A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae

PubMed Central

Soler-Lloréns, Pedro F.; Quance, Chris R.; Lawhon, Sara D.; Stuber, Tod P.; Edwards, John F.; Ficht, Thomas A.; Robbe-Austerman, Suelee; O'Callaghan, David; Keriel, Anne

2016-01-01

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog (Ceratophyrus ornate) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi. We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata-like strain BO2, with traits that depart significantly from those of the “classical” Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted “classical” species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella, but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required. PMID:27734009

A Brucella spp. Isolate from a Pac-Man Frog (Ceratophrys ornata) Reveals Characteristics Departing from Classical Brucellae.

PubMed

Soler-Lloréns, Pedro F; Quance, Chris R; Lawhon, Sara D; Stuber, Tod P; Edwards, John F; Ficht, Thomas A; Robbe-Austerman, Suelee; O'Callaghan, David; Keriel, Anne

2016-01-01

Brucella are highly infectious bacterial pathogens responsible for brucellosis, a frequent worldwide zoonosis. The Brucella genus has recently expanded from 6 to 11 species, all of which were associated with mammals; The natural host range recently expanded to amphibians after some reports of atypical strains from frogs. Here we describe the first in depth phenotypic and genetic characterization of a Brucella strains isolated from a frog. Strain B13-0095 was isolated from a Pac-Man frog ( Ceratophyrus ornate ) at a veterinary hospital in Texas and was initially misidentified as Ochrobactrum anthropi . We found that B13-0095 belongs to a group of early-diverging brucellae that includes Brucella inopinata strain BO1 and the B. inopinata -like strain BO2, with traits that depart significantly from those of the "classical" Brucella spp. Analysis of B13-0095 genome sequence revealed several specific features that suggest that this isolate represents an intermediate between a soil associated ancestor and the host adapted "classical" species. Like strain BO2, B13-0095 does not possess the genes required to produce the perosamine based LPS found in classical Brucella , but has a set of genes that could encode a rhamnose based O-antigen. Despite this, B13-0095 has a very fast intracellular replication rate in both epithelial cells and macrophages. Finally, another major finding in this study is the bacterial motility observed for strains B13-0095, BO1, and BO2, which is remarkable for this bacterial genus. This study thus highlights several novel characteristics in strains belonging to an emerging group within the Brucella genus. Accurate identification tools for such atypical Brucella isolates and careful evaluation of their zoonotic potential, are urgently required.

Brucella suis strain 2 vaccine is safe and protective against heterologous Brucella spp. infections.

PubMed

Zhu, Liangquan; Feng, Yu; Zhang, Ge; Jiang, Hui; Zhang, Zhen; Wang, Nan; Ding, Jiabo; Suo, Xun

2016-01-12

Brucellosis is a wide spread zoonotic disease that causes abortion and infertility in mammals and leads to debilitating, febrile illness in humans. Brucella abortus, Brucella melitensis and Brucella suis are the major pathogenic species to humans. Vaccination with live attenuated B. suis strain 2 (S2) vaccine is an essential and critical component in the control of brucellosis in China. The S2 vaccine is very effective in preventing brucellosis in goats, sheep, cattle and swine. However, there are still debates outside of China whether the S2 vaccine is able to provide protection against heterologous virulent Brucella species. We investigated the residual virulence, immunogenicity and protective efficacy of the S2 vaccine in BALB/c mice by determining bacteria persistence in spleen, serum antibody response, cellular immune response and protection against a heterologous virulent challenge. The S2 vaccine was of low virulence as there were no bacteria recovered in spleen four weeks post vaccination. The vaccinated mice developed Brucella-specific IgG in 2-3 weeks, and a burst production of IFN-γ at one week as well as a two-fold increase in TNF-α production. The S2 vaccine protected mice from a virulent challenge by B. melitensis M28, B. abortus 2308 and B. suis S1330, and the S2 vaccinated mice did not develop any clinical signs or tissue damage. Our study demonstrated that the S2 vaccine is of low virulence, stimulates good humoral and cellular immunity and protects animals against infection by heterologous, virulent Brucella species. Copyright © 2015 Elsevier Ltd. All rights reserved.

Epidemiology of brucellosis in domestic animals caused by Brucella melitensis, Brucella suis and Brucella abortus.

PubMed

Díaz Aparicio, E

2013-04-01

Brucellosis is a disease that causes severe economic losses for livestock farms worldwide. Brucella melitensis, B. abortus and B. suis, which are transmitted between animals both vertically and horizontally, cause abortion and infertility in their primary natural hosts - goats and sheep (B. melitensis), cows (B. abortus) and sows (B. suis). Brucella spp. infect not only their preferred hosts but also other domestic and wild animal species, which in turn can act as reservoirs of the disease for other animal species and humans. Brucellosis is therefore considered to be a major zoonosis transmitted by direct contact with animals and/or their secretions, or by consuming milk and dairy products.

Host response to Brucella infection: review and future perspective.

PubMed

Elfaki, Mohamed G; Alaidan, Alwaleed Abdullah; Al-Hokail, Abdullah Abdulrahman

2015-07-30

Brucellosis is a zoonotic and contagious infectious disease caused by infection with Brucella species. The infecting brucellae are capable of causing a devastating multi-organ disease in humans with serious health complications. The pathogenesis of Brucella infection is influenced largely by host factors, Brucella species/strain, and the ability of invading brucellae to survive and replicate within mononuclear phagocytic cells, preferentially macrophages (Mf). Consequently, the course of human infection may appear as an acute fatal or progress into chronic debilitating infection with periodical episodes that leads to bacteremia and death. The existence of brucellae inside Mf represents one of the strategies used by Brucella to evade the host immune response and is responsible for treatment failure in certain human populations treated with anti-Brucella drugs. Moreover, the persistence of brucellae inside Mf complicates the diagnosis and may affect the host cell signaling pathways with consequent alterations in both innate and adaptive immune responses. Therefore, there is an urgent need to pursue the development of novel drugs and/or vaccine targets against human brucellosis using high throughput technologies in genomics, proteomics, and immunology.

