot, although on immunoblot, the predicted protein was significantly smaller (27 kDa) than that of L. interrogans and L. kirschneri (32 kDa). Isolation was rare from humans (2/45 Leptospira isolates from 881 febrile patients sampled), but high titers of MAT antibodies against “Leptospira licerasiae” serovar Varillal were common (30%) among patients fulfilling serological criteria for acute leptospirosis in the Iquitos region, and uncommon (7%) elsewhere in Peru. This new leptospiral species reflects Amazonian biodiversity and has evolved to become an important cause of leptospirosis in the Peruvian Amazon. PMID:18382606

Salmonella enterica serovar Typhimurium mutants unable to convert malate to pyruvate and oxaloacetate are avirulent and immunogenic in BALB/c mice.

PubMed

Mercado-Lubo, Regino; Leatham, Mary P; Conway, Tyrrell; Cohen, Paul S

2009-04-01

Previously, we showed that the Salmonella enterica serovar Typhimurium SR-11 tricarboxylic acid (TCA) cycle must operate as a complete cycle for full virulence after oral infection of BALB/c mice (M. Tchawa Yimga, M. P. Leatham, J. H. Allen, D. C. Laux, T. Conway, and P. S. Cohen, Infect. Immun. 74:1130-1140, 2006). In the same study, we showed that for full virulence, malate must be converted to both oxaloacetate and pyruvate. Moreover, it was recently demonstrated that blocking conversion of succinyl-coenzyme A to succinate attenuates serovar Typhimurium SR-11 but does not make it avirulent; however, blocking conversion of succinate to fumarate renders it completely avirulent and protective against subsequent oral infection with the virulent serovar Typhimurium SR-11 wild-type strain (R. Mercado-Lubo, E. J. Gauger, M. P. Leatham, T. Conway, and P. S. Cohen, Infect. Immun. 76:1128-1134, 2008). Furthermore, the ability to convert succinate to fumarate appeared to be required only after serovar Typhimurium SR-11 became systemic. In the present study, evidence is presented that serovar Typhimurium SR-11 mutants that cannot convert fumarate to malate or that cannot convert malate to both oxaloacetate and pyruvate are also avirulent and protective in BALB/c mice. These results suggest that in BALB/c mice, the malate that is removed from the TCA cycle in serovar Typhimurium SR-11 for conversion to pyruvate must be replenished by succinate or one of its precursors, e.g., arginine or ornithine, which might be available in mouse phagocytes.

Clinical, serological and echocardiographic examination of healthy field dogs before and after vaccination with a commercial tetravalent leptospirosis vaccine.

PubMed

Spiri, Andrea M; Rodriguez-Campos, Sabrina; Matos, José M; Glaus, Tony M; Riond, Barbara; Reusch, Claudia E; Hofmann-Lehmann, Regina; Willi, Barbara

2017-05-25

Leptospirosis is a re-emerging bacterial zoonosis caused by spirochetes of the genus Leptospira. Severe disease has been reported in dogs in Europe despite vaccination with bivalent Leptospira vaccines. Recently, a tetravalent canine Leptospira vaccine (Nobivac® L4) was licenced in Europe. The goal of this study was to investigate clinical signs, microscopic agglutination test (MAT) titres, haematology, blood biochemistry, cardiac (c) Troponin I levels and echocardiography before and after vaccination with this tetravalent vaccine. Forty-eight healthy dogs were prospectively enrolled and vaccinated twice, 3-4 weeks apart (T0 and T1). Before vaccination (T0) and 16-31 days after the second vaccination (T2), MAT (n = 48), haematology (n = 48), blood biochemistry (n = 36) and cTroponin I measurements (n = 29) were performed, and MAT was repeated 347-413 days after the second vaccination (T3, n = 44). Echocardiography was performed before the first and second vaccination (T0 and T1, n = 24). Mild and transient clinical signs within 5 days following the first and second vaccination occurred in 23% and 10% of the dogs, respectively. Before the first vaccination (T0), all dogs showed negative MAT titres for the tested serovars except for Canicola (50% with titres 100-400). At T2, positive MAT titres to the serovars Canicola (100%), Australis (89%), Grippotyphosa (86%), Bratislava (60%), Autumnalis (58%), Copenhageni (42%), Pomona (12%), Pyrogenes (8%) and Icterohaemorrhagiae (2%) were found. Median to high titres (≥ 400) were most common to the serovar Canicola (92%) and less common to the serovars Australis (41%), Grippotyphosa (21%), Bratislava (12%), Autumnalis (4%), Pyrogenes (4%) and Pomona (2%). At T3, positive MAT titres (titre range: 100-400) were found in 2-18% of the dogs to serovars of the vaccine serogroups and in 2-18% of the dogs to the non-vaccine serovars Pomona, Autumnalis, Pyrogenes and Ballum. Haematology, blood biochemistry, c

A serological survey of leptospirosis in cattle of rural communities in the province of KwaZulu-Natal, South Africa.

PubMed

Hesterberg, U W; Bagnall, R; Bosch, B; Perrett, K; Horner, R; Gummow, B

2009-03-01

A serological survey of leptospirosis in cattle originating from rural communities of the province of KwaZulu-Natal (KZN) in South Africa was carried out between March 2001 and December 2003. The survey was designed as a 2-stage survey, using the local diptank as the primary sampling point. In total, 2021 animals from 379 diptanks in 33 magisterial districts were sampled and tested with the microscopic agglutination test (MAT). The apparent prevalence at district level was adjusted for clustering and diagnostic test sensitivity and specificity and displayed in maps. The prevalence of leptospirosis in cattle originating from communal grazing areas of KZN was found to be 19.4% with a 95% confidence interval of 14.8-24.1%. At district level the prevalence of leptospirosis varied from 0 to 63% of cattle. Bovine leptospirosis was found to occur in communal grazing areas throughout the province with the exception of 2 districts. The southeastern regions showed a higher prevalence than other areas of the province; while in some of the northern and western districts a lower prevalence was noted. Several serovars were detected by the MAT and although Leptospira interrogans serovar pomona occurred most frequently, serovars tarrasovi, bratislava, hardjo, canicola and icterohaemorrhagica were also frequently identified. The findings of the survey are discussed.

Complete Genome Sequences of 17 Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human, Animal, and Food Sources

PubMed Central

Labbé, Geneviève; Ziebell, Kim; Bekal, Sadjia; Parmley, E. Jane; Agunos, Agnes; Desruisseau, Andrea; Daignault, Danielle; Slavic, Durda; Hoang, Linda; Ramsay, Danielle; Pollari, Frank; Robertson, James; Nash, John H. E.

2016-01-01

Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S. Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain several drug resistance genes. PMID:27635008

Prevalence and characterization of multi-drug resistant Salmonella Enterica serovar Gallinarum biovar Pullorum and Gallinarum from chicken

PubMed Central

Parvej, Md. Shafiullah; Nazir, K. H. M. Nazmul Hussain; Rahman, M. Bahanur; Jahan, Mueena; Khan, Mohammad Ferdousur Rahman; Rahman, Marzia

2016-01-01

Aim: Salmonella is an important zoonotic pathogen responsible for animal and human diseases. The aim of the present study was to determine the prevalence and stereotyping of Salmonella isolates isolated from apparently healthy poultry. Furthermore, the clonal relatedness among the isolated Salmonella serovars was assessed. Materials and Methods: A total of 150 cloacal swab samples from apparently healthy chickens were collected, and were subjected for the isolation and identification of associated Salmonella organisms. The isolated colonies were identified and characterized on the basis of morphology, cultural characters, biochemical tests, slide agglutination test, polymerase chain reaction, and pulsed-field gel electrophoresis (PFGE). Antibiotic sensitivity patterns were also investigated using commonly used antibiotics. Results: Of the 150 samples, 11 (7.33%) produced characteristics pink colony with black center on XLD agar medium, and all were culturally and biochemically confirmed to be Salmonella. All possessed serovar-specific gene SpeF and reacted uniformly with group D antisera, suggesting that all of the isolates were Salmonella Enterica serovar Gallinarum, biovar Pullorum and/or Gallinarum. Antimicrobial susceptibility testing revealed that 54.54% of the isolated Salmonella Enterica serovars were highly sensitive to ciprofloxacin, whereas the 81.81% isolates were resistant to amoxycillin, doxycycline, kanamycin, gentamycin, and tetracycline. Pulsed-field gel electrophoresis of the XbaI-digested genomic DNA exhibited identical banding patterns, suggesting that the multidrug resistant Salmonella Enterica serovars occurring in commercial layers are highly clonal in Bangladesh. Conclusion: The present study was conducted to find out the prevalence of poultry Salmonella in layer chicken and to find out the clonal relationship among them. The data in this study suggest the prevalence of Salmonella Enterica, which is multidrug resistant and highly clonal for

DIVERSITY OF BAT-ASSOCIATED LEPTOSPIRA IN THE PERUVIAN AMAZON INFERRED BY BAYESIAN PHYLOGENETIC ANALYSIS OF 16S RIBOSOMAL DNA SEQUENCES

PubMed Central

MATTHIAS, MICHAEL A.; DÍAZ, M. MÓNICA; CAMPOS, KALINA J.; CALDERON, MARITZA; WILLIG, MICHAEL R.; PACHECO, VICTOR; GOTUZZO, EDUARDO; GILMAN, ROBERT H.; VINETZ, JOSEPH M.

2008-01-01

The role of bats as potential sources of transmission to humans or as maintenance hosts of leptospires is poorly understood. We quantified the prevalence of leptospiral colonization in bats in the Peruvian Amazon in the vicinity of Iquitos, an area of high biologic diversity. Of 589 analyzed bats, culture (3 of 589) and molecular evidence (20 of 589) of leptospiral colonization was found in the kidneys, yielding an overall colonization rate of 3.4%. Infection rates differed with habitat and location, and among different bat species. Bayesian analysis was used to infer phylogenic relationships of leptospiral 16S ribosomal DNA sequences. Tree topologies were consistent with groupings based on DNA-DNA hybridization studies. A diverse group of leptospires was found in peri-Iquitos bat populations including Leptospira interrogans (5 clones), L. kirschneri (1), L. borgpetersenii (4), L. fainei (1), and two previously undescribed leptospiral species (8). Although L. kirschenri and L. interrogans have been previously isolated from bats, this report is the first to describe L. borgpetersenii and L. fainei infection of bats. A wild animal reservoir of L. fainei has not been previously described. The detection in bats of the L. interrogans serovar Icterohemorrhagiae, a leptospire typically maintained by peridomestic rats, suggests a rodent-bat infection cycle. Bats in Iquitos maintain a genetically diverse group of leptospires. These results provide a solid basis for pursuing molecular epidemiologic studies of bat-associated Leptospira, a potentially new epidemiologic reservoir of transmission of leptospirosis to humans. PMID:16282313

Cyclical changes in seroprevalence of leptospirosis in California sea lions: endemic and epidemic disease in one host species?

PubMed Central

Lloyd-Smith, James O; Greig, Denise J; Hietala, Sharon; Ghneim, George S; Palmer, Lauren; St Leger, Judy; Grenfell, Bryan T; Gulland, Frances MD

2007-01-01

Background Leptospirosis is a zoonotic disease infecting a broad range of mammalian hosts, and is re-emerging globally. California sea lions (Zalophus californianus) have experienced recurrent outbreaks of leptospirosis since 1970, but it is unknown whether the pathogen persists in the sea lion population or is introduced repeatedly from external reservoirs. Methods We analyzed serum samples collected over an 11-year period from 1344 California sea lions that stranded alive on the California coast, using the microscopic agglutination test (MAT) for antibodies to Leptospira interrogans serovar Pomona. We evaluated seroprevalence among yearlings as a measure of incidence in the population, and characterized antibody persistence times based on temporal changes in the distribution of titer scores. We conducted multinomial logistic regression to determine individual risk factors for seropositivity with high and low titers. Results The serosurvey revealed cyclical patterns in seroprevalence to L. interrogans serovar Pomona, with 4–5 year periodicity and peak seroprevalence above 50%. Seroprevalence in yearling sea lions was an accurate index of exposure among all age classses, and indicated on-going exposure to leptospires in non-outbreak years. Analysis of titer decay rates showed that some individuals probably maintain high titers for more than a year following exposure. Conclusion This study presents results of an unprecedented long-term serosurveillance program in marine mammals. Our results suggest that leptospirosis is endemic in California sea lions, but also causes periodic epidemics of acute disease. The findings call into question the classical dichotomy between maintenance hosts of leptospirosis, which experience chronic but largely asymptomatic infections, and accidental hosts, which suffer acute illness or death as a result of disease spillover from reservoir species. PMID:17986335

Complete Genome Sequences of 17 Canadian Isolates of Salmonella enterica subsp. enterica Serovar Heidelberg from Human, Animal, and Food Sources.

PubMed

Labbé, Geneviève; Ziebell, Kim; Bekal, Sadjia; Macdonald, Kimberley A; Parmley, E Jane; Agunos, Agnes; Desruisseau, Andrea; Daignault, Danielle; Slavic, Durda; Hoang, Linda; Ramsay, Danielle; Pollari, Frank; Robertson, James; Nash, John H E; Johnson, Roger P

2016-09-15

Salmonella enterica subsp. enterica serovar Heidelberg is a highly clonal serovar frequently associated with foodborne illness. To facilitate subtyping efforts, we report fully assembled genome sequences of 17 Canadian S Heidelberg isolates including six pairs of epidemiologically related strains. The plasmid sequences of eight isolates contain several drug resistance genes. © Crown copyright 2016.

ramR mutations affecting fluoroquinolone susceptibility in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198

PubMed Central

Baucheron, Sylvie; Le Hello, Simon; Doublet, Benoît; Giraud, Etienne; Weill, François-Xavier; Cloeckaert, Axel

2013-01-01

A screening for non-target mutations affecting fluoroquinolone susceptibility was conducted in epidemic multidrug-resistant Salmonella enterica serovar Kentucky ST198. Among a panel of representative isolates (n = 27), covering the epidemic, only three showed distinct mutations in ramR resulting in enhanced expression of genes encoding the AcrAB-TolC efflux system and low increase in ciprofloxacin MIC. No mutations were detected in other regulatory regions of this efflux system. Ciprofloxacin resistance in serovar Kentucky ST198 is thus currently mainly due to multiple target gene mutations. PMID:23914184

Sero-prevalence of specific Leptospira serovars in fattening pigs from 5 provinces in Vietnam.

PubMed

Lee, Hu Suk; Khong, Nguyen Viet; Xuan, Huyen Nguyen; Nghia, Vuong Bui; Nguyen-Viet, Hung; Grace, Delia

2017-05-08

Leptospirosis is a zoonotic bacterial disease with a worldwide distribution. In Vietnam, leptospirosis is considered endemic. In pigs, leptospirosis can result in reproductive problems (such as abortion and infertility) which lead to economic loss. In addition, transmission to people presents a public health risk. In Vietnam, few national studies have been conducted on sero-prevalence of leptospirosis in pigs. The main objective of this study was to evaluate the sero-prevalence and incidence of presumptive infective leptospira serovars in fattening pigs from 5 provinces in Vietnam. Blood samples from fattening pigs were randomly collected at slaughterhouses. We collected 1959 sera samples from 5 provinces (Son La, Hanoi, Nghe An, Dak Lak and An Giang) between January and early June 2016. The microscopic agglutination test (MAT) was used to identify the serogroups/serovars. Overall, the sero-prevalence was 8.17% (95% CI: 6.99-9.47) and serovar Tarassovi Mitis (2.19%) had the highest prevalence followed by Australis (1.94%), Javanica (1.68%) and Autumnalis (1.17%) using a cutoff (≥ 1:100). The sero-prevalence among female pigs (5.28%, 95% CI: 3.94-6.93) was slightly higher than among male pigs (4.88%, 95% CI: 3.51-6.58), but this difference was not statistically significant. Leptospirosis in pigs may be a useful indicator of the human/animal burden in Vietnam and a risk assessment tool. The presence of some of the identified serovars suggests that wildlife may play an important role in the transmission of leptospirosis to domesticated pigs in Vietnam. Therefore, strengthened monitoring and surveillance systems are needed to better understand the epidemiology of the disease and prevent or reduce infection in humans and animals.

Serovars, bacteriophage types and antimicrobial sensitivities associated with salmonellosis in dogs in the UK (1954-2012).

PubMed

Philbey, A W; Mather, H A; Gibbons, J F; Thompson, H; Taylor, D J; Coia, J E

2014-01-25

Serovars and bacteriophage (phage) types were determined for 442 isolates of Salmonella enterica from dogs in the UK submitted to the Scottish Salmonella Reference Laboratory from 1954 to 2012. The most frequent serovars were Salmonella Typhimurium (196 isolates; 44.3 per cent), Dublin (40 isolates; 9.0 per cent), Enteritidis (28 isolates; 6.3 per cent), Montevideo (19 isolates; 4.3 per cent), Virchow (10 isolates; 2.3 per cent), Heidelberg (8 isolates; 1.8 per cent) and Derby (8 isolates; 1.8 per cent), along with 55 other recognised serovars among 127 other isolates, and six incompletely classified isolates. Serovars were frequently represented by strains commonly associated with poultry, cattle or pigs and their products. Among 196 Salmonella Typhimurium isolates from dogs, the most frequent phage types (definitive types) were the multiple antimicrobial-resistant strains DT104 (62 isolates), DT204c (18 isolates) and DT193 (8 isolates), along with antimicrobial sensitive wild finch strains DT40 (13 isolates) and DT56 variant (8 isolates). Eleven of 28 isolates of Salmonella Enteritidis were phage type 4. S enterica was frequently recovered from faecal or intestinal samples of dogs with diarrhoea, although many dogs had concurrent infection with other enteric pathogens. Salmonella Dublin was recovered from the brain and/or cerebrospinal fluid of two dogs with meningoencephalitis. Salmonella Kedougou was isolated from the joint fluid of a dog with septic arthritis. Salmonella Typhimurium and Salmonella Dublin were each recovered from the vaginas of bitches that had aborted. Isolates of Salmonella Enteritidis phage types 1, 4 and 8, Salmonella Typhimurium DT104, Salmonella Dublin and Salmonella Indiana were isolated from clinically healthy dogs in households where the same strains were recovered from human beings with diarrhoea. The pattern ampicillin-chloramphenicol-spectinomycin-streptomycin-sulfamethoxazole-tetracycline (ACSpSSuT) was the most frequent resistance

Prevalence and characterization of Salmonella serovars isolated from oysters served raw in restaurants.

PubMed

Brillhart, Crystal D; Joens, Lynn A

2011-06-01

To determine if Salmonella-contaminated oysters are reaching consumer tables, a survey of raw oysters served in eight Tucson restaurants was performed from October 2007 to September 2008. Salmonella spp. were isolated during 7 of the 8 months surveyed and were present in 1.2% of 2,281 oysters tested. This observed prevalence is lower than that seen in a previous study in which U.S. market oysters were purchased from producers at bays where oysters are harvested. To test whether the process of refrigerating oysters in restaurants for several days reduces Salmonella levels, oysters were artificially infected with Salmonella and kept at 4°C for up to 13 days. Direct plate counts of oyster homogenate showed that Salmonella levels within oysters did not decrease during refrigeration. Six different serovars of Salmonella enterica were found in the restaurant oysters, indicating multiple incidences of Salmonella contamination of U.S. oyster stocks. Of the 28 contaminated oysters, 12 (43%) contained a strain of S. enterica serovar Newport that matched by pulsed-field gel electrophoresis a serovar Newport strain seen predominantly in the study of bay oysters performed in 2002. The repeated occurrence of this strain in oyster surveys is concerning, since the strain was resistant to seven antimicrobials tested and thus presents a possible health risk to consumers of raw oysters.

Antimicrobial Drug Resistance of Salmonella enterica Serovar Typhi in Asia and Molecular Mechanism of Reduced Susceptibility to the Fluoroquinolones▿

PubMed Central

Chau, Tran Thuy; Campbell, James Ian; Galindo, Claudia M.; Van Minh Hoang, Nguyen; Diep, To Song; Nga, Tran Thu Thi; Van Vinh Chau, Nguyen; Tuan, Phung Quoc; Page, Anne Laure; Ochiai, R. Leon; Schultsz, Constance; Wain, John; Bhutta, Zulfiqar A.; Parry, Christopher M.; Bhattacharya, Sujit K.; Dutta, Shanta; Agtini, Magdarina; Dong, Baiqing; Honghui, Yang; Anh, Dang Duc; Canh, Do Gia; Naheed, Aliya; Albert, M. John; Phetsouvanh, Rattanaphone; Newton, Paul N.; Basnyat, Buddha; Arjyal, Amit; La, Tran Thi Phi; Rang, Nguyen Ngoc; Phuong, Le Thi; Van Be Bay, Phan; von Seidlein, Lorenz; Dougan, Gordon; Clemens, John D.; Vinh, Ha; Hien, Tran Tinh; Chinh, Nguyen Tran; Acosta, Camilo J.; Farrar, Jeremy; Dolecek, Christiane

2007-01-01

This study describes the pattern and extent of drug resistance in 1,774 strains of Salmonella enterica serovar Typhi isolated across Asia between 1993 and 2005 and characterizes the molecular mechanisms underlying the reduced susceptibilities to fluoroquinolones of these strains. For 1,393 serovar Typhi strains collected in southern Vietnam, the proportion of multidrug resistance has remained high since 1993 (50% in 2004) and there was a dramatic increase in nalidixic acid resistance between 1993 (4%) and 2005 (97%). In a cross-sectional sample of 381 serovar Typhi strains from 8 Asian countries, Bangladesh, China, India, Indonesia, Laos, Nepal, Pakistan, and central Vietnam, collected in 2002 to 2004, various rates of multidrug resistance (16 to 37%) and nalidixic acid resistance (5 to 51%) were found. The eight Asian countries involved in this study are home to approximately 80% of the world's typhoid fever cases. These results document the scale of drug resistance across Asia. The Ser83→Phe substitution in GyrA was the predominant alteration in serovar Typhi strains from Vietnam (117/127 isolates; 92.1%). No mutations in gyrB, parC, or parE were detected in 55 of these strains. In vitro time-kill experiments showed a reduction in the efficacy of ofloxacin against strains harboring a single-amino-acid substitution at codon 83 or 87 of GyrA; this effect was more marked against a strain with a double substitution. The 8-methoxy fluoroquinolone gatifloxacin showed rapid killing of serovar Typhi harboring both the single- and double-amino-acid substitutions. PMID:17908946

The Inositol Phosphatase SHIP Controls Salmonella enterica Serovar Typhimurium Infection In Vivo▿

PubMed Central

Bishop, Jennifer L.; Sly, Laura M.; Krystal, Gerald; Finlay, B. Brett

2008-01-01

The SH2 domain-containing inositol 5′-phosphatase, SHIP, negatively regulates various hematopoietic cell functions and is critical for maintaining immune homeostasis. However, whether SHIP plays a role in controlling bacterial infections in vivo remains unknown. Salmonella enterica causes human salmonellosis, a disease that ranges in severity from mild gastroenteritis to severe systemic illness, resulting in significant morbidity and mortality worldwide. The susceptibility of ship+/+and ship−/− mice and bone marrow-derived macrophages to S. enterica serovar Typhimurium infection was compared. ship−/− mice displayed an increased susceptibility to both oral and intraperitoneal serovar Typhimurium infection and had significantly higher bacterial loads in intestinal and systemic sites than ship+/+mice, indicating a role for SHIP in the gut-associated and systemic pathogenesis of serovar Typhimurium in vivo. Cytokine analysis of serum from orally infected mice showed that ship−/− mice produce lower levels of Th1 cytokines than do ship+/+ animals at 2 days postinfection, and in vitro analysis of supernatants taken from infected bone marrow-derived macrophages derived to mimic the in vivo ship−/− alternatively activated (M2) macrophage phenotype correlated with these data. M2 macrophages were the predominant population in vivo in both oral and intraperitoneal infections, since tissue macrophages within the small intestine and peritoneal macrophages from ship−/− mice showed elevated levels of the M2 macrophage markers Ym1 and Arginase 1 compared to ship+/+ cells. Based on these data, we propose that M2 macrophage skewing in ship−/− mice contributes to ineffective clearance of Salmonella in vivo. PMID:18426884

[Reconstruction of Leptospira interrogans lipL21 gene and characteristics of its expression product].

PubMed

Luo, Dong-jiao; Hu, Ye; Dennin, R H; Yan, Jie

2007-09-01

To reconstruct the nucleotide sequence of Leptospira interrogans lipL21 gene for increasing the output of prokaryotic expression and to understand the changes on immunogenicity of the expression products before and after reconstruction, and to determine the position of envelope lipoprotein LipL21 on the surface of leptospiral body. According to the preferred codons of E.coli, the nucleotide sequence of lipL21 gene was designed and synthesized, and then its prokaryotic expression system was constructed. By using SDS-PAGE plus BioRad agarose image analysor, the expression level changes of lipL21 genes before and after reconstruction were measured. A Western blot assay using rabbit anti-TR/Patoc I serum as the first antibody was performed to identify the immunoreactivity of the two target recombinant proteins rLipL21s before and after reconstruction. The changes of cross agglutination titers of antisera against two rLipL21s before and after reconstruction to the different leptospiral serogroups were demonstrated using microscope agglutination test (MAT). Immuno-electronmicroscopy was applied to confirm the location of LipL21s. The expression outputs of original and reconstructed lipL21 genes were 8.5 % and 46.5 % of the total bacterial proteins, respectively. Both the two rLipL21s could take place immune conjugation reaction with TR/Patoc I antiserum. After immunization with each of the two rLipL21s in rabbits, the animals could produce specific antibody. Similar MAT titers with 1:80 - 1:320 of the two antisera against rLipL21s were present. LipL21 was confirmed to locate on the surface of leptospiral envelope. LipL21 is a superficial antigen of Leptospira interrogans. The expression output of the reconstructed lipL21 gene is remarkably increased. The expression rLipL21 maintains fine antigenicity and immunoreactivity and its antibody still shows an extensive cross immunoagglutination activity. The high expression of the reconstructed lipL21 gene will offer a

Chlamydia trachomatis serovar distribution and other sexually transmitted coinfections in subjects attending an STD outpatients clinic in Italy.

PubMed

Marangoni, Antonella; Foschi, Claudio; Nardini, Paola; D'Antuono, Antonietta; Banzola, Nicoletta; Di Francesco, Antonietta; Ostanello, Fabio; Russo, Incoronata; Donati, Manuela; Cevenini, Roberto

2012-04-01

We studied the prevalence of Chlamydia trachomatis (CT) urogenital infection and the distribution of different genotypes in a non-selected STD population of 1625 patients, evaluating presence of coinfections with other sexually transmitted diseases. Each patient was bled to perform serological tests for syphilis and HIV, then urethral or endocervical swabs were obtained for the detection of CT and Neisseria gonorrhoeae by culture. DNA extracted from remnant positive swabs was amplified by omp1 Nested PCR and products were sequenced. Total prevalence of CT infection was 6.3% (103/1625), with strong differences between men and women (11.4% vs 3.9%, P<0.01). Clinical symptoms and coinfections were much more frequent in men than in women (P<0.01). The most common serovar was E (prevalence of 38.8%), followed by G (23.3%), F (13.5%) D/Da (11.6%) and J (4.8%). Serovars distribution was statistically different between men and women (P=0.042) and among patients with or without coinfection (P=0.035); patients infected by serovar D/Da showed the highest coinfection rate. This study can be considered a contribution in increasing knowledge on CT serovar distribution in Italy. Further studies are needed to better define molecular epidemiology of CT infection and to investigate its correlation with other STDs.

Prevalence and characterization of multidrug-resistant (type ACSSuT) Salmonella enterica serovar Typhimurium strains in isolates from four gosling farms and a hatchery farm.

PubMed

Yu, Chang-You; Chou, Shih-Jen; Yeh, Chia-Ming; Chao, Maw-Rong; Huang, Kwo-Ching; Chang, Yung-Fu; Chiou, Chien-Shun; Weill, Francois-Xavier; Chiu, Cheng-Hsun; Chu, Chi-Hong; Chu, Chishih

2008-02-01

Salmonella enterica serovar Typhimurium strains of phage types DT104 and U302 are often resistant to ampicillin, chloramphenicol, streptomycin, sulfonamides, and tetracycline (the ACSSuT resistance type) and are major zoonotic pathogens. Increased consumption of goose meat may enhance the risk of transferring S. enterica serovar Typhimurium and other enteric pathogens from geese to human due to the consumption of meats from infected geese or improper preparation of meats. Therefore, we characterized S. enterica serovar Typhimurium strains isolated from four goose farms (farms A, B, C, and D) and one hatchery farm (farm E) to determine the epidemic and genetic differences among them. Antibiotic susceptibility tests and multiplex PCR confirmed that 77.6% (52/67) of strains were ACSSuT strains isolated from farms A, C, and E. Antibiotic-susceptible strains were isolated mostly from farm B, and no strain was observed in farm D. All ACSSuT strains harbored a 94.7-kb virulence plasmid and contained one 1.1-kb conserved segment identical to that of Salmonella genomic island 1. Four genotypes were determined among these S. enterica serovar Typhimurium isolates by pulsed-field gel electrophoresis analysis of XbaI-digested DNA fragments. Most isolates (85.29%; 29/34) of major genotype Ib were ACSSuT strains isolated mainly from goslings of farm C and egg membranes of farm E, a hatchery farm, suggesting that S. enterica serovar Typhimurium strains in isolates from goslings might originate from its hatchery, from the egg membranes to the gosling fluff after hatching. Multiple phage types, types 8, 12, U283, DT104, and U302, were identified. In conclusion, geese were a reservoir of diverse multidrug-resistant (type ACSSuT) S. enterica serovar Typhimurium strains, and each farm was colonized with genetically closely related S. enterica serovar Typhimurium strains.

Serovar distribution, antimicrobial resistance profiles, and PFGE typing of Salmonella enterica strains isolated from 2007–2012 in Guangdong, China

PubMed Central

2014-01-01

Background Salmonella enterica includes the major serovars associated with human salmonellosis. In this study, 1764 clinical Salmonella enterica isolates from diarrhea outpatients were collected from fifteen cities in Guangdong province, China, between 2007 and 2012. These isolates represent all of the Salmonella isolates collected from the province during that period. Methods The isolates were characterized by serovar determination, antimicrobial susceptibility tests and PFGE fingerprint typing. Results The serovar distribution results demonstrated that Salmonella Typhimurium (n = 523, 29.65%) and Salmonella 4,5,12:i:- (n = 244, 13.83%) are the most common serovars causing infant salmonellosis, whereas Salmonella Enteritidis (n = 257, 14.57%) mainly causes human salmonellosis in adults. The serovar shift from Salmonella Enteritidis to Salmonella Typhimurium occurred in 2008. Antimicrobial susceptibility data showed a high burden of multidrug resistance (MDR) (n = 1128, 56.58%), and a 20%-30% increase in the number of isolates resistant to ciprofloxacin (n = 142, 8.05%) and third-generation cephalosporins (n = 88, 4.99%) from 2007–2012. Only 9.97% of isolates (n = 176) were fully susceptible to all agents tested. A high burden of MDR was observed in Salmonella Typhimurium and Salmonella 4,5,12:i:- for all age groups, and a reduced susceptibility to third-generation cephalosporins and quinolones occurred particularly in infants (≤6 years). The dominant PFGE patterns were JPXX01.GD0004, JEGX01.GD0006-7 and JNGX01.GD0006-7. ACSSuT was the predominant MDR profile in the Salmonella Typhimurium & 4,5,12:i:- complexes, while ASSuT-Nal and ASSu-Nal were the major MDR profiles in Salmonella Enteritidis. The predominant PFGE patterns of the Salmonella Typhimurium & 4,5,12:i:- complexes and Salmonella Stanley were most prevalent in infants (≤6 years). However, no obvious relationship was observed between these PFGE profiles and geographic

[Prokaryotic expression of trigeminy artificial fusion gene of Leptospira interrogans and the immunogenicity of its products].

PubMed

Luo, Dong-jiao; Qiu, Xiao-feng; Wang, Jiang; Yan, Jin; Wang, Hai-bin; Zhou, Jin-cheng; Yan, Jie

2008-11-01

To construct lipL32/1-lipL21-OmpL1/2 fusion gene of Leptospira interrogans and its prokaryotic expression system, and to identify the immunogenicity of its products. PCR using linking primers was applied to construct lipL32/1-lipL21-OmpL1/2 fusion gene and a prokaryotic expression system of the fusion gene was then established using routine genetic engineering technique. SDS-PAGE was used to examine output of the target recombinant protein rLipL32/1-LipL21-OmpL1/2. Double immunodiffusion and Western Blot assay were applied to identify immunogenicity of rLipL32/1-LipL21-OmpL1/2. lipL32/1-lipL21-OmpL1/2 fusion gene with correct sequence and its prokaryotic expression system E.coli BL21DE3pET42a-lipL32/1-lipL21-ompL1/2 was obtained in this study. The output of rLipL32/1-LipL21- OmpL1/2 after optimisation was 37.78 mg/L. The immunodiffusion titer of rabbit antiserum against rLipL32/1-LipL21-OmpL1/2 was 1:4. The rLipL32/1-LipL21-OmpL1/2 antiserum was able to recognize rLipL32/1-LipL21-OmpL1/2, rLipL32/1, rLipL21 and rOmpL1/2. Positive Western hybridization signals were found among rLipL32/1-LipL21-OmpL1/2 and rabbit antiserum against whole cell of strain 56601 and serum from patients infected with L.interrogans serogroups Icterohaemorrhagiae, Grippotyphosa, Autumnalis and Pomona. The fusion gene lipL32/1-lipL21-OmpL1/2 and its prokaryotic expression system were successfully constructed in this study. The expressed fusion protein can be used as the antigen for developing universal genetic engineering vaccine and universal serological tests of leptospirosis.

A multiplex real-time PCR assay for the identification and differentiation of Salmonella enterica serovar Typhimurium and monophasic serovar 4,[5],12:i:-.

PubMed

Prendergast, Deirdre M; Hand, Darren; Nί Ghallchóir, Eadaoin; McCabe, Evonne; Fanning, Seamus; Griffin, Margaret; Egan, John; Gutierrez, Montserrat

2013-08-16

Salmonella enterica subsp. enterica serovar 4,[5],12:i:- is considered to be a monophasic variant of Salmonella Typhimurium and is increasingly associated with human infections. The use of PCR for the unequivocal identification of strains identified by conventional serotyping as 4,[5],12:i:- has been recommended by the European Food Safety Authority (EFSA), in particular the conventional multiplex PCR developed by Tennant et al. (2010). An alternative protocol for the identification and differentiation of S. Typhimurium and S. Typhimurium-like strains, including its monophasic variants, based on a multiplex real-time PCR assay was developed in our laboratory. A panel of 206 Salmonella strains was used to validate our multiplex real-time PCR against the conventional multiplex PCR recommended by EFSA, i.e. 43 Salmonella strains of serovars other than Typhimurium and 163 routine isolates determined by slide agglutination serotyping to have an incomplete antigenic formula compatible with the S. Typhimurium formula 4,[5],12:i:1,2. Both methods correctly identified the 43 Salmonella strains as non S. Typhimurium. Among the 163 isolates of undetermined serovar by conventional serotyping, both PCR protocols identified 54 isolates as S. Typhimurium, 101 as monophasic S. Typhimurium and 8 as non-S. Typhimurium. Twenty isolates phenotypically lacking the phase-2 H antigen were positive for the fljB.1,2 gene. These strains have been recently described in the literature by other workers and have been referred to as "inconsistent" variants of S. Typhimurium. Antimicrobial resistance and phage typing were also performed on the S. Typhimurium isolates, including monophasic variants, and approximately half of the isolates identified as monophasic S. Typhimurium by our multiplex real-time PCR protocol were DT193 with the resistance pattern ASSuT. There was 100% concordance between the conventional PCR and the multiplex real-time PCR method developed in this study which proved that

Emergence and serovar profiling of non-typhoidal Salmonellae (NTS) isolated from gastroenteritis cases-A study from South India.

PubMed

Ballal, Mamatha; Devadas, Suganthi Martena; Shetty, Vignesh; Bangera, Sohan Rodney; Ramamurthy, Thandavarayan; Sarkar, Anirban

2016-01-01

Human infection with non-typhoidal Salmonella (NTS) serovars is often a neglected and undiagnosed infection in the developing world. Invasive NTS is now being established as having a new and emerging pathogenic role. There is not sufficient data on the prevalence of NTS serovars and their antibiotic susceptibility pattern from India. Faecal specimens collected from patients with acute gastroenteritis were processed to isolate Salmonella according to the standard protocol for a period from January 2011-December 2014. Salmonella isolates were serotyped and tested for antibiotic susceptibility. Of the total 320 (10.04%) bacterial enteric pathogens isolated, 64 (20%) were non-typhoidal Salmonella. Among the serogroup, O:4 (B) (n = 26; 40.6%) was found to be the commonest followed by O:7 (C1) (n = 11; 17.1%) and O:3,10 (E1) (n = 11; 17.1%). NTS infection in cancer patients could also be termed as nosocomial NTS diarrhoea due to primary community infection with prolonged incubation periods, consumption of contaminated food during hospital stay or Nosocomially acquired infection. Serovar Oslo has been predominant (9/17) in NTS isolates from cancer patients, whereas serovars Bovismorbificans, Wangata and Schleissheim have been reported for the first time in the country. The isolates were mostly susceptible to antibiotics except Salmonella ser Kentucky, which showed resistance to ciprofloxacin is reported for the first time in the country. Continuous surveillance is required to monitor resistance of NTS isolates.

Polynucleotide phosphorlyase (PNPase) is required for Salmonella enterica serovar Typhimurium colonization in swine

USDA-ARS?s Scientific Manuscript database

The pnp gene encodes polynucleotide phosphorylase, an exoribonuclease involved in RNA degradation. A mutation in the pnp gene was previously identified by our group in a signature-tagged mutagenesis screen designed to search for Salmonella enterica serovar Typhimurium genes required for survival in...

The Early Innate Response of Chickens to Salmonella enterica Is Dependent on the Presence of O-Antigen but Not on Serovar Classification

PubMed Central

Varmuzova, Karolina; Matulova, Marta Elsheimer; Sebkova, Alena; Sekelova, Zuzana; Havlickova, Hana; Sisak, Frantisek; Babak, Vladimir; Rychlik, Ivan

2014-01-01

Salmonella vaccines used in poultry in the EU are based on attenuated strains of either Salmonella serovar Enteritidis or Typhimurium which results in a decrease in S. Enteritidis and S. Typhimurium but may allow other Salmonella serovars to fill an empty ecological niche. In this study we were therefore interested in the early interactions of chicken immune system with S. Infantis compared to S. Enteritidis and S. Typhimurium, and a role of O-antigen in these interactions. To reach this aim, we orally infected newly hatched chickens with 7 wild type strains of Salmonella serovars Enteritidis, Typhimurium and Infantis as well as with their rfaL mutants and characterized the early Salmonella-chicken interactions. Inflammation was characterized in the cecum 4 days post-infection by measuring expression of 43 different genes. All wild type strains stimulated a greater inflammatory response than any of the rfaL mutants. However, there were large differences in chicken responses to different wild type strains not reflecting their serovar classification. The initial interaction between newly-hatched chickens and Salmonella was found to be dependent on the presence of O-antigen but not on its structure, i.e. not on serovar classification. In addition, we observed that the expression of calbindin or aquaporin 8 in the cecum did not change if inflammatory gene expression remained within a 10 fold fluctuation, indicating the buffering capacity of the cecum, preserving normal gut functions even in the presence of minor inflammatory stimuli. PMID:24763249

FlaA proteins in Leptospira interrogans are essential for motility and virulence but are not required for formation of the flagellum sheath.

PubMed

Lambert, Ambroise; Picardeau, Mathieu; Haake, David A; Sermswan, Rasana W; Srikram, Amporn; Adler, Ben; Murray, Gerald A

2012-06-01

Spirochetes have periplasmic flagella composed of a core surrounded by a sheath. The pathogen Leptospira interrogans has four flaB (proposed core subunit) and two flaA (proposed sheath subunit) genes. The flaA genes are organized in a locus with flaA2 immediately upstream of flaA1. In this study, flaA1 and flaA2 mutants were constructed by transposon mutagenesis. Both mutants still produced periplasmic flagella. The flaA1 mutant did not produce FlaA1 but continued to produce FlaA2 and retained normal morphology and virulence in a hamster model of infection but had reduced motility. The flaA2 mutant did not produce either the FlaA1 or the FlaA2 protein. Cells of the flaA2 mutant lacked the distinctive hook-shaped ends associated with L. interrogans and lacked translational motility in liquid and semisolid media. These observations were confirmed with a second, independent flaA2 mutant. The flaA2 mutant failed to cause disease in animal models of acute infection. Despite lacking FlaA proteins, the flagella of the flaA2 mutant were of the same thickness as wild-type flagella, as measured by electron microscopy, and exhibited a normal flagellum sheath, indicating that FlaA proteins are not essential for the synthesis of the flagellum sheath, as observed for other spirochetes. This study shows that FlaA subunits contribute to leptospiral translational motility, cellular shape, and virulence.

A 22-mer primer enhances discriminatory power of AP-PCR fingerprinting technique in characterization of leptospires.

PubMed

Roy, Subarna; Biswas, Debabrata; Vijayachari, P; Sugunan, A P; Sehgal, Subhash C

2004-11-01

To evaluate the discriminatory power and usefulness of arbitrarily primed-polymerase chain reaction (AP-PCR) characterization of leptospires with M16 primer. AP-PCR fingerprints of 20 reference strains of Leptospira representing 20 different serovars belonging to seven genospecies (Leptospira interrogans, 11; L. noguchii, 2; L. borgpetersenii, 1; L. santarosai, 2; L. biflexa, 2; L. kirschneri, 1; L. weilii, 1) were generated by employing M16 primer. Fingerprints generated with this primer were compared with those generated with two other commonly used primers PB1, and L10. An attempt was also made to type 20 leptospiral isolates with the M16 primer. Fingerprints with M16 primer could not only differentiate between strains of different genospecies, but also between strains of the same genospecies belonging to different serovars. While two commonly used primers (PB1 and L10) failed to discriminate between some of the different serovars belonging to the same genospecies, this primer was able to generate discriminatory fingerprints for all strains tested. All 20 Leptospira isolates, recovered from patients in Andaman Islands, could also be typed by fingerprints generated with the M16 primer. The discriminatory power of M16 primer adds more specificity to the rapidity of this system of characterization and can be used as an excellent tool in epidemiological studies on Leptospira.

Molecular identification of common Salmonella serovars using multiplex DNA sensor-based suspension array.

PubMed

Aydin, Muhsin; Carter-Conger, Jacqueline; Gao, Ning; Gilmore, David F; Ricke, Steven C; Ahn, Soohyoun

2018-04-01

Salmonella is one of major foodborne pathogens and the leading cause of foodborne illness-related hospitalizations and deaths. It is critical to develop a sensitive and rapid detection assay that can identify Salmonella to ensure food safety. In this study, a DNA sensor-based suspension array system of high multiplexing ability was developed to identify eight Salmonella serovars commonly associated with foodborne outbreaks to the serotype level. Each DNA sensor was prepared by activating pre-encoded microspheres with oligonucleotide probes that are targeting virulence genes and serovar-specific regions. The mixture of 12 different types of DNA sensors were loaded into a 96-well microplate and used as a 12-plex DNA sensor array platform. DNA isolated from Salmonella was amplified by multiplex polymerase chain reaction (mPCR), and the presence of Salmonella was determined by reading fluorescent signals from hybridization between probes on DNA sensors and fluorescently labeled target DNA using the Bio-Plex® system. The developed multiplex array was able to detect synthetic DNA at the concentration as low as 100 fM and various Salmonella serovars as low as 100 CFU/mL within 1 h post-PCR. Sensitivity of this assay was further improved to 1 CFU/mL with 6-h enrichment. The array system also correctly and specifically identified serotype of tested Salmonella strains without any cross-reactivity with other common foodborne pathogens. Our results indicate the developed DNA sensor suspension array can be a rapid and reliable high-throughput method for simultaneous detection and molecular identification of common Salmonella serotypes.

Inactivation of Salmonella serovars by Pseudomonas chlororaphis and Pseudomonas fluorescens strains on tomatoes

USDA-ARS?s Scientific Manuscript database

Salmonella enterica and its serovars have been associated with pathogen contamination of tomatoes and numerous outbreaks of Salmonellisis. To improve food safety, pathogen control is of immediate concern. The aim of this reserach was to: 1) Assess the populations of natural microflora (aerobic meso...