Identification of Brucella genus and eight Brucella species by Luminex bead-based suspension array.

PubMed

Lusk Pfefer, Tina S; Timme, Ruth; Kase, Julie A

2018-04-01

Globally, unpasteurized milk products are vehicles for the transmission of brucellosis, a zoonosis responsible for cases of foodborne illness in the United States and elsewhere. Existing PCR assays to detect Brucella species are restricted by the resolution of band sizes on a gel or the number of fluorescent channels in a single real-time system. The Luminex bead-based suspension array is performed in a 96-well plate allowing for high throughput screening of up to 100 targets in one sample with easily discernible results. We have developed an array using the Bio-Plex 200 to differentiate the most common Brucella species: B. abortus, B. melitensis, B. suis, B. suis bv5, B. canis, B. ovis, B. pinnipedia, and B. neotomae, as well as Brucella genus. All probes showed high specificity, with no cross-reaction with non-Brucella strains. We could detect pure DNA from B. abortus, B. melitensis, and genus-level Brucella at concentrations of ≤5 fg/μL. Pure DNA from all other species tested positive at concentrations well below 500 fg/μL and we positively identified B. neotomae in six artificially contaminated cheese and milk products. An intra-laboratory verification further demonstrated the assay's accuracy and robustness in the rapid screening (3-4 h including PCR) of DNA. Published by Elsevier Ltd.

Antibody Reactivity to Omp31 from Brucella melitensis in Human and Animal Infections by Smooth and Rough Brucellae

PubMed Central

Cassataro, Juliana; Pasquevich, Karina; Bruno, Laura; Wallach, Jorge C.; Fossati, Carlos A.; Baldi, Pablo C.

2004-01-01

Group 3 of outer membrane proteins (OMPs) of Brucella includes Omp25 and Omp31, which share 34% identity. Omp25 is highly conserved in Brucella species, and Omp31 is present in all Brucella species, except Brucella abortus. Antibodies to Brucella melitensis Omp31 have been sought only in infected sheep, and Western blotting of sera from infected sheep did not reveal anti-Omp31 reactivity. We obtained recombinant purified Omp31 (B. melitensis) and tested its recognition by sera from humans and animals suffering from brucellosis by an indirect enzyme-linked immunosorbent assay (ELISA). Serum samples from 74 patients, 57 sheep, and 47 dogs were analyzed; brucellosis was confirmed by bacteriological isolation in all ovine and canine cases and 31 human cases of brucellosis. Thirty-five patients (47%) were positive for antibodies to Omp31, including seven cases of Brucella suis infection, two cases of B. abortus infection, and three cases of B. melitensis infection. Of 39 sheep naturally infected with B. melitensis (biovars 1 and 3), 23 (59%) were positive for antibodies to Omp31. Anti-Omp31 antibodies were also detected in 12 of 18 rams (67%) in which Brucella ovis was isolated from semen. Antibodies to Omp31 were also found in 41 (87%) of the 47 dogs, including 13 with recent infection. These results suggest that an indirect ELISA using recombinant purified Omp31 from B. melitensis would be of limited value for the diagnosis of human and animal brucellosis. Nevertheless, the potential usefulness of this antigen in combination with other recombinant proteins from Brucella should not be dismissed.   PMID:14715555

[Evasion of anti-infectious immunity by Brucella - A review].

PubMed

Quan, Wurong; Yang, Yongjie

2016-05-04

Brucellosis, caused by Brucella species, is a worldwide zoonosis. As facultative intracellular pathogens, Brucella possess non-classical virulence factor, but its virulence is very powerful and can elicit chronic infections of both animals and humans. Evasion of host anti-infectious immunity is a prerequisite for chronic infections, this ability appears increasingly crucial for Brucella virulence. As successful pathogens, Brucella can escape or suppress innate immunity and modulate adaptive immunity to establish long lasting infections in host cells. In this review, we address the molecular mechanisms of Brucella to evade anti-infectious immunity. This will shed new insights on Brucella virulence and will, potentially, open new prophylactic avenues.

Brucella proteomes--a review.

PubMed

DelVecchio, Vito G; Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Estock, Frank; Elzer, Phil; Mujer, Cesar V

2002-12-20

The proteomes of selected Brucella spp. have been extensively analyzed by utilizing current proteomic technology involving 2-DE and MALDI-MS. In Brucella melitensis, more than 500 proteins were identified. The rapid and large-scale identification of proteins in this organism was accomplished by using the annotated B. melitensis genome which is now available in the GenBank. Coupled with new and powerful tools for data analysis, differentially expressed proteins were identified and categorized into several classes. A global overview of protein expression patterns emerged, thereby facilitating the simultaneous analysis of different metabolic pathways in B. melitensis. Such a global characterization would not have been possible by using time consuming and traditional biochemical approaches. The era of post-genomic technology offers new and exciting opportunities to understand the complete biology of different Brucella species.

Brucella

USDA-ARS?s Scientific Manuscript database

The genus Brucella encompasses a group of gram negative bacteria that survive almost exclusively in infected hosts with preference for localization in intracellular compartments of cells. The genus has traditionally been divided into species based on microbe characteristics and host preference, bu...

Brucella species survey in polar bears (ursus maritimus) of northern Alaska.

PubMed

O'Hara, Todd M; Holcomb, Darce; Elzer, Philip; Estepp, Jessica; Perry, Quinesha; Hagius, Sue; Kirk, Cassandra

2010-07-01

We report on the presence of specific antibodies to Brucella spp. and Yersinia enterocolitica in polar bears (Ursus maritimus) from northern Alaska (southern Beaufort Sea) during 2003-2006. Based on numerous known stressors (e.g., climate change and loss of sea ice habitat, contaminants), there is increased concern regarding the status of polar bears. Considering these changes, it is important to assess exposure to potentially pathogenic organisms and to improve understanding of transmission pathways. Brucella or specific antibodies to Brucella spp. has been reported in marine mammals. Various assays were used to elucidate the pathway or source of exposure (e.g., "marine" vs. "terrestrial" Brucella spp.) of northern Alaska polar bears to Brucella spp. The standard plate test (SPT) and the buffered Brucella antigen card test (BBA) were used for initial screening for antibodies specific to Brucella. We then evaluated positive reactors (presence of serum antibody specific for Brucella spp.) using immunoblots and competitive enzyme-linked immunosorbent assay (cELISA; based on pinniped-derived Brucella spp. antigen). Annual prevalence of antibody (BBA and SPT) for Brucella spp. ranged from 6.8% to 18.5% over 2003-2006, with an overall prevalence of 10.2%. Prevalence of Brucella spp. antibody did vary by age class. Western blot analyses indicated 17 samples were positive for Brucella spp. antibody; of these, 13 were negative by marine (pinniped) derived Brucella antigen cELISA and four were positive by marine cELISA. Of the four samples positive for Brucella antibody by marine cELISA, three cross-reacted with Y. enterocolitica and Brucella spp. (one sample was Brucella negative and Y. enterocolitica positive). It appears the polar bear antibody does not react with the antigens used on the marine cELISA assay, potentially indicating a terrestrial (nonpinniped) source of Brucella spp.

A review of Brucella infection in marine mammals, with special emphasis on Brucella pinnipedialis in the hooded seal (Cystophora cristata)

PubMed Central

2011-01-01

Brucella spp. were isolated from marine mammals for the first time in 1994. Two novel species were later included in the genus; Brucella ceti and Brucella pinnipedialis, with cetaceans and seals as their preferred hosts, respectively. Brucella spp. have since been isolated from a variety of marine mammals. Pathological changes, including lesions of the reproductive organs and associated abortions, have only been registered in cetaceans. The zoonotic potential differs among the marine mammal Brucella strains. Many techniques, both classical typing and molecular microbiology, have been utilised for characterisation of the marine mammal Brucella spp. and the change from the band-based approaches to the sequence-based approaches has greatly increased our knowledge about these strains. Several clusters have been identified within the B. ceti and B. pinnipedialis species, and multiple studies have shown that the hooded seal isolates differ from other pinniped isolates. We describe how different molecular methods have contributed to species identification and differentiation of B. ceti and B. pinnipedialis, with special emphasis on the hooded seal isolates. We further discuss the potential role of B. pinnipedialis for the declining Northwest Atlantic hooded seal population. PMID:21819589

Epizootiology of Brucella infection in Australian fur seals.

PubMed

Lynch, Michael; Duignan, Pádraig J; Taylor, Trevor; Nielsen, Ole; Kirkwood, Roger; Gibbens, John; Arnould, John P Y

2011-04-01

Novel members of the bacterial genus Brucella have recently emerged as pathogens of various marine mammal species and as potential zoonotic agents. We investigated the epizootiology of Brucella infection in Australian fur seals (Arctocephalus pusillus doriferus) by establishing demographic and temporal variations in antibody prevalence, attempting isolation of the causative agent, and determining whether this potential pathogen is involved in frequent abortions observed in this pinniped species. Two competitive enzyme-linked immunosorbent assays (cELISAs), an indirect ELISA, and a fluorescence polarization assay (FPA) were used to test sera for Brucella antibodies. The FPA and cELISA proved suitable for use in this species. Significant differences in antibody prevalence were found between age classes of seals sampled between 2007 and 2009 at one colony. Pups sampled at this site (n=134) were negative for Brucella antibodies by all serologic tests but 17 of 45 (38%) of juveniles were antibody-positive. Antibody prevalence in adult females was significantly higher than in juveniles (P=0.044). Antibody prevalence for adult females between 2003 and 2009 varied significantly over time (P=0.011), and for individuals sampled between 2003 and 2005, the likelihood of pregnancy was greater in individuals positive for Brucella antibodies (P=0.034). Inflammatory lesions suggestive of infectious agents were found in 14 of 39 aborted Australian fur seal pups, but pathologic changes were not uniformly consistent for Brucella infection. Culture and PCR investigations on fetal tissues were negative for Brucella. Culture and PCR on selected fresh or frozen tissues from 36 juvenile and adult animals were also negative. We suspect that the prevalence of active infection with Brucella in Australian fur seals is low relative to antibody prevalence.

Brucella dissociation is essential for macrophage egress and bacterial dissemination.

PubMed

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4-5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination.

Brucella dissociation is essential for macrophage egress and bacterial dissemination

PubMed Central

Pei, Jianwu; Kahl-McDonagh, Melissa; Ficht, Thomas A.