Vi Capsular Polysaccharide Produced by Recombinant Salmonella enterica Serovar Paratyphi A Confers Immunoprotection against Infection by Salmonella enterica Serovar Typhi

PubMed Central

Xiong, Kun; Zhu, Chunyue; Chen, Zhijin; Zheng, Chunping; Tan, Yong; Rao, Xiancai; Cong, Yanguang

2017-01-01

Enteric fever is predominantly caused by Salmonella enterica serovar Typhi and Salmonella enterica serovar Paratyphi A, and accounts for an annual global incidence of 26.9 millions. In recent years, the rate of S. Paratyphi A infection has progressively increased. Currently licensed vaccines for typhoid fever, live Ty21a vaccine, Vi subunit vaccine, and Vi-conjugate vaccine, confer inadequate cross immunoprotection against enteric fever caused by S. Paratyphi A. Therefore, development of bivalent vaccines against enteric fever is urgently required. The immunogenic Vi capsular polysaccharide is characteristically produced in S. Typhi, but it is absent in S. Paratyphi A. We propose that engineering synthesis of Vi in S. Paratyphi A live-attenuated vaccine may expand its protection range to cover S. Typhi. In this study, we cloned the viaB locus, which contains 10 genes responsible for Vi biosynthesis, and integrated into the chromosome of S. Paratyphi A CMCC 50093. Two virulence loci, htrA and phoPQ, were subsequently deleted to achieve a Vi-producing attenuated vaccine candidate. Our data showed that, despite more than 200 passages, the viaB locus was stably maintained in the chromosome of S. Paratyphi A and produced the Vi polysaccharide. Nasal immunization of the vaccine candidate stimulated high levels of Vi-specific and S. Paratyphi A-specific antibodies in mice sera as well as total sIgA in intestinal contents, and showed significant protection against wild-type challenge of S. Paratyphi A or S. Typhi. Our study show that the Vi-producing attenuated S. Paratyphi A is a promising bivalent vaccine candidate for the prevention of enteric fever. PMID:28484685

Persistence and clearance of different Salmonella serovars in buildings housing laying hens.

PubMed

Carrique-Mas, J J; Breslin, M; Snow, L; McLaren, I; Sayers, A R; Davies, R H

2009-06-01

We investigated factors associated with persistence of different Salmonella serovars in buildings housing laying hens in Great Britain using survival analysis. A total of 264 incidents of Salmonella detection occurring between July 1998 and August 2007 in 152 houses were recorded. For incidents involving Salmonella Enteritidis (SE), both the rodent score of the house and the type of house were positively associated with persistence. For non-SE serovars, only the type of house was associated with persistence. Persistence of SE in the houses was longest (>15 months) in step-cage and cage-scraper houses when high levels of rodents were present, and lowest in non-cage and cage-belt houses. We estimated that 42% (95% CI 23.3-63.1) of SE incidents may be cleared during the lay period, and this was related to elimination of rodents from the houses. From January 2009, EU legislation will ban the sale of fresh eggs from SE-positive and S. Typhimurium-positive flocks over their remaining lifespan. If infection is eliminated from such flocks, they would cease to represent a public health risk.

Comparison of advanced whole genome sequence-based methods to distinguish strains of Salmonella enterica serovar Heidelberg involved in foodborne outbreaks in Québec.

PubMed

Vincent, Caroline; Usongo, Valentine; Berry, Chrystal; Tremblay, Denise M; Moineau, Sylvain; Yousfi, Khadidja; Doualla-Bell, Florence; Fournier, Eric; Nadon, Céline; Goodridge, Lawrence; Bekal, Sadjia

2018-08-01

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. This serovar ranks second and third among serovars that cause human infections in Québec and Canada, respectively, and has been associated with severe infections. Traditional typing methods such as PFGE do not display adequate discrimination required to resolve outbreak investigations due to the low level of genetic diversity of isolates belonging to this serovar. This study evaluates the ability of four whole genome sequence (WGS)-based typing methods to differentiate among 145 S. Heidelberg strains involved in four distinct outbreak events and sporadic cases of salmonellosis that occurred in Québec between 2007 and 2016. Isolates from all outbreaks were indistinguishable by PFGE. The core genome single nucleotide variant (SNV), core genome multilocus sequence typing (MLST) and whole genome MLST approaches were highly discriminatory and separated outbreak strains into four distinct phylogenetic clusters that were concordant with the epidemiological data. The clustered regularly interspaced short palindromic repeats (CRISPR) typing method was less discriminatory. However, CRISPR typing may be used as a secondary method to differentiate isolates of S. Heidelberg that are genetically similar but epidemiologically unrelated to outbreak events. WGS-based typing methods provide a highly discriminatory alternative to PFGE for the laboratory investigation of foodborne outbreaks. Copyright © 2018 Elsevier Ltd. All rights reserved.

Influence of Temperature and Predation on Survival of Salmonella enterica Serovar Typhimurium and Expression of invA in Soil and Manure-Amended Soil▿

PubMed Central

García, R.; Bælum, J.; Fredslund, L.; Santorum, P.; Jacobsen, C. S.

2010-01-01

The effects of three temperatures (5, 15, and 25°C) on the survival of Salmonella enterica serovar Typhimurium in topsoil were investigated in small microcosms by three different techniques: plate counting, invA gene quantification, and invA mRNA quantification. Differences in survival were related to the effect of protozoan predation. Tetracycline-resistant Salmonella serovar Typhimurium was inoculated into soil and manure-amended soil at 1.5 × 108 cells g soil−1. Population densities were determined by plate counting and by molecular methods and monitored for 42 days. Simultaneous extraction of RNA and DNA, followed by quantitative PCR, was used to investigate invA gene levels and expression. Analysis by these three techniques showed that Salmonella serovar Typhimurium survived better at 5°C. Comparing DNA and CFU levels, significantly higher values were determined by DNA-based techniques. invA mRNA levels showed a fast decrease in activity, with no detectable mRNA after an incubation period of less than 4 days in any of the soil scenarios. A negative correlation was found between Salmonella serovar Typhimurium CFU levels and protozoan most probable numbers, and we propose the role of the predator-prey interaction as a factor to explain the die-off of the introduced strain by both culture- and DNA quantification-based methods. The results indicate that temperature, manure, and protozoan predation are important factors influencing the survival of Salmonella serovar Typhimurium in soil. PMID:20562283

Diversity of Salmonella serovars in feedyard and nonfeedyard playas of the Southern High Plains in the summer and winter.

PubMed

Purdy, Charles W; Straus, David C; Clark, R Nolan

2004-01-01

To compare Salmonella isolates cultured from feedyard and nonfeedyard (control) playas (ie, temporary shallow lakes) of the Southern High Plains. Water and muck (sediment) samples were obtained from 7 feedyard playas and 3 nonfeedyard playas in the winter and summer. Each water and muck sample was enriched with sulfur-brilliant-green broth and incubated in a shaker at 37 degrees C for 24 hours. A sample (100 mL) of the incubated bacterial-enriched broth was then mixed with 100 mL of fresh sulfur-brilliant-green enrichment broth and incubated in a shaker at 37 degrees C for 24 hours. After the second incubation, a swab sample was streaked on differential media. Suspect Salmonella isolates were further identified by use of biochemical tests, and Salmonella isolates were confirmed and serovar determinations made. Salmonella isolates were not recovered from the 3 control playas. Seven Salmonella enterica serovars were isolated from 5 of 7 feedyard playas in the summer, and 13 S. enterica serovars were isolated from 7 of 7 feedyard playas in the winter. In the summer, 296 isolates were cultured, and 47 were Salmonella organisms. In the winter, 288 isolates were cultured, and 171 were Salmonella organisms. Results indicated that feedyard playas are frequently contaminated with many Salmonella serovars. These pathogens should be considered whenever feedyard managers contemplate the use of water from these playas. Water from feedyard playas should not be used to cool cattle in the summer or for dust abatement.

Variable-Number Tandem Repeats That Are Useful in Genotyping Isolates of Salmonella enterica subsp. enterica Serovars Typhimurium and Newport▿

PubMed Central

Witonski, D. ; Stefanova, R.; Ranganathan, A.; Schutze, G. E.; Eisenach, K. D.; Cave, M. D.

2006-01-01

The genome of Salmonella enterica subsp. enterica serovar Typhimurium strain LT2 was analyzed for direct repeats, and 54 sequences containing variable-number tandem repeat loci were identified. Ten primer pairs that anneal upstream and downstream of each selected locus were designed and used to amplify PCR targets in isolates of S. enterica serovars Typhimurium and Newport. Four of the 10 loci did not show polymorphism in the length of products. Six loci were selected for analysis. Isolates of S. enterica serovars Typhimurium and Newport that were related to specific outbreaks and showed identical pulsed-field gel electrophoresis patterns were indistinguishable by the length of the six variable-number tandem repeats. Isolates that differed in their pulsed-field gel electrophoresis patterns showed polymorphism in variable-number tandem repeat profiles. Length of the products was confirmed by DNA sequence analysis. Only 2 of the 10 loci contained exact integers of the direct repeat. Eight loci contained partial copies. The partial copies were maintained at the ends of the variable-number tandem repeat loci in all isolates. In spite of having partial copies that were maintained in all isolates, the number of direct repeats at a locus was polymorphic. Six variable-number tandem repeat loci were useful in distinguishing isolates of S. enterica serovars Typhimurium and Newport that had different pulsed-field gel electrophoresis patterns and in identifying outbreak-associated cases that shared a common pulsed-field gel pattern. PMID:16943354

Utilization of a ts-sacB selection system for the generation of a Mycobacterium avium serovar-8 specific glycopeptidolipid allelic exchange mutant

PubMed Central

Irani, Vida R; Lee, Sun-Hwa; Eckstein, Torsten M; Inamine, Julia M; Belisle, John T; Maslow, Joel N

2004-01-01

Background Mycobacterium avium are ubiquitous environmental organisms and a cause of disseminated infection in patients with end-stage AIDS. The glycopeptidolipids (GPL) of M. avium are proposed to participate in the pathogenesis of this organism, however, establishment of a clear role for GPL in disease production has been limited by the inability to genetically manipulate M. avium. Methods To be able to study the role of the GPL in M. avium pathogenesis, a ts-sacB selection system, not previously used in M. avium, was employed as a means to achieve homologous recombination for the rhamnosyltransferase (rtfA) gene of a pathogenic serovar 8 strain of M. avium to prevent addition of serovar-specific sugars to rhamnose of the fatty acyl-peptide backbone of GPL. The genotype of the resultant rtfA mutant was confirmed by polymerase chain reaction and southern hybridization. Disruption in the proximal sugar of the haptenic oligosaccharide resulted in the loss of serovar specific GPL with no change in the pattern of non-serovar specific GPL moieties as shown by thin layer chromatography and gas chromatography/mass spectrometry. Complementation of wild type (wt) rtfA in trans through an integrative plasmid restored serovar-8 specific GPL expression identical to wt serovar 8 parent strain. Results In this study, we affirm our results that rtfA encodes an enzyme responsible for the transfer of Rha to 6d-Tal and provide evidence of a second allelic exchange mutagenesis system suitable for M. avium. Conclusion We report the second allelic exchange system for M. avium utilizing ts-sacB as double-negative and xylE as positive counter-selection markers, respectively. This system of allelic exchange would be especially useful for M. avium strains that demonstrate significant isoniazid (INH) resistance despite transformation with katG. Through the construction of mutants in GPL or other mycobacterial components, their roles in M. avium pathogenesis, biosynthesis, or drug

Complete genome sequence of salmonella enterica subsp. enterica Serovar Thompson Strain RM6836

USDA-ARS?s Scientific Manuscript database

Salmonella enterica subsp. enterica serovar Thompson (S. Thompson) strain RM6836 was isolated from lettuce in 2002. We report the complete sequence and annotation of the genome of S. Thompson strain RM6836. This is the first reported complete genome sequence for S. Thompson and will provide a point ...

Canine leptospirosis in Switzerland-A prospective cross-sectional study examining seroprevalence, risk factors and urinary shedding of pathogenic leptospires.

PubMed

Delaude, Alessandro; Rodriguez-Campos, Sabrina; Dreyfus, Anou; Counotte, Michel Jacques; Francey, Thierry; Schweighauser, Ariane; Lettry, Sophie; Schuller, Simone

2017-06-01

Leptospirosis is an important worldwide zoonosis. While human leptospirosis remains rare in Switzerland, the incidence of canine leptospirosis is unusually high compared to other European countries. The aims of this cross-sectional study were to determine the exposure of asymtomatic dogs to pathogenic Leptospira in Switzerland, to characterise risk factors associated with seropositivity and to determine the prevalence of urinary shedding. Sampling was stratified to cover the whole of Switzerland. Sera were tested by microscopic agglutination test for antibodies against a panel of 12 serovars. Urine was tested for pathogenic Leptospira using a LipL32 real-time PCR. Of 377 sera, 55.7% (95%CI 51.2-60.7) showed a reciprocal MAT titre of ≥1:40 and 24.9% (95%CI 20.7-29.4) of ≥1:100 to at least one serovar. Seropositivity (MAT ≥1:100) was most common to serovars Australis (14.9%; 95% CI 11.4-18.6) and Bratislava (8.8%; 95%CI 6.1-11.7), followed by Copenhageni (6.1%; 95%CI 3.7-8.5), Canicola (5%; 95%CI 2.9-7.4), Grippotyphosa (4.5%; 95%CI 2.7-6.9), Pomona (4%; 95%CI 2.1-6.1), Autumnalis (2.7%; 95%CI 1.3-4.2) and Icterohaemorrhagiae (1.6%; 95%CI 0.5-2.9). In unvaccinated dogs (n=84) the prevalence of a MAT titre ≥100 was 17.9% (95%CI 10.7-26.2), with a similar distribution of reactive serovars. Variables associated with seropositivity (≥1:40) to any serovar included age (OR 1.29/year; 95%CI: 1.1-1.5) and bioregion with higher risks in the regions Northern Alps (OR 14.5; 95%CI 2.2-292.7), Central Plateau (OR 12.3; 95%CI 2.0-244.1) and Jura (OR 11.2; 95%CI 1.7-226.7) compared to Southern Central Alps. Dogs living with horses were significantly more likely to have antibodies to serovar Bratislava (OR 4.68;95%CI 1.2-17.2). Hunting was a significant risk factor for seropositivtiy to serovar Grippotyphosa (OR 8.03; 95%CI 1.6-30.8). Urine qPCR positivity was uncommon (1/408 dogs; 0.2%; 95% CI0-0.7). These results demonstrate that dogs in Switzerland are commonly exposed

Effect of Water Activity on the Thermal Tolerance and Survival of Salmonella enterica Serovars Tennessee and Senftenberg in Goat's Milk Caramel.

PubMed

Acosta, Oscar; Usaga, Jessie; Churey, John J; Worobo, Randy W; Padilla-Zakour, Olga I

2017-06-01

The low thermal tolerance of Salmonella enterica in foods with intermediate moisture levels, such as caramel sauces, ensures that mild heat treatment is sufficient to achieve 5-log reductions of this pathogen. This treatment mitigates the risk posed by salmonellae in raw materials; however, recontamination might occur because of survival of the pathogen in products that are not heated before consumption. This study was conducted to evaluate the effect of water activity (a w ) on the thermal tolerance and survival of S. enterica serovars Tennessee and Senftenberg. The D-values at 76, 78, and 80°C, z-values, and survival at 20.0 ± 0.5°C for 32 weeks of these two serovars were determined in goat's milk caramel at three a w values (0.85, 0.90, and 0.93). The highest thermal tolerance was observed at a w = 0.85 for Salmonella Senftenberg (D 76°C = 2.9 ± 0.3 min), and the lowest was at a w = 0.93 for Salmonella Tennessee (D 80°C = 0.131 ± 0.007 min). After a logarithmic transformation of the z-values, a significant interaction between serovar and a w was found (P < 0.0001), but no consistent trends were observed at the three evaluated a w levels for either serovar. Survival response was modeled using two sigmoidal three-parameter models. A significant interaction was found between nominal variables a w and serovar when comparing inflection points of the resulting curves: P < 0.0016 for the logistic model (R 2 = 0.91) and P < 0.0014 for the Gompertz model (R 2 = 0.92). Although a >8-log reduction was observed at week 20 of storage, regardless of the product's a w and the serovar, low levels of salmonellae were found in the product up to week 32 of storage. Our findings may assist the food industry with the establishment of critical limits for the safe thermal treatment of milk- and sugar-based foods with intermediate moisture levels. The survival data presented here highlight the relevance of implementing and effectively maintaining good sanitation and hygiene

Distribution of Salmonella serovars and phage types on 80 Ontario swine farms in 2004

PubMed Central

Farzan, Abdolvahab; Friendship, Robert M.; Dewey, Catherine E.; Muckle, Anne C.; Gray, Jeff T.; Funk, Julie

2008-01-01

The objective of this study was to describe the distribution of Salmonella spp. on Ontario grower–finisher pig farms. Eighty swine farms were visited from January through July 2004. On each farm, fecal samples were collected from 5 pens, 2 rectal samples and 1 pooled sample from fresh manure on the floor per pen. Salmonella was isolated from 91 (11%) of the 800 rectal samples and 73 (18%) of the 397 pooled samples. Overall, Salmonella was recovered from 37 (46%) of the 80 farms. On each positive farm, Salmonella was cultured from 1 to 7 pigs or 1 to 5 pens. Of the 37 farms, 18, 13, 5, and 1 yielded 1, 2, 3, and 4 serovars, respectively. The most common serovars were S. Typhimurium var. Copenhagen, S. Infantis, S. Typhimurium, S. Derby, S. Agona, S. Havana, and S. enterica subsp. I:Rough-O. The 3 most frequent phage types were PT 104, PT 104a, and PT 104b. There was a statistically fair agreement between samples collected directly from pigs and pooled pen samples in determining the Salmonella status at the pen and farm level (κ = 0.6, P < 0.0001). However, in 62 pens, Salmonella status, serovars, or phage types differed between the pig and pooled pen samples. The distribution of Salmonella on the swine farms in this study indicates that, in developing an intervention strategy, priority should be given to farms positive for S. Typhimurium var. Copenhagen. Also, the variation in Salmonella status between pig and pooled pen samples deserves consideration in a sampling strategy. PMID:18214155

Microfluidic Chip-Based Detection and Intraspecies Strain Discrimination of Salmonella Serovars Derived from Whole Blood of Septic Mice

PubMed Central

Patterson, Adriana S.; Heithoff, Douglas M.; Ferguson, Brian S.; Soh, H. Tom; Mahan, Michael J.

2013-01-01

Salmonella is a zoonotic pathogen that poses a considerable public health and economic burden in the United States and worldwide. Resultant human diseases range from enterocolitis to bacteremia to sepsis and are acutely dependent on the particular serovar of Salmonella enterica subsp. enterica, which comprises over 99% of human-pathogenic S. enterica isolates. Point-of-care methods for detection and strain discrimination of Salmonella serovars would thus have considerable benefit to medical, veterinary, and field applications that safeguard public health and reduce industry-associated losses. Here we describe a single, disposable microfluidic chip that supports isothermal amplification and sequence-specific detection and discrimination of Salmonella serovars derived from whole blood of septic mice. The integrated microfluidic electrochemical DNA (IMED) chip consists of an amplification chamber that supports loop-mediated isothermal amplification (LAMP), a rapid, single-temperature amplification method as an alternative to PCR that offers advantages in terms of sensitivity, reaction speed, and amplicon yield. The amplification chamber is connected via a microchannel to a detection chamber containing a reagentless, multiplexed (here biplex) sensing array for sequence-specific electrochemical DNA (E-DNA) detection of the LAMP products. Validation of the IMED device was assessed by the detection and discrimination of S. enterica subsp. enterica serovars Typhimurium and Choleraesuis, the causative agents of enterocolitis and sepsis in humans, respectively. IMED chips conferred rapid (under 2 h) detection and discrimination of these strains at clinically relevant levels (<1,000 CFU/ml) from whole, unprocessed blood collected from septic animals. The IMED-based chip assay shows considerable promise as a rapid, inexpensive, and portable point-of-care diagnostic platform for the detection and strain-specific discrimination of microbial pathogens. PMID:23354710

Public health significance of major genotypes of Salmonella enterica serovar Enteritidis present in both human and chicken isolates in Korea.

PubMed

Kang, Min-Su; Oh, Jae-Young; Kwon, Yong-Kuk; Lee, Deog-Yong; Jeong, Ok-Mi; Choi, Byung-Kook; Youn, So-Youn; Jeon, Byung-Woo; Lee, Hye-Jin; Lee, Hee-Soo

2017-06-01

Salmonella enterica serovar Enteritidis is one of the most common serotypes implicated in Salmonella infections in both humans and poultry worldwide. It has been reported that human salmonellosis is mainly associated with the consumption of poultry products contaminated with serovar Enteritidis. The present study was to extensively analyze the public health risk of serovar Enteritidis isolates from chickens in Korea. A total of 127 chicken isolates were collected from clinical cases, on-farm feces, and chicken meat between 1998 and 2012 and 20 human clinical isolates were obtained from patients with diarrhea between 2000 and 2006 in Korea. To characterize the isolates from chickens and humans, we compared the pulsed-field gel electrophoresis (PFGE) patterns and multilocus variable-number tandem-repeat analysis (MLVA) profiles of the isolates. We further characterized representative isolates of different genotypes using a DNA microarray. PFGE revealed 28 patterns and MLVA identified 16 allelic profiles. The DNA microarray showed high genetic variability in plasmid regions and other fimbrial subunits of the isolates although the virulence gene contents of isolates from the same source and/or of the same genotype were unrelated. PFGE and MLVA showed that major genotypes were present in both human and chicken isolates. This result suggests that chickens in Korea pose a significant risk to public health as a source of serovar Enteritidis as has been noted in other countries. Copyright © 2017 Elsevier Ltd. All rights reserved.

A newly identified protein of Leptospira interrogans mediates binding to laminin.

PubMed

Longhi, Mariana T; Oliveira, Tatiane R; Romero, Eliete C; Gonçales, Amane P; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2009-10-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease that affects populations worldwide. The search for novel antigens that could be relevant in host-pathogen interactions is being pursued. These antigens have the potential to elicit several activities, including adhesion. This study focused on a hypothetical predicted lipoprotein of Leptospira, encoded by the gene LIC12895, thought to mediate attachment to extracellular matrix (ECM) components. The gene was cloned and expressed in Escherichia coli BL21 Star (DE3)pLys by using the expression vector pAE. The recombinant protein tagged with N-terminal hexahistidine was purified by metal-charged chromatography and characterized by circular dichroism spectroscopy. The capacity of the protein to mediate attachment to ECM components was evaluated by binding assays. The leptospiral protein encoded by LIC12895, named Lsa27 (leptospiral surface adhesin, 27 kDa), bound strongly to laminin in a dose-dependent and saturable fashion. Moreover, Lsa27 was recognized by antibodies from serum samples of confirmed leptospirosis specimens in both the initial and the convalescent phases of the disease. Lsa27 is most likely a surface protein of Leptospira as revealed in liquid-phase immunofluorescence assays with living organisms. Taken together, these data indicate that this newly identified membrane protein is expressed during natural infection and may play a role in mediating adhesion of L. interrogans to its host.

The Homolog of the Gene bstA of the BTP1 Phage from Salmonella enterica Serovar Typhimurium ST313 Is an Antivirulence Gene in Salmonella enterica Serovar Dublin.

PubMed

Herrero-Fresno, Ana; Espinel, Irene Cartas; Spiegelhauer, Malene Roed; Guerra, Priscila Regina; Andersen, Karsten Wiber; Olsen, John Elmerdahl

2018-01-01

In a previous study, a novel virulence gene, bstA , identified in a Salmonella enterica serovar Typhimurium sequence type 313 (ST313) strain was found to be conserved in all published Salmonella enterica serovar Dublin genomes. In order to analyze the role of this gene in the host-pathogen interaction in S Dublin, a mutant where this gene was deleted ( S Dublin Δ bstA ) and a mutant which was further genetically complemented with bstA ( S Dublin 3246-C) were constructed and tested in models of in vitro and in vivo infection as well as during growth competition assays in M9 medium, Luria-Bertani broth, and cattle blood. In contrast to the results obtained for a strain of S Typhimurium ST313, the lack of bstA was found to be associated with increased virulence in S Dublin. Thus, S Dublin Δ bstA showed higher levels of uptake than the wild-type strain during infection of mouse and cattle macrophages and higher net replication within human THP-1 cells. Furthermore, during mouse infections, S Dublin Δ bstA was more virulent than the wild type following a single intraperitoneal infection and showed an increased competitive index during competitive infection assays. Deletion of bstA did not affect either the amount of cytokines released by THP-1 macrophages or the cytotoxicity toward these cells. The histology of the livers and spleens of mice infected with the wild-type strain and the S Dublin Δ bstA mutant revealed similar levels of inflammation between the two groups. The gene was not important for adherence to or invasion of human epithelial cells and did not influence bacterial growth in rich medium, minimal medium, or cattle blood. In conclusion, a lack of bstA affects the pathogenicity of S Dublin by decreasing its virulence. Therefore, it might be regarded as an antivirulence gene in this serovar. Copyright © 2017 American Society for Microbiology.

ChpK and MazF of the toxin-antitoxin modules are involved in the virulence of Leptospira interrogans during infection.

PubMed

Komi, Komi Koukoura; Ge, Yu-Mei; Xin, Xiao-Yang; Ojcius, David M; Sun, Dexter; Hu, Wei-Lin; Zhao, Xin; Lin, Xu'ai; Yan, Jie

2015-01-01

Pathogenic Leptospira species are the causative agents of leptospirosis, a global zoonotic infectious disease. Toxin-antitoxin (TA) modules have been confirmed as stress-response elements that induce prokaryotic and eukaryotic cell-growth arrest or death, but their role in the virulence of Leptospira has not been reported. Here, we confirmed that all the tested leptospiral strains had the chpIK and mazEF TA modules with highly-conserved sequences. The transcription and expression of the chpI, chpK, mazE, and mazF genes of Leptospira interrogans strain Lai were significantly increased during infection of phorbol 12-myristate 13-acetate-induced human THP-1 macrophages. The toxic ChpK and MazF but not the antitoxic ChpI and MazE proteins were detectable in the cytoplasmic fraction of leptospire-infected THP-1 cells, indicating the external secretion of ChpK and MazF during infection. Transfection of the chpK or mazF gene caused decreased viability and necrosis in THP-1 cells, whereas the chpI or mazE gene transfection did not affect the viability of THP-1 cells but blocked the ChpK or MazF-induced toxicity. Deletion of the chpK or mazF gene also decreased the late-apoptotic and/or necrotic ratios of THP-1 cells at the late stages of infection. The recombinant protein MazF (rMazF) cleaved the RNAs but not the DNAs from Leptospira and THP-1 cells, and this RNA cleavage was blocked by rMazE. However, the rChpK had no RNA or DNA-degrading activity. All these findings indicate that the ChpK and MazF proteins in TA modules are involved in the virulence of L. interrogans during infection. Copyright © 2014 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

A comparative study on invasion, survival, modulation of oxidative burst, and nitric oxide responses of macrophages (HD11), and systemic infection in chickens by prevalent poultry Salmonella serovars

USDA-ARS?s Scientific Manuscript database

Poultry is a major reservoir for foodborne Salmonella serovars. Salmonella Typhimurium, S. Enteritidis, S. Heidelberg, S. Kentucky, and S. Senftenberg are the most prevalent serovars in poultry. Information concerning the interactions between different Salmonella species and host cells in poultry i...

Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins during in vitro Culture Attenuation

PubMed Central

Lehmann, Jason S.; Corey, Victoria C.; Ricaldi, Jessica N.; Vinetz, Joseph M.; Winzeler, Elizabeth A.; Matthias, Michael A.

2016-01-01

Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12–25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. PMID:26711524

Molecular analysis of leptospires from serogroup Sejroe obtained from asymptomatic cattle in Rio de Janeiro - Brazil reveals genetic proximity to serovar Guaricura.

PubMed

Loureiro, A P; Hamond, C; Pinto, P; Bremont, S; Bourhy, P; Lilenbaum, W

2016-04-01

Bovine leptospirosis causes substantial reproductive failure in cattle, mainly due to infections with serovar (sv) Hardjo infection. Notwithstanding, other serovars from the serogroup (sg) Sejroe could also have important roles in bovine leptospirosis. The objective was to investigate genetic diversity of serogroup Sejroe isolates obtained from asymptomatic cattle in the state of Rio de Janeiro, Brazil. Urine and vaginal fluid (VF) were collected from clinically healthy cattle immediately after slaughter. Five isolates were recovered and characterized (serogrouping) as belonging to sg Sejroe. Sequencing of rrs and secY genes further identified them as Leptospira santarosai. Analysis of secY sequences indicated a high level of sequence homology to sv Guaricura strains. Based on culture and sequence data, we inferred that other members of sg Sejroe may be important in bovine leptospiral infection, particularly genotypes of L. santarosai serovar Guaricura. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of LipL32-specific IgM by ELISA in sera of patients with a clinical diagnosis of leptospirosis

PubMed Central

Vedhagiri, Kumaresan; Velineni, Sridhar; Timoney, John F; Shanmughapriya, Santhanam; Vijayachari, Paluru; Narayanan, Ramasamy; Natarajaseenivasan, Kalimuthusamy

2013-01-01

Successful treatment of leptospirosis is heavily dependent on early diagnosis and prompt initiation of antibiotic therapy. An ELISA test to detect specific IgM antibodies against LipL32 for early diagnosis of leptospirosis is described and evaluated here. One thousand one hundred and eighty sera from clinically suspected leptospirosis cases were enrolled together with 109 healthy volunteers selected from an endemic area between October 2007 and January 2010. Patients were categorized based on their clinical signs and symptoms. Sera were screened for leptospiral antibodies by the microscopic agglutination test (MAT) using a panel of locally circulating serovars followed by enzyme-linked immunosorbent assay (ELISA) based on recombinant LipL32 from Leptospira interrogans serovar Autumnalis strain N2. The sensitivity and specificity of the ELISA test were determined to establish its diagnostic efficiency. The cut-off value was determined to be 0.205. Overall sensitivity and specificity compared to the MAT were found to be 96.4 and 90.4%, respectively. The LipL32-specific IgM ELISA had good sensitivity and acceptable specificity and may be a candidate for the early serodiagnosis of human leptospirosis. PMID:23683367

Leptospira infections in trappers from Ontario

PubMed Central

Warshawsky, Bryna; Lindsay, L Robbin; Artsob, Harvey

2000-01-01

BACKGROUND: Four trappers presented to the Middlesex-London Health Unit in November, 1997 with similar clinical presentations. All four complained of fever, chills and headache, and three of the four had severe muscle aches. All gave histories of trapping raccoons before the onset of illness. Three of the four men exhibited diagnostic seroconversions to Leptospira grippotyphosa. OBJECTIVE: To describe the four suspected cases of leptospira infections and to determine whether raccoons might serve as a reservoir of infection using field studies. DESIGN: Raccoon serology were undertaken using the microscopic agglutination test against eight serovars of Leptospira interrogans including L grippotyphosa. Raccoons were trapped using Tomahawk live traps, anaesthetized with intramuscular injection of ketamine and acepromazine, bled by cardiac puncture and released. RESULTS: Forty-two raccoons were trapped in Middlesex (n=36) and Kent counties (n=6) from April 25 to May 2, 1998, and 10 (23.8%) of these animals had antibodies to L grippotyphosa. CONCLUSIONS: Infections due to L grippotyphosa or a closely related serovar are a risk for trappers in Ontario, and raccoons are a likely reservoir of this bacterium. PMID:18159265

Attachment of Salmonella serovars and Listeria monocytogenes to stainless steel and plastic conveyor belts.

PubMed

Veluz, G A; Pitchiah, S; Alvarado, C Z

2012-08-01

In poultry industry, cross-contamination due to processing equipment and contact surfaces is very common. This study examined the extent of bacterial attachment to 6 different types and design of conveyor belts: stainless steel-single loop, stainless steel-balance weave, polyurethane with mono-polyester fabric, acetal, polypropylene mesh top, and polypropylene. Clean conveyor belts were immersed separately in either a cocktail of Salmonella serovars (Salmonella Typhimurium and Salmonella Enteritidis) or Listeria monocytogenes strains (Scott A, Brie 1, ATCC 6744) for 1 h at room temperature. Soiled conveyor chips were dipped in poultry rinses contaminated with Salmonella or Listeria cocktail and incubated at 10°C for 48 h. The polyurethane with mono-polyester fabric conveyor belt and chip exhibited a higher (P<0.05) mean number of attached Salmonella serovars (clean: 1.6 to 3.6 cfu/cm2; soiled: 0.8 to 2.4 cfu/cm2) and L. monocytogenes (clean: 4.0 to 4.3 cfu/cm2; soiled: 0.3 to 2.1 cfu/cm2) in both clean and soiled conditions. The stainless steel conveyor belt attached a lower (P<0.05) number of Salmonella serovars (clean: 0 to 2.6 cfu/cm2; soiled: 0.4 to 1.3 cfu/cm2) and L. monocytogenes (clean: 0.4 to 2.9 cfu/cm2; soiled: 0 to 0.7 cfu/cm2) than the polymeric materials, indicating weaker adhesion properties. Plastic conveyor belts exhibited stronger bacterial adhesion compared with stainless steel. The result suggests the importance of selecting the design and finishes of conveyor belt materials that are most resistant to bacterial attachment.

Complete Whole-Genome Sequence of Salmonella enterica subsp. enterica Serovar Java NCTC5706.

PubMed

Fazal, Mohammed-Abbas; Alexander, Sarah; Burnett, Edward; Deheer-Graham, Ana; Oliver, Karen; Holroyd, Nancy; Parkhill, Julian; Russell, Julie E

2016-11-03

Salmonellae are a significant cause of morbidity and mortality globally. Here, we report the first complete genome sequence for Salmonella enterica subsp. enterica serovar Java strain NCTC5706. This strain is of historical significance, having been isolated in the pre-antibiotic era and was deposited into the National Collection of Type Cultures in 1939. © Crown copyright 2016.

ESBL-Producing Salmonella enterica Serovar Typhi in Traveler Returning from Guatemala to Spain

PubMed Central

Piedra-Carrasco, Nuria; Salvador, Fernando; Rodríguez, Virginia; Sánchez-Montalvá, Adrián; Planes, Anna M.; Molina, Israel; Larrosa, M. Nieves

2014-01-01

We report a case of typhoid fever in a traveler returning to Spain from Guatemala that was caused by Salmonella enterica serovar Typhi which produced an extended-spectrum β-lactamase (ESBL). This finding demonstrates the presence of ESBL-producing S. enterica ser. Typhi strains in the Americas. Enhanced surveillance is necessary to prevent further spread. PMID:25340972

Survival of Salmonella enterica serovar infantis on and within stored table eggs.

PubMed

Lublin, Avishai; Maler, Ilana; Mechani, Sara; Pinto, Riky; Sela-Saldinger, Shlomo

2015-02-01

Contaminated table eggs are considered a primary source of foodborne salmonellosis globally. Recently, a single clone of Salmonella enterica serovar Infantis emerged in Israel and became the predominant serovar isolated in poultry. This clone is currently the most prevalent strain in poultry and is the leading cause of salmonellosis in humans. Because little is known regarding the potential transmission of this strain from contaminated eggs to humans, the objective of this study was to evaluate the ability of Salmonella Infantis to survive on the eggshell or within the egg during cold storage or at room temperature. Salmonella cells (5.7 log CFU per egg) were inoculated on the surface of 120 intact eggs or injected into the egg yolk (3.7 log CFU per egg) of another 120 eggs. Half of the eggs were stored at 5.5 ± 0.3°C and half at room temperature (25.5 ± 0.1°C) for up to 10 weeks. At both temperatures, the number of Salmonella cells on the shell declined by 2 log up to 4 weeks and remained constant thereafter. Yolk-inoculated Salmonella counts at cold storage declined by 1 log up to 4 weeks and remained constant, while room-temperature storage supported the growth of the pathogen to a level of 8 log CFU/ml of total egg content, as early as 4 weeks postinoculation. Examination of egg content following surface inoculation revealed the presence of Salmonella in a portion of the eggs at both temperatures up to 10 weeks, suggesting that this strain can also penetrate through the shell and survive within the egg. These findings imply that Salmonella enterica serovar Infantis is capable of survival both on the exterior and interior of table eggs and even multiply inside the egg at room temperature. Our findings support the need for prompt refrigeration to prevent Salmonella multiplication during storage of eggs at room temperature.

Genome and transcriptome adaptation accompanying emergence of the definitive type 2 host-restricted Salmonella enterica serovar Typhimurium pathovar.

PubMed

Kingsley, Robert A; Kay, Sally; Connor, Thomas; Barquist, Lars; Sait, Leanne; Holt, Kathryn E; Sivaraman, Karthi; Wileman, Thomas; Goulding, David; Clare, Simon; Hale, Christine; Seshasayee, Aswin; Harris, Simon; Thomson, Nicholas R; Gardner, Paul; Rabsch, Wolfgang; Wigley, Paul; Humphrey, Tom; Parkhill, Julian; Dougan, Gordon

2013-08-27

Salmonella enterica serovar Typhimurium definitive type 2 (DT2) is host restricted to Columba livia (rock or feral pigeon) but is also closely related to S. Typhimurium isolates that circulate in livestock and cause a zoonosis characterized by gastroenteritis in humans. DT2 isolates formed a distinct phylogenetic cluster within S. Typhimurium based on whole-genome-sequence polymorphisms. Comparative genome analysis of DT2 94-213 and S. Typhimurium SL1344, DT104, and D23580 identified few differences in gene content with the exception of variations within prophages. However, DT2 94-213 harbored 22 pseudogenes that were intact in other closely related S. Typhimurium strains. We report a novel in silico approach to identify single amino acid substitutions in proteins that have a high probability of a functional impact. One polymorphism identified using this method, a single-residue deletion in the Tar protein, abrogated chemotaxis to aspartate in vitro. DT2 94-213 also exhibited an altered transcriptional profile in response to culture at 42°C compared to that of SL1344. Such differentially regulated genes included a number involved in flagellum biosynthesis and motility. IMPORTANCE Whereas Salmonella enterica serovar Typhimurium can infect a wide range of animal species, some variants within this serovar exhibit a more limited host range and altered disease potential. Phylogenetic analysis based on whole-genome sequences can identify lineages associated with specific virulence traits, including host adaptation. This study represents one of the first to link pathogen-specific genetic signatures, including coding capacity, genome degradation, and transcriptional responses to host adaptation within a Salmonella serovar. We performed comparative genome analysis of reference and pigeon-adapted definitive type 2 (DT2) S. Typhimurium isolates alongside phenotypic and transcriptome analyses, to identify genetic signatures linked to host adaptation within the DT2 lineage.

Comparison of Chlamydia trachomatis serovar L2 growth in polarized genital epithelial cells grown in three-dimensional culture with non-polarized cells.

PubMed

Dessus-Babus, Sophie; Moore, Cheryl G; Whittimore, Judy D; Wyrick, Priscilla B

2008-04-01

A common model for studying Chlamydia trachomatis and growing chlamydial stocks uses Lymphogranuloma venereum serovar L2 and non-polarized HeLa cells. However, recent publications indicate that the growth rate and progeny yields can vary considerably for a particular strain depending on the cell line/type used, and seem to be partially related to cell tropism. In the present study, the growth of invasive serovar L2 was compared in endometrial HEC-1B and endocervical HeLa cells polarized on collagen-coated microcarrier beads, as well as in HeLa cells grown in tissue culture flasks. Microscopy analysis revealed no difference in chlamydial attachment/entry patterns or in inclusion development throughout the developmental cycle between cell lines. Very comparable growth curves in both cell lines were also found using real-time PCR analysis, with increases in chlamydial DNA content of 400-500-fold between 2 and 36 h post-inoculation. Similar progeny yields with comparable infectivity were recovered from HEC-1B and HeLa cell bead cultures, and no difference in chlamydial growth was found in polarized vs. non-polarized HeLa cells. In conclusion, unlike other C. trachomatis strains such as urogenital serovar E, invasive serovar L2 grows equally well in physiologically different endometrial and endocervical environments, regardless of the host cell polarization state.

Comparison of agglutinating and neutralizing antibodies to serovar hardjo in sows immunized with two commercial whole culture polivalent anti-leptospira bacterins

PubMed Central

Soto, Francisco Rafael Martins; Pinheiro, Sônia Regina; Morais, Zenaide Maria; Gonçales, Amane Paldês; de Azevedo, Sérgio Santos; Bernardi, Fernanda; Camargo, Sebastião Rodrigues; Vasconcellos, Silvio Arruda

2008-01-01

It was performed the comparison of the intensity and duration of agglutinating and neutralizing antibodies to serovar Hardjo in swines vaccinated with two commercial anti-leptospira bacterins. Sows no reactive to 24 Leptospira sp serovars in the microscopic agglutination test (MAT) were divided in three groups: Group A (n=08): received two vaccine A doses with 30 days interval, Group B (n=08) two vaccine B doses with 30 days interval and Group C (n=08): control no vaccinated against leptospirosis.Blood samples were collected each 30 days during six months following the first vaccination. The sera were tested by MAT and growth inhibition test (GIT) to serovar Hardjo in order to evaluate respectively agglutinating and neutralizing antibodies. It was found that neutralizing antibodies persisted for a longer time than the agglutinating ones and that the absence of agglutinating antibodies does not means in the absence of the neutralizing. The peaks of agglutinating antibodies was obtained at least 30 days earlier than that produced by neutralizing. The duration of both kinds of antibodies measured differed between the two bacterines tested. The period for inducing neutralizing antibodies against serovar Hardjo indicated that gilts must be immunized with two doses of whole culture anti-leptospira bacterines applied 30 days each other at least 90 days before the first mating. For the maintenance of hight levels of neutralizing antibodies the revaccinations must be performed every six months after the first vaccination. PMID:24031250

Swarm and swim motilities of Salmonella enterica serovar Typhimurium and role of osmoregulated periplasmic glucans

USDA-ARS?s Scientific Manuscript database

Background: Salmonella enterica serovar Typhimurium strains synthesize osmoregulated periplasmic glucans (OPGs) under low osmolarity conditions (< 70 mos mol l-1). OPG synthesis is not observed when cells are grown in iso- or hyper-osmotic media (> 400 mos mol l-1). Mutation in OPG structural gene...

Whole-genome sequencing of Salmonella enterica subsp. enterica serovar Cubana strains isolated from agricultural sources

USDA-ARS?s Scientific Manuscript database

We report draft genomes of Salmonella enterica subsp. enterica Serovar Cubana strain CVM42234 isolated from chick feed in 2012 and Salmonella Cubana strain 76814 isolated from swine in 2004. The genome sizes are 4,975,046 and 4,936,251 base pairs, respectively....

Novel insertion sequence- and transposon-mediated genetic rearrangements in genomic island SGI1 of Salmonella enterica serovar Kentucky.

PubMed

Doublet, Benoît; Praud, Karine; Bertrand, Sophie; Collard, Jean-Marc; Weill, François-Xavier; Cloeckaert, Axel

2008-10-01

Salmonella genomic island 1 (SGI1) is an integrative mobilizable element that harbors a multidrug resistance (MDR) gene cluster. Since its identification in epidemic Salmonella enterica serovar Typhimurium DT104 strains, variant SGI1 MDR gene clusters conferring different MDR phenotypes have been identified in several S. enterica serovars and classified as SGI1-A to -O. A study was undertaken to characterize SGI1 from serovar Kentucky strains isolated from travelers returning from Africa. Several strains tested were found to contain the partially characterized variant SGI1-K, recently described in a serovar Kentucky strain isolated in Australia. This variant contained only one cassette array, aac(3)-Id-aadA7, and an adjacent mercury resistance module. Here, the uncharacterized part of SGI1-K was sequenced. Downstream of the mer module similar to that found in Tn21, a mosaic genetic structure was found, comprising (i) part of Tn1721 containing the tetracycline resistance genes tetR and tet(A); (ii) part of Tn5393 containing the streptomycin resistance genes strAB, IS1133, and a truncated tnpR gene; and (iii) a Tn3-like region containing the tnpR gene and the beta-lactamase bla(TEM-1) gene flanked by two IS26 elements in opposite orientations. The rightmost IS26 element was shown to be inserted into the S044 open reading frame of the SGI1 backbone. This variant MDR region was named SGI1-K1 according to the previously described variant SGI1-K. Other SGI1-K MDR regions due to different IS26 locations, inversion, and partial deletions were characterized and named SGI1-K2 to -K5. Two new SGI1 variants named SGI1-P1 and -P2 contained only the Tn3-like region comprising the beta-lactamase bla(TEM-1) gene flanked by the two IS26 elements inserted into the SGI1 backbone. Three other new variants harbored only one IS26 element inserted in place of the MDR region of SGI1 and were named SGI1-Q1 to -Q3. Thus, in serovar Kentucky, the SGI1 MDR region undergoes recombinational and

Role for cis-acting RNA sequences in the temperature-dependent expression of the multiadhesive lig proteins in Leptospira interrogans.