2014-01-01

It has long been observed that smooth Brucella can dissociate into rough mutants that are cytotoxic to macrophages. However, the in vivo biological significance and/or mechanistic details of Brucella dissociation and cytotoxicity remain incomplete. In the current report, a plaque assay was developed using Brucella strains exhibiting varying degrees of cytotoxicity. Infected monolayers were observed daily using phase contrast microscopy for plaque formation while Brucella uptake and replication were monitored using an immunofluorescence assay (IFA). Visible plaques were detected at 4–5 days post infection (p.i.) with cytotoxic Brucella 16MΔmanBA at an MOI of 0.1. IFA staining demonstrated that the plaques consisted of macrophages with replicating Brucella. Visible plaques were not detected in monolayers infected with non-cytotoxic 16MΔmanBAΔvirB2 at an MOI of 0.1. However, IFA staining did reveal small groups of macrophages (foci) with replicating Brucella in the monolayers infected with 16MΔmanBAΔvirB2. The size of the foci observed in macrophage monolayers infected with rough Brucella correlated directly with cytotoxicity measured in liquid culture, suggesting that cytotoxicity was essential for Brucella egress and dissemination. In monolayers infected with 16M, small and large foci were observed. Double antibody staining revealed spontaneous rough mutants within the large, but not the small foci in 16M infected monolayers. Furthermore, plaque formation was observed in the large foci derived from 16M infections. Finally, the addition of gentamicin to the culture medium inhibited plaque formation, suggesting that cell-to-cell spread occurred only following release of the organisms from the cells. Taken together, these results demonstrate that Brucella-induced cytotoxicity is critical for Brucella egress and dissemination. PMID:24634889

Whole-genome analyses of speciation events in pathogenic Brucellae

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

Chain, Patrick S. G.; Comerci, Diego J.; Tolmasky, Marcelo E.

Despite their high DNA identity and a proposal to group classical Brucella species as biovars of Brucella melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters, as well as by a marked host range (e.g., Brucella suis for swine, B. melitensis for sheep and goats, and Brucella abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucella species and to B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions, and insertions supports previousmore » indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complements of both B. abortus strains are identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggests that these inactivations may play an important role in the establishment of host specificity and may have been a primary driver of speciation in the genus Brucella. Despite being nonmotile, the brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures and may contribute to differences in host specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g., glycogen, biotin, NAD, and choline) are consistent with adaptation of brucellae to an intracellular life-style.« less

Brucella papionis sp. nov., isolated from baboons (Papio spp.).

PubMed

Whatmore, Adrian M; Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S; Brew, Simon D; Perrett, Lorraine L; Koylass, Mark S; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C; Dick, Edward J; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E

2014-12-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60(T) and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60(T) and F8/08-61 could be distinguished clearly from all known species of the genus Brucella and their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucella suggested by the ICSP

Immunization with viable Brucella organisms*

PubMed Central

Spink, Wesley W.; Hall, James W.; Finstad, Joanne; Mallet, Edmund

1962-01-01

In many parts of the world contact with infected livestock may involve a serious risk of the spread of human brucellosis. Partial control of bovine brucellosis has been achieved by slaughter of infected herds and immunization of cattle with Brucella abortus strain 19 living vaccine. However, in areas where such measures are unpractical there remains a need for protection of humans. This study compares the safety of two living Brucella vaccine preparations in human volunteers. Some 32 healthy male volunteers with no evidence of past exposure to brucellosis were divided into two comparable groups; one group received 19-BA vaccine derived from Br. abortus and the other received Rev 1 vaccine derived from Br. melitensis. Detailed studies over a six-month period of the clinical effects, bacteraemia, Brucella agglutinin response and dermal hypersensitivity revealed striking differences between the two groups. Two of the 16 men in the 19-BA group developed acute brucellosis, and another had a positive blood culture. In the Rev 1 group, 11 of 16 developed acute brucellosis, and brucellae were recovered from 12. All 32 men developed Brucella agglutinins, the Rev 1 group having higher titres. Dermal hypersensitivity occurred in all of the Rev. 1 group but in only nine of the 19-BA group. Tetracycline treatment in all the Rev 1 group and in the two brucellosis cases in the 19-BA group resulted in complete recovery. The authors conclude from this study that neither the Rev 1 vaccine nor the 19-BA vaccine inoculated subcutaneously is sufficiently safe in the dosage used to warrant being used for vaccination of humans for prophylactic purposes. PMID:13915813

Comparative proteome analysis of laboratory grown Brucella abortus 2308 and Brucella melitensis 16M.

PubMed

Eschenbrenner, Michel; Horn, Troy A; Wagner, Mary Ann; Mujer, Cesar V; Miller-Scandle, Tabbi L; DelVecchio, Vito G

2006-07-01

Brucella species are pathogenic agents that cause brucellosis, a debilitating zoonotic disease that affects a large variety of domesticated animals and humans. Brucella melitensis and Brucella abortus are considered major health threats because of their highly infectious nature and worldwide occurrence. The availability of the annotated genomes for these two species has allowed a comparative proteomics study of laboratory grown B. melitensis 16M and B. abortus 2308 by two-dimensional (2-D) gel electrophoresis and peptide mass fingerprinting. Computer-assisted analysis of the different 2-D gel images of strains 16M and 2308 revealed significant quantitative and qualitative differences in their protein expression patterns. Proteins involved in membrane transport, particularly the high affinity amino acids binding proteins, and those involved in Sec-dependent secretion systems related to type IV and type V secretion systems, were differentially expressed. Differential expression of these proteins may be responsible for conferring specific host preference in the two strains 2308 and 16M.

Characterisation of North American Brucella isolates from marine mammals.

PubMed

Whatmore, Adrian M; Dawson, Claire; Muchowski, Jakub; Perrett, Lorraine L; Stubberfield, Emma; Koylass, Mark; Foster, Geoffrey; Davison, Nicholas J; Quance, Christine; Sidor, Inga F; Field, Cara L; St Leger, Judy

2017-01-01

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals.

Prevalence of Brucella spp in humans.

PubMed

Soares, Catharina de Paula Oliveira Cavalcanti; Teles, José Andreey Almeida; dos Santos, Aldenir Feitosa; Silva, Stemberg Oliveira Firmino; Cruz, Maria Vilma Rocha Andrade; da Silva-Júnior, Francisco Feliciano

2015-01-01

to determine the seroprevalence of Brucella spp in humans. this is an observational study, developed with 455 individuals between 18 and 64 years old, who use the Estratégia de Saúde da Família (Brazil's family health strategy). The serum samples of volunteers underwent buffered acid antigen tests, such as screening, agar gel immunodiffusion and slow seroagglutination test in tubes and 2-Mercaptoethanol. among the samples, 1.98% has responded to buffered-acid antigen, 2.85% to agar gel immunodiffusion test and 1.54% to the slow seroagglutination tests on tubes/2-Mercaptoethanol. The prevalence of Brucella spp was 4.4%, represented by the last two tests. the results of this research suggest that the studied population is exposed to Brucella spp infection.

Thermostable Cross-Protective Subunit Vaccine against Brucella Species

PubMed Central

Barabé, Nicole D.; Grigat, Michelle L.; Lee, William E.; Poirier, Robert T.; Jager, Scott J.; Berger, Bradley J.

2014-01-01

A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 105 CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. PMID:25320267

Thermostable cross-protective subunit vaccine against Brucella species.

PubMed

Cherwonogrodzky, John W; Barabé, Nicole D; Grigat, Michelle L; Lee, William E; Poirier, Robert T; Jager, Scott J; Berger, Bradley J

2014-12-01

A subunit vaccine candidate was produced from Brucella suis 145 (biovar 4; expressing both the A antigen of Brucella abortus and the M antigen of Brucella melitensis). The preparation consisted mostly of polysaccharide (PS; >90% [wt/wt]; both cell-associated PS and exo-PS were combined) and a small amount of protein (1 to 3%) with no apparent nucleic acids. Vaccinated mice were protected (these had a statistically significant reduction in bacterial colonization compared to that of unvaccinated controls) when challenged with representative strains of three Brucella species most pathogenic for humans, i.e., B. abortus, B. melitensis, and B. suis. As little as 1 ng of the vaccine, without added adjuvant, protected mice against B. suis 145 infection (5 × 10(5) CFU), and a single injection of 1 μg of this subunit vaccine protected mice from B. suis 145 challenge for at least 14 months. A single immunization induced a serum IgG response to Brucella antigens that remained elevated for up to 9 weeks. The use of heat (i.e., boiling-water bath, autoclaving) in the vaccine preparation showed that it was thermostable. This method also ensured safety and security. The vaccine produced was immunogenic and highly protective against multiple strains of Brucella and represents a promising candidate for further evaluation. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

MULTILOCUS SEQUENCE TYPING OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Chawjiraphan, Wireeya; Sonthayanon, Piengchan; Chanket, Phanita; Benjathummarak, Surachet; Kerdsin, Anusak; Kalambhaheti, Thareerat

2016-11-01

Although brucellosis outbreaks in Thailand are rare, they cause abortions and infertility in animals, resulting in significant economic loss. Because Brucella spp display > 90% DNA homology, multilocus sequence typing (MLST) was employed to categorize local Brucella isolates into sequence types (STs) and to determine their genetic relatedness. Brucella samples were isolated from vaginal secretion of cows and goats, and from blood cultures of infected individuals. Brucella species were determined by multiplex PCR of eight loci, in addition to MLST based on partial DNA sequences of nine house-keeping genes. MLST analysis of 36 isolates revealed 78 distinct novel allele types and 34 novel STs, while two isolates possessed the known ST8. Sequence alignments identified polymorphic sites in each allele, ranging from 2-6%, while overall genetic diversity was 3.6%. MLST analysis of the 36 Brucella isolates classified them into three species, namely, B. melitensis, B. abortus and B. suis, in agreement with multiplex PCR results. Genetic relatedness among ST members of B. melitensis and B. abortus determined by eBURST program revealed ST2 as founder of B. abortus isolates and ST8 the founder of B. melitensis isolates. ST 36, 41 and 50 of Thai Brucella isolates were identified as single locus variants of clonal cluster (CC) 8, while the majority of STs were diverse. The genetic diversity and relatedness identified using MLST revealed hitherto unexpected diversity among Thai Brucella isolates. Genetic classification of isolates could reveal the route of brucellosis transmission among humans and farm animals and also reveal their relationship with other isolates in the region and other parts of the world.

Brucella papionis sp. nov., isolated from baboons (Papio spp.)

PubMed Central

Davison, Nicholas; Cloeckaert, Axel; Al Dahouk, Sascha; Zygmunt, Michel S.; Brew, Simon D.; Perrett, Lorraine L.; Koylass, Mark S.; Vergnaud, Gilles; Quance, Christine; Scholz, Holger C.; Dick, Edward J.; Hubbard, Gene; Schlabritz-Loutsevitch, Natalia E.