PubMed

Matsunaga, James; Schlax, Paula J; Haake, David A

2013-11-01

The spirochete Leptospira interrogans causes a systemic infection that provokes a febrile illness. The putative lipoproteins LigA and LigB promote adhesion of Leptospira to host proteins, interfere with coagulation, and capture complement regulators. In this study, we demonstrate that the expression level of the LigA and LigB proteins was substantially higher when L. interrogans proliferated at 37°C instead of the standard culture temperature of 30°C. The RNA comprising the 175-nucleotide 5' untranslated region (UTR) and first six lig codons, whose sequence is identical in ligA and ligB, is predicted to fold into two distinct stem-loop structures separated by a single-stranded region. The ribosome-binding site is partially sequestered in double-stranded RNA within the second structure. Toeprint analysis revealed that in vitro formation of a 30S-tRNA(fMet)-mRNA ternary complex was inhibited unless a 5' deletion mutation disrupted the second stem-loop structure. To determine whether the lig sequence could mediate temperature-regulated gene expression in vivo, the 5' UTR and the first six codons were inserted between the Escherichia coli l-arabinose promoter and bgaB (β-galactosidase from Bacillus stearothermophilus) to create a translational fusion. The lig fragment successfully conferred thermoregulation upon the β-galactosidase reporter in E. coli. The second stem-loop structure was sufficient to confer thermoregulation on the reporter, while sequences further upstream in the 5' UTR slightly diminished expression at each temperature tested. Finally, the expression level of β-galactosidase was significantly higher when point mutations predicted to disrupt base pairs in the second structure were introduced into the stem. Compensatory mutations that maintained base pairing of the stem without restoring the wild-type sequence reinstated the inhibitory effect of the 5' UTR on expression. These results indicate that ligA and ligB expression is limited by double

Development of a LIBS assay for the detection of Salmonella enterica serovar Typhimurium from food.

PubMed

Barnett, Cleon; Bell, Courtneé; Vig, Komal; Akpovo, A C; Johnson, Lewis; Pillai, Shreekumar; Singh, Shree

2011-07-01

Laser-induced breakdown spectroscopy (LIBS) is used for the identification of the presence of hazardous bacteria in food. In this study, our main focus was centered on the identification of S. enterica serovar Typhimurium, a Gram-negative foodborne pathogen, in various liquids such as milk, chicken broth, and brain heart infusion due to the infection being most prevalent in raw meat and dairy products. A Nd:YAG laser of operating wavelength 266 nm was used to obtain the spectra from the artificially inoculated liquid samples. A series of experiments were performed to determine the effectiveness of LIBS to discriminate the bacteria from the background liquids. These results are compared with competing modern molecular methods of detection which include polymerase chain reaction (PCR) and quantitative real-time PCR. In addition to analyzing S. enterica serovar Typhimurium, another common Gram-negative, Escherichia coli, as well as Gram-positive pathogen, Staphlycoccus auerus, were used to determine the specificity of the LIBS technique.

The Vi Capsular Polysaccharide Enables Salmonella enterica Serovar Typhi to Evade Microbe-Guided Neutrophil Chemotaxis

PubMed Central

Wangdi, Tamding; Lee, Cheng-Yuk; Spees, Alanna M.; Yu, Chenzhou; Kingsbury, Dawn D.; Winter, Sebastian E.; Hastey, Christine J.; Wilson, R. Paul

2014-01-01

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis. PMID:25101794

A cross sectional observational study to estimate herd level risk factors for Leptospira spp. serovars in small holder dairy cattle farms in southern Chile

PubMed Central

2014-01-01

Background The south of Chile constitutes the main cattle milk producing area of the country. Regarding leptospirosis control in Chile, there is neither an official program nor an epidemiological characterization of smallholder dairy farms. This study was carried out to determine Leptospira seroprevalence and to evaluate risk factors associated with seropositivity at herd level in smallholder bovine dairy herds in southern Chile. A cross-sectional study was conducted, and a convenient sample of 1,537 apparently healthy dairy cows was included in the study. Individual blood samples were taken and examined for six selected reference Leptospira serovars by the Microscopic Agglutination Test (MAT). Results Of the included herds 75% (52/69) showed serological titers against one or more Leptospira serovar. Leptospira borgpetersenii serovar Hardjo was the serovar most frequently (81%) reported from animals with positive results. The variables considered risk factors for Leptospira seropositivity were calve natural breeding system, using a specific calving area and vaccination against Leptospira. Adult cows in contact with calves weaned, proved to be a protective factor against infection. Conclusions Herds neglecting the management practices mentioned in this study could represent an important source of Leptospira infection for other herds in the same geographic area, as well as for other animal species. PMID:24906684

A cross sectional observational study to estimate herd level risk factors for Leptospira spp. serovars in small holder dairy cattle farms in southern Chile.

PubMed

Salgado, Miguel; Otto, Barbara; Sandoval, Errol; Reinhardt, German; Boqvist, Sofia

2014-06-06

The south of Chile constitutes the main cattle milk producing area of the country. Regarding leptospirosis control in Chile, there is neither an official program nor an epidemiological characterization of smallholder dairy farms. This study was carried out to determine Leptospira seroprevalence and to evaluate risk factors associated with seropositivity at herd level in smallholder bovine dairy herds in southern Chile.A cross-sectional study was conducted, and a convenient sample of 1,537 apparently healthy dairy cows was included in the study. Individual blood samples were taken and examined for six selected reference Leptospira serovars by the Microscopic Agglutination Test (MAT). Of the included herds 75% (52/69) showed serological titers against one or more Leptospira serovar. Leptospira borgpetersenii serovar Hardjo was the serovar most frequently (81%) reported from animals with positive results. The variables considered risk factors for Leptospira seropositivity were calve natural breeding system, using a specific calving area and vaccination against Leptospira. Adult cows in contact with calves weaned, proved to be a protective factor against infection. Herds neglecting the management practices mentioned in this study could represent an important source of Leptospira infection for other herds in the same geographic area, as well as for other animal species.

ssrA (tmRNA) Plays a Role in Salmonella enterica Serovar Typhimurium Pathogenesis

PubMed Central

Julio, Steven M.; Heithoff, Douglas M.; Mahan, Michael J.

2000-01-01

Escherichia coli ssrA encodes a small stable RNA molecule, tmRNA, that has many diverse functions, including tagging abnormal proteins for degradation, supporting phage growth, and modulating the activity of DNA binding proteins. Here we show that ssrA plays a role in Salmonella enterica serovar Typhimurium pathogenesis and in the expression of several genes known to be induced during infection. Moreover, the phage-like attachment site, attL, encoded within ssrA, serves as the site of integration of a region of Salmonella-specific sequence; adjacent to the 5′ end of ssrA is another region of Salmonella-specific sequence with extensive homology to predicted proteins encoded within the unlinked Salmonella pathogenicity island SPI4. S. enterica serovar Typhimurium ssrA mutants fail to support the growth of phage P22 and are delayed in their ability to form viable phage particles following induction of a phage P22 lysogen. These data indicate that ssrA plays a role in the pathogenesis of Salmonella, serves as an attachment site for Salmonella-specific sequences, and is required for the growth of phage P22. PMID:10692360

[Leptospirosis in animal reproduction: III. Role of the hardjo serovar in bovine leptospirosis in Rio de Janeiro, Brazil].

PubMed

Lilenbaum, W; Dos Santos, M R

1995-01-01

Four hundred and five serum samples were drawn from cows with reproductive problems which were not vaccinated against leptospirosis from 21 dairy farms. Three distinct geographic regions were determined and the farms were also classified considering the production system, based on technological, zootechnical and sanitary resources. A total of 277 positive reactions were observed, corresponding to 68.39% of the samples. The predominant serovar was hardjo, reactive on 85 samples (20.98%), predominant on nine farms and observed on 17 farms (80.95%). It was observed the predominance of hardjo in all studied regions and on properties classified as type "A" (22 samples) and type "B" (49 samples). The role of this serovar on bovine leptospirosis in Brazil compared with other countries is discussed.

Immunogenicity of Salmonella enterica serovar Enteritidis virulence protein, InvH, and cross-reactivity of its antisera with Salmonella strains.

PubMed

Dehghani, Behzad; Rasooli, Iraj; Gargari, Seyed Latif Mousavi; Nadooshan, Mohammad Reza Jalali; Owlia, Parviz; Nazarian, Shahram

2013-02-22

Acellular vaccines containing bacterial immunodominant components such as surface proteins may be potent alternatives to live attenuated vaccines in order to reduce salmonellosis risk to human health. invH gene, an important part of needle complex in type three secretion system (TTSS) plays important role in efficient bacterial adherence and entry into epithelial cells. In this work we hypothesize that use of a 15 kDa recombinant InvH as Salmonella enterica serovar Enteritidis surface protein could provoke antibody production in mouse and would help us study feasibility of its potential for diagnosis and/or a recombinant vaccine. The purified InvH provoked significant rise of IgG in mice. Active protection induced by immunization with InvH against variable doses of S. enterica serovar Enteritidis, indicated that the immunized mice were completely protected against challenge with 10(4) LD(50). The immunoreaction of sera from immunized mice with other Salmonella strains or cross reaction with sera of Salmonella strains inoculated mice is indicative of possessing by Salmonella strains of the surface protein, InvH, that can be employed in both prophylactic and diagnostic measures against S. enterica. Bacteria free spleen and ileum of the immunized mice in this study indicate that the invH gene affects bacterial invasion. Efficacy of the virulence protein, InvH, in shuttling into host cells in injectisome of S. enterica serovar Enteritidis and inhibition of this phenomenon by active immunization was shown in this study. In conclusion immunization with InvH protein can develop protection against S. enterica serovar Enteritidis infections. InvH in Salmonella strains can be exploited in protective measures as well as a diagnostic tool in Salmonella infections. Copyright © 2012 Elsevier GmbH. All rights reserved.

Evolutionary genetic relationships of clones of Salmonella serovars that cause human typhoid and other enteric fevers.

PubMed Central

Selander, R K; Beltran, P; Smith, N H; Helmuth, R; Rubin, F A; Kopecko, D J; Ferris, K; Tall, B D; Cravioto, A; Musser, J M

1990-01-01

Multilocus enzyme electrophoresis was employed to measure chromosomal genotypic diversity and evolutionary relationships among 761 isolates of the serovars Salmonella typhi, S. paratyphi A, S. paratyphi B, S. paratyphi C, and S. sendai, which are human-adapted agents of enteric fever, and S. miami and S. java, which are serotypically similar to S. sendai and S. paratyphi B, respectively, but cause gastroenteritis in both humans and animals. To determine the phylogenetic positions of the clones of these forms within the context of the salmonellae of subspecies I, comparative data for 22 other common serovars were utilized. Except for S. paratyphi A and S. sendai, the analysis revealed no close phylogenetic relationships among clones of different human-adapted serovars, which implies convergence in host adaptation and virulence factors. Clones of S. miami are not allied with those of S. sendai or S. paratyphi A, being, instead, closely related to strains of S. panama. Clones of S. paratyphi B and S. java belong to a large phylogenetic complex that includes clones of S. typhimurium, S. heidelberg, S. saintpaul, and S. muenchen. Most strains of S. paratyphi B belong to a globally distributed clone that is highly polymorphic in biotype, bacteriophage type, and several other characters, whereas strains of S. java represent seven diverse lineages. The flagellar monophasic forms of S. java are genotypically more similar to clones of S. typhimurium than to other clones of S. java or S. paratyphi B. Clones of S. paratyphi C are related to those of S. choleraesuis. DNA probing with a segment of the viaB region specific for the Vi capsular antigen genes indicated that the frequent failure of isolates of S. paratyphi C to express Vi antigen is almost entirely attributable to regulatory processes rather than to an absence of the structural determinant genes themselves. Two clones of S. typhisuis are related to those of S. choleraesuis and S. paratyphi C, but a third clone is not

SALMONELLA ENTERICA SEROVAR ENTERITIDIS INFECTION MODULATES DIVERSE FUNCTIONAL PROCESSES OF CHICKEN MACROPHAGE AT THE TRANSCRIPTIONAL LEVEL

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar Enteritidis (SE) is a major etiologic agent of non-typhoid salmonellosis. The organisms colonize adult chicken hosts without causing overt clinical signs. The immunological mechanisms underlying the silent and persistent infection of chickens by SE are not clearly underst...

Epidemiology of a Salmonella enterica subsp. Enterica serovar Typhimurium strain associated with a songbird outbreak.

USGS Publications Warehouse

Blehert, David S.; Hernandez, Sonia M.; Keel, Kevin; Sanchez, Susan; Trees, Eija; ,

2012-01-01

Salmonella enterica subsp. enterica serovar Typhimurium is responsible for the majority of salmonellosis cases worldwide. This Salmonella serovar is also responsible for die-offs in songbird populations. In 2009, there was an S. Typhimurium epizootic reported in pine siskins in the eastern United States. At the time, there was also a human outbreak with this serovar that was associated with contaminated peanuts. As peanuts are also used in wild-bird food, it was hypothesized that the pine siskin epizootic was related to this human outbreak. A comparison of songbird and human S. Typhimurium pulsed-field gel electrophoresis (PFGE) patterns revealed that the epizootic was attributed not to the peanut-associated strain but, rather, to a songbird strain first characterized from an American goldfinch in 1998. This same S. Typhimurium strain (PFGE type A3) was also identified in the PulseNet USA database, accounting for 137 of 77,941 total S. Typhimurium PFGE entries. A second molecular typing method, multiple-locus variable-number tandem-repeat analysis (MLVA), confirmed that the same strain was responsible for the pine siskin epizootic in the eastern United States but was distinct from a genetically related strain isolated from pine siskins in Minnesota. The pine siskin A3 strain was first encountered in May 2008 in an American goldfinch and later in a northern cardinal at the start of the pine siskin epizootic. MLVA also confirmed the clonal nature of S. Typhimurium in songbirds and established that the pine siskin epizootic strain was unique to the finch family. For 2009, the distribution of PFGE type A3 in passerines and humans mirrored the highest population density of pine siskins for the East Coast.

Herd- and animal-level risk factors for bovine leptospirosis in Tanga region of Tanzania.

PubMed

Schoonman, L; Swai, Emanuel Senyael

2010-10-01

Leptospirosis is the zoonosis of worldwide distribution and common cause of economic loss and ill health among animals and human populations. A cross-sectional seroprevalence study, using a microscopic agglutination test (MAT) with a threshold titre of >or=1:160, to elucidate disease magnitude, distribution and associated risk factors in cattle in Tanga, Tanzania was conducted from May 2003 to January 2004. Serum (n = 655) samples collected from randomly selected herds (n = 130) were tested for antibodies against four different Leptospira interrogans serovars (Bataviae, Tarassovi, Hardjo and Pomona) used in the agglutination test. Positive titres were detected in 30.3% [95% confidence intervals (CI) = 26.7-33.9] of cattle and 58.5% (95% CI = 49.5-67.1) of herds, respectively. Of the 198 MAT positive serum samples, 98 (49.5%) were positive against serovar Hardjo, 80 (40.4%) were positive against serovar Tarassovi, 12 (6.1%) was positive against serovar Bataviae and eight (4%) were positive against serovar Pomona. Associations found to be statistically significant in univariate analyses (at P < 0.1) were assessed by multivariable logistic regression to control for confounding factors. The results showed that risk factors for cattle were pasture grazing [odd ratio (OR) = 2.83, 95% CI = 1.57-5.12, P = 0.001], presence of goats/sheep on the farm (OR = 1.73, 95% CI = 1.17-2.56, P = 0.001) and age of the animal (OR = 2.05, 95% CI = 1.42-2.96, P = 0.001), while concrete floor housing was protective (OR = 0.47, 95% CI = 0.30-0.74, P = 0.001). Herds managed under pasture grazing system were more likely to be sero-positive than those managed under zero grazed practices (OR = 9.31; 95% CI = 3.67-23.64 for grazing herd). We concluded that bovine leptospirosis is an endemic and locally widespread disease in Tanga and suggest that it may play a role in zoonotic transmission to humans.

Intermediate Susceptibility to Ciprofloxacin among Salmonella enterica Serovar Typhi Isolates in Lima, Peru

PubMed Central

Lejon, Veerle; Horna, Gertrudis; Astocondor, Lizeth; Vanhoof, Raymond; Bertrand, Sophie; Jacobs, Jan

2014-01-01

Thirty-three Salmonella enterica serovar Typhi blood isolates from Lima, Peru (2008 to 2012), were fully susceptible to trimethoprim-sulfamethoxazole, chloramphenicol, ceftriaxone, and tetracycline; 8/33 (24.2%) showed intermediate susceptibility to ciprofloxacin carrying mutations in the quinolone resistance-determining region of the gyrA gene (Ser83-Phe and Asp87-Asn) and in the gyrB gene (Ser464-Phe). PMID:24371234

Effect of selective growth media on the differentiation of Salmonella enterica serovars by Fourier-Transform Mid-Infrared Spectroscopy.

PubMed

Baldauf, Nathan A; Rodriguez-Romo, Luis A; Männig, Annegret; Yousef, Ahmed E; Rodriguez-Saona, Luis E

2007-01-01

Salmonella enterica serovars are prevalent foodborne pathogens responsible for high numbers of salmonellosis each year. Complex Fourier-transform infrared (FTIR) spectra offer unique biochemical fingerprints of bacteria with bands due to major cellular components. Growth media effects on discrimination of Salmonella serovars by FTIR spectroscopy were investigated and a novel sample preparation technique was developed. S. enterica strains from six serovars were grown on xylose lysine desoxycholate (XLD), Miller-Mallinson (MM), and plate count (PCA) agar as a control (37 degrees C, 24 h). Isolated colonies were suspended in 50% acetonitrile and centrifuged; the remaining pellet was placed on an AMTIR (attenuated total reflectance) crystal and dried under vacuum. Classification models (Soft Independent Modeling of Class Analogy, SIMCA), generated from derivatized infrared spectra (1300-900 cm-1 or 1200-900 cm-1), successfully discriminated among Salmonella strains with major discrimination from 1000-970 cm-1 associated to stretching modes of O-specific polysaccharide chains of lipopolysaccharides. Sample treatment with acetonitrile enhanced safe handling of the bacteria, removed interfering signals and improved the discriminating ability of SIMCA. All media were able to discriminate the S. enterica strains studied, varying in discriminating peaks and class distances in SIMCA classification. This methodology, with the production of large libraries of pathogenic bacteria, could be applied for the rapid monitoring of bacterial contamination in food with minimal sample manipulation.

Genetic and Phenotypic Characterization of a Salmonella enterica serovar Enteritidis Emerging Strain with Superior Intra-macrophage Replication Phenotype

PubMed Central

Shomer, Inna; Avisar, Alon; Desai, Prerak; Azriel, Shalhevet; Smollan, Gill; Belausov, Natasha; Keller, Nathan; Glikman, Daniel; Maor, Yasmin; Peretz, Avi; McClelland, Michael; Rahav, Galia; Gal-Mor, Ohad

2016-01-01

Salmonella enterica serovar Enteritidis (S. Enteritidis) is one of the ubiquitous Salmonella serovars worldwide and a major cause of food-born outbreaks, which are often associated with poultry and poultry derivatives. Here we report a nation-wide S. Enteritidis clonal outbreak that occurred in Israel during the last third of 2015. Pulsed field gel electrophoresis and whole genome sequencing identified genetically related strains that were circulating in Israel as early as 2008. Global comparison linked this outbreak strain to several clinical and marine environmental isolates that were previously isolated in California and Canada, indicating that similar strains are prevalent outside of Israel. Phenotypic comparison between the 2015 outbreak strain and other clinical and reference S. Enteritidis strains showed only limited intra-serovar phenotypic variation in growth in rich medium, invasion into Caco-2 cells, uptake by J774.1A macrophages, and host cell cytotoxicity. In contrast, significant phenotypic variation was shown among different S. Enteritidis isolates when biofilm-formation, motility, invasion into HeLa cells and uptake by THP-1 human macrophages were studied. Interestingly, the 2015 outbreak clone was found to possess superior intra-macrophage replication ability within both murine and human macrophages in comparison to the other S. Enteritidis strains studied. This phenotype is likely to play a role in the virulence and host-pathogen interactions of this emerging clone. PMID:27695450

mcr-1−Harboring Salmonella enterica Serovar Typhimurium Sequence Type 34 in Pigs, China

PubMed Central

Yi, Linxian; Wang, Jing; Gao, Yanling; Liu, Yiyun; Doi, Yohei; Wu, Renjie; Zeng, Zhenling; Liang, Zisen

2017-01-01

We detected the mcr-1 gene in 21 (14.8%) Salmonella isolates from pigs at slaughter; 19 were serovar Typhimurium sequence type 34. The gene was located on IncHI2-like plasmids that also harbored IncF replicons and lacked a conjugative transfer region. These findings highlight the need to prevent further spread of colistin resistance in animals and humans. PMID:28098547

Influence of the treatment of Listeria monocytogenes and Salmonella enterica serovar Typhimurium with citral on the efficacy of various antibiotics.

PubMed

Zanini, Surama F; Silva-Angulo, Angela B; Rosenthal, Amauri; Aliaga, Dolores Rodrigo; Martínez, Antonio

2014-04-01

The main goal of this work was to study the bacterial adaptive responses to antibiotics induced by sublethal concentration of citral on first-and second-generation cells of Listeria monocytogenes serovar 4b (CECT 4032) and Salmonella enterica serovar Typhimurium (CECT 443). The first-generation cells were not pretreated with citral, while the second-generation cells were obtained from cells previously exposed to citral during 5 h. The trials were conducted at 37°C. The presence of citral in the culture medium and the antibiotic strips resulted in a reduced minimum inhibitory concentration (MIC) for the first-generation cells of Listeria monocytogenes serovar 4b and Salmonella Typhimurium. This result was observed for almost all the antibiotics, compared with the same microorganisms of the control group (without citral), which could represent an additive effect. For Listeria serovar 4b, the second-generation cells of the test group maintained the same susceptibility to antibiotics compared with cells in the control group and in the test group of the first generation. The second-generation cells of the control group indicated that the Salmonella Typhimurium maintained the same sensitivity to the antibiotics tested compared with the first generation of this group, except in the case of erythromycin, which exhibited an increased MIC value. With respect to the second-generation cells of Salmonella Typhimurium, the presence of citral determined a decrease in the antibiotic susceptibility for almost all of the antibiotics, except colistin, compared with the first-generation of the test group, which can be seen by increase of MIC values. In conclusion, the presence of citral in the culture medium of Listeria 4b and Salmonella Typhimurium increased the antibiotic susceptibility of the first generations, while we observed an increase in antibiotic resistance in the second generation of Salmonella Typhimurium.

Emergence of Salmonella enterica serovar Indiana and California isolates with concurrent resistance to cefotaxime, amikacin and ciprofloxacin from chickens in China.

PubMed

Wang, Yongxiang; Zhang, Anyun; Yang, Yongqiang; Lei, Changwei; Jiang, Wei; Liu, Bihui; Shi, Hongping; Kong, Linghan; Cheng, Guangyang; Zhang, Xiuzhong; Yang, Xin; Wang, Hongning

2017-12-04

The aim of this study was to investigate the prevalence and characterization of Salmonella concerning the poultry industry in China. A total of 170 non-duplicate Salmonella isolates were recovered from the 1540 chicken samples. Among the Salmonella isolates from chickens, the predominant serovars were S. enterica serovar Enteritidis (S. Enteritidis) (49/170, 28.8%), S. enterica serovar Indiana (S. Indiana) (37/170, 21.8%) and S. enterica serovar California (S. California) (34/170, 20.0%). High antimicrobial resistance was observed for ciprofloxacin (68.2%), amikacin (48.2%) and cefotaxime (44.7%). Of particular concerns were the 18 S. Indiana and 17 S. California isolates, which were concurrently resistant to cefotaxime, amikacin and ciprofloxacin. The bla CTX-M genes, 16S rRNA methylase genes (armA, rmtD or rmtC) and five plasmid-mediated quinolone resistance (PMQR) determinants (aac(6')-Ib-cr, oqxAB, qnrB, qepA and qnrD) were identified in 18 S. Indiana and 17 S. California isolates. To clarify their genetic correlation, pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) were further conducted. PFGE profiles showed that the majority of S. Indiana and S. California isolates were clonally unrelated with a standard cut-off of 85%. The results of MLST demonstrated that ST17 and ST40 were the most common ST types in S. Indiana and S. California isolates, respectively. Our findings indicated that the multiple antibiotic resistant S. Indiana and S. California isolates were widespread in chicken in China and might pose a potential threat to public health. Copyright © 2017 Elsevier B.V. All rights reserved.

Preliminary Investigations on the Distribution of Leptospira Serovars in Domestic Animals in North-west Morocco.

PubMed

Benkirane, A; Noury, S; Hartskeerl, R A; Goris, M G A; Ahmed, A; Nally, J E

2016-04-01

Leptospirosis is a neglected zoonosis of global importance with a complex epidemiology that affects humans, domestic and wild mammals. However, due to the diversity of clinical signs and difficulties of establishing a confirmatory laboratory diagnosis, the disease remains poorly investigated, particularly in the developing world. In Morocco, a descriptive study of the seroprevalence of Leptospira infection in animals has never been undertaken. To fill this gap, the current study was conducted on a subset of animals in north-west Morocco as a preliminary step towards understanding the epidemiological patterns of animal leptospirosis in the country. The study was conducted on 289 serum samples collected between January and April 2012 from dogs, cattle, sheep, goats and donkeys in the areas of Rabat-Temara, Sidi Kacem and Oulmes. All serum samples were tested by the MAT with 14 reference strains of the most prevalent pathogenic serovars of Leptospira and two serovars of non-pathogenic Leptospira. The overall seroprevalence of Leptospira in cattle, sheep, goats, dogs and donkeys was 15%, 18%, 20%, 21% and 20%, respectively. The most prevalent serogroups found in each species were Ballum, Sejroe, and Australis in cattle, Ballum, Australis and Sejroe in sheep, Australis and Ballum in goats, Javanica and Australis in donkey and Australis, Ballum and Canicola in dogs. Of all the serogroups tested in this study, Icterohaemorrhagiae, the only serogroup which has been previously reported in humans in Morocco, was rarely reactive. The majority of reactive sera were collected from low land areas. A large number of sera samples classified as seronegative when tested against pathogenic leptospires were positive when tested against non-pathogenic leptospires; this is suggestive of possible novel, as yet unclassified, Leptospira serovars in Morocco. Eleven of thirteen sheep urine samples were positive by real-time PCR confirming their role as Leptospira carriers in Morocco. © 2014

Resuscitation by Ferrioxamine E of Stressed Salmonella enterica Serovar Typhimurium from Soil and Water Microcosms

PubMed Central

Reissbrodt, R.; Heier, H.; Tschäpe, H.; Kingsley, R. A.; Williams, P. H.

2000-01-01

Storage of Salmonella enterica serovar Typhimurium strains in soil and water microcosms resulted in loss of culturability on standard plating media. Prior incubation in buffered peptone water supplemented with ferrioxamine E markedly extended the time that bacteria were recoverable by plating, except in the case of mutants deficient in ferrioxamine E uptake. PMID:10966440

Chlortetracycline and florfenicol induce expression of genes associated with pathogenicity in multidrug-resistant Salmonella enterica serovar Typhimurium

USDA-ARS?s Scientific Manuscript database

Background Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium (S. Typhimurium) is a serious public health threat as infections caused by these strains are more difficult and expensive to treat. Livestock serve as a reservoir for MDR Salmonella, and the antibiotics chlortetracycline an...

Transcriptomic analysis of swarm motility phenotype of Salmonella enterica serovar Typhimurium mutant defective in periplasmic glucan synthesis

USDA-ARS?s Scientific Manuscript database

Movement of food-borne pathogens on moist surfaces enables them to migrate towards more favorable niches and facilitate their survival for extended periods of time. Salmonella enterica serovar Typhimurium mutants defective in OPG synthesis are unable to exhibit motility on moist surfaces (swarming) ...

Lack of efflux mediated quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi A

PubMed Central

Baucheron, Sylvie; Monchaux, Isabelle; Le Hello, Simon; Weill, François-Xavier; Cloeckaert, Axel

2014-01-01

Salmonella enterica serovars Typhi and Paratyphi A isolates from human patients in France displaying different levels of resistance to quinolones or fluoroquinolones were studied for resistance mechanisms to these antimicrobial agents. All resistant isolates carried either single or multiple target gene mutations (i.e., in gyrA, gyrB, or parC) correlating with the resistance levels observed. Active efflux, through upregulation of multipartite efflux systems, has also been previously reported as contributing mechanism for other serovars. Therefore, we investigated also the occurrence of non-target gene mutations in regulatory regions affecting efflux pump expression. However, no mutation was detected in these regions in both Typhi and Paratyphi isolates of this study. Besides, no overexpression of the major efflux systems was observed for these isolates. Nevertheless, a large deletion of 2334 bp was identified in the acrS-acrE region of all S. Typhi strains but which did not affect the resistance phenotype. As being specific to S. Typhi, this deletion could be used for specific molecular detection purposes. In conclusion, the different levels of quinolone or FQ resistance in both S. Typhi and S. Paratyphi A seem to rely only on target modifications. PMID:24478769

Prevalence, Virulence Genes and Antimicrobial Resistance Profiles of Salmonella Serovars from Retail Beef in Selangor, Malaysia

PubMed Central

Thung, Tze Y.; Radu, Son; Mahyudin, Nor A.; Rukayadi, Yaya; Zakaria, Zunita; Mazlan, Nurzafirah; Tan, Boon H.; Lee, Epeng; Yeoh, Soo L.; Chin, Yih Z.; Tan, Chia W.; Kuan, Chee H.; Basri, Dayang F.; Wan Mohamed Radzi, Che W. J.

2018-01-01

The aim of the present study was to investigate the prevalence of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium in retail beef from different retail markets of Selangor area, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 240 retail beef meat samples (chuck = 60; rib = 60; round = 60; sirloin = 60) were randomly collected. The multiplex polymerase chain reaction (mPCR) in combination with the most probable number (MPN) method was employed to detect Salmonella spp., S. Enteritidis and S. Typhimurium in the meat samples. The prevalence of Salmonella spp., S. Enteritidis and S. Typhimurium in 240 beef meat samples were 7.50, 1.25, and 0.83%, respectively. The microbial loads of total Salmonella was found in the range of <3 to 15 MPN/g. Eight different serovars of Salmonella were identified among the 23 isolates, and S. Agona was the predominant serovar (26.09%). Interestingly, all the Salmonella isolates were resistant to penicillin, erythromycin and vancomycin, but the sensitivity was observed for tetracycline, gentamicin and amoxicillin/clavulanic acid. All 23 isolates were resistant to at least three antibiotics. Two S. Typhimurium isolates (8.70%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.56 which shown resistance to nine antibiotics. PCR analysis of virulence genes showed that all Salmonella isolates (100%) were positive for the invA gene. Meanwhile, pefA was only identified in S. Enteritidis and S. Typhimurium. The findings in this study indicate that retail beef products tested were widely contaminated with multi-drug resistant (MDR) Salmonella and various virulence genes are present among the isolated Salmonella serovars. PMID:29379488

Prevalence, Virulence Genes and Antimicrobial Resistance Profiles of Salmonella Serovars from Retail Beef in Selangor, Malaysia.

PubMed

Thung, Tze Y; Radu, Son; Mahyudin, Nor A; Rukayadi, Yaya; Zakaria, Zunita; Mazlan, Nurzafirah; Tan, Boon H; Lee, Epeng; Yeoh, Soo L; Chin, Yih Z; Tan, Chia W; Kuan, Chee H; Basri, Dayang F; Wan Mohamed Radzi, Che W J

2017-01-01

The aim of the present study was to investigate the prevalence of Salmonella spp., Salmonella Enteritidis and Salmonella Typhimurium in retail beef from different retail markets of Selangor area, as well as, to assess their pathogenic potential and antimicrobial resistance. A total of 240 retail beef meat samples (chuck = 60; rib = 60; round = 60; sirloin = 60) were randomly collected. The multiplex polymerase chain reaction (mPCR) in combination with the most probable number (MPN) method was employed to detect Salmonella spp., S . Enteritidis and S . Typhimurium in the meat samples. The prevalence of Salmonella spp., S . Enteritidis and S . Typhimurium in 240 beef meat samples were 7.50, 1.25, and 0.83%, respectively. The microbial loads of total Salmonella was found in the range of <3 to 15 MPN/g. Eight different serovars of Salmonella were identified among the 23 isolates, and S . Agona was the predominant serovar (26.09%). Interestingly, all the Salmonella isolates were resistant to penicillin, erythromycin and vancomycin, but the sensitivity was observed for tetracycline, gentamicin and amoxicillin/clavulanic acid. All 23 isolates were resistant to at least three antibiotics. Two S . Typhimurium isolates (8.70%) exhibited the highest multiple antibiotic resistance (MAR) index value of 0.56 which shown resistance to nine antibiotics. PCR analysis of virulence genes showed that all Salmonella isolates (100%) were positive for the invA gene. Meanwhile, pefA was only identified in S . Enteritidis and S . Typhimurium. The findings in this study indicate that retail beef products tested were widely contaminated with multi-drug resistant (MDR) Salmonella and various virulence genes are present among the isolated Salmonella serovars.

Whole Genome Shotgun Sequencing Shows Selection on Leptospira Regulatory Proteins During in vitro Culture Attenuation.

PubMed

Lehmann, Jason S; Corey, Victoria C; Ricaldi, Jessica N; Vinetz, Joseph M; Winzeler, Elizabeth A; Matthias, Michael A

2016-02-01

Leptospirosis is the most common zoonotic disease worldwide with an estimated 500,000 severe cases reported annually, and case fatality rates of 12-25%, due primarily to acute kidney and lung injuries. Despite its prevalence, the molecular mechanisms underlying leptospirosis pathogenesis remain poorly understood. To identify virulence-related genes in Leptospira interrogans, we delineated cumulative genome changes that occurred during serial in vitro passage of a highly virulent strain of L. interrogans serovar Lai into a nearly avirulent isogenic derivative. Comparison of protein coding and computationally predicted noncoding RNA (ncRNA) genes between these two polyclonal strains identified 15 nonsynonymous single nucleotide variant (nsSNV) alleles that increased in frequency and 19 that decreased, whereas no changes in allelic frequency were observed among the ncRNA genes. Some of the nsSNV alleles were in six genes shown previously to be transcriptionally upregulated during exposure to in vivo-like conditions. Five of these nsSNVs were in evolutionarily conserved positions in genes related to signal transduction and metabolism. Frequency changes of minor nsSNV alleles identified in this study likely contributed to the loss of virulence during serial in vitro culture. The identification of new virulence-associated genes should spur additional experimental inquiry into their potential role in Leptospira pathogenesis. © The American Society of Tropical Medicine and Hygiene.

Lineage II (Serovar 1/2a and 1/2c) Human Listeria monocytogenes Pulsed-Field Gel Electrophoresis Types Divided into PFGE Groups Using the Band Patterns Below 145.5 kb.

PubMed

Lopez-Valladares, Gloria; Danielsson-Tham, Marie-Louise; Goering, Richard V; Tham, Wilhelm

2017-01-01

Among 504 clinical lineage II isolates of Listeria monocytogenes isolated during 1958-2010 in Sweden, 119 pulsed-field gel electrophoresis (PFGE) types (AscI) have been identified based on the number and distribution of all banding patterns in each DNA profile. In this study, these types were further divided into PFGE groups based on the configuration of small bands with sizes <145.5 kb. The 504 isolates included 483 serovar 1/2a isolates distributed into 114 PFGE types and 21 serovar 1/2c isolates distributed into 9 PFGE types; these were further divided into 21 PFGE groups. PFGE group, that is, configuration of small bands below 145.5 kb, and serovars were correlated. L. monocytogenes isolates belonging to PFGE groups A, B, C, E, F, H, K, L, M, S, V, W, Y, and Ö-6 to Ö-12 shared serovar 1/2a, with one exception. PFGE group E also included two PFGE types sharing serovar 1/2c and four PFGE types belonging to either serovar 1/2a or 1/2c. Isolates belonging to PFGE group N shared serovar 1/2c. In contrast to lineage I isolates, small fragments <33.3 kb were visible in all L. monocytogenes isolates belonging to lineage II. In the results from both the present and previous studies, the genomic region of small bands was genetically more conservative than in large bands. The distribution of these small bands established the relatedness of strains and defined a genetic marker for both lineages I and II, while also establishing their serogroup. The division of L. monocytogenes PFGE types into PFGE groups is advantageous as the profile of every new isolate can be identified easily and quickly through first studying the PFGE group affiliation of the isolate based on the smaller band patterns <145.5 kb, and then identifying the PFGE type based on the band patterns >145.5 kb.

Multilocus sequence typing (MLST) of leptospiral strains isolated from two geographic locations of Tamil Nadu, India.

PubMed

Kanagavel, Murugesan; Princy Margreat, Alphonse Asirvatham; Arunkumar, Manivel; Prabhakaran, Shanmugarajan Gnanasekaran; Shanmughapriya, Santhanam; Natarajaseenivasan, Kalimuthusamy

2016-01-01

Here the rodent carrier status for the transmission of human leptospirosis in Tiruchirappalli, district, Tamil Nadu, India was assessed. The predominantly circulating leptospiral STs were recognized by multilocus sequence typing (MLST). A total of 113 rodents were trapped from different provinces of the Tiruchirappalli district. The most prevalent rodent was Bandicota bengalensis (37.2%), and of the total, 52.2% (n=59) rodents were found to be positive for leptospiral 16S rRNA. These results were validated with a leptospiral culture positivity of 45.8% (n=27). Three isolates from Chennai (2 rodents and 1 human) and 1 human isolate from Tiruchirappalli were included to understand the spatial variations and to track the source of human leptospirosis. The serogroup, serovar, and species level identification of all 31 isolates identified 28 to be Leptospira borgpetersenii serovar Javanica and three as Leptospira interrogans serovar Autumnalis. MLST analysis defined all isolates to the existing ST profiles (ST145 and ST27) with the exception of 6 L. borgpetersenii (ST DR) isolates that showed variations in the sucA and pfkB loci. The DR ST was locally confined to Chatram province of Tiruchirappalli suggesting an epidemiological link. The predominant STs, ST145 and ST-DR form a group, indicating the presence of original strain that subsequently diverged evolutionarily into two STs. The variations between L. borgpetersenii in sucA and pfkB loci may be an indication that evolutionary changes transpired in Tiruchirappalli. Copyright © 2015 Elsevier B.V. All rights reserved.

Etiological agents causing leptospirosis in Sri Lanka: A review.

PubMed

Naotunna, Chamidri; Agampodi, Suneth Buddhika; Agampodi, Thilini Chanchala

2016-04-01

To systematically review the etiological agent causing human leptospirosis in Sri Lanka. Published articles on leptospirosis and Leptospira in Sri Lanka were all reviewed to determine serovar, strain and species level identification of Leptospira. After screening process, 74 full text articles/reports were reviewed and among of them, 12 published papers describing isolation of Leptospira from Sri Lankan patients/animals, 5 molecular epidemiology papers on newer typing methods citing Sri Lanka isolates, with a descriptions of the isolates and 6 published papers reporting PCR based species level identification were identified. Published literature showed that more than 40 strains classified under at least 20 serovars and 10 serogroups have been isolated from Sri Lanka. These isolates belong to four species, namely, Leptospira interrogans, Leptospira kirschneri, Leptospira borgpetersenii, and Leptospira santarosai. In addition, recent studies on direct patient samples without culture and isolation showed Leptospira from Leptospira weilli is also circulating in Sri Lanka. Multi locus sequence typing showed 13 genotypes of Leptospira from Sri Lankan isolates. This review shows the diversity of Leptospira in Sri Lanka, but culture isolation data has not been published in Sri Lanka during last 30 years. Copyright © 2016 Hainan Medical College. Production and hosting by Elsevier B.V. All rights reserved.

Evaluation of the recombinant LipL32 in enzyme-linked immunosorbent assay for the serodiagnosis of bovine leptospirosis.

PubMed

Bomfim, Maria Rosa Quaresma; Ko, Albert; Koury, Matilde Cota

2005-08-10

The recombinant leptospiral protein LipL32 was evaluated for use in the diagnosis of bovine leptospirosis by enzyme-linked immunosorbent assay (rLipL32 IgG ELISA). The microscopic agglutination test (MAT) of 150 serum samples from cattle suspected of leptospirosis showed that 125 (83.3%) samples had positive reciprocal agglutination titres, which ranged from 100 to 1600. The highest titres were observed for the serovars Hardjoprajitno and Bratislava. In the rLipL32 IgG ELISA, 83.3% of the samples were positive. The sensitivity of IgG ELISA for 125 bovine sera, which had MAT titres of greater than or equal to 100, was 100%. ELISA showed a specificity of 100% with 58 bovine sera, which were negative at a 1:50 dilution in the MAT for Leptospira interrogans serovars. When analytical specificity of the IgG ELISA was evaluted using 60 bovine serum samples from animals showing serum antibodies to other pathogens that cause abortion in cattle, such as Babesia sp., Anaplasma sp. and Brucella sp. and no cross-reaction was observed. The recombinant LipL32 IgG ELISA can be an alternative to the MAT for diagnosis of leptospiral infection in cattle.

[Demonstration of intraocular leptospira in 4 horses suffering from equine recurrent uveitis (ERU)].

PubMed

Brem, S; Gerhards, H; Wollanke, B; Meyer, P; Kopp, H

1998-01-01

Vitreous samples from 43 horses which underwent vitrectomy because of equine recurrent uveitis (ERU) were cultured for leptospires. Out of 4 vitreous samples (4/43 = 9%), leptospires could be isolated. In 3 cases, serovar grippotyphosa, and in one case, a serovar out of the serogroup Australis were identified. So for the first time, in several horses with ERU in vivo cultures of vitreous material were positive for leptospires. A strong evidence of association between leptospiral infection and uveitis is discussed for many years. In this investigation the leptospiral etiology is confirmed. Vitreous material from 42 and serum samples from 40 horses were tested for specific antibodies to leptospira by microagglutination test (MAT). In 34 vitreous samples (34/42 = 81%), leptospiral antibody titers of 1:50 or higher were detected. In 33 horses (33/40 = 83%) leptospiral antibody titers of 1:50 or higher could also be detected in the serum. Altogether, leptospiral antibodies were detected by the MAT in the serum and in the vitreous material of 39 of 43 horses (= 91%) subjected to vitrectomy. These results indicate, that ERU is probably often a sequel to systemic Leptospira interrogans infection. The presence of intact leptospires and specific antibodies in eyes affected with ERU indicates a local antibody production to leptospira organisms and/or their antigens.

Evidence for Wild Crocodiles as a Risk for Human Leptospirosis, Mexico.

PubMed

Pérez-Flores, Jonathan; Charruau, Pierre; Cedeño-Vázquez, Rogelio; Atilano, Daniel

2017-03-01

Sentinel species such as crocodilians are used to monitor the health of ecosystems. However, few studies have documented the presence of zoonotic diseases in wild populations of these reptiles. Herein we analyzed 48 serum samples from Crocodylus acutus (n = 34) and C. moreletii (n = 14) from different sites in the state of Quintana Roo (Mexico) to detect antibodies to Leptospira interrogans by means of a microscopic agglutination test (MAT). Crocodylus acutus and C. moreletii tested positive to 11 and 9 serovars, respectively, with Grippotyphosa being the serovar with the highest prevalence in Cozumel island (100%), Banco Chinchorro Biosphere Reserve (70.6%), and Río Hondo (100%), while in Chichankanab Lake, it was Bratislava (75%). Titers ranged from 1:50 to 1:3200, and the most frequent was 1:50 in all study sites. Leptospira is present in fresh and saltwater individuals due to the resistance of the bacterium in both environments. Cases of infected people involved with crocodile handling and egg collection suggest that these reptiles could play an important role in the transmission of leptospirosis. Preventive medicine programs should consider the monitoring of reptiles, and testing the soil and water, to prevent outbreaks of leptospirosis in facilities containing crocodiles.

Disease Surveillance of California Ground Squirrels ( Spermophilus beecheyi ) in a Drive-through Zoo in Oregon, USA.

PubMed

Beest, Julia Ter; Cushing, Andrew; McClean, Modesto; Hsu, Wendy; Bildfell, Robert

2017-07-01

Rodents and other small wild mammals are often considered to be pests and vectors for disease in zoos that house small populations of valuable threatened and endangered animals. In 2005, three nonhuman primates at a drive-through zoo in Oregon, US, acquired tularemia from an unknown source. Due to an abundance of California ground squirrels ( Spermophilus beecheyi ) on zoo grounds, we instituted serosurveillance of this species from July through September 2008 to determine the prevalence of antibodies against pathogens considered to be potentially transmissible to collection animals. Serologic testing was performed for Francisella tularensis ; Leptospira interrogans serovars Canicola, Grippotyphosa, Hardjo, Icterohemorrhagiae, and Pomona; Toxoplasma gondii ; and Yersinia pestis . All squirrels were seronegative for Yersinia pestis (0%; 0/45) and Toxoplasma gondii (0%; 0/20); there was a prevalence of 2% (1/45) for Francisella tularensis antibodies and 57% (24/42) were positive for various Leptospira serovars. Although it remains unclear whether ground squirrels present a significant risk for transmission of disease to zoo animals, vaccination of high-risk zoo animals against leptospirosis warrants consideration. Beyond this, continued vigilance and persistence with various forms of pest control may reduce the likelihood of disease transmission from wildlife hosts to animals in human care.