2014-01-01

Two Gram-negative, non-motile, non-spore-forming coccoid bacteria (strains F8/08-60T and F8/08-61) isolated from clinical specimens obtained from baboons (Papio spp.) that had delivered stillborn offspring were subjected to a polyphasic taxonomic study. On the basis of 16S rRNA gene sequence similarities, both strains, which possessed identical sequences, were assigned to the genus Brucella. This placement was confirmed by extended multilocus sequence analysis (MLSA), where both strains possessed identical sequences, and whole-genome sequencing of a representative isolate. All of the above analyses suggested that the two strains represent a novel lineage within the genus Brucella. The strains also possessed a unique profile when subjected to the phenotyping approach classically used to separate species of the genus Brucella, reacting only with Brucella A monospecific antiserum, being sensitive to the dyes thionin and fuchsin, being lysed by bacteriophage Wb, Bk2 and Fi phage at routine test dilution (RTD) but only partially sensitive to bacteriophage Tb, and with no requirement for CO2 and no production of H2S but strong urease activity. Biochemical profiling revealed a pattern of enzyme activity and metabolic capabilities distinct from existing species of the genus Brucella. Molecular analysis of the omp2 locus genes showed that both strains had a novel combination of two highly similar omp2b gene copies. The two strains shared a unique fingerprint profile of the multiple-copy Brucella-specific element IS711. Like MLSA, a multilocus variable number of tandem repeat analysis (MLVA) showed that the isolates clustered together very closely, but represent a distinct group within the genus Brucella. Isolates F8/08-60T and F8/08-61 could be distinguished clearly from all known species of the genus Brucellaand their biovars by both phenotypic and molecular properties. Therefore, by applying the species concept for the genus Brucellasuggested by the ICSP Subcommittee on

Brucella Infection in Asian Sea Otters (Enhydra lutris lutris) on Bering Island, Russia.

PubMed

Burgess, Tristan L; Johnson, Christine Kreuder; Burdin, Alexander; Gill, Verena A; Doroff, Angela M; Tuomi, Pamela; Smith, Woutrina A; Goldstein, Tracey

2017-10-01

Infection with Brucella spp., long known as a cause of abortion, infertility, and reproductive loss in domestic livestock, has increasingly been documented in marine mammals over the past two decades. We report molecular evidence of Brucella infection in Asian sea otters (Enhydra lutris lutris). Brucella DNA was detected in 3 of 78 (4%) rectal swab samples collected between 2004 and 2006 on Bering Island, Russia. These 78 animals had previously been documented to have a Brucella seroprevalence of 28%, markedly higher than the prevalence documented in sea otters (Enhydra lutris) in North America. All of the DNA sequences amplified were identical to one or more previously isolated Brucella spp. including strains from both terrestrial and marine hosts. Phylogenetic analysis of this sequence suggested that one animal was shedding Brucella spp. DNA with a sequence matching a Brucella abortus strain, whereas two animals yielded a sequence matching a group of strains including isolates classified as Brucella pinnipedialis and Brucella melitensis. Our results highlight the diversity of Brucella spp. within a single sea otter population.

Duplex PCR for differentiation of the vaccine strain Brucella suis S2 and B. suis biovar 1 from other strains of Brucella spp.

PubMed

Nan, Wenlong; Tan, Pengfei; Wang, Yong; Xu, Zouliang; Mao, Kairong; Peng, Daxin; Chen, Yiping

2014-09-01

Immunisation with attenuated Brucella spp. vaccines prevents brucellosis, but may also interfere with diagnosis. In this study, a duplex PCR was developed to distinguish Brucella suis vaccine strain S2 from field strains of B. suis biovar 1 and other Brucella spp. The PCR detected 60 fg genomic DNA of B. suis S2 or biovar 1 field strains and was able to distinguish B. suis S2 and wild-type strains of B. suis biovar 1 among 76 field isolates representing all the common species and biovars, as well as four vaccine strains, of Brucella. Copyright © 2014 Elsevier Ltd. All rights reserved.

Characterisation of North American Brucella isolates from marine mammals

PubMed Central

Dawson, Claire; Muchowski, Jakub; Perrett, Lorraine L.; Stubberfield, Emma; Koylass, Mark; Foster, Geoffrey; Davison, Nicholas J.; Quance, Christine; Sidor, Inga F.; Field, Cara L.; St. Leger, Judy

2017-01-01

Extension of known ecological niches of Brucella has included the description of two novel species from marine mammals. Brucella pinnipedialis is associated predominantly with seals, while two major Brucella ceti clades, most commonly associated with porpoises or dolphins respectively, have been identified. To date there has been limited characterisation of Brucella isolates obtained from marine mammals outside Northern European waters, including North American waters. To address this gap, and extend knowledge of the global population structure and host associations of these Brucella species, 61 isolates from marine mammals inhabiting North American waters were subject to molecular and phenotypic characterisation enabling comparison with existing European isolates. The majority of isolates represent genotypes previously described in Europe although novel genotypes were identified in both B. ceti clades. Harp seals were found to carry B. pinnipedialis genotypes previously confined to hooded seals among a diverse repertoire of sequence types (STs) associated with this species. For the first time Brucella isolates were characterised from beluga whales and found to represent a number of distinct B. pinnipedialis genotypes. In addition the known host range of ST27 was extended with the identification of this ST from California sea lion samples. Finally the performance of the frequently used diagnostic tool Bruce-ladder, in differentiating B. ceti and B. pinnipedialis, was critically assessed based on improved knowledge of the global population structure of Brucella associated with marine mammals. PMID:28934239

Pathogenesis and immunobiology of brucellosis: review of Brucella-host interactions.

PubMed

de Figueiredo, Paul; Ficht, Thomas A; Rice-Ficht, Allison; Rossetti, Carlos A; Adams, L Garry

2015-06-01

This review of Brucella-host interactions and immunobiology discusses recent discoveries as the basis for pathogenesis-informed rationales to prevent or treat brucellosis. Brucella spp., as animal pathogens, cause human brucellosis, a zoonosis that results in worldwide economic losses, human morbidity, and poverty. Although Brucella spp. infect humans as an incidental host, 500,000 new human infections occur annually, and no patient-friendly treatments or approved human vaccines are reported. Brucellae display strong tissue tropism for lymphoreticular and reproductive systems with an intracellular lifestyle that limits exposure to innate and adaptive immune responses, sequesters the organism from the effects of antibiotics, and drives clinical disease manifestations and pathology. Stealthy brucellae exploit strategies to establish infection, including i) evasion of intracellular destruction by restricting fusion of type IV secretion system-dependent Brucella-containing vacuoles with lysosomal compartments, ii) inhibition of apoptosis of infected mononuclear cells, and iii) prevention of dendritic cell maturation, antigen presentation, and activation of naive T cells, pathogenesis lessons that may be informative for other intracellular pathogens. Data sets of next-generation sequences of Brucella and host time-series global expression fused with proteomics and metabolomics data from in vitro and in vivo experiments now inform interactive cellular pathways and gene regulatory networks enabling full-scale systems biology analysis. The newly identified effector proteins of Brucella may represent targets for improved, safer brucellosis vaccines and therapeutics. Copyright © 2015 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.