Characterization of Leptospira isolates from humans and the environment in Uruguay.

PubMed

Meny, Paulina; Menéndez, Clara; Quintero, Jair; Hernández, Elba; Ríos, Cristina; Balassiano, Ilana Teruszkin; Trindade, Camilla Nunes Dos Reis; Vital-Brazil, Juliana Magalhães; Ramos, Tatiane Mendes Varela; Ashfield, Natalia; Feble, Camila; Avila, Esthefani; Schelotto, Felipe; Varela, Gustavo

2017-12-21

Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers' samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance.

PATHOGENIC LEPTOSPIRA SEROVARS IN FREE-LIVING SEA LIONS IN THE GULF OF CALIFORNIA AND ALONG THE BAJA CALIFORNIA COAST OF MEXICO.

PubMed

Avalos-Téllez, Rosalía; Carrillo-Casas, Erika M; Atilano-López, Daniel; Godínez-Reyes, Carlos R; Díaz-Aparicio, Efrén; Ramírez-Delgado, David; Ramírez-Echenique, María F; Leyva-Leyva, Margarita; Suzán, Gerardo; Suárez-Güemes, Francisco

2016-04-28

The California sea lion ( Zalophus californianus ), a permanent inhabitant of the Gulf of California in Mexico, is susceptible to pathogenic Leptospira spp. infection, which can result in hepatic and renal damage and may lead to renal failure and death. During summer 2013, we used the microscopic agglutination test (MAT) to investigate the prevalence of anti-Leptospira antibodies in blood of clinically healthy sea lion pups from seven rookery islands on the Pacific Coast of Baja California (Pacific Ocean) and in the Gulf of California. We also used PCR to examine blood for Leptospira DNA. Isolation of Leptospira in liquid media was unsuccessful. We found higher antibody prevalence in sea lions from the rookery islands in the gulf than in those from the Pacific Coast. Antibodies against 11 serovars were identified in the Gulf of California population; the most frequent reactions were against serovars Bataviae (90%), Pyrogenes (86%), Wolffi (86%), Celledoni (71%), and Pomona (65%). In the Pacific Ocean population, MAT was positive against eight serovars, where Wolffi (88%), Pomona (75%), and Bataviae (70%) were the most frequent. Serum samples agglutinated with more than one Leptospira serovar. The maximum titer was 3,200. Each island had a different serology profile, and islands combined showed a distinct profile for each region. We detected pathogenic Leptospira DNA in 63% of blood samples, but we found no saprophytic Leptospira. Positive PCR results were obtained in blood samples with high and low MAT titers. Together, these two methods enhance the diagnosis and interpretation of sea lion leptospirosis. Our results may be related to human activities or the presence of other reservoirs with which sea lions interact, and they may also be related to sea lion stranding.

Horizontal Acquisition of a Multidrug-Resistance Module (R-type ASSuT) Is Responsible for the Monophasic Phenotype in a Widespread Clone of Salmonella Serovar 4,[5],12:i:.

PubMed

García, Patricia; Malorny, Burkhard; Rodicio, M Rosario; Stephan, Roger; Hächler, Herbert; Guerra, Beatriz; Lucarelli, Claudia

2016-01-01

Salmonella enterica serovar 4,[5],12:i:- is a monophasic variant of S. Typhimurium incapable of expressing the second-phase flagellar antigen (fljAB operon), and it is recognized to be one of the most prevalent serovars causing human infections. A clonal lineage characterized by phage type DT193, PulseNet PFGE profile STYMXB.0131 and multidrug resistance to ampicillin, streptomycin, sulphonamides and tetracycline (R-type ASSuT) is commonly circulating in Europe. In this study we determined the deletions affecting the fljAB operon and the resistance region responsible for the R-type ASSuT in a strain of Salmonella enterica serovar 4,5,12:i:- DT193/STYMXB.0131, through an approach based on PCRs and Southern blot hybridization of genomic DNA. Using a set of nine specific PCRs, the prevalence of the resistance region was assessed in a collection of 144 S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains isolated from Germany, Switzerland and Italy. A 28 kb-region is embedded between the loci STM2759 and iroB, replacing the DNA located in between, including the fljAB operon. It encompasses the genes bla TEM-1, strA-strB, sul2 and tet(B) responsible for the R-type ASSuT together with genes involved in plasmid replication and orfs of unknown function characteristically located on IncH1 plasmids. Its location and internal structure is fairly conserved in S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 strains regardless of the isolation source or country. Hence, in the S. enterica serovar 4,[5],12:i:-/ASSuT/STYMXB.0131 clonal lineage widespread in Germany, Switzerland and Italy, a resistance region derived from IncH1 plasmids has replaced the chromosomal region encoding the second flagellar phase and is an example of the stabilization of new plasmid-derived genetic material due to integration into the bacterial chromosome.

[Eukaryotic expression of Leptospira interrogans lipL32/1-ompL1/1 fusion gene encoding genus-specific protein antigens and the immunoreactivity of expression products].

PubMed

Yan, Jie; Zhao, Shou-feng; Mao, Ya-fei; Ruan, Ping; Luo, Yi-hui; Li, Shu-ping; Li, Li-wei

2005-01-01

To construct the eukaryotic expression system of L.interrogans lipL32/1-ompL1/1 fusion gene and to identify the immunoreactivity of expression products. PCR with linking primer was used to construct the fusion gene lipL32/1-ompL1/1. The P.pastoris eukaryotic expression system of the fusion gene, pPIC9K-lipL32/1-ompL1/1-P. pastorisGS115, was constructed after the fusion gene was cloned and sequenced. Colony with phenotype His(+)Mut(+) was isolated by using MD and MM plates and His(+) Mut(+) transformant with high resistance to G418 was screened out by using YPD plate. Using lysate of His(+) Mut(+) colony with high copies of the target gene digested with yeast lyase as the template and 5'AOX1 and 3'AOX1 as the primers, the target fusion gene in chromosome DNA of the constructed P. pastoris engineering strain was detected by PCR. Methanol in BMMY medium was used to induce the target recombinant protein rLipL32/1-rOmpL1/1 expression. rLipL32/1-rOmpL1/1 in the medium supernatant was extracted by using ammonium sulfate precipitation and Ni-NTA affinity chromatography. Output and immunoreactivity of rLipL32/1-rOmpL1/1 were measured by SDS-PAGE and Western blot methods, respectively. Amplification fragments of the obtained fusion gene lipL32/1-ompL1/1 was 1794 bp in size. The homogeneity of nucleotide and putative amino acid sequences of the fusion gene were as high as 99.94 % and 100 %, respectively, compared with the sequences of original lipL32/1 and ompL1/1 genotypes. The constructed eukaryotic expression system was able to secrete rLipL32/1-rOmpL1/1 with an output of 10 % of the total proteins in the supernatant, which located the expected position after SDS-PAGE. The rabbit anti-rLipL32/1 and anti-rOmpL1/1 sera could combine the expressed rLipL32/1-rOmpL1/1. An eukaryotic expression system with high efficiency in P.pastoris of L.interrogans lipL32/1-ompL1/1 fusion gene was successfully constructed in this study. The expressed fusion protein shows specific

Whole genome sequencing of multidrug-resistant Salmonella enterica serovar Typhimurium isolated from humans and poultry in Burkina Faso

USDA-ARS?s Scientific Manuscript database

Background. Multidrug-resistant Salmonella is an important cause of morbidity and mortality in developing countries. The aim of this study was to characterize and compare multidrug-resistant Salmonella enterica serovar Typhimurium isolates from patients and poultry feces. Methods. Salmonella strains...

Draft Genome Sequences of 18 Salmonella enterica subsp. enterica Serovar Oranienburg Strains Isolated from Rivers in Northwestern Mexico

PubMed Central

Casteñeda-Ruelas, Gloria M.; Carreón-Gaxiola, César; Castelán-Sánchez, Hugo G.; Acatzi-Silva, Abraham; Romero-Martínez, Salvador; García-Molina, Alejandra

2017-01-01

ABSTRACT Salmonella enterica subsp. enterica serovar Oranienburg is recognized as a foodborne pathogen widely distributed in the environment. Here, we report 18 draft genomes of S. Oranienburg strains isolated from rivers in the northwestern region of Mexico. PMID:28280020

Prevalence of nontyphoidal Salmonella and Salmonella strains with conjugative antimicrobial-resistant serovars contaminating animal feed in Texas

USDA-ARS?s Scientific Manuscript database

The objective of this study was to characterize 365 nontyphoidal Salmonella enterica isolates from animal feed. Among the 365 isolates, 78 serovars were identified. Twenty-four isolates (7.0%) were recovered from three of six medicated feed types. Three of these isolates derived from the medicate...

MARTX Toxin in the Zoonotic Serovar of Vibrio vulnificus Triggers an Early Cytokine Storm in Mice

PubMed Central

Murciano, Celia; Lee, Chung-Te; Fernández-Bravo, Ana; Hsieh, Tsung-Han; Fouz, Belén; Hor, Lien-I; Amaro, Carmen

2017-01-01

Vibrio vulnificus biotype 2-serovar E is a zoonotic clonal complex that can cause death by sepsis in humans and fish. Unlike other biotypes, Bt2 produces a unique type of MARTXVv (Multifunctional-Autoprocessive-Repeats-in-Toxin; RtxA13), which is encoded by a gene duplicated in the pVvBt2 plasmid and chromosome II. In this work, we analyzed the activity of this toxin and its role in human sepsis by performing in vitro, ex vivo, and in vivo assays. First, we demonstrated that the ACD domain, present exclusively in this toxin variant, effectively has an actin-cross-linking activity. Second, we determined that the whole toxin caused death of human endotheliocytes and monocytes by lysis and apoptosis, respectively. Finally, we tested the hypothesis that RtxA13 contributes to human death caused by this zoonotic serovar by triggering an early cytokine storm in blood. To this end, we used a Bt2-SerE strain (R99) together with its rtxA13 deficient mutant, and a Bt1 strain (YJ016) producing RtxA11 (the most studied MARTXVv) together with its rtxA11 deficient mutant, as controls. Our results showed that RtxA13 was essential for virulence, as R99ΔΔrtxA13 was completely avirulent in our murine model of infection, and that R99, but not strain YJ016, induced an early, strong and dysregulated immune response involving the up-regulation of a high number of genes. This dysregulated immune response was directly linked to RtxA13. Based on these results and those obtained ex vivo (human blood), we propose a model of infection for the zoonotic serovar of V. vulnificus, in which RtxA13 would act as a sepsis-inducing toxin. PMID:28775962

Role of anionic charges of osmoregulated periplasmic glucans of Salmonella enterica Serovar Typhimurium SL1344 in mice virulence

USDA-ARS?s Scientific Manuscript database

Osmoregulated periplasmic glucans (OPGs) are important periplasmic constituents of Salmonella spp. and are required for optimal growth in hypoosmotic environments such as irrigation and vegetable wash waters as well as for mice virulence. opgB gene of Salmonella enterica serovar Typhimurium was ide...

Development and Evaluation of a Multiplex Real-Time Polymerase Chain Reaction Procedure to Clinically Type Prevalent Salmonella enterica Serovars

PubMed Central

Muñoz, Nélida; Diaz-Osorio, Miguel; Moreno, Jaime; Sánchez-Jiménez, Miryan; Cardona-Castro, Nora

2010-01-01

A multiplex real-time polymerase chain reaction procedure was developed to identify the most prevalent clinical isolates of Salmonella enterica subsp. enterica. Genes from the rfb, fliC, fljB, and viaB groups that encode the O, H, and Vi antigens were used to design 15 primer pairs and TaqMan probes specific for the genes rfbJ, wzx, fliC, fljB, wcdB, the sdf-l sequence, and invA, which was used as an internal amplification control. The primers and probes were variously combined into six sets. The first round of reactions used two of these sets to detect Salmonella O:4, O:9, O:7, O:8, and O:3,10 serogroups. Once the serogroups were identified, the results of a second round of reactions that used primers and probes for the flagellar antigen l genes, 1,2; e,h; g,m; d; e,n,x; and z10, and the Vi gene were used to identify individual serovars. The procedure was standardized using 18 Salmonella reference strains and other enterobacteria. The procedure's reliability and sensitivity was evaluated using 267 randomly chosen serotyped Salmonella clinical isolates. The procedure had a sensitivity of 95.5% and was 100% specific. Thus, our technique is a quick, sensitive, reliable, and specific means of identifying S. enterica serovars and can be used in conjunction with traditional serotyping. Other primer and probe combinations could be used to increase the number of identifiable serovars. PMID:20110454

The relationship between the immune response and susceptibility to Salmonella enterica serovar Enteritidis infection in the laying hen

USDA-ARS?s Scientific Manuscript database

Chicken eggs are one of the main sources of human salmonellosis, with Salmonella enterica serovar Enteritidis the most frequent cause of human non-typhoid salmonellosis. Laying hens colonized with S. Enteritidis generally do not show clinical signs. The bacteria colonize and invade the intestinal ...

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence

PubMed Central

Eshghi, Azad; Henderson, Jeremy; Trent, M. Stephen

2015-01-01

Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. PMID:26283339

Draft Genome Sequences of 18 Salmonella enterica subsp. enterica Serovar Oranienburg Strains Isolated from Rivers in Northwestern Mexico.

PubMed

Casteñeda-Ruelas, Gloria M; Carreón-Gaxiola, César; Castelán-Sánchez, Hugo G; Acatzi-Silva, Abraham; Romero-Martínez, Salvador; García-Molina, Alejandra; Jiménez-Edeza, Maribel

2017-03-09

Salmonella enterica subsp. enterica serovar Oranienburg is recognized as a foodborne pathogen widely distributed in the environment. Here, we report 18 draft genomes of S  Oranienburg strains isolated from rivers in the northwestern region of Mexico. Copyright © 2017 Casteñeda-Ruelas et al.

Effect of Challenge Temperature and Solute Type on Heat Tolerance of Salmonella Serovars at Low Water Activity

PubMed Central

Mattick, K. L.; Jørgensen, F.; Wang, P.; Pound, J.; Vandeven, M. H.; Ward, L. R.; Legan, J. D.; Lappin-Scott, H. M.; Humphrey, T. J.

2001-01-01

Salmonella spp. are reported to have an increased heat tolerance at low water activity (aw; measured by relative vapor pressure [rvp]), achieved either by drying or by incorporating solutes. Much of the published data, however, cover only a narrow treatment range and have been analyzed by assuming first-order death kinetics. In this study, the death of Salmonella enterica serovar Typhimurium DT104 when exposed to 54 combinations of temperature (55 to 80°C) and aw (rvp 0.65 to 0.90, reduced using glucose-fructose) was investigated. The Weibull model (LogS = −btn) was used to describe microbial inactivation, and surface response models were developed to predict death rates for serovar Typhimurium at all points within the design surface. The models were evaluated with data generated by using six different Salmonella strains in place of serovar Typhimurium DT104 strain 30, two different solutes in place of glucose-fructose to reduce aw, or six low-aw foods artificially contaminated with Salmonella in place of the sugar broths. The data demonstrate that, at temperatures of ≥70°C, Salmonella cells at low aw were more heat tolerant than those at a higher aw but below 65°C the reverse was true. The same patterns were generated when sucrose (rvp 0.80 compared with 0.90) or NaCl (0.75 compared with 0.90) was used to reduce aw, but the extent of the protection afforded varied with solute type. The predictions of thermal death rates in the low-aw foods were usually fail-safe, but the few exceptions highlight the importance of validating models with specific foods that may have additional factors affecting survival. PMID:11526015

Longitudinal Study of Distributions of Similar Antimicrobial-Resistant Salmonella Serovars in Pigs and Their Environment in Two Distinct Swine Production Systems

PubMed Central

Keelara, Shivaramu; Scott, H. Morgan; Morrow, William M.; Gebreyes, Wondwossen A.; Correa, Maria; Nayak, Rajesh; Stefanova, Rossina

2013-01-01

The aim of this longitudinal study was to determine and compare the prevalences and genotypic profiles of antimicrobial-resistant (AR) Salmonella isolates from pigs reared in antimicrobial-free (ABF) and conventional production systems at farm, at slaughter, and in their environment. We collected 2,889 pig fecal and 2,122 environmental (feed, water, soil, lagoon, truck, and floor swabs) samples from 10 conventional and eight ABF longitudinal cohorts at different stages of production (farrowing, nursery, finishing) and slaughter (postevisceration, postchill, and mesenteric lymph nodes [MLN]). In addition, we collected 1,363 carcass swabs and 205 lairage and truck samples at slaughter. A total of 1,090 Salmonella isolates were recovered from the samples; these were isolated with a significantly higher prevalence in conventionally reared pigs (4.0%; n = 66) and their environment (11.7%; n = 156) than in ABF pigs (0.2%; n = 2) and their environment (0.6%; n = 5) (P < 0.001). Salmonella was isolated from all stages at slaughter, including the postchill step, in the two production systems. Salmonella prevalence was significantly higher in MLN extracted from conventional carcasses than those extracted from ABF carcasses (P < 0.001). We identified a total of 24 different serotypes, with Salmonella enterica serovar Typhimurium, Salmonella enterica serovar Anatum, Salmonella enterica serovar Infantis, and Salmonella enterica serovar Derby being predominant. The highest frequencies of antimicrobial resistance (AR) were exhibited to tetracycline (71%), sulfisoxazole (42%), and streptomycin (17%). Multidrug resistance (resistance to ≥3 antimicrobials; MDR) was detected in 27% (n = 254) of the Salmonella isolates from the conventional system. Our study reports a low prevalence of Salmonella in both production systems in pigs on farms, while a higher prevalence was detected among the carcasses at slaughter. The dynamics of Salmonella prevalence in pigs and carcasses were

Transposon Mutagenesis of Salmonella enterica Serovar Enteritidis Identifies Genes That Contribute to Invasiveness in Human and Chicken Cells and Survival in Egg Albumen

PubMed Central

Zhou, Xiaohui; Kim, Hye-Young; Call, Douglas R.; Guard, Jean

2012-01-01

Salmonella enterica serovar Enteritidis is an important food-borne pathogen, and chickens are a primary reservoir of human infection. While most knowledge about Salmonella pathogenesis is based on research conducted on Salmonella enterica serovar Typhimurium, S. Enteritidis is known to have pathobiology specific to chickens that impacts epidemiology in humans. Therefore, more information is needed about S. Enteritidis pathobiology in comparison to that of S. Typhimurium. We used transposon mutagenesis to identify S. Enteritidis virulence genes by assay of invasiveness in human intestinal epithelial (Caco-2) cells and chicken liver (LMH) cells and survival within chicken (HD-11) macrophages as a surrogate marker for virulence. A total of 4,330 transposon insertion mutants of an invasive G1 Nalr strain were screened using Caco-2 cells. This led to the identification of attenuating mutations in a total of 33 different loci, many of which include genes previously known to contribute to enteric infection (e.g., Salmonella pathogenicity island 1 [SPI-1], SPI-4, SPI-5, CS54, fliH, fljB, csgB, spvR, and rfbMN) in S. Enteritidis and other Salmonella serovars. Several genes or genomic islands that have not been reported previously (e.g., SPI-14, ksgA, SEN0034, SEN2278, and SEN3503) or that are absent in S. Typhimurium or in most other Salmonella serovars (e.g., pegD, SEN1152, SEN1393, and SEN1966) were also identified. Most mutants with reduced Caco-2 cell invasiveness also showed significantly reduced invasiveness in chicken liver cells and impaired survival in chicken macrophages and in egg albumen. Consequently, these genes may play an important role during infection of the chicken host and also contribute to successful egg contamination by S. Enteritidis. PMID:22988017

Genome Sequences of Multidrug-Resistant Salmonella enterica subsp. enterica Serovar Infantis Strains from Broiler Chicks in Hungary

PubMed Central

Wilk, Tímea; Szabó, Móni; Szmolka, Ama; Kiss, János; Barta, Endre; Nagy, Tibor

2016-01-01

Three strains of Salmonella enterica serovar Infantis isolated from healthy broiler chickens from 2012 to 2013 have been sequenced. Comparison of these and previously published S. Infantis genome sequences of broiler origin in 1996 and 2004 will provide new insight into the genome evolution and recent spread of S. Infantis in poultry. PMID:27979950

Complete Genome Sequences of Salmonella enterica Serovars Anatum and Anatum var. 15+, Isolated from Retail Ground Turkey

PubMed Central

Marasini, Daya; Abo-Shama, Usama H.

2016-01-01

The complete genome sequences of two isolates of Salmonella enterica serovars Anatum and Anatum var. 15+ revealed the presence of two plasmids of 112 kb and 3 kb in size in each. The chromosome of Salmonella Anatum (4.83 Mb) was slightly smaller than that of Salmonella Anatum var. 15+ (4.88 Mb). PMID:26798111

Resistance to essential oils affects survival of Salmonella enterica serovars in growing and harvested basil.

PubMed

Kisluk, Guy; Kalily, Emmanuel; Yaron, Sima

2013-10-01

The number of outbreaks of food-borne illness associated with consumption of fresh products has increased. A recent and noteworthy outbreak occurred in 2007. Basil contaminated with Salmonella enterica serovar Senftenberg was the source of this outbreak. Since basil produces high levels of antibacterial compounds the aim of this study was to investigate if the emerging outbreak reflects ecological changes that occurred as a result of development of resistance to ingredients of the basil oil. We irrigated basil plants with contaminated water containing two Salmonella serovars, Typhimurium and Senftenberg, and showed that Salmonella can survive on the basil plants for at least 100 days. S. Senftenberg counts in the phyllosphere were significantly higher than S. Typhimurium, moreover, S. Senftenberg was able to grow on stored harvested basil leaves. Susceptibility experiments demonstrated that S. Senftenberg is more resistant to basil oil and to its antimicrobial constituents: linalool, estragole and eugenol. This may indicate that S. Senftenberg had adapted to the basil environment by developing resistance to the basil oil. The emergence of resistant pathogens has a significant potential to change the ecology, and opens the way for pathogens to survive in new niches in the environment such as basil and other plants. © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology.

Influence of ethanol adaptation on Salmonella enterica serovar Enteritidis survival in acidic environments and expression of acid tolerance-related genes

USDA-ARS?s Scientific Manuscript database

Aims: Salmonella enterica serovar Enteritidis (S. Enteritidis) can encounter mild ethanol stress during its life cycle. However, adaptation to a stressful condition may affect bacterial resistance to subsequent stresses. Hence, this work was undertaken to investigate the influences of ethanol adapta...

Salmonella serovars and their distribution in Nigerian commercial chicken layer farms

PubMed Central

Fagbamila, Idowu Oluwabunmi; Barco, Lisa; Mancin, Marzia; Kwaga, Jacob; Ngulukun, Sati Samuel; Zavagnin, Paola; Lettini, Antonia Anna; Lorenzetto, Monica; Abdu, Paul Ayuba; Kabir, Junaidu; Umoh, Jarlath; Ricci, Antonia; Muhammad, Maryam

2017-01-01

Commercial poultry farms (n° 523), located in all the six regions of Nigeria were sampled with a view to generate baseline information about the distribution of Salmonella serovars in this country. Five different matrices (litter, dust, faeces, feed and water) were collected from each visited farm. Salmonella was isolated from at least one of the five matrices in 228 farms, with a farm prevalence of 43.6% (CI95[39.7–48.3%]). Altogether, 370 of 2615 samples collected (14.1%, CI95[12.8; 15.5%]) contained Salmonella. Considering the number of positive farms and the number of positive samples, it was evident that for the majority of the sampled farms, few samples were positive for Salmonella. With regard to the matrices, there was no difference in Salmonella prevalence among the five matrices considered. Of the 370 isolates serotyped, eighty-two different serotypes were identified and Salmonella Kentucky was identified as having the highest isolation rate in all the matrices sampled (16.2%), followed by S. Poona and S. Elisabethville. S. Kentucky was distributed across the country, whereas the other less frequent serovars had a more circumscribed diffusion. This is one of few comprehensive studies on the occurrence and distribution of Salmonella in commercial chicken layer farms from all the six regions of Nigeria. The relatively high prevalence rate documented in this study may be attributed to the generally poor infrastructure and low biosecurity measures in controlling stray animals, rodents and humans. Data collected could be valuable for instituting effective intervention strategies for Salmonella control in Nigeria and also in other developing countries with a similar poultry industry structure, with the final aim of reducing Salmonella spread in animals and ultimately in humans. PMID:28278292

Salmonella serovars and their distribution in Nigerian commercial chicken layer farms.

PubMed

Fagbamila, Idowu Oluwabunmi; Barco, Lisa; Mancin, Marzia; Kwaga, Jacob; Ngulukun, Sati Samuel; Zavagnin, Paola; Lettini, Antonia Anna; Lorenzetto, Monica; Abdu, Paul Ayuba; Kabir, Junaidu; Umoh, Jarlath; Ricci, Antonia; Muhammad, Maryam

2017-01-01

Commercial poultry farms (n° 523), located in all the six regions of Nigeria were sampled with a view to generate baseline information about the distribution of Salmonella serovars in this country. Five different matrices (litter, dust, faeces, feed and water) were collected from each visited farm. Salmonella was isolated from at least one of the five matrices in 228 farms, with a farm prevalence of 43.6% (CI95[39.7-48.3%]). Altogether, 370 of 2615 samples collected (14.1%, CI95[12.8; 15.5%]) contained Salmonella. Considering the number of positive farms and the number of positive samples, it was evident that for the majority of the sampled farms, few samples were positive for Salmonella. With regard to the matrices, there was no difference in Salmonella prevalence among the five matrices considered. Of the 370 isolates serotyped, eighty-two different serotypes were identified and Salmonella Kentucky was identified as having the highest isolation rate in all the matrices sampled (16.2%), followed by S. Poona and S. Elisabethville. S. Kentucky was distributed across the country, whereas the other less frequent serovars had a more circumscribed diffusion. This is one of few comprehensive studies on the occurrence and distribution of Salmonella in commercial chicken layer farms from all the six regions of Nigeria. The relatively high prevalence rate documented in this study may be attributed to the generally poor infrastructure and low biosecurity measures in controlling stray animals, rodents and humans. Data collected could be valuable for instituting effective intervention strategies for Salmonella control in Nigeria and also in other developing countries with a similar poultry industry structure, with the final aim of reducing Salmonella spread in animals and ultimately in humans.

Counts, serovars, and antimicrobial resistance phenotypes of Salmonella on raw chicken meat at retail in Colombia.

PubMed

Donado-Godoy, Pilar; Clavijo, Viviana; León, Maribel; Arevalo, Alejandra; Castellanos, Ricardo; Bernal, Johan; Tafur, Mc Allister; Ovalle, Maria Victoria; Alali, Walid Q; Hume, Michael; Romero-Zuñiga, Juan Jose; Walls, Isabel; Doyle, Michael P

2014-02-01

The objective of this study was to determine Salmonella counts, serovars, and antimicrobial-resistant phenotypes on retail raw chicken carcasses in Colombia. A total of 301 chicken carcasses were collected from six departments (one city per department) in Colombia. Samples were analyzed for Salmonella counts using the most-probable-number method as recommended by the U.S. Department of Agriculture, Food Safety Inspection Service protocol. A total of 378 isolates (268 from our previous study) were serotyped and tested for antimicrobial susceptibility. The overall Salmonella count (mean log most probable number per carcass ± 95% confidence interval) and prevalence were 2.1 (2.0 to 2.3) and 37%, respectively. There were significant differences (P < 0.05) by Salmonella levels (i.e., counts and prevalence) by storage temperature (i.e., frozen, chilled, or ambient), retail store type (wet markets, supermarkets, and independent markets), and poultry company (chicken produced by integrated or nonintegrated company). Frozen chicken had the lowest Salmonella levels compared with chicken stored at other temperatures, chickens from wet markets had higher levels than those from other retail store types, and chicken produced by integrated companies had lower levels than nonintegrated companies. Thirty-one Salmonella serovars were identified among 378 isolates, with Salmonella Paratyphi B tartrate-positive (i.e., Salmonella Paratyphi B dT+) the most prevalent (44.7%), followed by Heidelberg (19%), Enteritidis (17.7%), Typhimurium (5.3%), and Anatum (2.1%). Of all the Salmonella isolates, 35.2% were resistant to 1 to 5 antimicrobial agents, 24.6% to 6 to 10, and 33.9% to 11 to 15. Among all the serovars obtained, Salmonella Paratyphi B dT+ and Salmonella Heidelberg were the most antimicrobial resistant. Salmonella prevalence was determined to be high, whereas cell numbers were relatively low. These data can be used in developing risk assessment models for preventing the

Osmotic regulation of expression of two extracellular matrix-binding proteins and a haemolysin of Leptospira interrogans: differential effects on LigA and Sph2 extracellular release.

PubMed

Matsunaga, James; Medeiros, Marco A; Sanchez, Yolanda; Werneid, Kristian F; Ko, Albert I

2007-10-01

The life cycle of the pathogen Leptospira interrogans involves stages outside and inside the host. Entry of L. interrogans from moist environments into the host is likely to be accompanied by the induction of genes encoding virulence determinants and the concomitant repression of genes encoding products required for survival outside of the host. The expression of the adhesin LigA, the haemolysin Sph2 (Lk73.5) and the outer-membrane lipoprotein LipL36 of pathogenic Leptospira species have been reported to be regulated by mammalian host signals. A previous study demonstrated that raising the osmolarity of the leptospiral growth medium to physiological levels encountered in the host by addition of various salts enhanced the levels of cell-associated LigA and LigB and extracellular LigA. In this study, we systematically examined the effects of osmotic upshift with ionic and non-ionic solutes on expression of the known mammalian host-regulated leptospiral genes. The levels of cell-associated LigA, LigB and Sph2 increased at physiological osmolarity, whereas LipL36 levels decreased, corresponding to changes in specific transcript levels. These changes in expression occurred irrespective of whether sodium chloride or sucrose was used as the solute. The increase of cellular LigA, LigB and Sph2 protein levels occurred within hours of adding sodium chloride. Extracellular Sph2 levels increased when either sodium chloride or sucrose was added to achieve physiological osmolarity. In contrast, enhanced levels of extracellular LigA were observed only with an increase in ionic strength. These results indicate that the mechanisms for release of LigA and Sph2 differ during host infection. Thus, osmolarity not only affects leptospiral gene expression by affecting transcript levels of putative virulence determinants but also affects the release of such proteins into the surroundings.

Leptospira interrogans lpxD Homologue Is Required for Thermal Acclimatization and Virulence.

PubMed

Eshghi, Azad; Henderson, Jeremy; Trent, M Stephen; Picardeau, Mathieu

2015-11-01

Leptospirosis is an emerging disease with an annual occurrence of over 1 million human cases worldwide. Pathogenic Leptospira bacteria are maintained in zoonotic cycles involving a diverse array of mammals, with the capacity to survive outside the host in aquatic environments. Survival in the diverse environments encountered by Leptospira likely requires various adaptive mechanisms. Little is known about Leptospira outer membrane modification systems, which may contribute to the capacity of these bacteria to successfully inhabit and colonize diverse environments and animal hosts. Leptospira bacteria carry two genes annotated as UDP-3-O-[3-hydroxymyristoyl] glucosamine N-acyltransferase genes (la0512 and la4326 [lpxD1 and lpxD2]) that in other bacteria are involved in the early steps of biosynthesis of lipid A, the membrane lipid anchor of lipopolysaccharide. Inactivation of only one of these genes, la0512/lpxD1, imparted sensitivity to the host physiological temperature (37°C) and rendered the bacteria avirulent in an animal infection model. Polymyxin B sensitivity assays revealed compromised outer membrane integrity in the lpxD1 mutant at host physiological temperature, but structural analysis of lipid A in the mutant revealed only minor changes in the lipid A moiety compared to that found in the wild-type strain. In accordance with this, an in trans complementation restored the phenotypes to a level comparable to that of the wild-type strain. These results suggest that the gene annotated as lpxD1 in Leptospira interrogans plays an important role in temperature adaptation and virulence in the animal infection model. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Human Infections Attributable to the d-Tartrate-Fermenting Variant of Salmonella enterica Serovar Paratyphi B in Germany Originate in Reptiles and, on Rare Occasions, Poultry

PubMed Central

Toboldt, Anne; Tietze, Erhard; Helmuth, Reiner; Fruth, Angelika; Junker, Ernst

2012-01-01

In this study, the population structure, incidence, and potential sources of human infection caused by the d-tartrate-fermenting variant of Salmonella enterica serovar Paratyphi B [S. Paratyphi B (dT+)] was investigated. In Germany, the serovar is frequently isolated from broilers. Therefore, a selection of 108 epidemiologically unrelated S. enterica serovar Paratyphi B (dT+) strains isolated in Germany between 2002 and 2010 especially from humans, poultry/poultry meat, and reptiles was investigated by phenotypic and genotypic methods. Strains isolated from poultry and products thereof were strongly associated with multilocus sequence type ST28 and showed antimicrobial multiresistance profiles. Pulsed-field gel electrophoresis XbaI profiles were highly homogeneous, with only a few minor XbaI profile variants. All strains isolated from reptiles, except one, were strongly associated with ST88, another distantly related type. Most of the strains were susceptible to antimicrobial agents, and XbaI profiles were heterogeneous. Strains isolated from humans yielded seven sequence types (STs) clustering in three distantly related lineages. The first lineage, comprising five STs, represented mainly strains belonging to ST43 and ST149. The other two lineages were represented only by one ST each, ST28 and ST88. The relatedness of strains based on the pathogenicity gene repertoire (102 markers tested) was mostly in agreement with the multilocus sequence type. Because ST28 was frequently isolated from poultry but rarely in humans over the 9-year period investigated, overall, this study indicates that in Germany S. enterica serovar Paratyphi B (dT+) poses a health risk preferentially by contact with reptiles and, to a less extent, by exposure to poultry or poultry meat. PMID:22885742

Characterization of a multidrug-resistant Salmonella enterica serovar Heidelberg outbreak strain in commercial turkeys: Colonization, transmission, and host transcriptional response

USDA-ARS?s Scientific Manuscript database

In recent years, multidrug-resistant (MDR) Salmonella enterica serovar Heidelberg has been associated with numerous human foodborne illness outbreaks due to consumption of poultry. For example, in 2011, an MDR S. Heidelberg outbreak associated with ground turkey sickened 136 individuals and resulted...

Genomic diversity and adaptation of Salmonella enterica serovar Typhimurium from analysis of six genomes of different phage types

PubMed Central

2013-01-01

Background Salmonella enterica serovar Typhimurium (or simply Typhimurium) is the most common serovar in both human infections and farm animals in Australia and many other countries. Typhimurium is a broad host range serovar but has also evolved into host-adapted variants (i.e. isolated from a particular host such as pigeons). Six Typhimurium strains of different phage types (defined by patterns of susceptibility to lysis by a set of bacteriophages) were analysed using Illumina high-throughput genome sequencing. Results Variations between strains were mainly due to single nucleotide polymorphisms (SNPs) with an average of 611 SNPs per strain, ranging from 391 SNPs to 922 SNPs. There were seven insertions/deletions (indels) involving whole or partial gene deletions, four inactivation events due to IS200 insertion and 15 pseudogenes due to early termination. Four of these inactivated or deleted genes may be virulence related. Nine prophage or prophage remnants were identified in the six strains. Gifsy-1, Gifsy-2 and the sopE2 and sspH2 phage remnants were present in all six genomes while Fels-1, Fels-2, ST64B, ST104 and CP4-57 were variably present. Four strains carried the 90-kb plasmid pSLT which contains several known virulence genes. However, two strains were found to lack the plasmid. In addition, one strain had a novel plasmid similar to Typhi strain CT18 plasmid pHCM2. Conclusion The genome data suggest that variations between strains were mainly due to accumulation of SNPs, some of which resulted in gene inactivation. Unique genetic elements that were common between host-adapted phage types were not found. This study advanced our understanding on the evolution and adaptation of Typhimurium at genomic level. PMID:24138507

rpoS-Regulated Core Genes Involved in the Competitive Fitness of Salmonella enterica Serovar Kentucky in the Intestines of Chickens

PubMed Central

Cheng, Ying; Pedroso, Adriana Ayres; Porwollik, Steffen; McClelland, Michael; Lee, Margie D.; Kwan, Tiffany; Zamperini, Katherine; Soni, Vivek; Sellers, Holly S.; Russell, Scott M.

2014-01-01

Salmonella enterica serovar Kentucky has become the most frequently isolated serovar from poultry in the United States over the past decade. Despite its prevalence in poultry, it causes few human illnesses in the United States. The dominance of S. Kentucky in poultry does not appear to be due to single introduction of a clonal strain, and its reduced virulence appears to correlate with the absence of virulence genes grvA, sseI, sopE, and sodC1. S. Kentucky's prevalence in poultry is possibly attributable to its metabolic adaptation to the chicken cecum. While there were no difference in the growth rate of S. Kentucky and S. Typhimurium grown microaerophilically in cecal contents, S. Kentucky persisted longer when chickens were coinfected with S. Typhimurium. The in vivo advantage that S. Kentucky has over S. Typhimurium appears to be due to differential regulation of core Salmonella genes via the stationary-phase sigma factor rpoS. Microarray analysis of Salmonella grown in cecal contents in vitro identified several metabolic genes and motility and adherence genes that are differentially activated in S. Kentucky. The contributions of four of these operons (mgl, prp, nar, and csg) to Salmonella colonization in chickens were assessed. Deletion of mgl and csg reduced S. Kentucky persistence in competition studies in chickens infected with wild-type or mutant strains. Subtle mutations affecting differential regulation of core Salmonella genes appear to be important in Salmonella's adaptation to its animal host and especially for S. Kentucky's emergence as the dominant serovar in poultry. PMID:25362062

Evaluation of a Multiplex PCR Assay for the Identification of Salmonella Serovars Enteritidis and Typhimurium Using Retail and Abattoir Samples.

PubMed

Ogunremi, Dele; Nadin-Davis, Susan; Dupras, Andrée Ann; Márquez, Imelda Gálvan; Omidi, Katayoun; Pope, Louise; Devenish, John; Burke, Teresa; Allain, Ray; Leclair, Daniel

2017-02-01

A multiplex PCR was developed to identify the two most common serovars of Salmonella causing foodborne illness in Canada, namely, serovars Enteritidis and Typhimurium. The PCR was designed to amplify DNA fragments from four Salmonella genes, namely, invA gene (211-bp fragment), iroB gene (309-bp fragment), Typhimurium STM 4497 (523-bp fragment), and Enteritidis SE147228 (612-bp fragment). In addition, a 1,026-bp ribosomal DNA (rDNA) fragment universally present in bacterial species was included in the assay as an internal control fragment. The detection rate of the PCR was 100% among Salmonella Enteritidis (n = 92) and Salmonella Typhimurium (n = 33) isolates. All tested Salmonella isolates (n = 194) were successfully identified based on the amplification of at least one Salmonella -specific DNA fragment. None of the four Salmonella DNA amplicons were detected in any of the non- Salmonella isolates (n = 126), indicating an exclusivity rate of 100%. When applied to crude extracts of 2,001 field isolates of Salmonella obtained during the course of a national microbiological baseline study in broiler chickens and chicken products sampled from abattoir and retail outlets, 163 isolates, or 8.1%, tested positive for Salmonella Enteritidis and another 80 isolates, or 4.0%, tested as Salmonella Typhimurium. All isolates identified by serological testing as Salmonella Enteritidis in the microbiological study were also identified by using the multiplex PCR. The new test can be used to identify or confirm pure isolates of the two serovars and is also amenable for integration into existing culture procedures for accurate detection of Salmonella colonies.

A novel prophage identified in strains from Salmonella enterica serovar Enteritidis is a phylogenetic signature of the lineage ST-1974

PubMed Central

D'Alessandro, Bruno; Pérez Escanda, Victoria; Balestrazzi, Lucía; Iriarte, Andrés; Pickard, Derek; Yim, Lucía; Chabalgoity, José Alejandro; Betancor, Laura

2018-01-01

Salmonella enterica serovar Enteritidis is a major agent of foodborne diseases worldwide. In Uruguay, this serovar was almost negligible until the mid 1990s but since then it has become the most prevalent. Previously, we characterized a collection of strains isolated from 1988 to 2005 and found that the two oldest strains were the most genetically divergent. In order to further characterize these strains, we sequenced and annotated eight genomes including those of the two oldest isolates. We report on the identification and characterization of a novel 44 kbp Salmonella prophage found exclusively in these two genomes. Sequence analysis reveals that the prophage is a mosaic, with homologous regions in different Salmonella prophages. It contains 60 coding sequences, including two genes, gogB and sseK3, involved in virulence and modulation of host immune response. Analysis of serovar Enteritidis genomes available in public databases confirmed that this prophage is absent in most of them, with the exception of a group of 154 genomes. All 154 strains carrying this prophage belong to the same sequence type (ST-1974), suggesting that its acquisition occurred in a common ancestor. We tested this by phylogenetic analysis of 203 genomes representative of the intraserovar diversity. The ST-1974 forms a distinctive monophyletic lineage, and the newly described prophage is a phylogenetic signature of this lineage that could be used as a molecular marker. The phylogenetic analysis also shows that the major ST (ST-11) is polyphyletic and might have given rise to almost all other STs, including ST-1974. PMID:29509137

Neutral Genomic Microevolution of a Recently Emerged Pathogen, Salmonella enterica Serovar Agona

PubMed Central

Litrup, Eva; Murphy, Ronan; Cormican, Martin; Fanning, Seamus; Brown, Derek; Guttman, David S.; Brisse, Sylvain; Achtman, Mark

2013-01-01

Salmonella enterica serovar Agona has caused multiple food-borne outbreaks of gastroenteritis since it was first isolated in 1952. We analyzed the genomes of 73 isolates from global sources, comparing five distinct outbreaks with sporadic infections as well as food contamination and the environment. Agona consists of three lineages with minimal mutational diversity: only 846 single nucleotide polymorphisms (SNPs) have accumulated in the non-repetitive, core genome since Agona evolved in 1932 and subsequently underwent a major population expansion in the 1960s. Homologous recombination with other serovars of S. enterica imported 42 recombinational tracts (360 kb) in 5/143 nodes within the genealogy, which resulted in 3,164 additional SNPs. In contrast to this paucity of genetic diversity, Agona is highly diverse according to pulsed-field gel electrophoresis (PFGE), which is used to assign isolates to outbreaks. PFGE diversity reflects a highly dynamic accessory genome associated with the gain or loss (indels) of 51 bacteriophages, 10 plasmids, and 6 integrative conjugational elements (ICE/IMEs), but did not correlate uniquely with outbreaks. Unlike the core genome, indels occurred repeatedly in independent nodes (homoplasies), resulting in inaccurate PFGE genealogies. The accessory genome contained only few cargo genes relevant to infection, other than antibiotic resistance. Thus, most of the genetic diversity within this recently emerged pathogen reflects changes in the accessory genome, or is due to recombination, but these changes seemed to reflect neutral processes rather than Darwinian selection. Each outbreak was caused by an independent clade, without universal, outbreak-associated genomic features, and none of the variable genes in the pan-genome seemed to be associated with an ability to cause outbreaks. PMID:23637636

Complete genome sequence of the thermotolerant foodborne pathogen Salmonella enterica serovar Senftenberg ATCC 43845 and phylogenetic analysis of loci encoding thermotolerance

USDA-ARS?s Scientific Manuscript database

Introduction: Previous studies in Cronobacter sakazakii, Klebsiella spp., and Escherichia coli have identified a genomic island that confers thermotolerance to its hosts. This island has recently been identified in Salmonella enterica serovar Senfentenberg ATCC 43845, a historically important, heat ...

Characterization of Leptospira isolates from humans and the environment in Uruguay

PubMed Central

Meny, Paulina; Menéndez, Clara; Quintero, Jair; Hernández, Elba; Ríos, Cristina; Balassiano, Ilana Teruszkin; Trindade, Camilla Nunes Dos Reis; Vital-Brazil, Juliana Magalhães; Ramos, Tatiane Mendes Varela; Ashfield, Natalia; Feble, Camila; Avila, Esthefani; Schelotto, Felipe; Varela, Gustavo

2017-01-01

ABSTRACT Laboratory diagnosis of human leptospirosis usually relies on indirect methods exploring specific immune response. Isolation and identification of the involved strains are cumbersome, but can provide biological resources for pathogenic studies and relevant information for guiding prevention and control measures. The aim of the research we are hereby reporting was the characterization of Leptospira isolates obtained from humans and the environment in Uruguay. Blood cultures were performed from early samples of 302 Uruguayan patients, mainly rural workers, and from 36 water samples taken from their living or working environments. Eight human isolates and seven environmental isolates were obtained and analyzed by end point Polymerase Chain Reaction (PCR), Multilocus Variable Number of Tandem Repeat Analysis (MLVA) and other molecular methods. Human isolates corresponded to several serogroups and serovars of Leptospira interrogans and Leptospira kirschneri species, probably reflecting the infection with similar involved Leptospira species and serovars of an extended animal reservoir in rural settings of the country, mostly dedicated to meat and dairy production. Culture-positive patients were older than usually affected workers, and presented signs and symptoms of severe illness. A high organic and circulating bacterial burden may explain an easier positive result from these workers’ samples. Environmental isolates were mainly identified as Leptospira biflexa strains, with a single L. meyeri isolate of uncertain significance. PMID:29267587

Heterologous Expression, Purification and Characterization of an Oligopeptidase A from the Pathogen Leptospira interrogans.

PubMed

Anu, Prasannan V; Madanan, Madathiparambil G; Nair, Ananthakrishnan J; Nair, Gangaprasad A; Nair, Govinda Pillai M; Sudhakaran, Perumana R; Satheeshkumar, Padikara K

2018-04-01

Oligopeptidases are enzymes involved in the degradation of short peptides (generally less than 30 amino acids in size) which help pathogens evade the host defence mechanisms. Leptospira is a zoonotic pathogen and causes leptospirosis in mammals. Proteome analysis of Leptospira revealed the presence of oligopeptidase A (OpdA) among other membrane proteins. To study the role of oligopeptidase in leptospirosis, the OpdA of L. interrogans was cloned and expressed in Escherichia coli with a histidine tag (His-tag). The protein showed maximum expression at 37 °C with 0.5 mM of IPTG after 2 h of induction. Recombinant OpdA protein was purified to homogeneity using Ni-affinity chromatography. The purified OpdA showed more than 80% inhibition with a serine protease inhibitor but the activity was reduced to 30% with the cysteine protease inhibitor. The peptidase activity was increased significantly in the presence of Zn 2+ at a neutral pH. Inhibitor assay indicate the presence of more than one active sites for peptidase activity as reported with the OpdA of E. coli and Salmonella. Over-expression of OpdA in E. coli BL21 (DE3) did not cause any negative effects on normal cell growth and viability. The role of OpdA as virulence factor in Leptospira and its potential as a therapeutic and diagnostic target in leptospirosis is yet to be identified.

Novel Leptospira interrogans protein Lsa32 is expressed during infection and binds laminin and plasminogen.

PubMed

Domingos, Renan F; Fernandes, Luis G; Romero, Eliete C; de Morais, Zenaide M; Vasconcellos, Silvio A; Nascimento, Ana L T O

2015-04-01

Pathogenic Leptospira is the aetiological agent of leptospirosis, a life-threatening disease of human and veterinary concern. The quest for novel antigens that could mediate host-pathogen interactions is being pursued. Owing to their location, these antigens have the potential to elicit numerous activities, including immune response and adhesion. This study focuses on a hypothetical protein of Leptospira, encoded by the gene LIC11089, and its three derived fragments: the N-terminal, intermediate and C terminus regions. The gene coding for the full-length protein and fragments was cloned and expressed in Escherichia coli BL21(SI) strain by using the expression vector pAE. The recombinant protein and fragments tagged with hexahistidine at the N terminus were purified by metal affinity chromatography. The leptospiral full-length protein, named Lsa32 (leptospiral surface adhesin, 32 kDa), adheres to laminin, with the C terminus region being responsible for this interaction. Lsa32 binds to plasminogen in a dose-dependent fashion, generating plasmin when an activator is provided. Moreover, antibodies present in leptospirosis serum samples were able to recognize Lsa32. Lsa32 is most likely a new surface protein of Leptospira, as revealed by proteinase K susceptibility. Altogether, our data suggest that this multifaceted protein is expressed during infection and may play a role in host-L. interrogans interactions. © 2015 The Authors.

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Infantis Strain SPE101, Isolated from a Chronic Human Infection

PubMed Central

Iriarte, Andrés; Giner-Lamia, Joaquín; Betancor, Laura; Astocondor, Lizeth; Cestero, Juan J.; Ochoa, Theresa; García, Coralith; Puente, José L.; Chabalgoity, José A.

2017-01-01

ABSTRACT We report a 4.99-Mb draft genome sequence of Salmonella enterica subsp. enterica serovar Infantis strain SPE101, isolated from feces of a 5-month-old breast-fed female showing diarrhea associated with severe dehydration and malnutrition. The infection prolonged for 6 months despite antibiotic treatment. PMID:28729277

Prevalence of antimicrobial resistance of non-typhoidal Salmonella serovars in retail aquaculture products.

PubMed

Zhang, Jianmin; Yang, Xiaowei; Kuang, Dai; Shi, Xianming; Xiao, Wenjia; Zhang, Jing; Gu, Zhen; Xu, Xuebin; Meng, Jianghong

2015-10-01

Aquaculture products can become sources of Salmonella by exposure to contaminated water or through processing practices, thus representing a public health hazard. A study was conducted on Salmonella contamination in aquaculture products sampled from marketplaces and retailers in Shanghai, China. A total of 730 samples (including fish, shellfish, bullfrog, clam, shrimp and others) were obtained from 2006 to 2011. Among them, 217 (29.7%) were positive for Salmonella. Thirty-eight serovars were identified in the 217 Salmonella isolates. The most prevalent were Salmonella Aberdeen (18.4%), S. Wandsworth (12.0%), S. Thompson (9.2%), S. Singapore (5.5%), S. Stanley (4.6%), S. Schwarzengrund (4.6%), S. Hvittingfoss (4.1%) and S. Typhimurium (4.1%). Many resistant isolates were detected, with 69.6% resistant to at least one antimicrobial drug. We observed high resistance to sulfonamides (56.5%), tetracycline (34.1%), streptomycin (28.6%), ampicillin (23.5%) and nalidixic acid (21.2%). Lower levels of resistance were found for gentamicin (3.2%), ciprofloxacin (2.3%), ceftiofur (1.3%), cefotaxime (0.9%), ceftazidime (0.5%) and cefepime (0.5%). A total of 43.3% of the Salmonella isolates were multidrug-resistant and 44 different resistance patterns were found. This study provided data on the prevalence, serovars and antimicrobial resistance of Salmonella from retail aquaculture products in Shanghai, and indicated the need for monitoring programs for microbiologic safety in such projects and for more prudent drug use in aquaculture production in order to reduce the risk of development and spread of antimicrobial resistance. Copyright © 2015 Elsevier B.V. All rights reserved.

Modulation of systemic and mucosal immunity against an inactivated vaccine of Newcastle disease virus by oral co-administration of live attenuated Salmonella enterica serovar Typhimurium expressing chicken interleukin-18 and interferon-α

PubMed Central

RAHMAN, Md. Masudur; UYANGAA, Erdenebelig; HAN, Young Woo; HUR, Jin; PARK, Sang-Youel; LEE, John Hwa; KIM, Koanhoi; EO, Seong Kug

2014-01-01

Newcastle disease (ND) is a highly contagious disease of chickens causing significant economic losses worldwide. Due to limitations in the efficacy against currently circulating ND viruses, existing vaccination strategies require improvements, and incorporating immunomodulatory cytokines with existing vaccines might be a novel approach. Here, we investigated the systemic and mucosal immunomodulatory properties of oral co-administration of chicken interleukin-18 (chIL-18) and chicken interferon-α (chIFN-α) using attenuated Salmonella enterica serovar Typhimurium on an inactivated ND vaccine. Our results demonstrate that oral administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α provided enhanced systemic and mucosal immune responses, as determined by serum hemagglutination inhibition antibody and NDV Ag-specific IgG as well as NDV Ag-specific IgA in lung and duodenal lavages of chickens immunized with inactivated ND vaccine via the intramuscular or intranasal route. Notably, combined oral administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α significantly enhanced systemic and mucosal immunity in ND-vaccinated chickens, compared to single administration of S. enterica serovar Typhimurium expressing chIL-18 or chIFN-α. In addition, oral co-administration of S. enterica serovar Typhimurium expressing chIL-18 and chIFN-α provided enhanced NDV Ag-specific proliferation of peripheral blood mononuclear cells and Th1-biased cell-mediated immunity, compared to single administration of either construct. Therefore, our results provide valuable insight into the modulation of systemic and mucosal immunity by incorporation of immunomodulatory chIL-18 and chIFN-α using Salmonella vaccines into existing ND vaccines. PMID:25502364

Infection of the reproductive tract and eggs with Salmonella enterica serovar pullorum in the chicken is associated with suppression of cellular immunity at sexual maturity.

PubMed

Wigley, Paul; Hulme, Scott D; Powers, Claire; Beal, Richard K; Berchieri, Angelo; Smith, Adrian; Barrow, Paul

2005-05-01

Salmonella enterica serovar Pullorum causes persistent infections in laying hens. Splenic macrophages are the main site of persistence. At sexual maturity, numbers of bacteria increase and spread to the reproductive tract, which may result in vertical transmission to eggs or chicks. In this study we demonstrate that both male and female chickens may develop a carrier state following infection but that the increases in bacterial numbers and spread to the reproductive tract are phenomena restricted to hens, indicating that such changes are likely to be related to the onset of egg laying. The immunological responses during the carrier state and through the onset of laying in hens were determined. These indicate that chickens produce both humoral and T-cell responses to infection, but at the onset of laying both the T-cell response to Salmonella and nonspecific responses to mitogenic stimulation fall sharply in both infected and noninfected birds. The fall in T-cell responsiveness coincided with the increase in numbers of Salmonella serovar Pullorum and its spread to the reproductive tract. Three weeks after the onset of egg laying, T-cell responsiveness began to increase and bacterial numbers declined. Specific antibody levels changed little at the onset of laying but increased following the rise in bacterial numbers in a manner reminiscent of a secondary antibody response to rechallenge. These findings indicate that a nonspecific suppression of cellular responses occurs at the onset of laying and plays a major role the ability of Salmonella serovar Pullorum to infect the reproductive tract, leading to transmission to eggs. The loss of T-cell activity at the point of laying also has implications for Salmonella enterica serovar Enteritidis infection and transmission to eggs, along with its control by vaccination offering a "window of opportunity" in which infection may occur.

Demonstration of persistent contamination of a cooked egg product production facility with Salmonella enterica serovar Tennessee and characterization of the persistent strain.

PubMed

Jakočiūnė, D; Bisgaard, M; Pedersen, K; Olsen, J E

2014-08-01

The aim of this study was to investigate whether continuous contamination of light pasteurized egg products with Salmonella enterica serovar Tennessee (S. Tennessee) at a large European producer of industrial egg products was caused by persistent contamination of the production facility and to characterize the persistent strains. Seventy-three S. Tennessee isolates collected from products over a 3-year period with intermittent contamination, and 15 control strains were compared by pulsed field gel electrophoresis (PFGE) using two enzymes. Forty-five case isolates distributed throughout the full period were shown to belong to one profile type. Isolates representing different PFGE profiles were all assigned to ST 319 by multilocus sequence typing (MLST). The case isolates did not show a higher ability to form biofilm on a plastic surface than noncase isolates. Characteristically, members of the persistent clone were weak producers of H2 S in laboratory medium. S. Tennessee isolated from the case was able to grow better in pasteurized egg product compared with other serovars investigated. It was concluded that the contamination was caused by a persistent strain in the production facility and that this strain apparently had adapted to grow in the relevant egg product. S. Tennessee has previously been associated with persistence in hatching facilities. This is the first report of persistent contamination of an egg production facility with this serovar. © 2014 The Society for Applied Microbiology.

Prevalence, serovars, phage types, and antibiotic susceptibilities of Salmonella strains isolated from animals in the United Arab Emirates from 1996 to 2009.

PubMed

Münch, Sebastian; Braun, Peggy; Wernery, Ulrich; Kinne, Jörg; Pees, Michael; Flieger, Antje; Tietze, Erhard; Rabsch, Wolfgang

2012-10-01

The aim of this study was to give some insights into the prevalence, serovars, phage types, and antibiotic resistances of Salmonella from animal origin in the United Arab Emirates. Data on diagnostic samples from animals (n = 20,871) examined for Salmonella between 1996 and 2009 were extracted from the databases of the Central Veterinary Research Laboratory in Dubai and from typed strains (n = 1052) from the Robert Koch Institute, Wernigerode Branch in Germany and analyzed for general and animal-specific trends. Salmonella was isolated from 1,928 (9 %) of the 20,871 samples examined. Among the 1,052 typed strains, most were from camels (n = 232), falcons (n = 166), bustards (n = 101), antelopes (n = 66), and horses (n = 63). The predominant serovars were Salmonella Typhimurium (25 %), Salmonella Kentucky (8 %), followed by Salmonella Frintrop (7 %), and Salmonella Hindmarsh (5 %). When analyzed by animal species, the most frequent serovars in camels were Salmonella Frintrop (28 %) and Salmonella Hindmarsh (21 %), in falcons Salmonella Typhimurium (32 %), in bustards Salmonella Kentucky (19 %), in antelopes Salmonella Typhimurium (9 %), and in horses Salmonella Typhimurium (17 %) and S. Kentucky (16 %). Resistance of all typed Salmonella strains (n = 1052) was most often seen to tetracycline (23 %), streptomycin (22 %), nalidixic acid (18 %), and ampicillin (15 %). These data show trends in the epidemiology of Salmonella in different animal species which can be used as a base for future prevention, control, and therapy strategies.

Asymptomatic and chronic carriage of Leptospira interrogans serovar Pomona in California sea lions (Zalophus californianus)

USDA-ARS?s Scientific Manuscript database

Since 1970, periodic outbreaks of leptospirosis, caused by pathogenic spirochetes in the genus Leptospira, have caused morbidity and mortality of California sea lions (Zalophus californianus) along the Pacific coast of North America. Yearly seasonal epizootics of varying magnitude occur between the ...

The agricultural antibiotic carbadox induces prophage and antibiotic resistance gene transfer in multidrug-resistant salmonella enterica serovar typhimurium DT104

USDA-ARS?s Scientific Manuscript database

Non-typhoidal Salmonella strains cause ~1 million cases of foodborne disease each year in the U.S. and are a leading cause of food-related deaths. The prevalence of multidrug-resistant (MDR) Salmonella serovars has increased over the last few decades, and infection with these strains has an increase...

Draft Genome Sequence of Salmonella enterica subsp. enterica Serovar Infantis Strain SPE101, Isolated from a Chronic Human Infection.

PubMed

Iriarte, Andrés; Giner-Lamia, Joaquín; Silva, Claudia; Betancor, Laura; Astocondor, Lizeth; Cestero, Juan J; Ochoa, Theresa; García, Coralith; Puente, José L; Chabalgoity, José A; García-Del Portillo, Francisco

2017-07-20

We report a 4.99-Mb draft genome sequence of Salmonella enterica subsp. enterica serovar Infantis strain SPE101, isolated from feces of a 5-month-old breast-fed female showing diarrhea associated with severe dehydration and malnutrition. The infection prolonged for 6 months despite antibiotic treatment. Copyright © 2017 Iriarte et al.

[Construction of the eukaryotic recombinant vector and expression of the outer membrane protein LipL32 gene from Leptospira serovar Lai].

PubMed

Huang, Bi; Bao, Lang; Zhong, Qi; Shang, Zheng-ling; Zhang, Hui-dong; Zhang, Ying

2008-02-01

To construct the eukaryotic experssion vector of LipL32 gene from Leptospira serovar Lai and express the recombinant plasmid in COS-7 cell. The LipL32 gene was amplified from Leptospira strain 017 genomic DNA by PCR and cloned into pcDNA3.1, through restriction nuclease enzyme digestion. Then the recombinant plasmid was transformed into E.coli DH5alpha. After identified by nuclease digestion, PCR and sequencing analysis, the recombinant vector was transfected into COS-7 cell with lipsome. The expression of the target gene was detected by RT-PCR and Western blot. The eukaryotic experssion vector pcDNA3.1-LipL32 was successfully constructed and stably expressed in COS-7 cell. The eukaryotic recombinant vector of outer membrane protein LipL32 gene from Leptospira serovar Lai can be expressed in mammalian cell, which provides an experimental basis for the application of the Leptospira DNA vaccine.

Chimeric epitope vaccine against Leptospira interrogans infection and induced specific immunity in guinea pigs.

PubMed

Lin, Xu'ai; Xiao, Guohui; Luo, Dongjiao; Kong, Liangliang; Chen, Xu; Sun, Dexter; Yan, Jie

2016-10-14

Leptospirosis is an important reemerging zoonosis, with more than half a million cases reported annually, and is caused by pathogenic Leptospira species. Development of a universal vaccine is one of the major strategic goals to overcome the disease burden of leptospirosis. In this study, a chimeric multi-epitope protein-based vaccine was designed and tested for its potency to induce a specific immune response and provide protection against L. interrogans infection. The protein, containing four repeats of six T- and B-cell combined epitopes from the leptospiral outer membrane proteins, OmpL1, LipL32 and LipL21, was expressed and purified. Western blot analysis showed that the recombinant protein (named r4R) mainly expressed in a soluble pattern, and reacted with antibodies raised in rabbit against heat-killed Leptospira and in guinea pigs against the r4R vaccine. Microscopic agglutination tests showed that r4R antisera was immunological cross-reactive with a range of Chinese standard reference strains of Leptospira belonging to different serogroups. In guinea pigs, the r4R vaccine induced a Th1-biased immune response, as reflected by the IgG2a/IgG1 ratio and cytokine production of stimulated splenocytes derived from immunized animals. Finally, r4R-immunized guinea pigs showed increased survival of lethal Leptospira challenges compared with PBS-immunized animals and tissue damage and leptospiral colonization of the kidney were reduced. The multi-epitope chimeric r4R protein is a promising antigen for the development of a universal cross-reactive vaccine against leptospirosis.

Analysis of LexA binding sites and transcriptomics in response to genotoxic stress in Leptospira interrogans.

PubMed

Schons-Fonseca, Luciane; da Silva, Josefa B; Milanez, Juliana S; Domingos, Renan H; Smith, Janet L; Nakaya, Helder I; Grossman, Alan D; Ho, Paulo L; da Costa, Renata M A

2016-02-18

We determined the effects of DNA damage caused by ultraviolet radiation on gene expression in Leptospira interrogans using DNA microarrays. These data were integrated with DNA binding in vivo of LexA1, a regulator of the DNA damage response, assessed by chromatin immunoprecipitation and massively parallel DNA sequencing (ChIP-seq). In response to DNA damage, Leptospira induced expression of genes involved in DNA metabolism, in mobile genetic elements and defective prophages. The DNA repair genes involved in removal of photo-damage (e.g. nucleotide excision repair uvrABC, recombinases recBCD and resolvases ruvABC) were not induced. Genes involved in various metabolic pathways were down regulated, including genes involved in cell growth, RNA metabolism and the tricarboxylic acid cycle. From ChIP-seq data, we observed 24 LexA1 binding sites located throughout chromosome 1 and one binding site in chromosome 2. Expression of many, but not all, genes near those sites was increased following DNA damage. Binding sites were found as far as 550 bp upstream from the start codon, or 1 kb into the coding sequence. Our findings indicate that there is a shift in gene expression following DNA damage that represses genes involved in cell growth and virulence, and induces genes involved in mutagenesis and recombination. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Prevalence of Salmonella serovars and antimicrobial resistance profiles in poultry of Savar area, Bangladesh.

PubMed

Mahmud, Md Showkat; Bari, Md Latiful; Hossain, M Anwar

2011-10-01

Salmonellosis is one of the major concerns in the poultry industry and some serovars of Salmonella involve in zoonosis. This study determines the seroprevalence of Salmonella in poultry and their drug-resistant patterns, variability in infectivity and mortality rate of birds, and predilection of some serovars to cause zoonoses. The average seroprevalance of Salmonella in three different age groups was found to be 37.9%. A total of 503 samples were examined over a period of 1 year from five different poultry farms of a semiurban area of Savar, Dhaka, Bangladesh. The prevalence of Salmonella was recorded to be 21.1%. Salmonella was found high in dead birds (31.2%) than live birds (18.1%). Salmonella infection was higher (23.6%) in summer than in winter (12.9%) season. Among the 106 isolates, 46 belong to serogroup B (43%) and 60 isolates to serogroup D (57%). The highest Salmonella infection was recorded as 47.9% on the 30-35-week-old birds. A total of 106 Salmonella isolates were used for antimicrobial susceptibility test against 10 common antibiotics and 17 multiple drug resistance patterns were found. Among the isolates, 69 (65%) harbored plasmids 1-4 with size variation between >1.63 and >40 kb and rest 37 (35%) isolates were plasmid free but showed resistance against 5-10 antibiotics. The results of the present investigation suggested that multiple drug resistance is common among the Salmonella isolates of poultry and some of these isolates may have zoonotic implications.

Effect of farm type on within-herd Salmonella prevalence, serovar distribution, and antimicrobial resistance.

PubMed

Rasschaert, G; Michiels, J; Arijs, D; Wildemauwe, C; De Smet, S; Heyndrickx, M

2012-05-01

Salmonella represents a major challenge to the pig industry, as pork presents a risk for human salmonellosis. In this study, we have examined the effect of farm type on the prevalence of fattening pigs shedding Salmonella on 12 farms at risk for harboring Salmonella. On six open (grow-to-finish) and six closed (farrow-to-finish) farms, the prevalence of pigs shedding Salmonella was determined on two occasions approximately 2 months apart. The serovar, phage type, and antimicrobial resistance of the obtained Salmonella isolates were determined. On all farms, pigs shedding Salmonella were detected on at least one of the two sampling days. The mean within-herd prevalence was 7.8%. Closed farms were two times less likely to have pigs shedding Salmonella than open farms. On open farms, the odds of finding Salmonella shedding in pigs were 1.9 times higher when sampling was performed at slaughter age than when samples were taken halfway through the fattening period. Salmonella enterica serovar Typhimurium was the most predominant serotype, with a prevalence of 62 to 63% on both farm types. Of all the Salmonella Typhimurium isolates, 65% had the tetraresistant profile ASSuT (ampicillin, streptomycin, sulfonamide, and tetracycline) with or without additional resistance to trimethoprim-sulfonamide. Phage type DT120 seemed to be especially associated with this antimicrobial-resistant profile. The prevalence of Salmonella Typhimurium isolates showing resistance to ampicillin, streptomycin, tetracycline, sulfonamide, trimethoprim-sulfonamide, and lincomycin hydrochloride and spectinomycin sulfate tetrahydrate was significantly higher on open farms than on closed farms.

Prevalence of ColE1-like plasmids and kanamycin resistance genes in Salmonella enterica serovars.

PubMed

Chen, Chin-Yi; Lindsey, Rebecca L; Strobaugh, Terence P; Frye, Jonathan G; Meinersmann, Richard J

2010-10-01

Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kan(r)) phenotypes, 102 Kan(r) Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kan(r) Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3')-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group.

Salmonella enterica Serovar Enteritidis, England and Wales, 1945–2011

PubMed Central

Lane, Christopher R.; LeBaigue, Susan; Esan, Oluwaseun B.; Awofisyo, Adedoyin A.; Adams, Natalie L.; Fisher, Ian S.T.; Grant, Kathie A.; Peters, Tansy M.; Larkin, Lesley; Davies, Robert H.

2014-01-01

In England and Wales, the emergence of Salmonella enterica serovar Enteritidis resulted in the largest and most persistent epidemic of foodborne infection attributable to a single subtype of any pathogen since systematic national microbiological surveillance was established. We reviewed 67 years of surveillance data to examine the features, underlying causes, and overall effects of S. enterica ser. Enteritidis. The epidemic was associated with the consumption of contaminated chicken meat and eggs, and a decline in the number of infections began after the adoption of vaccination and other measures in production and distribution of chicken meat and eggs. We estimate that >525,000 persons became ill during the course of the epidemic, which caused a total of 6,750,000 days of illness, 27,000 hospitalizations, and 2,000 deaths. Measures undertaken to control the epidemic have resulted in a major reduction in foodborne disease in England and Wales. PMID:24960614

Comparison of Salmonella enterica Serovars Typhi and Typhimurium Reveals Typhoidal Serovar-Specific Responses to Bile

PubMed Central

Johnson, Rebecca; Ravenhall, Matt; Pickard, Derek; Dougan, Gordon; Byrne, Alexander

2017-01-01

ABSTRACT Salmonella enterica serovars Typhi and Typhimurium cause typhoid fever and gastroenteritis, respectively. A unique feature of typhoid infection is asymptomatic carriage within the gallbladder, which is linked with S. Typhi transmission. Despite this, S. Typhi responses to bile have been poorly studied. Transcriptome sequencing (RNA-Seq) of S. Typhi Ty2 and a clinical S. Typhi isolate belonging to the globally dominant H58 lineage (strain 129-0238), as well as S. Typhimurium 14028, revealed that 249, 389, and 453 genes, respectively, were differentially expressed in the presence of 3% bile compared to control cultures lacking bile. fad genes, the actP-acs operon, and putative sialic acid uptake and metabolism genes (t1787 to t1790) were upregulated in all strains following bile exposure, which may represent adaptation to the small intestine environment. Genes within the Salmonella pathogenicity island 1 (SPI-1), those encoding a type IIII secretion system (T3SS), and motility genes were significantly upregulated in both S. Typhi strains in bile but downregulated in S. Typhimurium. Western blots of the SPI-1 proteins SipC, SipD, SopB, and SopE validated the gene expression data. Consistent with this, bile significantly increased S. Typhi HeLa cell invasion, while S. Typhimurium invasion was significantly repressed. Protein stability assays demonstrated that in S. Typhi the half-life of HilD, the dominant regulator of SPI-1, is three times longer in the presence of bile; this increase in stability was independent of the acetyltransferase Pat. Overall, we found that S. Typhi exhibits a specific response to bile, especially with regard to virulence gene expression, which could impact pathogenesis and transmission. PMID:29229736

Validation of Baking To Control Salmonella Serovars in Hamburger Bun Manufacturing, and Evaluation of Enterococcus faecium ATCC 8459 and Saccharomyces cerevisiae as Nonpathogenic Surrogate Indicators.

PubMed

Channaiah, Lakshmikantha H; Holmgren, Elizabeth S; Michael, Minto; Sevart, Nicholas J; Milke, Donka; Schwan, Carla L; Krug, Matthew; Wilder, Amanda; Phebus, Randall K; Thippareddi, Harshavardhan; Milliken, George

2016-04-01

This study was conducted to validate a simulated commercial baking process for hamburger buns to destroy Salmonella serovars and to determine the appropriateness of using nonpathogenic surrogates (Enterococcus faecium ATCC 8459 or Saccharomyces cerevisiae) for in-plant process validation studies. Wheat flour was inoculated (∼6 log CFU/g) with three Salmonella serovars (Typhimurium, Newport, or Senftenberg 775W) or with E. faecium. Dough was formed, proofed, and baked to mimic commercial manufacturing conditions. Buns were baked for up to 13 min in a conventional oven (218.3°C), with internal crumb temperature increasing to ∼100°C during the first 8 min of baking and remaining at this temperature until removal from the oven. Salmonella and E. faecium populations were undetectable by enrichment (>6-log CFU/g reductions) after 9.0 and 11.5 min of baking, respectively, and ≥5-log-cycle reductions were achieved by 6.0 and 7.75 min, respectively. D-values of Salmonella (three-serovar cocktail) and E. faecium 8459 in dough were 28.64 and 133.33, 7.61 and 55.67, and 3.14 and 14.72 min at 55, 58, and 61°C, respectively, whereas D-values of S. cerevisiae were 18.73, 5.67, and 1.03 min at 52, 55, and 58°C, respectivly. The z-values of Salmonella, E. faecium, and S. cerevisiae were 6.58, 6.25, and 4.74°C, respectively. A high level of thermal lethality was observed for baking of typical hamburger bun dough, resulting in rapid elimination of high levels of the three-strain Salmonella cocktail; however, the lethality and microbial destruction kinetics should not be extrapolated to other bakery products without further research. E. faecium demonstrated greater thermal resistance compared with Salmonella during bun baking and could serve as a conservative surrogate to validate thermal process lethality in commercial bun baking operations. Low thermal tolerance of S. cerevisiae relative to Salmonella serovars limits its usefulness as a surrogate for process validations.

Polymerase chain reaction for detection of Leptospira spp. in clinical samples.

PubMed Central

Mérien, F; Amouriaux, P; Perolat, P; Baranton, G; Saint Girons, I

1992-01-01

A sensitive assay for Leptospira spp., the causative agent of leptospirosis, was developed on the basis of the polymerase chain reaction (PCR). A 331-bp sequence from the Leptospira interrogans serovar canicola rrs (16S) gene was amplified, and the PCR products were analyzed by DNA-DNA hybridization by using a 289-bp fragment internal to the amplified DNA. Specific PCR products also were obtained with DNA from the closely related nonpathogenic Leptospira biflexa but not with DNA from other spirochetes, such as Borrelia burgdorferi, Borrelia hermsii, Treponema denticola, Treponema pallidum, Spirochaeta aurantia, or more distant organisms such as Escherichia coli, Staphylococcus aureus, Mycobacterium tuberculosis, and Proteus mirabilis. The assay was able to detect as few as 10 bacteria. Leptospira DNA was detected in urine from experimentally infected mice. In addition, the test was found to be suitable for diagnosing leptospirosis in humans. Cerebrospinal fluid and urine from patients with leptospirosis were positive, whereas samples from control uninfected patients were negative. Images PMID:1400983

A Phylogenetic and Phenotypic Analysis of Salmonella enterica Serovar Weltevreden, an Emerging Agent of Diarrheal Disease in Tropical Regions

PubMed Central

Makendi, Carine; Page, Andrew J.; Wren, Brendan W.; Le Thi Phuong, Tu; Clare, Simon; Hale, Christine; Goulding, David; Klemm, Elizabeth J.; Pickard, Derek; Okoro, Chinyere; Hunt, Martin; Thompson, Corinne N.; Phu Huong Lan, Nguyen; Tran Do Hoang, Nhu; Thwaites, Guy E.; Le Hello, Simon; Brisabois, Anne; Weill, François-Xavier; Baker, Stephen; Dougan, Gordon

2016-01-01

Salmonella enterica serovar Weltevreden (S. Weltevreden) is an emerging cause of diarrheal and invasive disease in humans residing in tropical regions. Despite the regional and international emergence of this Salmonella serovar, relatively little is known about its genetic diversity, genomics or virulence potential in model systems. Here we used whole genome sequencing and bioinformatics analyses to define the phylogenetic structure of a diverse global selection of S. Weltevreden. Phylogenetic analysis of more than 100 isolates demonstrated that the population of S. Weltevreden can be segregated into two main phylogenetic clusters, one associated predominantly with continental Southeast Asia and the other more internationally dispersed. Subcluster analysis suggested the local evolution of S. Weltevreden within specific geographical regions. Four of the isolates were sequenced using long read sequencing to produce high quality reference genomes. Phenotypic analysis in Hep-2 cells and in a murine infection model indicated that S. Weltevreden were significantly attenuated in these models compared to the classical S. Typhimurium reference strain SL1344. Our work outlines novel insights into this important emerging pathogen and provides a baseline understanding for future research studies. PMID:26867150

Identification of novel Haemophilus parasuis serovar 5 vaccine candidates using an immunoproteomic approach.

PubMed

Li, Gang; Xie, Fang; Li, Jianjun; Liu, Jiao; Li, Dapeng; Zhang, Yanhe; Langford, Paul R; Li, Yanwen; Liu, Siguo; Wang, Chunlai

2017-06-23

Haemophilus parasuis is the aetiological agent of Glässer's disease, which is responsible for cases of fibrinous polyserositis, polyarthritis and meningitis. No vaccine is known that provides cross-protection against all serovars. The identification of novel immunoprotective antigens would undoubtedly contribute to the development of efficient subunit vaccines. In the present study, an immunoproteomic approach was used to analyze secreted proteins of H. parasuis and six proteins with high immunogenicity were identified. Five of them were successfully expressed, and their immunogenicity and protective efficacy were assessed in a mouse challenge model. All five proteins elicited strong humoral antibody and cellular immune responses in mice. They all effectively reduced the growth of H. parasuis in mouse organs and conferred different levels of protection (40-80%) against challenge. IgG subtype analysis revealed that the five proteins induce a bias toward a Th1-type immune response, and a significant increase was observed in the cytokine levels of IL-2, IFN-γ and Th2-specific IL-4 in the culture supernatants of splenocytes isolated from immunized mice. The results suggest that both Th1 and Th2 responses are involved in mediating protection. These data suggest that the five proteins could be potential subunit vaccine candidates for use to prevent H. parasuis infection. Haemophilus parasuis can cause huge financial loss in the swine industry worldwide. There are still no vaccines which can provide cross-protection against all serovars. To address this need, we applied an immunoproteomic approach involving 2-DE, MALDI-TOF/TOF MS and Western-blot to identify the secreted proteins which may be able to provide immunoprotection to this disease. We identified six immunogenic proteins, and the immunogenicity and protective efficacy were validated. This result provides a foundation for developing novel subunit vaccines against Haemophilus parasuis. Copyright © 2017

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities

USDA-ARS?s Scientific Manuscript database

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been significantly associated with egg-transmitted illness. Contamination of the edible conten...

MALDI-TOF mass spectrometry provides high accuracy in identification of Salmonella at species level but is limited to type or subtype Salmonella serovars.

PubMed

Kang, Lin; Li, Nan; Li, Ping; Zhou, Yang; Gao, Shan; Gao, Hongwei; Xin, Wenwen; Wang, Jinglin

2017-04-01

Salmonella can cause global foodborne illnesses in humans and many animals. The current diagnostic gold standard used for detecting Salmonella infection is microbiological culture followed by serological confirmation tests. However, these methods are complicated and time-consuming. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis offers some advantages in rapid identification, for example, simple and fast sample preparation, fast and automated measurement, and robust and reliable identification up to genus and species levels, possibly even to the strain level. In this study, we established a reference database for species identification using whole-cell MALDI-TOF MS; the database consisted of 12 obtained main spectra of the Salmonella culture collection strains belonged to seven serotypes. Eighty-two clinical isolates of Salmonella were identified using established database, and partial 16S rDNA gene sequencing and serological method were used as comparison. We found that MALDI-TOF mass spectrometry provided high accuracy in identification of Salmonella at species level but was limited to type or subtype Salmonella serovars. We also tried to find serovar-specific biomarkers and failed. Our study demonstrated that (a) MALDI-TOF MS was suitable for identification of Salmonella at species level with high accuracy and (b) that MALDI-TOF MS method presented in this study was not useful for serovar assignment of Salmonella currently, because of its low matching with serological method and (c) MALDI-TOF MS method presented in this study was not suitable to subtype S. typhimurium because of its low discriminatory ability.

A new multivalent (DHPPi/L4R) canine combination vaccine prevents infection, shedding and clinical signs following experimental challenge with four Leptospira serovars.

PubMed

Wilson, Stephen; Stirling, Catrina; Thomas, Anne; King, Vickie; Plevová, Edita; Chromá, Ludmila; Siedek, Elisabeth; Illambas, Joanna; Salt, Jeremy; Sture, Gordon

2013-06-28

Although effective vaccines have been developed against the common Leptospira serovars, they are still reported in clinical cases, while others are increasingly prevalent. The results from four challenge studies following vaccination of dogs with a new combination vaccine (DHPPi/L4R) containing inactivated L. serovars, L. canicola, L. icterohaemorrhagiae, L. bratislava and L. grippotyphosa conducted to satisfy the requirements of the European Pharmacopoeia monograph (01/2008:0447), are reported. Six week old dogs received two vaccinations, three weeks apart, and were challenged 25 days later with different isolates of the L. serovars. Clinical observations were recorded, and blood, urine and tissue samples were collected for analysis. Following challenge, non-vaccinated dogs demonstrated various clinical signs, while no vaccinated dogs were affected; significant differences in mean clinical scores were observed. Measurable antibody titres to each Leptospira antigen were seen in vaccinated dogs 21 days following the first vaccination, with further increases in antibody titres observed following challenge with the respective Leptospira strain. Non-vaccinated dogs remained seronegative until challenge. Leptospira were re-isolated from the blood, urine, kidney and liver of all non-vaccinated dogs following challenge. In contrast no vaccinated dogs had Leptospira re-isolated from the same tissues. Significant differences were seen in number of days with positive isolation (blood and urine) and in number of dogs with positive samples (kidney and liver). In conclusion, vaccination of dogs with the new vaccine induces protective immunity 25 days after second vaccination with protection against infection, renal infection and clinical signs following challenge. Copyright © 2013 Elsevier Ltd. All rights reserved.

[Optimization of prokaryotic expression conditions of Leptospira interrogans trigeminy genus-specific protein antigen based on surface response analysis].

PubMed

Wang, Jiang; Luo, Dongjiao; Sun, Aihua; Yan, Jie

2008-07-01

Lipoproteins LipL32 and LipL21 and transmembrane protein OMPL1 have been confirmed as the superficial genus-specific antigens of Leptospira interrogans, which can be used as antigens for developing a universal genetic engineering vaccine. In order to obtain high expression of an artificial fusion gene lipL32/1-lipL21-ompL1/2, we optimized prokaryotic expression conditions. We used surface response analysis based on the central composite design to optimize culture conditions of a new antigen protein by recombinant Escherichia coli DE3.The culture conditions included initial pH, induction start time, post-induction time, Isopropyl beta-D-thiogalactopyranoside (IPTG) concentration, and temperature. The maximal production of antigen protein was 37.78 mg/l. The optimal culture conditions for high recombinant fusion protein was determined: initial pH 7.9, induction start time 2.5 h, a post-induction time of 5.38 h, 0.20 mM IPTG, and a post-induction temperature of 31 degrees C. Surface response analysis based on CCD increased the target production. This statistical method reduced the number of experiments required for optimization and enabled rapid identification and integration of the key culture condition parameters for optimizing recombinant protein expression.

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015

PubMed Central

Peters, Tansy; Bertrand, Sophie; Björkman, Jonas T; Brandal, Lin T; Brown, Derek J; Erdõsi, Tímea; Heck, Max; Ibrahem, Salha; Johansson, Karin; Kornschober, Christian; Kotila, Saara M; Le Hello, Simon; Lienemann, Taru; Mattheus, Wesley; Nielsen, Eva Møller; Ragimbeau, Catherine; Rumore, Jillian; Sabol, Ashley; Torpdahl, Mia; Trees, Eija; Tuohy, Alma; de Pinna, Elizabeth

2017-01-01

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data. PMID:28277220

Multi-laboratory validation study of multilocus variable-number tandem repeat analysis (MLVA) for Salmonella enterica serovar Enteritidis, 2015.

PubMed

Peters, Tansy; Bertrand, Sophie; Björkman, Jonas T; Brandal, Lin T; Brown, Derek J; Erdõsi, Tímea; Heck, Max; Ibrahem, Salha; Johansson, Karin; Kornschober, Christian; Kotila, Saara M; Le Hello, Simon; Lienemann, Taru; Mattheus, Wesley; Nielsen, Eva Møller; Ragimbeau, Catherine; Rumore, Jillian; Sabol, Ashley; Torpdahl, Mia; Trees, Eija; Tuohy, Alma; de Pinna, Elizabeth

2017-03-02

Multilocus variable-number tandem repeat analysis (MLVA) is a rapid and reproducible typing method that is an important tool for investigation, as well as detection, of national and multinational outbreaks of a range of food-borne pathogens. Salmonella enterica serovar Enteritidis is the most common Salmonella serovar associated with human salmonellosis in the European Union/European Economic Area and North America. Fourteen laboratories from 13 countries in Europe and North America participated in a validation study for MLVA of S. Enteritidis targeting five loci. Following normalisation of fragment sizes using a set of reference strains, a blinded set of 24 strains with known allele sizes was analysed by each participant. The S. Enteritidis 5-loci MLVA protocol was shown to produce internationally comparable results as more than 90% of the participants reported less than 5% discrepant MLVA profiles. All 14 participating laboratories performed well, even those where experience with this typing method was limited. The raw fragment length data were consistent throughout, and the inter-laboratory validation helped to standardise the conversion of raw data to repeat numbers with at least two countries updating their internal procedures. However, differences in assigned MLVA profiles remain between well-established protocols and should be taken into account when exchanging data. This article is copyright of The Authors, 2017.

Colonization of internal organs by Salmonella serovars Heidelberg and Typhimurium in experimentally infected laying hens housed in enriched colony cages at different stocking densities

USDA-ARS?s Scientific Manuscript database

Contaminated eggs produced by infected commercial laying flocks are often implicated as sources of human infections with Salmonella Enteritidis, but Salmonella serovars Heidelberg and Typhimurium have also been associated with egg-transmitted illness. Contamination of the edible contents of eggs is ...

Prevalence of ColE1-Like Plasmids and Kanamycin Resistance Genes in Salmonella enterica Serovars ▿

PubMed Central

Chen, Chin-Yi; Lindsey, Rebecca L.; Strobaugh, Terence P.; Frye, Jonathan G.; Meinersmann, Richard J.

2010-01-01

Multi-antimicrobial-resistant Salmonella enterica strains frequently carry resistance genes on plasmids. Recent studies focus heavily on large conjugative plasmids, and the role that small plasmids play in resistance gene transfer is largely unknown. To expand our previous studies in assessing the prevalence of the isolates harboring ColE1-like plasmids carrying the aph gene responsible for kanamycin resistance (Kanr) phenotypes, 102 Kanr Salmonella isolates collected through the National Antimicrobial Resistance Monitoring System (NARMS) in 2005 were screened by PCR using ColE1 primer sets. Thirty isolates were found to be positive for ColE1-like replicon. Plasmids from 23 isolates were able to propagate in Escherichia coli and were subjected to further characterization. Restriction mapping revealed three major plasmid groups found in three or more isolates, with each group consisting of two to three subtypes. The aph genes from the Kanr Salmonella isolates were amplified by PCR, sequenced, and showed four different aph(3′)-I genes. The distribution of the ColE1 plasmid groups in association with the aph gene, Salmonella serovar, and isolate source demonstrated a strong linkage of the plasmid with S. enterica serovar Typhimurium DT104. Due to their high copy number and mobility, the ColE1-like plasmids may play a critical role in transmission of antibiotic resistance genes among enteric pathogens, and these findings warrant a close monitoring of this plasmid incompatibility group. PMID:20693446

The Salmonella enterica serovar Typhimurium QseB Response Regulator Negatively Regulates Bacterial Motility and Swine Colonization in the Absence of the QseC Sensor Kinase

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar Typhimurium (S. Typhimurium) responds to the catecholamine, norepinephrine by increasing bacterial growth and enhancing motility. In this study, iron with or without the siderophore, ferrioxamine E also enhanced bacterial motility. Iron-enhanced motility was growth-rate ...

Isolation rates, serovars, and toxin genotypes of nicotinamide adenine dinucleotide-independent Actinobacillus pleuropneumoniae among pigs suffering from pleuropneumonia in Spain.

PubMed

Maldonado, Jaime; Valls, Laura; Martínez, Eva; Riera, Pere

2009-11-01

Actinobacillus pleuropneumoniae is the etiologic agent of swine pleuropneumonia, a major production-limiting disease in the pig industry. In the current study, 2,171 lung specimens obtained from pigs housed in 870 Spanish pig farms in regions of substantial pig production were examined. Conventional microbiology, coupled with species-specific polymerase chain reaction, identified 127 biovar 2 isolates, accounting for 25.3% of all A. pleuropneumoniae (n = 502) detected. Most isolates (79%) were recovered as pure primary cultures or as the predominant bacteria from lungs exhibiting lesions typical of acute swine pleuropneumonia. Coagglutination testing identified the isolates as belonging to serovars 2 (4.7%), 4 (4.7%), 7 (68.5%), and 11 (1.6%); however, 26 isolates were nontypeable. All biovar 2 isolates showed genes of the apxII operon alone, which encodes the corresponding ApxII exotoxin, leading to a different gene pattern for isolates in serovars 2, 4, and 11 compared with those of biovar 1. From this survey, it can be concluded that A. pleuropneumoniae biovar 2 infections are common in pigs in Spain, and they may be a common cause of respiratory disease in swine.