Molecular investigation of virulence factors of Brucella melitensis and Brucella abortus strains isolated from clinical and non-clinical samples.

PubMed

Mirnejad, Reza; Jazi, Faramarz Masjedian; Mostafaei, Shayan; Sedighi, Mansour

2017-08-01

Brucella is zoonotic pathogen that induces abortion and sterility in domestic mammals and chronic infections in humans called Malta fever. It is a facultative intracellular potential pathogen with high infectivity. The virulence of Brucella is dependent upon its potential virulence factors such as enzymes and cell envelope associated virulence genes. The aim of this study was to investigate the Brucella virulence factors among strains isolated from humans and animals in different parts of Iran. Seventy eight strains of Brucella species isolated from suspected human and animal cases from several provinces of Iran during 2015-2016 and identified by phenotypic and molecular methods. The multiplex-PCR (M-PCR) assay was performed in order to detect the ure, wbkA, omp19, mviN, manA and perA genes by using gene specific primers. Out of 78 isolates of Brucella spp., 57 (73%) and 21 (27%) isolates were detected as B. melitensis and B. abortus, respectively, by molecular method. The relative frequency of virulence genes ure, wbkA, omp19, mviN, manA and perA were 74.4%, 89.7%, 93.6%, 94.9%, 100% and 92.3%, respectively. Our results indicate that the most of Brucella strains isolated from this region possess high percent of virulence factor genes (ure, wbkA, omp19, mviN, manA and perA) in their genome. So, each step of infection can be mediated by a number of virulence factors and each strain may have a unique combination of these factors that affected the rate of bacterial pathogenesis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Analyzing the molecular mechanism of lipoprotein localization in Brucella

PubMed Central

Goolab, Shivani; Roth, Robyn L.; van Heerden, Henriette; Crampton, Michael C.

2015-01-01

Bacterial lipoproteins possess diverse structure and functionality, ranging from bacterial physiology to pathogenic processes. As such many lipoproteins, originating from Brucella are exploited as potential vaccines to countermeasure brucellosis infection in the host. These membrane proteins are translocated from the cytoplasm to the cell membrane where they are anchored peripherally by a multifaceted targeting mechanism. Although much research has focused on the identification and classification of Brucella lipoproteins and their potential use as vaccine candidates for the treatment of Brucellosis, the underlying route for the translocation of these lipoproteins to the outer surface of the Brucella (and other pathogens) outer membrane (OM) remains mostly unknown. This is partly due to the complexity of the organism and evasive tactics used to escape the host immune system, the variation in biological structure and activity of lipoproteins, combined with the complex nature of the translocation machinery. The biosynthetic pathway of Brucella lipoproteins involves a distinct secretion system aiding translocation from the cytoplasm, where they are modified by lipidation, sorted by the lipoprotein localization machinery pathway and thereafter equipped for export to the OM. Surface localized lipoproteins in Brucella may employ a lipoprotein flippase or the β-barrel assembly complex for translocation. This review provides an overview of the characterized Brucella OM proteins that form part of the OM, including a handful of other characterized bacterial lipoproteins and their mechanisms of translocation. Lipoprotein localization pathways in gram negative bacteria will be used as a model to identify gaps in Brucella lipoprotein localization and infer a potential pathway. Of particular interest are the dual topology lipoproteins identified in Escherichia coli and Haemophilus influenza. The localization and topology of these lipoproteins from other gram negative bacteria

Brucella endocarditis: an occupational hazard!

PubMed

Agarwal, Sanjeev Kumar; Rajani, Ali Raza; Hussain, Kosar; Dande, Mangesh Manoharrao

2013-04-22

A young man presented with a 2-month history of fever and malaise. Cardiac auscultation revealed the presence of a diastolic murmur. Subsequently, a cardiac echocardiogram was done, which showed a large vegetation adherent to an anterior mitral leaflet. The blood culture was positive for Brucella species. The patient was given antibiotic therapy for brucellosis and referred for surgery. Brucella endocarditis is one of the rarest, yet most notorious complications of this infection. This condition requires a high degree of clinical suspicion in order to facilitate prompt diagnosis and treatment.

In vitro assay for the anti-brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis *

PubMed Central

Motamedi, Hossein; Darabpour, Esmaeil; Gholipour, Mahnaz; Seyyed Nejad, Seyyed Mansour

2010-01-01

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50–400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study. PMID:20593515

In vitro assay for the anti-Brucella activity of medicinal plants against tetracycline-resistant Brucella melitensis.