Molecular Epidemiology of Salmonella enterica Serovar Typhimurium Isolates Determined by Pulsed-Field Gel Electrophoresis: Comparison of Isolates from Avian Wildlife, Domestic Animals, and the Environment in Norway

PubMed Central

Refsum, Thorbjørn; Heir, Even; Kapperud, Georg; Vardund, Traute; Holstad, Gudmund

2002-01-01

The molecular epidemiology of 142 isolates of Salmonella enterica serovar Typhimurium from avian wildlife, domestic animals, and the environment in Norway was investigated using pulsed-field gel electrophoresis (PFGE) and computerized numerical analysis of the data. The bacterial isolates comprised 79 isolates from wild-living birds, including 46 small passerines and 26 gulls, and 63 isolates of nonavian origin, including 50 domestic animals and 13 environmental samples. Thirteen main clusters were discernible at the 90% similarity level. Most of the isolates (83%) were grouped into three main clusters. These were further divided into 20 subclusters at the 95% similarity level. Isolates from passerines, gulls, and pigeons dominated within five subclusters, whereas isolates from domestic animals and the environment belonged to many different subclusters with no predominance. The results support earlier results that passerines constitute an important source of infection to humans in Norway, whereas it is suggested that gulls and pigeons, based on PFGE analysis, represent only a minor source of human serovar Typhimurium infections. Passerines, gulls, and pigeons may also constitute a source of infection of domestic animals and feed plants or vice versa. Three isolates from cattle and a grain source, of which two were multiresistant, were confirmed as serovar Typhimurium phage type DT 104. These represent the first reported phage type DT 104 isolates from other sources than humans in Norway. PMID:12406755

Interaction of the putative tyrosine recombinases RipX (UU145), XerC (UU222), and CodV (UU529) of Ureaplasma parvum serovar 3 with specific DNA

PubMed Central

Zimmerman, Carl-Ulrich R; Rosengarten, Renate; Spergser, Joachim

2013-01-01

Phase variation of two loci (‘mba locus’ and ‘UU172 phase-variable element’) in Ureaplasma parvum serovar 3 has been suggested as result of site-specific DNA inversion occurring at short inverted repeats. Three potential tyrosine recombinases (RipX, XerC, and CodV encoded by the genes UU145, UU222, and UU529) have been annotated in the genome of U. parvum serovar 3, which could be mediators in the proposed recombination event. We document that only orthologs of the gene xerC are present in all strains that show phase variation in the two loci. We demonstrate in vitro binding of recombinant maltose-binding protein fusions of XerC to the inverted repeats of the phase-variable loci, of RipX to a direct repeat that flanks a 20-kbp region, which has been proposed as putative pathogenicity island, and of CodV to a putative dif site. Co-transformation of the model organism Mycoplasma pneumoniae M129 with both the ‘mba locus’ and the recombinase gene xerC behind an active promoter region resulted in DNA inversion in the ‘mba locus’. Results suggest that XerC of U. parvum serovar 3 is a mediator in the proposed DNA inversion event of the two phase-variable loci. PMID:23305333

Identification of lymphogranuloma venereum-associated Chlamydia trachomatis serovars by fluorescence in situ hybridisation--a proof-of-principle analysis.

PubMed

Frickmann, Hagen; Essig, Andreas; Poppert, Sven

2014-04-01

We describe a proof-of-principle evaluation of a fluorescence in situ hybridisation (FISH) procedure to identify Chlamydia trachomatis serovars L1-L3, the causative agents of lymphogranuloma venereum, in cell cultures based on newly designed DNA probes. Rapid and easy-to-perform FISH could facilitate the diagnosis of lymphogranuloma venereum without nucleic acid amplification or serotyping, but requires broader evaluation studies, for example, in tropical high-endemicity regions. © 2014 John Wiley & Sons Ltd.

Impairment of Swimming Motility by Antidiarrheic Lactobacillus acidophilus Strain LB Retards Internalization of Salmonella enterica Serovar Typhimurium within Human Enterocyte-Like Cells▿

PubMed Central

Liévin-Le Moal, Vanessa; Amsellem, Raymonde; Servin, Alain L.

2011-01-01

We report that both culture and the cell-free culture supernatant (CFCS) of Lactobacillus acidophilus strain LB (Lactéol Boucard) have the ability (i) to delay the appearance of Salmonella enterica serovar Typhimurium strain SL1344-induced mobilization of F-actin and, subsequently, (ii) to retard cell entry by S. Typhimurium SL1344. Time-lapse imaging and Western immunoblotting showed that S. Typhimurium SL1344 swimming motility, as represented by cell tracks of various types, was rapidly but temporarily blocked without affecting the expression of FliC flagellar propeller protein. We show that the product(s) secreted by L. acidophilus LB that supports the inhibitory activity is heat stable and of low molecular weight. The product(s) caused rapid depolarization of the S. Typhimurium SL1344 cytoplasmic membrane without affecting bacterial viability. We identified inhibition of swimming motility as a newly discovered mechanism by which the secreted product(s) of L. acidophilus strain LB retards the internalization of the diarrhea-associated pathogen S. enterica serovar Typhimurium within cultured human enterocyte-like cells. PMID:21825295

FUNCTIONS EXERTED BY THE VIRULENCE ASSOCIATED TYPE THREE SECRETION SYSTEMS DURING SALMONELLA ENTERICA SEROVAR ENTERITIDIS INFECTION OF CHICKEN OVIDUCT EPITHELIAL CELLS AND MACROPHAGES

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar, Enteritidis (SE) infection of chicken is a major contributing factor to non-typhoidal salmonellosis. The roles of the type three secretion systems (T3SS-1 and T3SS-2) in the pathogenesis of SE infection of chickens are poorly understood. In this study, the functions exer...

Aggregation via the Red, Dry, and Rough Morphotype Is Not a Virulence Adaptation in Salmonella enterica Serovar Typhimurium▿

PubMed Central

White, A. P.; Gibson, D. L.; Grassl, G. A.; Kay, W. W.; Finlay, B. B.; Vallance, B. A.; Surette, M. G.

2008-01-01

The Salmonella rdar (red, dry, and rough) morphotype is an aggregative and resistant physiology that has been linked to survival in nutrient-limited environments. Growth of Salmonella enterica serovar Typhimurium was analyzed in a variety of nutrient-limiting conditions to determine whether aggregation would occur at low cell densities and whether the rdar morphotype was involved in this process. The resulting cultures consisted of two populations of cells, aggregated and nonaggregated, with the aggregated cells preferentially displaying rdar morphotype gene expression. The two groups of cells could be separated based on the principle that aggregated cells were producing greater amounts of thin aggregative fimbriae (Tafi or curli). In addition, the aggregated cells retained some physiological characteristics of the rdar morphotype, such as increased resistance to sodium hypochlorite. Competitive infection experiments in mice showed that nonaggregative ΔagfA cells outcompeted rdar-positive wild-type cells in all tissues analyzed, indicating that aggregation via the rdar morphotype was not a virulence adaptation in Salmonella enterica serovar Typhimurium. Furthermore, in vivo imaging experiments showed that Tafi genes were not expressed during infection but were expressed once Salmonella was passed out of the mice into the feces. We hypothesize that the primary role of the rdar morphotype is to enhance Salmonella survival outside the host, thereby aiding in transmission. PMID:18195033

A defective mutant of Salmonella enterica Serovar Gallinarum in cobalamin biosynthesis is avirulent in chickens

PubMed Central

de Paiva, Jacqueline Boldrin; Penha Filho, Rafael Antonio Casarin; Arguello, Yuli Melisa Sierra; Berchieri Junior, Ângelo; Lemos, Manuel Victor Franco; Barrow, Paul A.

2009-01-01

Salmonella enterica serovar Gallinarum (SG) is a fowl typhoid agent in chickens and is a severe disease with worldwide economic impact as its mortality may reach up to 80%. It is one of a small group of serovars that typically produces typhoid-like infections in a narrow range of host species and which therefore represents a good model for human typhoid. The survival mechanisms are not considered to be virulent mechanisms but are essential for the life of the bacterium. Mutants of Salmonella Gallinarum containing defective genes, related to cobalamin biosynthesis and which Salmonella spp. has to be produced to survive when it is in an anaerobic environment, were produced in this study. Salmonella Gallinarum is an intracellular parasite. Therefore, this study could provide information about whether vitamin B12 biosynthesis might be essential to its survival in the host. The results showed that the singular deletion in cbiA or cobS genes did not interfere in the life of Salmonella Gallinarum in the host, perhaps because single deletion is not enough to impede vitamin B12 biosynthesis. It was noticed that diluted SG mutants with single deletion produced higher mortality than the wild strain of SG. When double mutation was carried out, the Salmonella Gallinarum mutant was unable to provoke mortality in susceptible chickens. This work showed that B12 biosynthesis is a very important step in the metabolism of Salmonella Gallinarum during the infection of the chickens. Further research on bacterium physiology should be carried out to elucidate the events described in this research and to assess the mutant as a vaccine strain. PMID:24031393

A scalable method for O-antigen purification applied to various Salmonella serovars

PubMed Central

Micoli, F.; Rondini, S.; Gavini, M.; Pisoni, I.; Lanzilao, L.; Colucci, A.M.; Giannelli, C.; Pippi, F.; Sollai, L.; Pinto, V.; Berti, F.; MacLennan, C.A.; Martin, L.B.; Saul, A.

2014-01-01

The surface lipopolysaccharide of gram-negative bacteria is both a virulence factor and a B cell antigen. Antibodies against O-antigen of lipopolysaccharide may confer protection against infection, and O-antigen conjugates have been designed against multiple pathogens. Here, we describe a simplified methodology for extraction and purification of the O-antigen core portion of Salmonella lipopolysaccharide, suitable for large-scale production. Lipopolysaccharide extraction and delipidation are performed by acetic acid hydrolysis of whole bacterial culture and can take place directly in a bioreactor, without previous isolation and inactivation of bacteria. Further O-antigen core purification consists of rapid filtration and precipitation steps, without using enzymes or hazardous chemicals. The process was successfully applied to various Salmonella enterica serovars (Paratyphi A, Typhimurium, and Enteritidis), obtaining good yields of high-quality material, suitable for conjugate vaccine preparations. PMID:23142430

Complete Genome Sequencing of a Multidrug-Resistant and Human-Invasive Salmonella enterica Serovar Typhimurium Strain of the Emerging Sequence Type 213 Genotype

PubMed Central

Calva, Edmundo; Zaidi, Mussaret B.; Sanchez-Flores, Alejandro; Estrada, Karel; Silva, Genivaldo G. Z.; Soto-Jiménez, Luz M.; Wiesner, Magdalena; Fernández-Mora, Marcos; Edwards, Robert A.

2015-01-01

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids. PMID:26089426

Complete genome sequencing of a multidrug-resistant and human-invasive Salmonella enterica serovar Typhimurium strain of the emerging sequence type 213 genotype

DOE Office of Scientific and Technical Information (OSTI.GOV)

Calva, Edmundo; Silva, Claudia; Zaidi, Mussaret B.

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids.

Complete genome sequencing of a multidrug-resistant and human-invasive Salmonella enterica serovar Typhimurium strain of the emerging sequence type 213 genotype

DOE PAGES

Calva, Edmundo; Silva, Claudia; Zaidi, Mussaret B.; ...

2015-06-18

Salmonella enterica subsp. enterica serovar Typhimurium strain YU39 was isolated in 2005 in the state of Yucatán, Mexico, from a human systemic infection. The YU39 strain is representative of the multidrug-resistant emergent sequence type 213 (ST213) genotype. The YU39 complete genome is composed of a chromosome and seven plasmids.

Transcriptional response of Leptospira interrogans to iron limitation and characterization of a PerR homolog.

PubMed

Lo, Miranda; Murray, Gerald L; Khoo, Chen Ai; Haake, David A; Zuerner, Richard L; Adler, Ben

2010-11-01

Leptospirosis is a globally significant zoonosis caused by Leptospira spp. Iron is essential for growth of most bacterial species. Since iron availability is low in the host, pathogens have evolved complex iron acquisition mechanisms to survive and establish infection. In many bacteria, expression of iron uptake and storage proteins is regulated by Fur. L. interrogans encodes four predicted Fur homologs; we have constructed a mutation in one of these, la1857. We conducted microarray analysis to identify iron-responsive genes and to study the effects of la1857 mutation on gene expression. Under iron-limiting conditions, 43 genes were upregulated and 49 genes were downregulated in the wild type. Genes encoding proteins with predicted involvement in inorganic ion transport and metabolism (including TonB-dependent proteins and outer membrane transport proteins) were overrepresented in the upregulated list, while 54% of differentially expressed genes had no known function. There were 16 upregulated genes of unknown function which are absent from the saprophyte L. biflexa and which therefore may encode virulence-associated factors. Expression of iron-responsive genes was not significantly affected by mutagenesis of la1857, indicating that LA1857 is not a global regulator of iron homeostasis. Upregulation of heme biosynthetic genes and a putative catalase in the mutant suggested that LA1857 is more similar to PerR, a regulator of the oxidative stress response. Indeed, the la1857 mutant was more resistant to peroxide stress than the wild type. Our results provide insights into the role of iron in leptospiral metabolism and regulation of the oxidative stress response, including genes likely to be important for virulence.

Lsa21, a novel leptospiral protein binding adhesive matrix molecules and present during human infection

PubMed Central

Atzingen, Marina V; Barbosa, Angela S; De Brito, Thales; Vasconcellos, Silvio A; de Morais, Zenáide M; Lima, Dirce MC; Abreu, Patricia AE; Nascimento, Ana LTO

2008-01-01

Background It has been well documented over past decades that interaction of pathogens with the extracellular matrix (ECM) plays a primary role in host cell attachment and invasion. Adherence to host tissues is mediated by surface-exposed proteins expressed by the microorganisms during infection. The mechanisms by which pathogenic leptospires invade and colonize the host remain poorly understood since few virulence factors contributing to the pathogenesis of the disease have been identified. Whole-genome sequencing analysis of L. interrogans allowed identification of a repertoire of putative leptospiral surface proteins. Results Here, we report the identification and characterization of a new leptospiral protein that exhibits extracellular matrix-binding properties, called as Lsa21 (leptospiral surface adhesin, 21 kDa). Compatible with its role in adhesion, the protein was shown to be surface-exposed by indirect immunofluorescence. Attachment of Lsa21 to laminin, collagen IV, and plasma fibronectin was specific and dose dependent. Laminin oxidation by sodium metaperiodate reduced the protein-laminin interaction in a concentration-dependent manner, indicating that laminin sugar moieties are crucial for this interaction. The gene coding for Lsa21 is present in pathogenic strains belonging to the L. interrogans species but was not found in the saprophytic L. biflexa serovar Patoc strain Patoc 1. Loss of gene expression occurs upon culture attenuation of pathogenic strains. Environmental factors such as osmolarity and temperature affect Lsa21 expression at the transcriptional level. Moreover, anti-Lsa21 serum labeled liver and kidney tissues of human fatal cases of leptospirosis. Conclusion Our data suggest a role of Lsa21 in the pathogenesis of leptospirosis. PMID:18445272

Survey for selected pathogens in wild pigs (Sus scrofa) from Guam, Marianna Islands, USA.

PubMed

Cleveland, Christopher A; DeNicola, Anthony; Dubey, J P; Hill, Dolores E; Berghaus, Roy D; Yabsley, Michael J

2017-06-01

Pigs (Sus scrofa) were introduced to Guam in the 1600's and are now present in high densities throughout the island. Wild pigs are reservoirs for pathogens of concern to domestic animals and humans. Exposure to porcine parvovirus, transmissible gastroenteritis, and Leptospira interrogans has been documented in domestic swine but data from wild pigs are lacking. The close proximity of humans, domestic animals, and wild pigs, combined with the liberal hunting of wild pigs, results in frequent opportunities for pathogen transmission. From February-March 2015, blood, tissue and ectoparasite samples were collected from 47 wild pigs. Serologic testing found exposure to Brucella spp. (2%), Toxoplasma gondii (11%), porcine reproductive and respiratory syndrome (PRRS) virus (13%), porcine circovirus type 2 (36%), pseudorabies virus (64%), Actinobacillus pleuropneumoniae (93%), Lawsonia intracellularis (93%), and porcine parvovirus (94%). Eleven (24%) samples had low titers (1:100) to Leptospira interrogans serovars Bratislava (n=6), Icterohaemorrhagiae (n=6), Pomona (n=2), and Hardjo (n=1). Kidney samples from nine pigs with Leptospira antibodies were negative for Leptospira antigens. Numerous pigs had Metastrongylus lungworms and three had Stephanurus dentatus. Lice (Hematopinus suis) and ticks (Amblyomma breviscutatum) were also detected. No antibodies to Influenza A viruses were detected. In contrast to the previous domestic swine survey, we found evidence of numerous pathogens in wild pigs including new reports of pseudorabies virus, PRRS virus, Brucella, and Leptospira in pigs on Guam. These findings highlight that domestic swine-wild pig interactions should be prevented and precautions are needed when handling wild pigs to minimize the risk of pathogen transmission. Copyright © 2017 Elsevier B.V. All rights reserved.

The molecular adjuvant mC3d enhances the immunogenicity of FimA from type I fimbriae of Salmonella enterica serovar Enteritidis.

PubMed

Musa, Hassan-Hussein; Zhang, Wei-Juan; Lv, Jing; Duan, Xiao-Li; Yang, Yang; Zhu, Chun-Hong; Li, Hui-Fang; Chen, Kuan-Wei; Meng, Xia; Zhu, Guo-Qiang

2014-02-01

The fimbriae of Salmonella enterica serovar Enteritidis are used for colonization and invasion into host cells, and have drawn considerable interest because fimbriae can serve as potential immunogens against many pathogenic bacteria that colonize on epithelial surfaces. The purpose of the study is to use a molecular adjuvant, C3d, to enhance the immunogenicity of FimA proteins against Salmonella enterica serovar Enteritidis. FimA of type I fimbriae from Salmonella enteritidis and FimA with one copy of mC3d, two copies of mC3d2 and three copies of mC3d3 were cloned into the expression vector pCold-TF. Soluble fusion proteins of FimA with different copy of mC3d were induced by IPTG and expressed into Escherichia coli BL21 (DE3). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) showed that the recombinant proteins from pCold-TF-fimA, TF-fimA-mC3d, TF-fimA-mC3d2, TF-fimA-mC3d3 were 70 kDa, 100 kDa, 130 kDa and 160 kDa, respectively. The fusion protein was recognized by rabbit anti-fimbriae polyclonal antibodies, and then visualized by goat anti-rabbit polyclonal antibodies with a chrome appearance by enzyme-subtract interaction. The recombinant proteins were purified by Ni-TED (tris-carboxymethyl ethylene diamine), immobilized metal ion affinity chromatography (IMAC). Balb/c mice were subcutaneously immunized with the purified proteins and the immune response was monitored by an enzyme-linked immunosorbent assay (ELISA) for FimA-specific antibody. The immunized mice were challenged with a 10-fold LD50 dose (i.e., 100 CFU) of Salmonella enterica serovar Enteritidis standard strain (SD-2) 1 week after the second immunization. The immunized mice with the fusion proteins FimA-mC3d2 and FimA-mC3d3 had increased levels of ELISA titer of antibody that were 2 and 4 logs, respectively, more immunogenic than the TF-FimA protein alone. The challenge results showed that immune protection rate in the mice immunized with 10 μg of FimA, FimA-mC3d2, and FimA-mC3d3

A Comparative Genomic Analysis Provides Novel Insights Into the Ecological Success of the Monophasic Salmonella Serovar 4,[5],12:i:-

PubMed Central

Mastrorilli, Eleonora; Pietrucci, Daniele; Barco, Lisa; Ammendola, Serena; Petrin, Sara; Longo, Alessandra; Mantovani, Claudio; Battistoni, Andrea; Ricci, Antonia; Desideri, Alessandro; Losasso, Carmen

2018-01-01

Over the past decades, Salmonella 4,[5],12:i:- has rapidly emerged and it is isolated with high frequency in the swine food chain. Although many studies have documented the epidemiological success of this serovar, few investigations have tried to explain this phenomenon from a genetic perspective. Here a comparative whole-genome analysis of 50 epidemiologically unrelated S. 4,[5],12:i:-, isolated in Italy from 2010 to 2016 was performed, characterizing them in terms of genetic elements potentially conferring resistance, tolerance and persistence characteristics. Phylogenetic analyses indicated interesting distinctions among the investigated isolates. The most striking genetic trait characterizing the analyzed isolates is the widespread presence of heavy metals tolerance gene cassettes: most of the strains possess genes expected to confer resistance to copper and silver, whereas about half of the isolates also contain the mercury tolerance gene merA. A functional assay showed that these genes might be useful for preventing the toxic effects of metals, thus supporting the hypothesis that they can contribute to the success of S. 4,[5],12:i:- in farming environments. In addition, the analysis of the distribution of type II toxin-antitoxin families indicated that these elements are abundant in this serovar, suggesting that this is another factor that might favor its successful spread. PMID:29719530

Genomics of an emerging clone of Salmonella serovar Typhimurium ST313 from Nigeria and the Democratic Republic of Congo.

PubMed

Leekitcharoenphon, Pimlapas; Friis, Carsten; Zankari, Ea; Svendsen, Christina Aaby; Price, Lance B; Rahmani, Maral; Herrero-Fresno, Ana; Fashae, Kayode; Vandenberg, Olivier; Aarestrup, Frank M; Hendriksen, Rene S

2013-10-15

Salmonella enterica serovar Typhimurium ST313 is an invasive and phylogenetically distinct lineage present in sub-Saharan Africa. We report the presence of S. Typhimurium ST313 from patients in the Democratic Republic of Congo and Nigeria. Eighteen S. Typhimurium ST313 isolates were characterized by antimicrobial susceptibility testing, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST). Additionally, six of the isolates were characterized by whole genome sequence typing (WGST). The presence of a putative virulence determinant was examined in 177 Salmonella isolates belonging to 57 different serovars. All S. Typhimurium ST313 isolates harbored resistant genes encoded by blaTEM1b, catA1, strA/B, sul1, and dfrA1. Additionally, aac(6')1aa gene was detected. Phylogenetic analyses revealed close genetic relationships among Congolese and Nigerian isolates from both blood and stool. Comparative genomic analyses identified a putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and S. Dublin. We showed in a limited number of isolates that S. Typhimurium ST313 is a prevalent sequence-type causing gastrointestinal diseases and septicemia in patients from Nigeria and DRC. We found three distinct phylogenetic clusters based on the origin of isolation suggesting some spatial evolution. Comparative genomics showed an interesting putative virulence fragment (ST313-TD) unique to S. Typhimurium ST313 and invasive S. Dublin.

Differences in Pathogenesis for Salmonella enterica serovar Typhimurium in the Mouse Versus the Swine Model Identify Bacterial Gene Products Required for Systemic but not Gastrointestinal Disease

USDA-ARS?s Scientific Manuscript database

Over the last several decades, the mouse model of Typhoid fever has been an extremely productive model to investigate Salmonella enterica serovar Typhimurium pathogenesis. The mouse is the paradigm for investigating systemic disease due to infection by Salmonella; however, the swine model of gastro...

Salmonella on Raw Poultry in Retail Markets in Guatemala: Levels, Antibiotic Susceptibility, and Serovar Distribution.

PubMed

Jarquin, Claudia; Alvarez, Danilo; Morales, Oneida; Morales, Ana Judith; López, Beatriz; Donado, Pilar; Valencia, Maria F; Arévalo, Alejandra; Muñoz, Fredy; Walls, Isabel; Doyle, Michael P; Alali, Walid Q

2015-09-01

The objective of this study was to determine Salmonella numbers on retail raw chicken carcasses in Guatemala and to phenotypically characterize the isolates (serotyping and antibiotic susceptibility). In total, 300 chicken carcasses were collected from seven departments in Guatemala. Salmonella numbers were determined using the most-probable-number method following the U. S. Department of Agriculture's Food Safety and Inspection Service protocol. In total, 103 isolates were obtained, all of which were tested for antibiotic susceptibility, whereas 46 isolates were serotyped. Overall, Salmonella prevalence and mean number (mean log most probable number per carcass) was 34.3% and 2.3 (95% confidence interval: 2.1 to 2.5), respectively. Significant differences (P < 0.05) in Salmonella prevalence were found by storage condition (refrigerated or ambient temperature), market type (wet markets, supermarkets, and independent poultry stores), chicken production system (integrated or nonintegrated production company), and chicken skin color (white or yellow). Chickens produced by integrated companies had lower Salmonella numbers (P < 0.05) than nonintegrated companies, and white-skin carcasses had lower numbers (P < 0.05) than yellow-skin carcasses. Among 13 different Salmonella serovars identified, Paratyphi B (34.8%) was most prevalent, followed by Heidelberg (16.3%) and Derby (11.6%). Of all the Salmonella isolates, 59.2% were resistant to one to three antibiotics and 13.6% to four or more antibiotics. Among all the serovars obtained, Salmonella Paratyphi B and Heidelberg were the most resistant to the antibiotics tested. Salmonella levels and antibiotic resistant profiles among isolates from raw poultry at the retail market level were high relative to other reports from North and South America. These data can be used by Guatemalan stakeholders to develop risk assessment models and support further research opportunities to control transmission of Salmonella spp. and

Fifteen-month duration of immunity for the serovar Grippotyphosa fraction of a tetravalent canine leptospirosis vaccine.

PubMed

Grosenbaugh, Deborah A; Pardo, Maria Camila

2018-06-09

Forty-four specific pathogen-free beagles, median age 65 days, received two subcutaneous doses of either a commercially available, five-way combination vaccine or the same vaccine in combination with a tetravalent Leptospira bacterin (Canicola, Grippotyphosa, Icterohaemorrhagiae, Pomona). They were subsequently challenged with a pathogenic strain L kirschneri serovar Grippotyphosa 470 days following completion of the vaccination protocol. Titres of agglutinating serum antibodies were determined at various time points before and after both vaccination and challenge, along with postchallenge reisolation of the challenge organisms from blood and urine, and evaluation of renal histopathology. Clinical signs of generalised leptospirosis were not observed in any of the dogs after challenge. In order to demonstrate efficacy, leptospirosis was defined as having at least one positive urine sample and a positive renal histopathology score; or, in the absence of renal pathology, multiple positive urine samples. Leptospiremia was not demonstrated in any of the vaccinated dogs versus 27 per cent of the controls; leptospiruria was noted in 5 per cent of the vaccinates compared with 76 per cent of controls; and renal lesions were observed in 15 per cent of the vaccinates and 65 per cent controls. Using these criteria, the vaccine was able to significantly prevent leptospirosis (P=0.0001) in the vaccinated animals. This study establishes duration of immunity of at least 15 months for the prevention of disease and renal excretion of leptospires for the Leptospira serovar Grippotyphosa fraction of a quadrivalent Leptospira vaccine. © British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Receptor binding proteins of Listeria monocytogenes bacteriophages A118 and P35 recognize serovar-specific teichoic acids

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bielmann, Regula; Habann, Matthias; Eugster, Marcel R.

Adsorption of a bacteriophage to the host requires recognition of a cell wall-associated receptor by a receptor binding protein (RBP). This recognition is specific, and high affinity binding is essential for efficient virus attachment. The molecular details of phage adsorption to the Gram-positive cell are poorly understood. We present the first description of receptor binding proteins and a tail tip structure for the siphovirus group infecting Listeria monocytogenes. The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. Two proteins were identified as RBPs in phage A118. Rhamnose residues in wallmore » teichoic acids represent the binding ligands for both proteins. In phage P35, protein gp16 could be identified as RBP and the role of both rhamnose and N-acetylglucosamine in phage adsorption was confirmed. Immunogold-labeling and transmission electron microscopy allowed the creation of a topological model of the A118 phage tail. - Highlights: • We present the first description of receptor binding proteins and a tail tip structure for the Siphovirus group infecting Listeria monocytogenes. • The host-range determining factors in two phages, A118 and P35 specific for L. monocytogenes serovar 1/2 have been determined. • Rhamnose residues in wall teichoic acids represent the binding ligands for both receptor binding proteins in phage A118. • Rhamnose and N-acetylglucosamine are required for adsorption of phage P35. • We preset a topological model of the A118 phage tail.« less

Salmonella serovars and antimicrobial resistance in strains isolated from wild animals in captivity in Sinaloa, Mexico.

PubMed

Silva-Hidalgo, Gabriela; López-Valenzuela, Martin; Juárez-Barranco, Felipe; Montiel-Vázquez, Edith; Valenzuela-Sánchez, Beatriz

2014-08-01

The aim of the present study was to evaluate the frequency of antibiotic resistance in Salmonella spp. strains from wild animals in captivity at the Culiacan Zoo and the Mazatlan Aquarium in Sinaloa, Mexico. We identified 17 different Salmonella enterica serovars at a prevalence of 19.90% (Culiacan Zoo) and 6.25% (Mazatlan Aquarium). Antibiotic sensitivity tests revealed that, of the 83 strains studied, 100% were multidrug resistant (MDR). The drugs against which the greatest resistance was observed were: penicillin, erythromycin, dicloxacillin, ampicillin, cephalothin, and chloramphenicol. We therefore conclude that MDR is common among Salmonella isolates originating from wild animals in captivity in Sinaloa.

Impact of the choice of reference genome on the ability of the core genome SNV methodology to distinguish strains of Salmonella enterica serovar Heidelberg.

PubMed

Usongo, Valentine; Berry, Chrystal; Yousfi, Khadidja; Doualla-Bell, Florence; Labbé, Genevieve; Johnson, Roger; Fournier, Eric; Nadon, Celine; Goodridge, Lawrence; Bekal, Sadjia

2018-01-01

Salmonella enterica serovar Heidelberg (S. Heidelberg) is one of the top serovars causing human salmonellosis. The core genome single nucleotide variant pipeline (cgSNV) is one of several whole genome based sequence typing methods used for the laboratory investigation of foodborne pathogens. SNV detection using this method requires a reference genome. The purpose of this study was to investigate the impact of the choice of the reference genome on the cgSNV-informed phylogenetic clustering and inferred isolate relationships. We found that using a draft or closed genome of S. Heidelberg as reference did not impact the ability of the cgSNV methodology to differentiate among 145 S. Heidelberg isolates involved in foodborne outbreaks. We also found that using a distantly related genome such as S. Dublin as choice of reference led to a loss in resolution since some sporadic isolates were found to cluster together with outbreak isolates. In addition, the genetic distances between outbreak isolates as well as between outbreak and sporadic isolates were overall reduced when S. Dublin was used as the reference genome as opposed to S. Heidelberg.

The Legacy of Genetic Analysis Advances Contemporary Research with Escherichia coli K-12 and Salmonella enterica serovar Typhimurium LT2.

PubMed

Stewart, Valley

2017-04-01

Escherichia coli K-12 and Salmonella enterica serovar Typhimurium LT2 became standard organisms for genetic analysis during the Truman administration. Half a century later, genetic analysis with these strains had become an art form, interpreted through 23 articles in the ambitious two-volume masterpiece edited by the late Fred Neidhardt and colleagues. These legacy articles now are available through EcoSal Plus , so as to inform and inspire contemporary genetic analyses in these standard organisms and their relatives.

Features of Two New Proteins with OmpA-Like Domains Identified in the Genome Sequences of Leptospira interrogans

PubMed Central

Teixeira, Aline F.; de Morais, Zenaide M.; Kirchgatter, Karin; Romero, Eliete C.; Vasconcellos, Silvio A.; Nascimento, Ana Lucia T. O.

2015-01-01

Leptospirosis is an acute febrile disease caused by pathogenic spirochetes of the genus Leptospira. It is considered an important re-emerging infectious disease that affects humans worldwide. The knowledge about the mechanisms by which pathogenic leptospires invade and colonize the host remains limited since very few virulence factors contributing to the pathogenesis of the disease have been identified. Here, we report the identification and characterization of two new leptospiral proteins with OmpA-like domains. The recombinant proteins, which exhibit extracellular matrix-binding properties, are called Lsa46 - LIC13479 and Lsa77 - LIC10050 (Leptospiral surface adhesins of 46 and 77 kDa, respectively). Attachment of Lsa46 and Lsa77 to laminin was specific, dose dependent and saturable, with KD values of 24.3 ± 17.0 and 53.0 ± 17.5 nM, respectively. Lsa46 and Lsa77 also bind plasma fibronectin, and both adhesins are plasminogen (PLG)-interacting proteins, capable of generating plasmin (PLA) and as such, increase the proteolytic ability of leptospires. The proteins corresponding to Lsa46 and Lsa77 are present in virulent L. interrogans L1-130 and in saprophyte L. biflexa Patoc 1 strains, as detected by immunofluorescence. The adhesins are recognized by human leptospirosis serum samples at the onset and convalescent phases of the disease, suggesting that they are expressed during infection. Taken together, our data could offer valuable information to the understanding of leptospiral pathogenesis. PMID:25849456

ACUTE CLINICAL LEPTOSPIROSIS (GRIPPOTYPHOSA SEROVAR) IN AN ADULT DROMEDARY CAMEL (CAMELUS DROMEDARIUS).

PubMed

Gyimesi, Zoltan S; Burns, Roy B; Erol, Erdal; Bolin, Steven R

2015-09-01

A 9-yr-old castrated male dromedary camel (Camelus dromedarius) presented with lethargy and partial anorexia. A diagnostic examination revealed fever, and further workup revealed a neutrophilia, hyperfibrinogenemia, renal azotemia, and a rapid onset of a high Leptospira antibody titer during the acute clinical period (Grippotyphosa serovar). The camel responded clinically to antimicrobial treatment with ceftiofur crystalline free acid injections, but renal azotemia persisted, presumably secondary to chronic renal damage. Subsequent Leptospira polymerase chain reaction testing on urine samples obtained over the following 4 mo revealed no evidence of urinary shedding, so a persistent infection was unlikely. Although often mentioned as a potential cause of reproductive loss, well-documented case reports of clinical leptospirosis in camelids are very rare. In this case, native wildlife contamination of a small watering hole is suspected to have been the source of infection. In response to this experience, the camel and two conspecifics were prescribed a vaccination regimen using an inactivated pentavalent Leptospira vaccine licensed for cattle.

Microgravity as a novel environmental signal affecting Salmonella enterica serovar Typhimurium virulence

NASA Technical Reports Server (NTRS)

Nickerson, C. A.; Ott, C. M.; Mister, S. J.; Morrow, B. J.; Burns-Keliher, L.; Pierson, D. L.

2000-01-01

The effects of spaceflight on the infectious disease process have only been studied at the level of the host immune response and indicate a blunting of the immune mechanism in humans and animals. Accordingly, it is necessary to assess potential changes in microbial virulence associated with spaceflight which may impact the probability of in-flight infectious disease. In this study, we investigated the effect of altered gravitational vectors on Salmonella virulence in mice. Salmonella enterica serovar Typhimurium grown under modeled microgravity (MMG) were more virulent and were recovered in higher numbers from the murine spleen and liver following oral infection compared to organisms grown under normal gravity. Furthermore, MMG-grown salmonellae were more resistant to acid stress and macrophage killing and exhibited significant differences in protein synthesis than did normal-gravity-grown cells. Our results indicate that the environment created by simulated microgravity represents a novel environmental regulatory factor of Salmonella virulence.

A Mutation in the PoxA Gene of Salmonella enterica Serovar Typhimurium Results in Altered Protein Production, Elevated Susceptibility to Environmental Challenges, and Decreased Swine Colonization

USDA-ARS?s Scientific Manuscript database

Using signature-tagged mutagenesis of Salmonella enterica serovar Typhimurium (S. Typhimurium), a mutation in the poxA gene (STM4344; yjeA; poxR), encoding a putative lysyl-tRNA synthetase, was previously identified by our research group which caused decreased survival in an ex vivo swine stomach co...

In vivo gene expression and immunoreactivity of Leptospira collagenase.

PubMed

Janwitthayanan, Weena; Keelawat, Somboon; Payungporn, Sunchai; Lowanitchapat, Alisa; Suwancharoen, Duangjai; Poovorawan, Yong; Chirathaworn, Chintana

2013-06-12

Pulmonary hemorrhage is an increasing cause of death of leptospirosis patients. Bacterial collagenase has been shown to be involved in lung hemorrhage induced by various infectious agents. According to Leptospira whole genome study, colA, a gene suggested to code for bacterial collagenase has been identified. We investigated colA gene expression in lung tissues of Leptospira infected hamsters. Golden Syrian Hamsters were injected intraperitoneally with Leptospira interrogans serovar Pyrogenes. The hamsters were sacrificed on days 3, 5 and 7 post-infection and lung tissues were collected for histological examination and RNA extraction. Lung pathologies including atelectasis and hemorrhage were observed. Expression of colA gene in lung tissues was demonstrated by both RT-PCR and real time PCR. In addition, ColA protein was cloned and the purified protein could react with sera from leptospirosis patients. Leptospira ColA protein may play a role in Leptospira survival or pathogenesis in vivo. Its reaction with leptospirosis sera suggests that this protein is immunogenic and could be another candidate for vaccine development. Copyright © 2012 Elsevier GmbH. All rights reserved.

Leptospirosis Outbreak in Sri Lanka in 2008: Lessons for Assessing the Global Burden of Disease

PubMed Central

Agampodi, Suneth B.; Peacock, Sharon J.; Thevanesam, Vasanthi; Nugegoda, Danaseela B.; Smythe, Lee; Thaipadungpanit, Janjira; Craig, Scott B.; Burns, Mary Ann; Dohnt, Michael; Boonsilp, Siriphan; Senaratne, Thamarasi; Kumara, Athula; Palihawadana, Paba; Perera, Sahan; Vinetz, Joseph M.

2011-01-01

Global leptospirosis disease burden estimates are hampered by the lack of scientifically sound data from countries with probable high endemicity and limited diagnostic capacities. We describe the seroepidemiologic and clinical characteristics of the leptospirosis outbreak in 2008 in Sri Lanka. Definitive/presumptive case definitions proposed by the World Health Organization Leptospirosis Epidemiology Reference Group were used for case confirmation. Of the 404 possible cases, 155 were confirmed to have leptospirosis. Highest titers of patient seum samples reacted with serovars Pyrogenes (28.7%), Hardjo (18.8%), Javanica (11.5%), and Hebdomadis (11.5%). Sequencing of the 16S ribosomal DNA gene identified six infections: five with Leptospira interrogans and one with L. weilli. In this patient population, acute renal failure was the main complication (14.8%), followed by myocarditis (7.1%) and heart failure (3.9%). The case-fatality rate was 1.3%. This report strengthens the urgent need for increasing laboratory diagnostic capabilities to determine the causes of epidemic and endemic infectious diseases in Sri Lanka, a finding relevant to other tropical regions. PMID:21896807

Of guinea pigs and men--an unusual case of jaundice.

PubMed

Pischke, S; Ehmer, U; Schedel, I; Gratz, W F; Wedemeyer, H; Ziesing, S; Bange, F C; Burchard, G D; Manns, M P; Bahr, M J; Strassburg, C P

2010-01-01

A 21-year-old male presented at the emergency room with jaundice, itching, dry cough, malaise and weight loss of 10 kg during the preceding four weeks. Eighteen months earlier, the patient had suffered an automobile accident leading to polytrauma. Serological markers for viral or other causes of hepatitis were absent. For suspected secondary sclerosing cholangitis, ultrasound and ERCP were performed but failed to reveal pathological findings. A liver biopsy showed cholestatic liver disease without signs of portal field-associated hepatitis. Hepato-biliary scintigraphy demonstrated hepatocellular dysfunction. The patient finally mentioned his guinea pig farm with around 50 animals, 20 of which had recently died for unknown reasons. The patient and three of his guinea pigs were subsequently tested for serological evidence of leptospirosis. IgG and IgM antibodies reacting with Leptospira interrogans were detected in the patient's serum, and all 3 guinea pigs were serologically positive for serovar Bratislava. Bacterial culture was not successful, and also PCR tests remained negative. The clinical symptoms quickly resolved after the initiation of antibiotic therapy with amoxicillin.

The Use of a Combined Bioinformatics Approach to Locate Antibiotic Resistance Genes on Plasmids From Whole Genome Sequences of Salmonella enterica Serovars From Humans in Ghana.

PubMed

Kudirkiene, Egle; Andoh, Linda A; Ahmed, Shahana; Herrero-Fresno, Ana; Dalsgaard, Anders; Obiri-Danso, Kwasi; Olsen, John E

2018-01-01

In the current study, we identified plasmids carrying antimicrobial resistance genes in draft whole genome sequences of 16 selected Salmonella enterica isolates representing six different serovars from humans in Ghana. The plasmids and the location of resistance genes in the genomes were predicted using a combination of PlasmidFinder, ResFinder, plasmidSPAdes and BLAST genomic analysis tools. Subsequently, S1-PFGE was employed for analysis of plasmid profiles. Whole genome sequencing confirmed the presence of antimicrobial resistance genes in Salmonella isolates showing multidrug resistance phenotypically. ESBL, either bla TEM52-B or bla CTX-M15 were present in two cephalosporin resistant isolates of S . Virchow and S . Poona, respectively. The systematic genome analysis revealed the presence of different plasmids in different serovars, with or without insertion of antimicrobial resistance genes. In S . Enteritidis, resistance genes were carried predominantly on plasmids of IncN type, in S . Typhimurium on plasmids of IncFII(S)/IncFIB(S)/IncQ1 type. In S . Virchow and in S . Poona, resistance genes were detected on plasmids of IncX1 and TrfA/IncHI2/IncHI2A type, respectively. The latter two plasmids were described for the first time in these serovars. The combination of genomic analytical tools allowed nearly full mapping of the resistance plasmids in all Salmonella strains analyzed. The results suggest that the improved analytical approach used in the current study may be used to identify plasmids that are specifically associated with resistance phenotypes in whole genome sequences. Such knowledge would allow the development of rapid multidrug resistance tracking tools in Salmonella populations using WGS.

Homologous stress adaptation, antibiotic resistance, and biofilm forming ability of Salmonella enterica serovar Heidelberg (ATCC8326) on different food-contact surfaces following exposure to sub-lethal chlorine concentrations

USDA-ARS?s Scientific Manuscript database

Salmonella enterica serovar Heidelberg (American Type Culture Collection; ATCC 8326) was examined for the ability to adapt to the homologous stress of chlorine through exposure to increasing chlorine concentrations (25 ppm daily increments) in tryptic soy broth (TSB). The tested strain exhibited an ...

Genome-Wide Transposon Mutagenesis in Pathogenic Leptospira Species▿ ‡

PubMed Central

Murray, Gerald L.; Morel, Viviane; Cerqueira, Gustavo M.; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I.; Dellagostin, Odir A.; Bulach, Dieter M.; Sermswan, Rasana W.; Adler, Ben; Picardeau, Mathieu

2009-01-01

Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans. PMID:19047402

Genome-wide transposon mutagenesis in pathogenic Leptospira species.

PubMed

Murray, Gerald L; Morel, Viviane; Cerqueira, Gustavo M; Croda, Julio; Srikram, Amporn; Henry, Rebekah; Ko, Albert I; Dellagostin, Odir A; Bulach, Dieter M; Sermswan, Rasana W; Adler, Ben; Picardeau, Mathieu

2009-02-01

Leptospira interrogans is the most common cause of leptospirosis in humans and animals. Genetic analysis of L. interrogans has been severely hindered by a lack of tools for genetic manipulation. Recently we developed the mariner-based transposon Himar1 to generate the first defined mutants in L. interrogans. In this study, a total of 929 independent transposon mutants were obtained and the location of insertion determined. Of these mutants, 721 were located in the protein coding regions of 551 different genes. While sequence analysis of transposon insertion sites indicated that transposition occurred in an essentially random fashion in the genome, 25 unique transposon mutants were found to exhibit insertions into genes encoding 16S or 23S rRNAs, suggesting these genes are insertional hot spots in the L. interrogans genome. In contrast, loci containing notionally essential genes involved in lipopolysaccharide and heme biosynthesis showed few transposon insertions. The effect of gene disruption on the virulence of a selected set of defined mutants was investigated using the hamster model of leptospirosis. Two attenuated mutants with disruptions in hypothetical genes were identified, thus validating the use of transposon mutagenesis for the identification of novel virulence factors in L. interrogans. This library provides a valuable resource for the study of gene function in L. interrogans. Combined with the genome sequences of L. interrogans, this provides an opportunity to investigate genes that contribute to pathogenesis and will provide a better understanding of the biology of L. interrogans.

Occurrence of plasmid-mediated quinolone resistance determinants and rmtB gene in Salmonella enterica serovar enteritidis and Typhimurium isolated from food-animal products in Tunisia.

PubMed

Al-Gallas, Nazek; Abbassi, Mohamed Salah; Gharbi, Becher; Manai, Molka; Ben Fayala, Mohamed N; Bichihi, Raghda; Al-Gallas, Amna; Ben Aissa, Ridha

2013-09-01

Four hundred and thirty Salmonella isolates, recovered from various food-animal products, were tested for nalidixic acid resistance, plasmid-mediated quinolone resistance, and genetic relationship. One hundred fifteen isolates (113 Salmonella serovar Enteritidis and two Salmonella serovar Typhimurium isolates) of 430 (26.7%) Salmonella isolates exhibited nalidixic acid resistance. Polymerase chain reaction screening for qnrA, qnrB, qnrS, qepA (encoding fluoroquinolones resistance) and rmtB (encoding aminoglycosides resistance) showed that 5 (1.16%) isolates were positive for qnr- and qepA-type genes, and the aac(6')-Ib-cr gene was observed in two (1.7%) Enteritidis isolates concomitantly with qnrA or qnrB. The co-occurrence of qepA and rmtB in one Typhimurium isolate is noteworthy. Pulsed-field gel electrophoresis revealed a high genetic homogeneity of nalidixic-resistant isolates and the persistence of clonal clusters over 4 years in different regions in Tunisia and from various food-animal products. To the best of our knowledge, this is the first report of co-occurrence of qepA and rmtB in a Salmonella strain.

A rapid method to identify Salmonella enterica serovar Gallinarum biovar Pullorum using a specific target gene ipaJ.

PubMed

Xu, Lijuan; Liu, Zijian; Li, Yang; Yin, Chao; Hu, Yachen; Xie, Xiaolei; Li, Qiuchun; Jiao, Xinan

2018-06-01

Salmonella enterica serovar Gallinarum biovar Pullorum (S. Pullorum) is the pathogen of pullorum disease, which leads to severe economic losses in many developing countries. Traditional methods to identify S. enterica have relied on biochemical reactions and serotyping, which are time-consuming with accurate identification if properly carried out. In this study, we developed a rapid polymerase chain reaction (PCR) method targeting the specific gene ipaJ to detect S. Pullorum. Among the 650 S. Pullorum strains isolated from 1962 to 2016 all over China, 644 strains were identified to harbour ipaJ gene in the plasmid pSPI12, accounting for a detection rate of 99.08%. Six strains were ipaJ negative because pSPI12 was not found in these strains according to whole genome sequencing results. There was no cross-reaction with other Salmonella serotypes, including Salmonella enterica serovar Gallinarum biovar Gallinarum (S. Gallinarum), which show a close genetic relationship with S. Pullorum. This shows that the PCR method could distinguish S. Gallinarum from S. Pullorum in one-step PCR without complicated biochemical identification. The limit of detection of this PCR method was as low as 90 fg/μl or 10 2 CFU, which shows a high sensitivity. Moreover, this method was applied to identify Salmonella isolated from the chicken farm and the results were consistent with what we obtained from biochemical reactions and serotyping. Together, all the results demonstrated that this one-step PCR method is simple and feasible to efficiently identify S. Pullorum.

Polyamines are essential for virulence in Salmonella enterica serovar Gallinarum despite evolutionary decay of polyamine biosynthesis genes.

PubMed

Schroll, Casper; Christensen, Jens P; Christensen, Henrik; Pors, Susanne E; Thorndahl, Lotte; Jensen, Peter R; Olsen, John E; Jelsbak, Lotte

2014-05-14

Serovars of Salmonella enterica exhibit different host-specificities where some have broad host-ranges and others, like S. Gallinarum and S. Typhi, are host-specific for poultry and humans, respectively. With the recent availability of whole genome sequences it has been reported that host-specificity coincides with accumulation of pseudogenes, indicating adaptation of host-restricted serovars to their narrow niches. Polyamines are small cationic amines and in Salmonella they can be synthesized through two alternative pathways directly from l-ornithine to putrescine and from l-arginine via agmatine to putrescine. The first pathway is not active in S. Gallinarum and S. Typhi, and this prompted us to investigate the importance of polyamines for virulence in S. Gallinarum. Bioinformatic analysis of all sequenced genomes of Salmonella revealed that pseudogene formation of the speC gene was exclusive for S. Typhi and S. Gallinarum and happened through independent events. The remaining polyamine biosynthesis pathway was found to be essential for oral infection with S. Gallinarum since single and double mutants in speB and speE, encoding the pathways from agmatine to putrescine and from putrescine to spermidine, were attenuated. In contrast, speB was dispensable after intraperitoneal challenge, suggesting that putrescine was less important for the systemic phase of the disease. In support of this hypothesis, a ΔspeE;ΔpotCD mutant, unable to synthesize and import spermidine, but with retained ability to import and synthesize putrescine, was attenuated after intraperitoneal infection. We therefore conclude that polyamines are essential for virulence of S. Gallinarum. Furthermore, our results point to distinct roles for putrescine and spermidine during systemic infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Characteristic features of intracellular pathogenic Leptospira in infected murine macrophages.

PubMed

Toma, Claudia; Okura, Nobuhiko; Takayama, Chitoshi; Suzuki, Toshihiko

2011-11-01

Leptospira interrogans is a spirochaete responsible for a zoonotic disease known as leptospirosis. Leptospires are able to penetrate the abraded skin and mucous membranes and rapidly disseminate to target organs such as the liver, lungs and kidneys. How this pathogen escape from innate immune cells and spread to target organs remains poorly understood. In this paper, the intracellular trafficking undertaken by non-pathogenic Leptospira biflexa and pathogenic L. interrogans in mouse bone marrow-derived macrophages was compared. The delayed in the clearance of L. interrogans was observed. Furthermore, the acquisition of lysosomal markers by L. interrogans-containing phagosomes lagged behind that of L. biflexa-containing phagosomes, and although bone marrow-derived macrophages could degrade L. biflexa as well as L. interrogans, a population of L. interrogans was able to survive and replicate. Intact leptospires were found within vacuoles at 24 h post infection, suggesting that bacterial replication occurs within a membrane-bound compartment. In contrast, L. biflexa were completely degraded at 24 h post infection. Furthermore, L. interrogans but not L. biflexa, were released to the extracellular milieu. These results suggest that pathogenic leptospires are able to survive, replicate and exit from mouse macrophages, enabling their eventual spread to target organs. © 2011 Blackwell Publishing Ltd.

Respiratory Hydrogen Use by Salmonella enterica Serovar Typhimurium Is Essential for Virulence

PubMed Central

Maier, R. J.; Olczak, A.; Maier, S.; Soni, S.; Gunn, J.

2004-01-01

Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H2-using hydrogenase enzymes. These enzymes split molecular H2, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H2 oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H2 per mol of O2 used in the H2-O2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 μM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H2 is only 2.1 μM, so it is expected that H2-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H2 and cause morbidity in mice within 11 days of

Respiratory hydrogen use by Salmonella enterica serovar Typhimurium is essential for virulence.

PubMed

Maier, R J; Olczak, A; Maier, S; Soni, S; Gunn, J

2004-11-01

Based on available annotated gene sequence information, the enteric pathogen salmonella, like other enteric bacteria, contains three putative membrane-associated H2-using hydrogenase enzymes. These enzymes split molecular H2, releasing low-potential electrons that are used to reduce quinone or heme-containing components of the respiratory chain. Here we show that each of the three distinct membrane-associated hydrogenases of Salmonella enterica serovar Typhimurium is coupled to a respiratory pathway that uses oxygen as the terminal electron acceptor. Cells grown in a blood-based medium expressed four times the amount of hydrogenase (H2 oxidation) activity that cells grown on Luria Bertani medium did. Cells suspended in phosphate-buffered saline consumed 2 mol of H2 per mol of O2 used in the H2-O2 respiratory pathway, and the activity was inhibited by the respiration inhibitor cyanide. Molecular hydrogen levels averaging over 40 microM were measured in organs (i.e., livers and spleens) of live mice, and levels within the intestinal tract (the presumed origin of the gas) were four times greater than this. The half-saturation affinity of S. enterica serovar Typhimurium for H2 is only 2.1 microM, so it is expected that H2-utilizing hydrogenase enzymes are saturated with the reducing substrate in vivo. All three hydrogenase enzymes contribute to the virulence of the bacterium in a typhoid fever-mouse model, based on results from strains with mutations in each of the three hydrogenase genes. The introduced mutations are nonpolar, and growth of the mutant strains was like that of the parent strain. The combined removal of all three hydrogenases resulted in a strain that is avirulent and (in contrast to the parent strain) one that is unable to invade liver or spleen tissue. The introduction of one of the hydrogenase genes into the triple mutant strain on a low-copy-number plasmid resulted in a strain that was able to both oxidize H2 and cause morbidity in mice within 11

PCR Method To Identify Salmonella enterica Serovars Typhi, Paratyphi A, and Paratyphi B among Salmonella Isolates from the Blood of Patients with Clinical Enteric Fever▿

PubMed Central

Levy, Haim; Diallo, Souleymane; Tennant, Sharon M.; Livio, Sofie; Sow, Samba O.; Tapia, Milagritos; Fields, Patricia I.; Mikoleit, Matthew; Tamboura, Boubou; Kotloff, Karen L.; Lagos, Rosanna; Nataro, James P.; Galen, James E.; Levine, Myron M.

2008-01-01

PCR methodology was developed to identify Salmonella enterica serovars Typhi, Paratyphi A, and Paratyphi B. One multiplex PCR identifies serogroup D, A, and B and Vi-positive strains; another confirms flagellar antigen “d,” “a,” or “b.” Blinded testing of 664 Malian and Chilean Salmonella blood isolates demonstrated 100% sensitivity and specificity. PMID:18367574

Production of reactive oxygen (H2O2) and nitrogen (NO) intermediates and tnf-α in mice genetically selected for high (H) and low (L) antibody response and experimentally infected with Leptospira serovar pomona

PubMed Central

Haanwinckel, Maria Cristina Santos; de Oliveira, Silvio Luis

2011-01-01

The aim of the present study was to evaluate the activity of macrophages, and the production of TNF-α and antibodies against experimental infection by Leptospira serovar Pomona in mice genetically selected for High (H) or Low (L) humoral immune response. To evaluate macrophagic activity, peritoneal and splenic lavages were performed for determination of oxygen (H2O2) and nitrogen (NO) intermediates. The production of the tumor necrosis factor (TNF-α) was investigated through bioassays in serum and homogenates of splenic and hepatic cells of control and infected animals, as was as specific antibodies production. The immune response against serovar Pomona in those lines, was characterized by high antibody production, especially in later periods of the infectious process, whereas values of bacterial recovery in culture medium were lower. The production of reactives oxygen and nitrogen intermediate, also helped to eliminate Leptospira Pomona in both lines; H2O2 production an important factor in HIV-A, as well as NO production in LIV-A, especially in later post-inoculation periods. The same was detected for TNF-α. Results suggest that such lines could be an important model to investigate the pathogenesis and the immune response of animals against the several Leptospira serovars. PMID:24031688

Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A

PubMed Central

Thieu, Nga Tran Vu; Dolecek, Christiane; Karkey, Abhilasha; Gupta, Ruchi; Turner, Paul; Dance, David; Maude, Rapeephan R.; Ha, Vinh; Tran, Chinh Nguyen; Thi, Phuong Le; Be, Bay Pham Van; Phi, La Tran Thi; Ngoc, Rang Nguyen; Ghose, Aniruddha; Dongol, Sabina; Campbell, James I.; Thanh, Duy Pham; Thanh, Tuyen Ha; Moore, Catrin E.; Sona, Soeng; Gaind, Rajni; Deb, Monorama; Anh, Ho Van; Van, Sach Nguyen; Tinh, Hien Tran; Day, Nicholas P. J.; Dondorp, Arjen; Thwaites, Guy; Faiz, Mohamed Abul; Phetsouvanh, Rattanaphone; Newton, Paul; Basnyat, Buddha; Farrar, Jeremy J.; Baker, Stephen

2015-01-01

Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤16 μg/ml or disk inhibition zone size of ≥13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A. PMID:25733500

Ureaplasma parvum Serovar 3 Multiple Banded Antigen Size Variation after Chronic Intra-Amniotic Infection/Colonization

PubMed Central

Robinson, James W.; Dando, Samantha J.; Nitsos, Ilias; Newnham, John; Polglase, Graeme R.; Kallapur, Suhas G.; Pillow, J. Jane; Kramer, Boris W.; Jobe, Alan H.; Payton, Diane; Knox, Christine L.

2013-01-01

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2×107 colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145–150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation. PMID:23638142

Ureaplasma parvum serovar 3 multiple banded antigen size variation after chronic intra-amniotic infection/colonization.

PubMed

Robinson, James W; Dando, Samantha J; Nitsos, Ilias; Newnham, John; Polglase, Graeme R; Kallapur, Suhas G; Pillow, J Jane; Kramer, Boris W; Jobe, Alan H; Payton, Diane; Knox, Christine L

2013-01-01

Ureaplasma species are the microorganisms most frequently associated with adverse pregnancy outcomes. The multiple banded antigen (MBA), a surface-exposed lipoprotein, is a key virulence factor of ureaplasmas. The MBA demonstrates size variation, which we have shown previously to be correlated with the severity of chorioamnion inflammation. We aimed to investigate U. parvum serovar 3 pathogenesis in vivo, using a sheep model, by investigating: MBA variation after long term (chronic) and short term (acute) durations of in utero ureaplasma infections, and the severity of chorioamnionitis and inflammation in other fetal tissues. Inocula of 2 × 10(7) colony-forming-units (CFU) of U. parvum serovar 3 (Up) or media controls (C) were injected intra-amniotically into pregnant ewes at one of three time points: day 55 (69d Up, n = 8; C69, n = 4); day 117 (7d Up, n = 8; C7, n = 2); and day 121 (3d Up, n = 8; C3, n = 2) of gestation (term = 145-150d). At day 124, preterm fetuses were delivered surgically. Samples of chorioamnion, fetal lung, and umbilical cord were: (i) snap frozen for subsequent ureaplasma culture, and (ii) fixed, embedded, sectioned and stained by haematoxylin and eosin stain for histological analysis. Selected fetal lung clinical ureaplasma isolates were cloned and filtered to obtain cultures from a single CFU. Passage 1 and clone 2 ureaplasma cultures were tested by western blot to demonstrate MBA variation. In acute durations of ureaplasma infection no MBA variants (3d Up) or very few MBA variants (7d Up) were present when compared to the original inoculum. However, numerous MBA size variants were generated in vivo (alike within contiguous tissues, amniotic fluid and fetal lung, but different variants were present within chorioamnion), during chronic, 69d exposure to ureaplasma infection. For the first time we have shown that the degree of ureaplasma MBA variation in vivo increased with the duration of gestation.

Quantification, serovars, and antibiotic resistance of salmonella isolated from retail raw chicken meat in Vietnam.

PubMed

Ta, Yen T; Nguyen, Trung Thanh; To, Phuong Bich; Pham, Da Xuan; Le, Hao Thi Hong; Thi, Giang Nguyen; Alali, Walid Q; Walls, Isabel; Doyle, Michael P

2014-01-01

The objectives of this study were to quantify Salmonella counts on retail raw poultry meat in Vietnam and to phenotypically characterize (serovars and antibiotic resistance) the isolates. A total of 300 chicken carcasses were collected from two cities and two provinces in Vietnam. Salmonella counts on the samples were determined according to the most-probable-number (MPN) method of the U.S. Department of Agriculture, Food Safety and Inspection Service (USDA-FSIS). A total of 457 isolates were serotyped and tested for antibiotic susceptibility. Overall, 48.7% of chicken samples were Salmonella positive with a count of 2.0 log MPN per carcass. There were no significant differences (P > 0.05) in log MPN per carcass by the study variables (market type, storage condition, and chicken production system). There was a significant difference (P < 0.05) in Salmonella-positive prevalence by chicken production system. Among the 22 Salmonella serovars identified, Albany was the most frequent (34.1%), followed by Agona (15.5%) and Dabou (8.8%). Resistance to at least one antibiotic was common (i.e., 73.3%), with high resistance to tetracycline (59.1%) and ampicillin (41.6%). Resistance to three antibiotics was the most frequently found multidrug resistance profile (17.7%, n = 81); the profile that was resistant to the highest number of drugs was resistant to nine antibiotics (0.7%, n = 3). Only Salmonella Albany posed phenotypic resistance to ceftriaxone (a drug of choice to treat severe cases of salmonellosis). The data revealed that, whereas Salmonella prevalence on raw poultry was high (48.7%), counts were low, which suggests that the exposure risk to Salmonella is low. However, improper storage of raw chicken meat and cross-contamination may increase Salmonella cell counts and pose a greater risk for infection. These data may be helpful in developing risk assessment models and preventing the transmission of foodborne Salmonella from poultry to humans in Vietnam.

Analysis of a Spontaneous Non-Motile and Avirulent Mutant Shows That FliM Is Required for Full Endoflagella Assembly in Leptospira interrogans.

PubMed

Fontana, Célia; Lambert, Ambroise; Benaroudj, Nadia; Gasparini, David; Gorgette, Olivier; Cachet, Nathalie; Bomchil, Natalia; Picardeau, Mathieu

2016-01-01

Pathogenic Leptospira strains are responsible for leptospirosis, a worldwide emerging zoonotic disease. These spirochetes are unique amongst bacteria because of their corkscrew-like cell morphology and their periplasmic flagella. Motility is reported as an important virulence determinant, probably favoring entry and dissemination of pathogenic Leptospira in the host. However, proteins constituting the periplasmic flagella and their role in cell shape, motility and virulence remain poorly described. In this study, we characterized a spontaneous L. interrogans mutant strain lacking motility, correlated with the loss of the characteristic hook-shaped ends, and virulence in the animal model. Whole genome sequencing allowed the identification of one nucleotide deletion in the fliM gene resulting in a premature stop codon, thereby preventing the production of flagellar motor switch protein FliM. Genetic complementation restored cell morphology, motility and virulence comparable to those of wild type cells. Analyses of purified periplasmic flagella revealed a defect in flagella assembly, resulting in shortened flagella compared to the wild type strain. This also correlated with a lower amount of major filament proteins FlaA and FlaB. Altogether, these findings demonstrate that FliM is required for full and correct assembly of the flagella which is essential for motility and virulence.

TolC is important for bacterial survival and oxidative stress response in Salmonella enterica serovar Choleraesuis in an acidic environment.

PubMed

Lee, Jen-Jie; Wu, Ying-Chen; Kuo, Chih-Jung; Hsuan, Shih-Ling; Chen, Ter-Hsin

2016-09-25

The outer membrane protein TolC, which is one of the key components of several multidrug efflux pumps, is thought to be involved in various independent systems in Enterobacteriaceae. Since the acidic environment of the stomach is an important protection barrier against foodborne pathogen infections in hosts, we evaluated whether TolC played a role in the acid tolerance of Salmonella enterica serovar Choleraesuis. Comparison of the acid tolerance of the tolC mutant and the parental wild-type strain showed that the absence of TolC limits the ability of Salmonella to sustain life under extreme acidic conditions. Additionally, the mutant exhibited morphological changes during growth in an acidic medium, leading to the conflicting results of cell viability measured by spectrophotometry and colony-forming unit counting. Reverse-transcriptional-PCR analysis indicated that acid-related molecules, apparatus, or enzymes and oxidation-induced factors were significantly affected by the acidic environment in the null-tolC mutant. The elongated cellular morphology was restored by adding antioxidants to the culture medium. Furthermore, we found that increased cellular antioxidative activity provides an overlapping protection against acid killing, demonstrating the complexity of the bacterial acid stress response. Our findings reinforce the multifunctional characteristics of TolC in acid tolerance or oxidative stress resistance and support the correlative protection mechanism between oxygen- and acid-mediated stress responses in Salmonella enterica serovar Choleraesuis. Copyright © 2016 Elsevier B.V. All rights reserved.

Complete Genome Sequence of Salmonella enterica Serovar Typhimurium Strain YU15 (Sequence Type 19) Harboring the Salmonella Genomic Island 1 and Virulence Plasmid pSTV

PubMed Central

Calva, Edmundo; Puente, José L.; Zaidi, Mussaret B.

2016-01-01

The complete genome of Salmonella enterica subsp. enterica serovar Typhimurium sequence type 19 (ST19) strain YU15, isolated in Yucatán, Mexico, from a human baby stool culture, was determined using PacBio technology. The chromosome contains five intact prophages and the Salmonella genomic island 1 (SGI1). This strain carries the Salmonella virulence plasmid pSTV. PMID:27081132

Diversity of pulsed-field gel electrophoresis pulsotypes, serovars, and antibiotic resistance among Salmonella isolates from wild amphibians and reptiles in the California Central Coast.

PubMed

Gorski, Lisa; Jay-Russell, Michele T; Liang, Anita S; Walker, Samarpita; Bengson, Yingjia; Govoni, Jessica; Mandrell, Robert E

2013-06-01

A survey of cold-blooded vertebrates and associated surface waters in a produce-growing region on the Central California Coast was done between May and September 2011 to determine the diversity of Salmonella. Samples from 460 amphibians and reptiles and 119 water samples were collected and cultured for Salmonella. Animals sampled were frogs (n=331), lizards (n=59), newts (n=5), salamanders (n=6), snakes (n=39), and toads (n=20). Salmonella was isolated from 37 individual animals, including frogs, lizards, snakes, and toads. Snakes were the most likely to contain Salmonella, with 59% testing positive followed by 15.3% of lizards, 5% of toads, and 1.2% of frogs. Fifteen water samples (12.6%) were positive. Twenty-two different serovars were identified, and the majority of isolates were S. enterica subsp. IIIb, with subsp. I, II, and IIIa also found. The serovar isolated most frequently was S. enterica subsp. IIIb 16:z₁₀:e,n,x,z₁₅, from snakes and frogs in five different locations. S. enterica subsp. I serovar Typhimurium and the monophasic I 6,8:d:- were isolated from water, and subspecies I Duisburg and its variants were found in animals and water. Some samples contained more than one type of Salmonella. Analysis of pulsed-field gel electrophoresis pulsotypes indicated that some strains persisted in animals and water collected from the same location. Sixty-six isolates displayed antibiotic resistance, with 27 isolates resistant to more than one antibiotic, including a subspecies IIIb isolate from snake having resistance to five different antibiotics. Twenty-three isolates were resistant to more than one class of antibiotic, and six isolates were resistant to three classes. While these subspecies of IIIa and IIIb cause fewer instances of human illness, they may serve as reservoirs of antibiotic resistance, determinants in the environment, and be sources of contamination of leafy greens associated with product recalls.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota.

PubMed

Drumo, Rosanna; Pesciaroli, Michele; Ruggeri, Jessica; Tarantino, Michela; Chirullo, Barbara; Pistoia, Claudia; Petrucci, Paola; Martinelli, Nicola; Moscati, Livia; Manuali, Elisabetta; Pavone, Silvia; Picciolini, Matteo; Ammendola, Serena; Gabai, Gianfranco; Battistoni, Andrea; Pezzotti, Giovanni; Alborali, Giovanni L; Napolioni, Valerio; Pasquali, Paolo; Magistrali, Chiara F

2015-01-01

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota.

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Modify Swine Intestinal Microbiota

PubMed Central

Drumo, Rosanna; Pesciaroli, Michele; Ruggeri, Jessica; Tarantino, Michela; Chirullo, Barbara; Pistoia, Claudia; Petrucci, Paola; Martinelli, Nicola; Moscati, Livia; Manuali, Elisabetta; Pavone, Silvia; Picciolini, Matteo; Ammendola, Serena; Gabai, Gianfranco; Battistoni, Andrea; Pezzotti, Giovanni; Alborali, Giovanni L.; Napolioni, Valerio; Pasquali, Paolo; Magistrali, Chiara F.

2016-01-01

Salmonella enterica serovar Typhimurium is an important zoonotic gastrointestinal pathogen responsible for foodborne disease worldwide. It is a successful enteric pathogen because it has developed virulence strategies allowing it to survive in a highly inflamed intestinal environment exploiting inflammation to overcome colonization resistance provided by intestinal microbiota. In this study, we used piglets featuring an intact microbiota, which naturally develop gastroenteritis, as model for salmonellosis. We compared the effects on the intestinal microbiota induced by a wild type and an attenuated S. Typhimurium in order to evaluate whether the modifications are correlated with the virulence of the strain. This study showed that Salmonella alters microbiota in a virulence-dependent manner. We found that the wild type S. Typhimurium induced inflammation and a reduction of specific protecting microbiota species (SCFA-producing bacteria) normally involved in providing a barrier against pathogens. Both these effects could contribute to impair colonization resistance, increasing the host susceptibility to wild type S. Typhimurium colonization. In contrast, the attenuated S. Typhimurium, which is characterized by a reduced ability to colonize the intestine, and by a very mild inflammatory response, was unable to successfully sustain competition with the microbiota. PMID:26835435

Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins.

PubMed

Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Krzyżewska, Eva; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-07-11

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg ( S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins' patterns. The strategy of bacterial membrane proteins' analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species.

Relationship of Triamine-Biocide Tolerance of Salmonella enterica Serovar Senftenberg to Antimicrobial Susceptibility, Serum Resistance and Outer Membrane Proteins

PubMed Central

Futoma-Kołoch, Bożena; Dudek, Bartłomiej; Kapczyńska, Katarzyna; Wańczyk, Martyna; Korzekwa, Kamila; Rybka, Jacek; Klausa, Elżbieta; Bugla-Płoskońska, Gabriela

2017-01-01

A new emerging phenomenon is the association between the incorrect use of biocides in the process of disinfection in farms and the emergence of cross-resistance in Salmonella populations. Adaptation of the microorganisms to the sub-inhibitory concentrations of the disinfectants is not clear, but may result in an increase of sensitivity or resistance to antibiotics, depending on the biocide used and the challenged Salmonella serovar. Exposure of five Salmonella enterica subsp. enterica serovar Senftenberg (S. Senftenberg) strains to triamine-containing disinfectant did not result in variants with resistance to antibiotics, but has changed their susceptibility to normal human serum (NHS). Three biocide variants developed reduced sensitivity to NHS in comparison to the sensitive parental strains, while two isolates lost their resistance to serum. For S. Senftenberg, which exhibited the highest triamine tolerance (6 × MIC) and intrinsic sensitivity to 22.5% and 45% NHS, a downregulation of flagellin and enolase has been demonstrated, which might suggest a lower adhesion and virulence of the bacteria. This is the first report demonstrating the influence of biocide tolerance on NHS resistance. In conclusion, there was a potential in S. Senftenberg to adjust to the conditions, where the biocide containing triamine was present. However, the adaptation did not result in the increase of antibiotic resistance, but manifested in changes within outer membrane proteins’ patterns. The strategy of bacterial membrane proteins’ analysis provides an opportunity to adjust the ways of infection treatments, especially when it is connected to the life-threating bacteremia caused by Salmonella species. PMID:28696348

Salmonella enterica serovar Typhimurium in Mauritius linked to consumption of marlin mousse.

PubMed

Issack, Mohammad I; Hendriksen, Rene S; Lun, Phimy Lan Keng; Lutchun, Ram K S; Aarestrup, Frank M

2009-01-01

We report the first outbreak of salmonellosis caused by consumption of contaminated marlin mousse. Between 29 October and 5 November 2008, at least 53 persons developed diarrheal illness, all with a history of eating marlin mousse. Salmonella spp. that did not produce gas from glucose was isolated from stools of 26 affected patients and blood culture from one patient. Salmonella sp. isolates with the same phenotype were isolated in three samples of marlin mousse manufactured on 27 October 2008. The constituents of the mousse were smoked marlin, raw eggs, bovine gelatin, oil, and cream. A laboratory investigation of one sample of marlin mousse manufactured 3 days later, and the individual ingredients sampled a week after production of the contaminated batch were all negative for Salmonella. Serotyping and minimum inhibitory concentration determination were performed on 12 patient isolates related to the outbreak and two mousse isolates. All isolates belonged to Salmonella serovar Typhimurium and were pansusceptible to all antimicrobials tested. Pulsed-field gel electrophoresis revealed that all the isolates were indistinguishable, thus implicating the mousse as the vehicle of the outbreak.

Analysis of antimicrobial resistance genes detected in multidrug-resistant Salmonella enterica serovar Typhimurium isolated from food animals.

PubMed

Glenn, LaShanda M; Lindsey, Rebecca L; Frank, Joseph F; Meinersmann, Richard J; Englen, Mark D; Fedorka-Cray, Paula J; Frye, Jonathan G

2011-09-01

Multidrug-resistant (MDR) Salmonella enterica serovar Typhimurium is the most prevalent penta-resistant serovar isolated from animals by the U.S. National Antimicrobial Resistance Monitoring System. Penta-resistant isolates are often resistant to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline. To investigate MDR in Salmonella Typhimurium (including variant 5-), one isolate each from cattle, poultry, and swine with at least the ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline phenotype were selected for each year from 1997 to 2007 (n = 33) for microarray analysis of antimicrobial resistance, incompatibility IncA/C, and HI1 plasmid genes. Cluster analysis based on these data separated 31 of the isolates into two groups A and B (15 and 16 isolates, respectively). Isolates in group A were phage type DT104 or U302 and were mostly swine isolates (7/15). Genes detected included intI1, bla(PSE-1), floR, aadA, sulI, tet(G), and tetR, which are often found in Salmonella Genomic Island I. Isolates in group B had numerous IncA/C plasmid genes detected and were mostly cattle isolates (9/16). Genes detected included bla(CMY-2), floR, aac(3), aadA, aphA1, strA, strB, sulI, sulII, dfrA, dhf, tet(A)(B)(C)(D), and tetR, which are often found on MDR-AmpC IncA/C plasmids. The IncA/C replicon was also detected in all group B isolates. The two remaining isolates did not cluster with any others and both had many HI1 plasmid genes detected. Linkage disequilibrium analysis detected significant associations between plasmid replicon type, phage type, and animal source. These data suggest that MDR in Salmonella Typhimurium is associated with DT104/Salmonella Genomic Island I or IncA/C MDR-AmpC encoding plasmids and these genetic elements have persisted throughout the study period.

Complete genome sequence of a ciprofloxacin resistant Salmonella enterica subsp. enterica serovar Kentucky sequence of a ciprofloxacin strain, PU131, isolated from a human patient in Washington State.

USDA-ARS?s Scientific Manuscript database

A ciprofloxacin resistant (CipR) Salmonella enterica subsp. enterica serovar Kentucky ST198 has rapidly and extensively disseminated globally to become a major food-safety and public health concern. Here, we report a complete genome sequence of a CipR S. Kentucky ST198 strain PU131 isolated from a ...

Plurality of Leptospira strains on slaughtered animals suggest a broader concept of adaptability of leptospires to cattle.

PubMed

Pinto, Priscila S; Pestana, Cristiane; Medeiros, Marco A; Lilenbaum, Walter

2017-08-01

Leptospirosis in bovines is in majority determined by the host-adapted serovars, mainly Hardjo (types Hardjoprajitno and Hardjobovis), that belong to the serogroup Sejroe. Members of other serogroups as Pomona and Tarassovi have been eventually reported, mainly when outbreaks occurs. Nevertheless, the real role of other strains (non-Hardjo) on determining disease or being transmitted by cattle free of apparent clinical signs of acute infection remains to be elucidated. In that context, the aim of the present study was to investigate the hypothesis that strains of serovars/serogroups other than Hardjo may also be maintained and shed by cattle free of clinical signs. Samples of urine and/or vaginal fluid were collected from 697 bovines from a slaughterhouse located close to Rio de Janeiro, Brazil. Culturing yielded 19 isolates what represents the largest number ever obtained in Brazil on similar studies. These strains were serogrouped and genetically characterized. Fifteen of those were described in other papers and four are first described on the present study. Isolates belong to three different species (Leptospira santarosai, L. alstonii and L. interrogans) and five serogroups (Sarmin, Tarassovi, Shermani, Grippotyphosa and Sejroe). The majority (84.2%) of the isolates belongs to the species L. santarosai, the most prevalent species on cattle in the studied region. Non-Hardjo (non-Sejroe) strains represent 57.9% of the isolates, what indicates an unexpected high diversity of serogroups obtained from these cattle. This suggest that non-Hardjo (non-Sejroe) strains may also be maintained and shed by cattle and that finding must be considered in the epidemiology and control of the disease. Copyright © 2017 Elsevier B.V. All rights reserved.

Interlaboratory and between-specimen comparisons of diagnostic tests for leptospirosis in sheep and cattle.

PubMed

Fang, Fang; Collins-Emerson, Julie M; Heuer, Cord; Hill, Fraser I; Tisdall, David J; Wilson, Peter R; Benschop, Jackie

2014-11-01

A study was performed to investigate interlaboratory test agreement between a research and a commercial veterinary diagnostic laboratory on blood and urine samples, and to investigate test agreement between blood, urine, and kidney samples (research laboratory) for leptospirosis diagnosis. Samples were sourced from 399 sheep and 146 beef cattle from a local abattoir. Interlaboratory agreement for real-time quantitative polymerase chain reaction (qPCR) results on urine samples was almost perfect (kappa = 0.90), despite the use of different amplification targets (DNA gyrase subunit B gene vs. 16s ribosomal RNA gene), chemistries (SYTO9 vs. TaqMan probe), and pre-PCR processing. Interlaboratory agreement for microscopic agglutination test (MAT) positivity was almost perfect (kappa = 0.93) for Leptospira borgpetersenii serovar Hardjo subtype Hardjobovis (Hardjobovis) but moderate (kappa = 0.53) for Leptospira interrogans serovar Pomona (Pomona). Among animals that had different titers recorded, higher Hardjobovis and lower Pomona titers were reported by the commercial laboratory than by the research laboratory (P < 0.005). These interlaboratory comparisons can assist researchers and diagnosticians in interpreting the sometimes discrepant test results. Within the research laboratory, the comparison of qPCR results on urine and kidney showed almost perfect agreement (kappa = 0.84), suggesting that the qPCR on these 2 specimens can be used interchangeably. The agreement between MAT positivity and urine and kidney qPCR results was fair (kappa = 0.32 and kappa = 0.33, respectively). However, the prevalence ratio of urine and kidney qPCR positivity in Hardjobovis-seropositive versus Hardjobovis-seronegative sheep indicated that Hardjobovis seropositivity found in sheep may be able to predict shedding or renal carriage. © 2014 The Author(s).

Evidence for Lack of Acquisition of Tolerance in Salmonella enterica Serovar Typhimurium ATCC 14028 after Exposure to Subinhibitory Amounts of Origanum vulgare L. Essential Oil and Carvacrol

PubMed Central

Luz, Isabelle da Silva; Gomes Neto, Nelson Justino; Tavares, Adassa Gama; Nunes, Pollyana Campos; Magnani, Marciane

2012-01-01

Overnight exposure of Salmonella enterica serovar Typhimurium to sublethal amounts of Origanum vulgare essential oil (OV) and carvacrol (CAR) did not result in direct and cross-bacterial protection. Cells subcultured with increasing amounts of OV or CAR survived up to the MIC of either compound, revealing few significant changes in bacterial susceptibility. PMID:22544235

Inhibition of the early stage of Salmonella enterica serovar Enteritidis biofilm development on stainless steel by cell-free supernatant of a Hafnia alvei culture.

PubMed

Chorianopoulos, Nikos G; Giaouris, Efstathios D; Kourkoutas, Yiannis; Nychas, George-John E

2010-03-01

Compounds present in Hafnia alvei cell-free culture supernatant cumulatively negatively influence the early stage of biofilm development by Salmonella enterica serovar Enteritidis on stainless steel while they also reduce the overall metabolic activity of S. Enteritidis planktonic cells. Although acylhomoserine lactones (AHLs) were detected among these compounds, the use of several synthetic AHLs was not able to affect the initial stage of biofilm formation by this pathogen.

Aneurysm of the cranial mesenteric artery as a site of carriage of Salmonella enterica subsp. enterica serovar Abortusequi in the horse.

PubMed

Niwa, Hidekazu; Hobo, Seiji; Kinoshita, Yuta; Muranaka, Masanori; Ochi, Akihiro; Ueno, Takanori; Oku, Kazuomi; Hariu, Kazuhisa; Katayama, Yoshinari

2016-07-01

Salmonella enterica subsp. enterica serovar Abortusequi is a pathogen restricted to horses. Our investigation targeted 4 draft horses (9-10 months old) kept on a Japanese farm that had suffered an outbreak of S. Abortusequi abortion. The 4 horses were suspected to be carriers of the bacterium owing to their high agglutination titers (≥1:2,560) in tube agglutination testing. The owners' on-farm observations confirmed that the horses had no apparent abnormalities, and S. Abortusequi was not isolated from their blood, rectal swabs, or sternal bone marrow fluid at antemortem investigation. However, at autopsy, all horses displayed the following: suppurative aneurysm of the cranial mesenteric artery with heavy infection with Strongylus vulgaris larvae; heavy intestinal parasitic infection with Gasterophilus intestinalis, Parascaris equorum, Anoplocephala perfoliata, and S. vulgaris; and enlargement of the systemic lymph nodes. In each case, large numbers of S. Abortusequi were isolated from the anterior mesenteric artery thrombus. The thrombus isolates harbored a single virulence plasmid, and the pulsed-field gel electrophoresis profiles of the isolates were identical not only to each other but also to those of Japanese enzootic strains of S. Abortusequi. These results reveal that parasitic aneurysms of the cranial mesenteric artery should be considered an important possible site of carriage of S. Abortusequi in horses. The results also suggest high clonality of the isolated serovar in the horse population in Japan. © 2016 The Author(s).

Distribution of Salmonella serovars in breeding, nursery, and grow-to-finish pigs, and risk factors for shedding in ten farrow-to-finish swine farms in Alberta and Saskatchewan.

PubMed

Wilkins, Wendy; Rajić, Andrijana; Waldner, Cheryl; McFall, Margaret; Chow, Eva; Muckle, Anne; Rosengren, Leigh

2010-04-01

The study objectives were to investigate Salmonella prevalence, serovar distribution, and risk factors for shedding in 10 purposively selected farrow-to-finish farms in Saskatchewan and Alberta. Pooled fecal samples from the breeding and grow-finish phases and individual fecal samples from breeding, nursery, and grow-finish pigs were cultured for Salmonella; serotyping of isolates was performed. Pig and pen characteristics were recorded for each pig and pen sampled.Overall, 407/1143 (36%) of samples were Salmonella positive; within-farm prevalence ranged from 1% to 79%. Sows, nursery, and grow-finish pigs accounted for 43%, 29%, and 28% of positive samples, respectively. More Salmonella were detected in pooled pen than individual pig samples (P < 0.001). Among 418 Salmonella isolates, there were 19 distinct serovars; the most common were S. Derby (28.5%), S. Typhimurium, var. Copenhagen (19.1%), S. Putten (11.8%), S. Infantis (6.8%), and S. Mbandaka (6.1%). Sows were more likely to shed Salmonella than nursery or grow-finisher (OR 2.9, P < 0.001) pigs. Pelleted feed (OR 8.2, P < 0.001) and nose-to-nose pig contact through pens (OR 2.2, P = 0.005) were associated with increased Salmonella prevalence. Significant differences in serovar distribution were detected among production phases. The use of pooled pen samples is recommended as a more efficient means for accurate evaluation of Salmonella status in different phases of pig production. The breeding herd might be an important source of Salmonella persistence within farrow-to-finish farms and should be targeted in control efforts. The latter might also apply to the use of pelleted feed, which remains the most consistently reported significant risk factor for Salmonella shedding in pigs.

Predicting adhesion and biofilm formation boundaries on stainless steel surfaces by five Salmonella enterica strains belonging to different serovars as a function of pH, temperature and NaCl concentration.

PubMed

Moraes, Juliana O; Cruz, Ellen A; Souza, Enio G F; Oliveira, Tereza C M; Alvarenga, Verônica O; Peña, Wilmer E L; Sant'Ana, Anderson S; Magnani, Marciane

2018-05-26

This study aimed to assess the capability of 97 epidemic S. enterica strains belonging to 18 serovars to form biofilm. Five strains characterized as strong biofilm-producers, belonging to distinct serovars (S. Enteritidis 132, S. Infantis 176, S. Typhimurium 177, S. Heidelberg 281 and S. Corvallis 297) were assayed for adhesion/biofilm formation on stainless steel surfaces. The experiments were conducted in different combinations of NaCl (0, 2, 4, 5, 6, 8 and 10% w/v), pH (4, 5, 6 and 7) and temperatures (8 °C, 12 °C, 20 °C and 35 °C). Only adhesion was assumed to occur when S. enterica counts were ≥3 and <5 log CFU/cm 2 , whereas biofilm formation was defined as when the counts were ≥5 log CFU/cm 2 . The binary responses were used to develop models to predict the probability of adhesion/biofilm formation on stainless steel surfaces by five strains belonging to different S. enterica serovars. A total of 99% (96/97) of the tested S. enterica strains were characterized as biofilm-producers in the microtiter plate assays. The ability to form biofilm varied (P < 0.05) within and among the different serovars. Among the biofilm-producers, 21% (20/96), 45% (43/96), and 35% (34/96) were weak, moderate and strong biofilm-producers, respectively. The capability for adhesion/biofilm formation on stainless steel surfaces under the experimental conditions studied varied among the strains studied, and distinct secondary models were obtained to describe the behavior of the five S. enterica tested. All strains showed adhesion at pH 4 up to 4% of NaCl and at 20 °C and 35 °C. The probability of adhesion decreased when NaCl concentrations were >8% and at 8 °C, as well as in pH values ≤ 5 and NaCl concentrations > 6%, for all tested strains. At pH 7 and 6, biofilm formation for S. Enteritidis, S. Infantis, S. Typhimurium, S. Heidelberg was observed up to 6% of NaCl at 35 °C and 20 °C. The predicted boundaries for adhesion were p

Characterization of antimicrobial resistance, molecular and phage types of Salmonella enterica serovar Typhi isolations.

PubMed

Demczuk, W H B; Finley, R; Nadon, C; Spencer, A; Gilmour, M; Ng, L-K

2010-10-01

Isolation rates in Canada of Salmonella enterica serovar Typhi increased from 0.29 to 0.55 isolations/100,000 population during 2000-2006. Although no ciprofloxacin resistance was detected, nalidixic acid resistance increased from 41% to 80%. Multidrug-resistant S. Typhi represented 18% of the strains tested. Pulsed-field gel electrophoresis (PFGE) analysis of 222 isolates resulted in 91 distinct patterns clustering into four major genetic similarity groups. The five most frequently occurring PFGE patterns accounted for 46% of the isolates. Drug-resistant isolates predominantly occurred in one PFGE similarity group. There were 39 phage types identified in 826 isolates analysed with 60% described by five phage types; 134 were untypable. The phage types associated with multidrug resistance were phage types 53, B1, D1, E1, E9, G3 and M1. Improved integration of epidemiological and laboratory case data will facilitate the protection of public health in Canada during an era of increasing travel and globalization.

Simultaneous detection of antibodies to five Actinobacillus pleuropneumoniae serovars using bead-based multiplex analysis.

PubMed

Berger, Sanne Schou; Lauritsen, Klara Tølbøll; Boas, Ulrik; Lind, Peter; Andresen, Lars Ole

2017-11-01

We developed and made a preliminary validation of a bead-based multiplexed immunoassay for simultaneous detection of porcine serum antibodies to Actinobacillus pleuropneumoniae serovars 1, 2, 6, 7, and 12. Magnetic fluorescent beads were coupled with A. pleuropneumoniae antigens and tested with a panel of serum samples from experimentally infected pigs and with serum samples from uninfected and naturally infected pigs. The multiplex assay was compared to in-house ELISAs and complement fixation (CF) tests, which have been used for decades as tools for herd classification in the Danish Specific Pathogen Free system. Assay specificities and sensitivities as well as the corresponding cutoff values were determined using receiver operating characteristic (ROC) curve analysis, and the A. pleuropneumoniae multiplex assay showed good correlation with the in-house ELISAs and CF tests with areas under ROC curves ≥ 0.988. Benefits of multiplexed assays compared to ELISAs and CF tests include reduced serum sample volumes needed for analysis, less labor, and shorter assay time.

An extended genotyping framework for Salmonella enterica serovar Typhi, the cause of human typhoid

PubMed Central

Wong, Vanessa K.; Baker, Stephen; Connor, Thomas R.; Pickard, Derek; Page, Andrew J.; Dave, Jayshree; Murphy, Niamh; Holliman, Richard; Sefton, Armine; Millar, Michael; Dyson, Zoe A.; Dougan, Gordon; Holt, Kathryn E.; Parkhill, Julian; Feasey, Nicholas A.; Kingsley, Robert A.; Thomson, Nicholas R.; Keane, Jacqueline A.; Weill, François- Xavier; Le Hello, Simon; Hawkey, Jane; Edwards, David J.; Harris, Simon R.; Cain, Amy K.; Hadfield, James; Hart, Peter J.; Thieu, Nga Tran Vu; Klemm, Elizabeth J.; Breiman, Robert F.; Watson, Conall H.; Edmunds, W. John; Kariuki, Samuel; Gordon, Melita A.; Heyderman, Robert S.; Okoro, Chinyere; Jacobs, Jan; Lunguya, Octavie; Msefula, Chisomo; Chabalgoity, Jose A.; Kama, Mike; Jenkins, Kylie; Dutta, Shanta; Marks, Florian; Campos, Josefina; Thompson, Corinne; Obaro, Stephen; MacLennan, Calman A.; Dolecek, Christiane; Keddy, Karen H.; Smith, Anthony M.; Parry, Christopher M.; Karkey, Abhilasha; Dongol, Sabina; Basnyat, Buddha; Arjyal, Amit; Mulholland, E. Kim; Campbell, James I.; Dufour, Muriel; Bandaranayake, Don; Toleafoa, Take N.; Singh, Shalini Pravin; Hatta, Mochammad; Newton, Paul N.; Dance, David; Davong, Viengmon; Onsare, Robert S.; Isaia, Lupeoletalalelei; Thwaites, Guy; Wijedoru, Lalith; Crump, John A.; De Pinna, Elizabeth; Nair, Satheesh; Nilles, Eric J.; Thanh, Duy Pham; Turner, Paul; Soeng, Sona; Valcanis, Mary; Powling, Joan; Dimovski, Karolina; Hogg, Geoff; Farrar, Jeremy; Mather, Alison E.; Amos, Ben

2016-01-01

The population of Salmonella enterica serovar Typhi (S. Typhi), the causative agent of typhoid fever, exhibits limited DNA sequence variation, which complicates efforts to rationally discriminate individual isolates. Here we utilize data from whole-genome sequences (WGS) of nearly 2,000 isolates sourced from over 60 countries to generate a robust genotyping scheme that is phylogenetically informative and compatible with a range of assays. These data show that, with the exception of the rapidly disseminating H58 subclade (now designated genotype 4.3.1), the global S. Typhi population is highly structured and includes dozens of subclades that display geographical restriction. The genotyping approach presented here can be used to interrogate local S. Typhi populations and help identify recent introductions of S. Typhi into new or previously endemic locations, providing information on their likely geographical source. This approach can be used to classify clinical isolates and provides a universal framework for further experimental investigations. PMID:27703135

The Rcs-Regulated Colanic Acid Capsule Maintains Membrane Potential in Salmonella enterica serovar Typhimurium

PubMed Central

Pando, Jasmine M.; Karlinsey, Joyce E.; Lara, Jimmie C.; Libby, Stephen J.

2017-01-01

ABSTRACT The Rcs phosphorelay and Psp (phage shock protein) systems are envelope stress responses that are highly conserved in gammaproteobacteria. The Rcs regulon was found to be strongly induced during metal deprivation of Salmonella enterica serovar Typhimurium lacking the Psp response. Nineteen genes activated by the RcsA-RcsB response regulator make up an operon responsible for the production of colanic acid capsular polysaccharide, which promotes biofilm development. Despite more than half a century of research, the physiological function of colanic acid has remained elusive. Here we show that Rcs-dependent colanic acid production maintains the transmembrane electrical potential and proton motive force in cooperation with the Psp response. Production of negatively charged exopolysaccharide covalently bound to the outer membrane may enhance the surface potential by increasing the local proton concentration. This provides a unifying mechanism to account for diverse Rcs/colanic acid-related phenotypes, including susceptibility to membrane-damaging agents and biofilm formation. PMID:28588134

Assessment of contamination potential of lettuce by Salmonella enterica serovar Newport added to the plant growing medium.

PubMed

Bernstein, Nirit; Sela, Shlomo; Neder-Lavon, Sarit

2007-07-01

The capacity of Salmonella enterica serovar Newport to contaminate Romaine lettuce (Lactuca sativa L. cv. Nogal) via the root system was evaluated in 17-, 20-, and 33-day-old plants. Apparent internalization of Salmonella via the root to the above-ground parts was identified in 33- but not 17- or 20-day-old plants and was stimulated by root decapitation. Leaves of lettuce plants with intact and damaged roots harbored Salmonella at 500 +/- 120 and 5,130 +/- 440 CFU/g of leaf, respectively, at 2 days postinoculation but not 5 days later. These findings are first to suggest that Salmonella Newport can translocate from contaminated roots to the aerial parts of lettuce seedlings and propose that the process is dependent on the developmental stage of the plant.

Characterization of Foodborne Outbreaks of Salmonella enterica Serovar Enteritidis with Whole-Genome Sequencing Single Nucleotide Polymorphism-Based Analysis for Surveillance and Outbreak Detection.

PubMed

Taylor, Angela J; Lappi, Victoria; Wolfgang, William J; Lapierre, Pascal; Palumbo, Michael J; Medus, Carlota; Boxrud, David

2015-10-01

Salmonella enterica serovar Enteritidis is a significant cause of gastrointestinal illness in the United States; however, current molecular subtyping methods lack resolution for this highly clonal serovar. Advances in next-generation sequencing technologies have made it possible to examine whole-genome sequencing (WGS) as a potential molecular subtyping tool for outbreak detection and source trace back. Here, we conducted a retrospective analysis of S. Enteritidis isolates from seven epidemiologically confirmed foodborne outbreaks and sporadic isolates (not epidemiologically linked) to determine the utility of WGS to identify outbreaks. A collection of 55 epidemiologically characterized clinical and environmental S. Enteritidis isolates were sequenced. Single nucleotide polymorphism (SNP)-based cluster analysis of the S. Enteritidis genomes revealed well supported clades, with less than four-SNP pairwise diversity, that were concordant with epidemiologically defined outbreaks. Sporadic isolates were an average of 42.5 SNPs distant from the outbreak clusters. Isolates collected from the same patient over several weeks differed by only two SNPs. Our findings show that WGS provided greater resolution between outbreak, sporadic, and suspect isolates than the current gold standard subtyping method, pulsed-field gel electrophoresis (PFGE). Furthermore, results could be obtained in a time frame suitable for surveillance activities, supporting the use of WGS as an outbreak detection and characterization method for S. Enteritidis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Genetic Relatedness of Salmonella Serovars Isolated from Catfish (Clarias gariepinus) and Tilapia (Tilapia mossambica) Obtained from Wet Markets and Ponds in Penang, Malaysia.

PubMed

Budiati, Titik; Rusul, Gulam; Wan-Abdullah, Wan Nadiah; Chuah, Li-Oon; Ahmad, Rosma; Thong, Kwai Lin

2016-04-01

A total of 43 Salmonella enterica isolates belonging to different serovars (Salmonella Albany, Salmonella Agona, Salmonella Corvallis, Salmonella Stanley, Salmonella Typhimurium, Salmonella Mikawasima, and Salmonella Bovismorbificans) were isolated from catfish (Clarias gariepinus) and tilapia (Tilapia mossambica) obtained from nine wet markets and eight ponds in Penang, Malaysia. Thirteen, 19, and 11 isolates were isolated from 9 of 32 catfish, 14 of 32 tilapia, and 11 of 44 water samples, respectively. Fish reared in ponds were fed chicken offal, spoiled eggs, and commercial fish feed. The genetic relatedness of these Salmonella isolates was determined by random amplified polymorphic DNA PCR (RAPD-PCR) using primer OPC2, repetitive extragenic palindromic PCR (REP-PCR), and pulsed-field gel electrophoresis (PFGE). Composite analysis of the RAPD-PCR, REP-PCR, and PFGE results showed that the Salmonella serovars could be differentiated into six clusters and 15 singletons. RAPD-PCR differentiated the Salmonella isolates into 11 clusters and 10 singletons, while REP-PCR differentiated them into 4 clusters and 1 singleton. PFGE differentiated the Salmonella isolates into seven clusters and seven singletons. The close genetic relationship of Salmonella isolates from catfish or tilapia obtained from different ponds, irrespective of the type of feed given, may be caused by several factors, such as the quality of the water, density of fish, and size of ponds.

A Mutation in the Putative Lysl-tRNA Synthetase Gene, PoxR of Salmonella enterica Serovar Typhimurium Results in Altered Protein Production, Elevated Susceptibility to Environmental Challenges and Decreased Swine Colonization

USDA-ARS?s Scientific Manuscript database

Using signature-tagged mutagenesis, a mutation in the poxR gene of Salmonella enterica serovar Typhimurium was identified with decreased survival in an ex vivo swine stomach content assay(Bearson et al. Appl Environ Microbiol. 72:2829-36). Gastrointestinal colonization and fecal shedding of the pox...

Direct ROS scavenging activity of CueP from Salmonella enterica serovar Typhimurium.

PubMed

Yoon, Bo-Young; Yeom, Ji-Hyun; Kim, Jin-Sik; Um, Si-Hyeon; Jo, Inseong; Lee, Kangseok; Kim, Yong-Hak; Ha, Nam-Chul

2014-02-01

Salmonella enterica serovar Typhimurium (S. Typhimurium) is an intracellular pathogen that has evolved to survive in the phagosome of macrophages. The periplasmic copper-binding protein CueP was initially known to confer copper resistance to S. Typhimurium. Crystal structure and biochemical studies on CueP revealed a putative copper binding site surrounded by the conserved cysteine and histidine residues. A recent study reported that CueP supplies copper ions to periplasmic Cu, Zn-superoxide dismutase (SodCII) at a low copper concentration and thus enables the sustained SodCII activity in the periplasm. In this study, we investigated the role of CueP in copper resistance at a high copper concentration. We observed that the survival of a cueP-deleted strain of Salmonella in macrophage phagosome was significantly reduced. Subsequent biochemical experiments revealed that CueP specifically mediates the reduction of copper ion using electrons released during the formation of the disulfide bond. We observed that the copper ion-mediated Fenton reaction in the presence of hydrogen peroxide was blocked by CueP. This study provides insight into how CueP confers copper resistance to S. Typhimurium in copper-rich environments such as the phagosome of macrophages.

Salmonella Enterica Serovar Typhimurium BipA Exhibits Two Distinct Ribosome Binding Modes

DOE Office of Scientific and Technical Information (OSTI.GOV)

deLivron, M.; Robinson, V

BipA is a highly conserved prokaryotic GTPase that functions to influence numerous cellular processes in bacteria. In Escherichia coli and Salmonella enterica serovar Typhimurium, BipA has been implicated in controlling bacterial motility, modulating attachment and effacement processes, and upregulating the expression of virulence genes and is also responsible for avoidance of host defense mechanisms. In addition, BipA is thought to be involved in bacterial stress responses, such as those associated with virulence, temperature, and symbiosis. Thus, BipA is necessary for securing bacterial survival and successful invasion of the host. Steady-state kinetic analysis and pelleting assays were used to assess themore » GTPase and ribosome-binding properties of S. enterica BipA. Under normal bacterial growth, BipA associates with the ribosome in the GTP-bound state. However, using sucrose density gradients, we demonstrate that the association of BipA and the ribosome is altered under stress conditions in bacteria similar to those experienced during virulence. The data show that this differential binding is brought about by the presence of ppGpp, an alarmone that signals the onset of stress-related events in bacteria.« less

Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China.

PubMed

Yang, Xiaojuan; Wu, Qingping; Zhang, Jumei; Huang, Jiahui; Guo, Weipeng; Cai, Shuzhen

2015-01-01

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more

Prevalence and Characterization of Monophasic Salmonella Serovar 1,4,[5],12:i:- of Food Origin in China

PubMed Central

Yang, Xiaojuan; Wu, Qingping; Zhang, Jumei; Huang, Jiahui; Guo, Weipeng; Cai, Shuzhen

2015-01-01

Salmonella enterica subsp. enterica serovar 1,4,[5],12:i:- is a monophasic variant of Salmonella Typhimurium, which has recently been recognized as an emerging cause of infection worldwide. This bacterium has also ranked among the four most frequent serovars causing human salmonellosis in China. However, there are no reports on its contamination in Chinese food. Serotyping, polymerase chain reaction, antibiotic resistance, virulotyping, and multilocus sequence typing (MLST) assays were used to investigate the prevalence of this serological variant in food products in China, and to determine phenotypic and genotypic difference of monophasic isolates and Salmonella Typhimurium isolated over the same period. Salmonella 1,4,[5],12:i:- was prevalent in various food sources, including beef, pork, chicken, and pigeon. The study also confirmed the high prevalence (53.8%) of resistance to ampicillin, streptomycin, sulfonamides, and tetracycline in Salmonella 1,4,[5],12:i:-, which was higher than that in Salmonella Typhimurium. Moreover, Salmonella 1,4,[5],12:i:- isolates in our study were different from Salmonella Typhimurium isolates by the absence of three plasmid-borne genes (spvC, pefA, and rck) and the presence of gipA in all isolates. All Salmonella 1,4,[5],12:i:- isolates demonstrated MLST pattern ST34. Genomic deletions within the fljBA operon and surrounding genes were only found in Salmonella 1,4,[5],12:i:- isolates, with all isolates containing a deletion of fljB. However, hin and iroB were identified in all Salmonella 1,4,[5],12:i:- isolates. Three different deletion profiles were observed and two of them were different from the reported Salmonella 1,4,[5],12:i:- clones from Spain, America, and Italy, which provided some new evidence on the independent evolution of the multiple successful monophasic clones from Salmonella Typhimurium ancestors. This study is the first report of Salmonella 1,4,[5],12:i:- in food products from China. The data are more

Enhancement of Th1-biased protective immunity against avian influenza H9N2 virus via oral co-administration of attenuated Salmonella enterica serovar Typhimurium expressing chicken interferon-α and interleukin-18 along with an inactivated vaccine

PubMed Central

2012-01-01

Background Control of currently circulating re-assorted low-pathogenicity avian influenza (LPAI) H9N2 is a major concern for both animal and human health. Thus, an improved LPAI H9N2 vaccination strategy is needed to induce complete immunity in chickens against LPAI H9N2 virus strains. Cytokines play a crucial role in mounting both the type and extent of an immune response generated following infection with a pathogen or after vaccination. To improve the efficacy of inactivated LPAI H9N2 vaccine, attenuated Salmonella enterica serovar Typhimurium was used for oral co-administration of chicken interferon-α (chIFN-α) and chicken interleukin-18 (chIL-18) as natural immunomodulators. Results Oral co-administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18, prior to vaccination with inactivated AI H9N2 vaccine, modulated the immune response of chickens against the vaccine antigen through enhanced humoral and Th1-biased cell-mediated immunity, compared to chickens that received single administration of S. enterica serovar Typhimurium expressing either chIFN-α or chIL-18. To further test the protective efficacy of this improved vaccination regimen, immunized chickens were intra-tracheally challenged with a high dose of LPAI H9N2 virus. Combined administration of S. enterica serovar Typhimurium expressing chIFN-α and chIL-18 showed markedly enhanced protection compared to single administration of the construct, as determined by mortality, clinical severity, and feed and water intake. This enhancement of protective immunity was further confirmed by reduced rectal shedding and replication of AIV H9N2 in different tissues of challenged chickens. Conclusions Our results indicate the value of combined administration of chIFN-α and chIL-18 using a Salmonella vaccine strain to generate an effective immunization strategy in chickens against LPAI H9N2. PMID:22776696

Molecular and cellular characterization of a Salmonella enterica serovar Paratyphi a outbreak strain and the human immune response to infection.

PubMed

Gal-Mor, Ohad; Suez, Jotham; Elhadad, Dana; Porwollik, Steffen; Leshem, Eyal; Valinsky, Lea; McClelland, Michael; Schwartz, Eliezer; Rahav, Galia

2012-02-01

Enteric fever is an invasive life-threatening systemic disease caused by the Salmonella enterica human-adapted serovars Typhi and Paratyphi. Increasing incidence of infections with Salmonella enterica serovar Paratyphi A and the spreading of its antibiotic-resistant derivates pose a significant health concern in some areas of the world. Herein, we describe a molecular and phenotypic characterization of an S. Paratyphi A strain accounted for a recent paratyphoid outbreak in Nepal that affected at least 37 travelers. Pulsed-field gel electrophoresis analysis of the outbreak isolates revealed one genetic clone (pulsotype), confirming a single infecting source. Genetic profiling of the outbreak strain demonstrated the contribution of specific bacteriophages as a prime source of genetic diversity among clinical isolates of S. Paratyphi A. Phenotypic characterization in comparison with the S. Paratyphi A ATCC 9150 reference sequenced strain showed differences in flagellar morphology and increased abilities of the outbreak strain with respect to its motility, invasion into nonphagocytic cells, intracellular multiplication, survival within macrophages, and higher induction of interleukin-8 (IL-8) secreted by host cells. Collectively, these differences suggest an enhanced virulence potential of this strain and demonstrate an interesting phenotypic variation among S. Paratyphi A isolates. In vivo profiling of 16 inflammatory cytokines in patients infected with the outbreak strain revealed a common profile of a remarkable gamma interferon (IFN-γ) induction together with elevated concentrations of tumor necrosis factor alpha (TNF-α), IL-6, IL-8, IL-10, and IL-15, but not IL-12, which was previously demonstrated as elevated in nontyphoidal Salmonella infections. This apparent profile implies a distinct immune response to paratyphoid infections.

Sensitive Leptospira DNA detection using tapered optical fiber sensor.

PubMed

Zainuddin, Nurul H; Chee, Hui Y; Ahmad, Muhammad Z; Mahdi, Mohd A; Abu Bakar, Muhammad H; Yaacob, Mohd H

2018-03-23

This paper presents the development of tapered optical fiber sensor to detect a specific Leptospira bacteria DNA. The bacteria causes Leptospirosis, a deadly disease but with common early flu-like symptoms. Optical single mode fiber (SMF) of 125 μm diameter is tapered to produce 12 μm waist diameter and 15 cm length. The novel DNA-based optical fiber sensor is functionalized by incubating the tapered region with sodium hydroxide (NaOH), (3-Aminopropyl) triethoxysilane and glutaraldehyde. Probe DNA is immobilized onto the tapered region and subsequently hybridized by its complementary DNA (cDNA). The transmission spectra of the DNA-based optical fiber sensor are measured in the 1500 to 1600 nm wavelength range. It is discovered that the shift of the wavelength in the SMF sensor is linearly proportional with the increase in the cDNA concentrations from 0.1 to 1.0 nM. The sensitivity of the sensor toward DNA is measured to be 1.2862 nm/nM and able to detect as low as 0.1 fM. The sensor indicates high specificity when only minimal shift is detected for non-cDNA testing. The developed sensor is able to distinguish between actual DNA of Leptospira serovars (Canicola and Copenhageni) against Clostridium difficile (control sample) at very low (femtomolar) target concentrations. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

A pulsed field gel electrophoresis (PFGE) study that suggests a major world-wide clone of Salmonella enterica serovar Enteritidis.

PubMed

Pang, Jen-Chieh; Chiu, Tsai-Hsin; Helmuth, Reiner; Schroeter, Andreas; Guerra, Beatriz; Tsen, Hau-Yang

2007-05-30

Since human infections by Salmonella enterica serovar Enteritidis (Salmonella Enteritidis) have been increasing world-wide over the past years and epidemiological studies have implicated the consumption of meat, poultry, eggs and egg products, elucidation of the predominant subtypes for this Salmonella spp. is important. In this study, 107 poultry and food isolates of Salmonella Enteritidis obtained from Germany were analyzed by pulsed field gel electrophoresis (PFGE), and the subtypes were compared with those of the 124 human isolates obtained in Taiwan. Results showed that for these 107 poultry and food isolates, when XbaI, SpeI and NotI were used for chromosomal DNA digestion followed by PFGE analysis, a total of 19, 20 and 19 PFGE patterns, respectively, were identified. Of them, 51 (47.7%), 52 (48.6%) and 42 (39.3%) strains belong to a single pattern of X3, S3 and N3, respectively, and 34 strains belong to a pattern combination of X3S3N3, which was the major subtype. When PFGE patterns of these 107 German isolates were compared with those of the 124 human isolates obtained in Taiwan, pattern combination of X3S3N3 was found as the most common pattern shared by isolates from both areas. PT4 is a major phage type for German and Taiwan isolates. Although most of the X3S3N3 strains are of this phage type, some strains of other PFGE patterns are also of this phage type. Since strains used in this study were unrelated, i.e., they were isolated from different origins in areas geographically far apart from each other, the PFGE study suggests a major world-wide clone of S. enterica serovar Enteritidis.

Microarray-based detection of Salmonella enterica serovar Enteritidis genes involved in chicken reproductive tract colonization.

PubMed

Raspoet, R; Appia-Ayme, C; Shearer, N; Martel, A; Pasmans, F; Haesebrouck, F; Ducatelle, R; Thompson, A; Van Immerseel, F

2014-12-01

Salmonella enterica serovar Enteritidis has developed the potential to contaminate table eggs internally, by colonization of the chicken reproductive tract and internalization in the forming egg. The serotype Enteritidis has developed mechanisms to colonize the chicken oviduct more successfully than other serotypes. Until now, the strategies exploited by Salmonella Enteritidis to do so have remained largely unknown. For that reason, a microarray-based transposon library screen was used to identify genes that are essential for the persistence of Salmonella Enteritidis inside primary chicken oviduct gland cells in vitro and inside the reproductive tract in vivo. A total of 81 genes with a potential role in persistence in both the oviduct cells and the oviduct tissue were identified. Major groups of importance include the Salmonella pathogenicity islands 1 and 2, genes involved in stress responses, cell wall, and lipopolysaccharide structure, and the region-of-difference genomic islands 9, 21, and 40. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

A cross-sectional epidemiological study of domestic animals related to human leptospirosis cases in Nicaragua.

PubMed

Flores, Byron J; Pérez-Sánchez, Tania; Fuertes, Héctor; Sheleby-Elías, Jessica; Múzquiz, José Luis; Jirón, William; Duttmann, Christianne; Halaihel, Nabil

2017-06-01

Leptospirosis is one of the most extended zoonosis worldwide and humans become infected most commonly through contact with the urine of carrier animals, either directly or via contaminated water or soil. The aim in this study was to analyse the epidemiological behaviour of Leptospira spp., from domestic animals around the sites of human leptospirosis cases in Nicaragua, from 2007 through 2013. We report the results of a cross-sectional epidemiological study with a non-probability sampling of blood (n=3050) and urine (n=299) from Domestic Animals (DA) around the sites of human leptospirosis cases in Nicaragua. We analysed data obtained through Microscopic Agglutination Test (MAT), in-vitro culture, real time PCR and sequencing of lfb1 locus. Frequencies of 30.31% (95% CI: 28.66-31.95) and 15.38% (95% CI: 11.12-19.64) were obtained from serological test and from in-vitro culture, respectively. Although similar frequencies from serology test (P≥0.05) were found in DA species, in-vitro culture frequencies were significantly higher from bovine, equine and sheep (P<0.05) in comparison with swine and canine species. Ten serogroups of pathogenic Leptospira spp. were encountered, with the highest presence of Icterohaemorrhagiae serogroup 34.65% (95% CI: 29.35-39.94). We identified 7 samples homologous to L. interrogans species Pyrogenes serovar and 3 samples as L. noguchii Louisiana or Panama serovars by analysis of lfb1 sequences. We were able to establish a temporal and spatial correlation from DA and cumulative incidence of human cases. Therefore an effective epidemiological surveillance should be implemented with a specific control program toward DA in order to reduce human leptospirosis incidence. Copyright © 2017 Elsevier B.V. All rights reserved.

Role of Environmental Factors in Shaping Spatial Distribution of Salmonella enterica Serovar Typhi, Fiji.

PubMed

de Alwis, Ruklanthi; Watson, Conall; Nikolay, Birgit; Lowry, John H; Thieu, Nga Tran Vu; Van, Tan Trinh; Ngoc, Dung Tran Thi; Rawalai, Kitione; Taufa, Mere; Coriakula, Jerimaia; Lau, Colleen L; Nilles, Eric J; Edmunds, W John; Kama, Mike; Baker, Stephen; Cano, Jorge

2018-02-01

Fiji recently experienced a sharp increase in reported typhoid fever cases. To investigate geographic distribution and environmental risk factors associated with Salmonella enterica serovar Typhi infection, we conducted a cross-sectional cluster survey with associated serologic testing for Vi capsular antigen-specific antibodies (a marker for exposure to Salmonella Typhi in Fiji in 2013. Hotspots with high seroprevalence of Vi-specific antibodies were identified in northeastern mainland Fiji. Risk for Vi seropositivity increased with increased annual rainfall (odds ratio [OR] 1.26/quintile increase, 95% CI 1.12-1.42), and decreased with increased distance from major rivers and creeks (OR 0.89/km increase, 95% CI 0.80-0.99) and distance to modeled flood-risk areas (OR 0.80/quintile increase, 95% CI 0.69-0.92) after being adjusted for age, typhoid fever vaccination, and home toilet type. Risk for exposure to Salmonella Typhi and its spatial distribution in Fiji are driven by environmental factors. Our findings can directly affect typhoid fever control efforts in Fiji.

Role of Environmental Factors in Shaping Spatial Distribution of Salmonella enterica Serovar Typhi, Fiji

PubMed Central

Watson, Conall; Nikolay, Birgit; Lowry, John H.; Thieu, Nga Tran Vu; Van, Tan Trinh; Ngoc, Dung Tran Thi; Rawalai, Kitione; Taufa, Mere; Coriakula, Jerimaia; Lau, Colleen L.; Nilles, Eric J.; Edmunds, W. John; Kama, Mike; Baker, Stephen; Cano, Jorge

2018-01-01

Fiji recently experienced a sharp increase in reported typhoid fever cases. To investigate geographic distribution and environmental risk factors associated with Salmonella enterica serovar Typhi infection, we conducted a cross-sectional cluster survey with associated serologic testing for Vi capsular antigen–specific antibodies (a marker for exposure to Salmonella Typhi in Fiji in 2013. Hotspots with high seroprevalence of Vi-specific antibodies were identified in northeastern mainland Fiji. Risk for Vi seropositivity increased with increased annual rainfall (odds ratio [OR] 1.26/quintile increase, 95% CI 1.12–1.42), and decreased with increased distance from major rivers and creeks (OR 0.89/km increase, 95% CI 0.80–0.99) and distance to modeled flood-risk areas (OR 0.80/quintile increase, 95% CI 0.69–0.92) after being adjusted for age, typhoid fever vaccination, and home toilet type. Risk for exposure to Salmonella Typhi and its spatial distribution in Fiji are driven by environmental factors. Our findings can directly affect typhoid fever control efforts in Fiji. PMID:29350150

Type I interferon induces necroptosis in macrophages during infection with Salmonella enterica serovar Typhimurium

PubMed Central

Robinson, Nirmal; McComb, Scott; Mulligan, Rebecca; Dudani, Renu; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Salmonella enterica serovar Typhimurium (S. Typhimurium) is a virulent pathogen that induces rapid host death. Here we observed that host survival after infection with S. Typhimurium was enhanced in the absence of type I interferon signaling, with improved survival of mice deficient in the receptor for type I interferons (Ifnar1−/− mice) that was attributed to macrophages. Although there was no impairment in cytokine expression or inflammasome activation in Ifnar1−/− macrophages, they were highly resistant to S. Typhimurium–induced cell death. Specific inhibition of the kinase RIP1or knockdown of the gene encoding the kinase RIP3 prevented the death of wild-type macrophages, which indicated that necroptosis was a mechanism of cell death. Finally, RIP3-deficient macrophages, which cannot undergo necroptosis, had similarly less death and enhanced control of S. Typhimurium in vivo. Thus, we propose that S. Typhimurium induces the production of type I interferon, which drives necroptosis of macrophages and allows them to evade the immune response. PMID:22922364

Salmonella enterica Serovar Typhimurium Exploits Inflammation to Compete with the Intestinal Microbiota

PubMed Central

Stecher, Bärbel; Westendorf, Astrid M; Barthel, Manja; Kremer, Marcus; Chaffron, Samuel; Macpherson, Andrew J; Buer, Jan; Parkhill, Julian; Dougan, Gordon; von Mering, Christian; Hardt, Wolf-Dietrich

2007-01-01

Most mucosal surfaces of the mammalian body are colonized by microbial communities (“microbiota”). A high density of commensal microbiota inhabits the intestine and shields from infection (“colonization resistance”). The virulence strategies allowing enteropathogenic bacteria to successfully compete with the microbiota and overcome colonization resistance are poorly understood. Here, we investigated manipulation of the intestinal microbiota by the enteropathogenic bacterium Salmonella enterica subspecies 1 serovar Typhimurium (S. Tm) in a mouse colitis model: we found that inflammatory host responses induced by S. Tm changed microbiota composition and suppressed its growth. In contrast to wild-type S. Tm, an avirulent invGsseD mutant failing to trigger colitis was outcompeted by the microbiota. This competitive defect was reverted if inflammation was provided concomitantly by mixed infection with wild-type S. Tm or in mice (IL10−/−, VILLIN-HACL4-CD8) with inflammatory bowel disease. Thus, inflammation is necessary and sufficient for overcoming colonization resistance. This reveals a new concept in infectious disease: in contrast to current thinking, inflammation is not always detrimental for the pathogen. Triggering the host's immune defence can shift the balance between the protective microbiota and the pathogen in favour of the pathogen. PMID:17760501

Characterization of Novel OmpA-Like Protein of Leptospira interrogans That Binds Extracellular Matrix Molecules and Plasminogen

PubMed Central

Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L. T. O.

2011-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K D, 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K D of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date. PMID:21755014

Characterization of novel OmpA-like protein of Leptospira interrogans that binds extracellular matrix molecules and plasminogen.

PubMed

Oliveira, Rosane; de Morais, Zenaide Maria; Gonçales, Amane Paldes; Romero, Eliete Caló; Vasconcellos, Silvio Arruda; Nascimento, Ana L T O

2011-01-01

Leptospira interrogans is the etiological agent of leptospirosis, a zoonotic disease of human and veterinary concern. The identification of novel proteins that mediate host-pathogen interactions is important for understanding the bacterial pathogenesis as well as to identify protective antigens that would help fight the disease. We describe in this work the cloning, expression, purification and characterization of three predicted leptospiral membrane proteins, LIC10258, LIC12880 (Lp30) and LIC12238. We have employed Escherichia coli BL21 (SI) strain as a host expression system. Recently, we have identified LIC12238 as a plasminogen (PLG)-binding receptor. We show now that Lp30 and rLIC10258 are also PLG-receptors of Leptospira, both exhibiting dose-dependent and saturating binding (K(D), 68.8±25.2 nM and 167.39±60.1 nM, for rLIC10258 and rLIC12880, respectively). In addition, LIC10258, which is a novel OmpA-like protein, binds laminin and plasma fibronectin ECM molecules and hence, it was named Lsa66 (Leptospiral surface adhesin of 66 kDa). Binding of Lsa66 to ECM components was determined to be specific, dose-dependent and saturable, with a K(D) of 55.4±15.9 nM to laminin and of 290.8±11.8 nM to plasma fibronectin. Binding of the recombinant proteins to PLG or ECM components was assessed by using antibodies against each of the recombinant proteins obtained in mice and confirmed by monoclonal anti-polyhistidine antibodies. Lsa66 caused partial inhibition on leptospiral adherence to immobilized ECM and PLG. Moreover, this adhesin and rLIC12238 are recognized by antibodies in serum samples of confirmed leptospirosis cases. Thus, Lsa66 is a novel OmpA-like protein with dual activity that may promote the attachment of Leptospira to host tissues and may contribute to the leptospiral invasion. To our knowledge, this is the first leptospiral protein with ECM and PLG binding properties reported to date.

Optimization of inactivated H5N9 highly pathogenic avian influenza vaccine and inactivated Salmonella enterica serovar Typhimurium vaccine with antigen dose and prime-boost regimen in domestic ducks.

PubMed

Yuk, Seong-Su; To, Eredene-Ochir; Kwon, Jung-Hoon; Noh, Jin-Yong; Hong, Woo-Tack; Jeong, Jei-Hyun; Gwon, Gyeong-Bin; Song, Chang-Seon

2017-09-01

Owing to the increase in the number of diseases affecting ducks and the demand for food safety by consumers, vaccination has become one of the factors that influence duck meat productivity. The highly pathogenic avian influenza (HPAI) virus is one of the most prevalent and causes one of the most lethal diseases in domestic ducks, and Salmonella enterica serovar Typhimurium is a food-borne pathogen persistent in the domestic duck population. To better understand the optimal usage of HPAI and S. enterica serovar Typhimurium vaccines, we aimed to determine antigen dose, oil and gel adjuvant usage with prime-boost regimen, and vaccination age, inducing the best immune response in ducks, without an effect on body weight gain. In the case of the inactivated H5N9 vaccine, a single dose of vaccine was inadequate to induce proper antibody titer when administered to day-old ducks, which necessitates boost vaccination. Administration of the oil-adjuvanted H5N9 vaccine administration in day-old and 2-week-old ducks resulted in a lower body weight at the time of slaughtering, compared to that of gel-adjuvanted H5N9 vaccine. However, gel-adjuvanted H5N9 vaccine failed to induce proper immune response to an extent recommend by OIE-World Organization for Animal Health. In the case of the Salmonella enterica serovar Typhimurium vaccine, a moderate or low dose of vaccine was appropriate for day-old ducks receiving the gel prime-oil boost vaccination. Single vaccination with oil adjuvants affects the mean body weight of 7-week-old ducks, suggesting that the gel adjuvant is more suitable for meat production. We expect that the use of adjuvants in a prime-boost regimen and at antigen doses set in this study will be helpful to maximize body weight in the case of domestic duck production at the actual farm site. © 2017 Poultry Science Association Inc.

Two Novel Salmonella Bivalent Vaccines Confer Dual Protection against Two Salmonella Serovars in Mice

PubMed Central

Zhao, Xinxin; Dai, Qinlong; Jia, Renyong; Zhu, Dekang; Liu, Mafeng; Wang, Mingshu; Chen, Shun; Sun, Kunfeng; Yang, Qiao; Wu, Ying; Cheng, Anchun

2017-01-01

Non-typhoidal Salmonella includes thousands of serovars that are leading causes of foodborne diarrheal illness worldwide. In this study, we constructed three bivalent vaccines for preventing both Salmonella Typhimurium and Salmonella Newport infections by using the aspartate semialdehyde dehydrogenase (Asd)-based balanced-lethal vector-host system. The constructed Asd+ plasmid pCZ11 carrying a subset of the Salmonella Newport O-antigen gene cluster including the wzx-wbaR-wbaL-wbaQ-wzy-wbaW-wbaZ genes was introduced into three Salmonella Typhimurium mutants: SLT19 (Δasd) with a smooth LPS phenotype, SLT20 (Δasd ΔrfbN) with a rough LPS phenotype, and SLT22 (Δasd ΔrfbN ΔpagL::T araC PBAD rfbN) with a smooth LPS phenotype when grown with arabinose. Immunoblotting demonstrated that SLT19 harboring pCZ11 [termed SLT19 (pCZ11)] co-expressed the homologous and heterologous O-antigens; SLT20 (pCZ11) exclusively expressed the heterologous O-antigen; and when arabinose was available, SLT22 (pCZ11) expressed both types of O-antigens, while in the absence of arabinose, SLT22 (pCZ11) expressed only the heterologous O-antigen. Exclusive expression of the heterologous O-antigen in Salmonella Typhimurium decreased the swimming ability of the bacterium and its susceptibility to polymyxin B. Next, the crp gene was deleted from the three recombinant strains for attenuation purposes, generating the three bivalent vaccine strains SLT25 (pCZ11), SLT26 (pCZ11), and SLT27 (pCZ11), respectively. Groups of BALB/c mice (12 mice/group) were orally immunized with 109 CFU of each vaccine strain twice at an interval of 4 weeks. Compared with a mock immunization, immunization with all three vaccine strains induced significant serum IgG responses against both Salmonella Typhimurium and Salmonella Newport LPS. The bacterial loads in the mouse tissues were significantly lower in the three vaccine-strain-immunized groups than in the mock group after either Salmonella Typhimurium or Salmonella

Determining risk for severe leptospirosis by molecular analysis of environmental surface waters for pathogenic Leptospira.

PubMed

Ganoza, Christian A; Matthias, Michael A; Collins-Richards, Devon; Brouwer, Kimberly C; Cunningham, Calaveras B; Segura, Eddy R; Gilman, Robert H; Gotuzzo, Eduardo; Vinetz, Joseph M

2006-08-01

Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters. A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (approximately 10(3) leptospires/ml versus 0.5 x 10(2) leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources. Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure and transmission. This

Determining Risk for Severe Leptospirosis by Molecular Analysis of Environmental Surface Waters for Pathogenic Leptospira

PubMed Central

Collins-Richards, Devon; Brouwer, Kimberly C; Cunningham, Calaveras B; Segura, Eddy R; Gilman, Robert H; Gotuzzo, Eduardo; Vinetz, Joseph M

2006-01-01

Background Although previous data indicate that the overall incidence of human leptospirosis in the Peruvian Amazon is similar in urban and rural sites, severe leptospirosis has been observed only in the urban context. As a potential explanation for this epidemiological observation, we tested the hypothesis that concentrations of more virulent Leptospira would be higher in urban than in rural environmental surface waters. Methods and Findings A quantitative real-time PCR assay was used to compare levels of Leptospira in urban and rural environmental surface waters in sites in the Peruvian Amazon region of Iquitos. Molecular taxonomic analysis of a 1,200-bp segment of the leptospiral 16S ribosomal RNA gene was used to identify Leptospira to the species level. Pathogenic Leptospira species were found only in urban slum water sources (Fisher's exact test; p = 0.013). The concentration of pathogen-related Leptospira was higher in urban than rural water sources (~103 leptospires/ml versus 0.5 × 102 leptospires/ml; F = 8.406, p < 0.05). Identical 16S rRNA gene sequences from Leptospira interrogans serovar Icterohaemorrhagiae were found in urban slum market area gutter water and in human isolates, suggesting a specific mode of transmission from rats to humans. In a prospective, population-based study of patients presenting with acute febrile illness, isolation of L. interrogans-related leptospires from humans was significantly associated with urban acquisition (75% of urban isolates); human isolates of other leptospiral species were associated with rural acquisition (78% of rural isolates) (chi-square analysis; p < 0.01). This distribution of human leptospiral isolates mirrored the distribution of leptospiral 16S ribosomal gene sequences in urban and rural water sources. Conclusions Our findings data support the hypothesis that urban severe leptospirosis in the Peruvian Amazon is associated with higher concentrations of more pathogenic leptospires at sites of exposure

Comparison of Genotypes of Salmonella enterica Serovar Enteritidis Phage Type 30 and 9c Strains Isolated during Three Outbreaks Associated with Raw Almonds▿

PubMed Central

Parker, Craig T.; Huynh, Steven; Quiñones, Beatriz; Harris, Linda J.; Mandrell, Robert E.

2010-01-01

In 2000 to 2001, 2003 to 2004, and 2005 to 2006, three outbreaks of Salmonella enterica serovar Enteritidis were linked with the consumption of raw almonds. The S. Enteritidis strains from these outbreaks had rare phage types (PT), PT30 and PT9c. Clinical and environmental S. Enteritidis strains were subjected to pulsed-field gel electrophoresis (PFGE), multilocus variable-number tandem repeat analysis (MLVA), and DNA microarray-based comparative genomic indexing (CGI) to evaluate their genetic relatedness. All three methods differentiated these S. Enteritidis strains in a manner that correlated with PT. The CGI analysis confirmed that the majority of the differences between the S. Enteritidis PT9c and PT30 strains corresponded to bacteriophage-related genes present in the sequenced genomes of S. Enteritidis PT4 and S. enterica serovar Typhimurium LT2. However, PFGE, MLVA, and CGI failed to discriminate between S. Enteritidis PT30 strains related to outbreaks from unrelated clinical strains or between strains separated by up to 5 years. However, metabolic fingerprinting demonstrated that S. Enteritidis PT4, PT8, PT13a, and clinical PT30 strains metabolized l-aspartic acid, l-glutamic acid, l-proline, l-alanine, and d-alanine amino acids more efficiently than S. Enteritidis PT30 strains isolated from orchards. These data indicate that S. Enteritidis PT9c and 30 strains are highly related genetically and that PT30 orchard strains differ from clinical PT30 strains metabolically, possibly due to fitness adaptations. PMID:20363782

Role of outer membrane lipopolysaccharides in the protection of Salmonella enterica serovar Typhimurium from desiccation damage.

PubMed

Garmiri, Penelope; Coles, Karen E; Humphrey, Tom J; Cogan, Tristan A

2008-04-01

The ability to survive desiccation between hosts is often essential to the success of pathogenic bacteria. The bacterial outer membrane is both the cellular interface with hostile environments and the focus of much of the drying-induced damage. This study examined the contribution of outer membrane-associated polysaccharides to the survival of Salmonella enterica serovar Typhimurium in air-dried blood droplets following growth in high and low osmolarity medium and under conditions known to induce expression of these polysaccharides. Strains lacking the O polysaccharide (OPS) element of the outer membrane lipopolysaccharide were more sensitive to desiccation. Lipopolysaccharide core mutation further to OPS loss did not result in increased susceptibility to drying. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed lipopolysaccharide profiles that supported the hypothesis that OPS expression is required for optimal drying resistance in S. Typhimurium. The role of O antigen in Salmonella spp. in maintaining a hydrated layer around the dried cell or in slowing the rate of dehydration and rehydration is discussed.

In vitro evaluation of anti-infective activity of a Lactobacillus plantarum strain against Salmonella enterica serovar Enteritidis

PubMed Central

2013-01-01

Background Salmonella enterica serovar Enteritidis infections are known to exhibit worldwide prevalence with increased morbidity and mortality. The conventional strategies like antibiotic therapy and vaccination have not only proved to be of sub-optimal efficacy but also led to the development of multidrug resistant strains of Salmonella. Antimicrobial activities of probiotics against various enteropathogens and other health promoting effects have assumed greater significance in recent years. The present study aims to evaluate the efficacy of a Lactobacillus plantarum strain (KSBT 56, isolated from a traditional food product of India), in preventing Salmonella enterica serovar Enteritidis growth and pathogenicity in vitro. Methods and results The cell free culture supernatant (CFCS) of KSBT 56 strain notably inhibited the growth of Salmonella Enteritidis without affecting the growth of other gram-positive lactic acid bacteria. The isolated KSBT 56 strain produces lactic acid similar to other standard probiotic strains like Lactobacillus plantarum MTCC 1407. The free radical production by KSBT 56 strain was studied by using sodC mutant of S. Enteritidis, which exhibited reduced growth in the presence of CFCS of the KSBT 56 strain, indicating the inhibitory activity of free radicals on the growth of S. Enteritidis. Our results also showed a significant reduction in the biofilm forming ability of Salmonella Enteritidis in the presence of the KSBT 56 strain (2 log cfu/ml, p = 0.01). Further, the anti-infective characteristics of KSBT 56 strain was validated by gentamicin protection assay which revealed 80% reduction in the invasion of Salmonella Enteritidis to HCT-116 cell line (Salmonella Enteritidis and KSBT 56 in a 1:1 ratio) and delayed addition of Salmonella Enteritidis by 1 h. Similarly, the reduced adhesion of Salmonella to the HCT-116 cells was observed along with the down regulation of hilA gene of Salmonella Pathogenicity Island 1 (SPI1) indicating that they

Biofilm formation ability of Salmonella enterica serovar Typhimurium acrAB mutants.

PubMed

Schlisselberg, Dov B; Kler, Edna; Kisluk, Guy; Shachar, Dina; Yaron, Sima

2015-10-01

Recent studies offer contradictory findings about the role of multidrug efflux pumps in bacterial biofilm development. Thus, the aim of this study was to investigate the involvement of the AcrAB efflux pump in biofilm formation by investigating the ability of AcrB and AcrAB null mutants of Salmonella enterica serovar Typhimurium to produce biofilms. Three models were used to compare the ability of S. Typhimurium wild-type and its mutants to form biofilms: formation of biofilm on polystyrene surfaces; production of biofilm (mat model) on the air/liquid interface; and expression of curli and cellulose on Congo red-supplemented agar plates. All three investigated genotypes formed biofilms with similar characteristics. However, upon exposure to chloramphenicol, formation of biofilms on solid surfaces as well as the production of curli were either reduced or were delayed more significantly in both mutants, whilst there was no visible effect on pellicle formation. It can be concluded that when no selective pressure is applied, S. Typhimurium is able to produce biofilms even when the AcrAB efflux pumps are inactivated, implying that the use of efflux pump inhibitors to prevent biofilm formation is not a general solution and that combined treatments might be more efficient. Other factors that affect the ability to produce biofilms depending on efflux pump activity are yet to be identified. Copyright © 2015 Elsevier B.V. and the International Society of Chemotherapy. All rights reserved.

Adhesion and growth inhibitory effect of chicken egg yolk antibody (IgY) on Salmonella enterica serovars Enteritidis and Typhimurium in vitro.

PubMed

Chalghoumi, Raja; Théwis, André; Beckers, Yves; Marcq, Christopher; Portetelle, Daniel; Schneider, Yves-Jacques

2009-06-01

The protective effects of powder preparation of egg yolk immunoglobulin Y (IgY), specific to Salmonella Enteritidis and Salmonella Typhimurium outer membrane proteins (OMP), against these two Salmonella sp. serovars were investigated in vitro in two different assays: adhesion-prevention and growth-inhibition. The adhesion-prevention assay was conducted using polarized monolayers of the human intestinal epithelial Caco-2 cell line. First, the conditions of Salmonella adherence to Caco-2 cells were optimized, and interferences of bacteria with the transepithelial electrical resistance (TER) of fully differentiated Caco-2 cell monolayers and the lactate dehydrogenase release upon exposure of the cells to Salmonella were evaluated. Both Salmonella sp. serovars were able to adhere to Caco-2 cells and decreased TER. Results from the adhesion-prevention assay demonstrated that specific IgY reduced the decrease in TER of the infected Caco-2 cell monolayers and blocked the Salmonella sp. adhesion in a concentration-dependent manner (p < 0.05). Nonspecific IgY also exhibited an inhibitory effect on these two parameters, but to a lesser extent than that of the specific IgY (p < 0.05). The protective effect of nonspecific IgY could be attributed to the low-density lipoprotein component of the water-soluble fraction of egg yolks that may not have been eliminated during ultrafiltration. The growth-inhibition assay revealed that specific IgY had an inhibitory effect on the bacterial growth, markedly during the late exponential phase, whereas nonspecific IgY failed to do so. Taken together, these results suggest that the in vitro growth inhibitory effect of specific IgY on Salmonella spp. resulted from the specific binding activity of these IgY to Salmonella sp. OMP. Passive immunization with Salmonella sp. OMP-specific IgY could thus be useful to prevent Salmonella colonization in broiler chickens and the subsequent carcass contamination during processing.