PubMed

Motamedi, Hossein; Darabpour, Esmaeil; Gholipour, Mahnaz; Seyyed Nejad, Seyyed Mansour

2010-07-01

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. Brucella melitensis is the main causative agent of brucellosis in both human and small ruminants. As an alternative to conventional antibiotics, medicinal plants are valuable resources for new agents against antibiotic-resistant strains. The aim of this study was to investigate the usage of native plants for brucellosis treatment. For this purpose, the anti-brucella activities of ethanolic and methanolic extracts of Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, Plantago ovata, Cordia myxa, and Crocus sativus were assessed. The activity against a resistant Br. melitensis strain was determined by disc diffusion method at various concentrations from 50-400 mg/ml. Antibiotic discs were also used as a control. Among the evaluated herbs, six plant (Salvia sclarea, Oliveria decumbens, Ferulago angulata, Vitex pseudo-negundo, Teucrium polium, and Crocus sativus) showed anti-brucella activity. Oliveria decumbens was chosen as the most effective plant for further studies. A tested isolate exhibited resistance to tetracycline, nafcillin, oxacillin, methicillin, and colistin. Minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) values for Oliveria decumbens against resistant Br. melitensis were the same (5 mg/ml), and for gentamicin they were both 2 mg/ml. Time-kill kinetics for a methanolic extract of Oliveria decumbens was 7 h whereas for an ethanolic extract it was 28 h. Also, Oliveria decumbens extracts showed a synergistic effect in combination with doxycycline and tetracycline. In general, the similar values of MIC and MBC for Oliveria decumbens suggest that these extracts could act as bactericidal agents against Br. melitensis. In addition to Oliveria decumbens, Crocus sativus and Salvia sclarea also had good anti-brucella activity and these should be considered for further study.

The changing nature of the Brucella-containing vacuole.

PubMed

Celli, Jean

2015-07-01

Bacteria of the genus Brucella are intracellular vacuolar pathogens of mammals that cause the worldwide zoonosis brucellosis, and reside within phagocytes of infected hosts to promote their survival, persistence and proliferation. These traits are essential to the bacterium's ability to cause disease and have been the subject of much investigation to gain an understanding of Brucella pathogenic mechanisms. Although the endoplasmic reticulum-derived nature of the Brucella replicative niche has been long known, major strides have recently been made in deciphering the molecular mechanisms of its biogenesis, including the identification of bacterial determinants and host cellular pathways involved in this process. Here I will review and discuss the most recent advances in our knowledge of Brucella intracellular pathogenesis, with an emphasis on bacterial exploitation of the host endoplasmic reticulum-associated functions, and how autophagy-related processes contribute to the bacterium's intracellular cycle. © 2015 John Wiley & Sons Ltd.

Macrophage activation induced by Brucella DNA suppresses bacterial intracellular replication via enhancing NO production.

PubMed

Liu, Ning; Wang, Lin; Sun, Changjiang; Yang, Li; Tang, Bin; Sun, Wanchun; Peng, Qisheng

2015-12-01

Brucella DNA can be sensed by TLR9 on endosomal membrane and by cytosolic AIM2-inflammasome to induce proinflammatory cytokine production that contributes to partially activate innate immunity. Additionally, Brucella DNA has been identified to be able to act as a major bacterial component to induce type I IFN. However, the role of Brucella DNA in Brucella intracellular growth remains unknown. Here, we showed that stimulation with Brucella DNA promote macrophage activation in TLR9-dependent manner. Activated macrophages can suppresses wild type Brucella intracellular replication at early stage of infection via enhancing NO production. We also reported that activated macrophage promotes bactericidal function of macrophages infected with VirB-deficient Brucella at the early or late stage of infection. This study uncovers a novel function of Brucella DNA, which can help us further elucidate the mechanism of Brucella intracellular survival. Copyright © 2015 Elsevier Ltd. All rights reserved.

Knowledge of Brucella as a food-borne pathogen

USDA-ARS?s Scientific Manuscript database

Although Brucella spp. are known for causing reproductive losses in domestic livestock, they are also capable of infecting humans and causing clinical disease. Human infection with Brucella is almost exclusively a result of direct contact with infected animals or consumption of products made from un...

Isolation of Brucella pinnipedialis from Grey Seals (Halichoerus grypus) in the Baltic Sea.

PubMed

Hirvelä-Koski, Varpu; Nylund, Minna; Skrzypczak, Teresa; Heikkinen, Petra; Kauhala, Kaarina; Jay, Maryne; Isomursu, Marja

2017-10-01

Brucella infection in seals was reported for the first time in 1994 around the coast of Scotland. Since then, marine mammal Brucella infections were found to be widely distributed in the northern hemisphere. Two Brucella species affect marine mammals: Brucella pinnipedialis in pinnipeds and Brucella ceti in cetaceans. We examined the livers of Baltic grey seals (Halichoerus grypus) from the Finnish coast (n=122) hunted, found dead, or killed as by-catch in fishing gear in 2013-15 as part of population health monitoring. We detected B. pinnipedialis in the livers of three grey seals. The bacterium was isolated from livers displaying parasitic cholangitis. We also detected Brucella DNA in liver flukes (Pseudamphistomum truncatum) obtained from a Brucella-infected grey seal, suggesting that flukes might be possible vectors of this pathogen in the marine environment.

Type IV secretion system of Brucella spp. and its effectors

PubMed Central

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis. PMID:26528442

Type IV secretion system of Brucella spp. and its effectors.

PubMed

Ke, Yuehua; Wang, Yufei; Li, Wengfeng; Chen, Zeliang

2015-01-01

Brucella spp. are intracellular bacterial pathogens that cause infection in domestic and wild animals. They are often used as model organisms to study intracellular bacterial infections. Brucella VirB T4SS is a key virulence factor that plays important roles in mediating intracellular survival and manipulating host immune response to infection. In this review, we discuss the roles of Brucella VirB T4SS and 15 effectors that are proposed to be crucial for Brucella pathogenesis. VirB T4SS regulates the inflammation response and manipulates vesicle trafficking inside host cells. VirB T4SS also plays crucial roles in the inhibition of the host immune response and intracellular survival during infection. Here, we list the key molecular events in the intracellular life cycle of Brucella that are potentially targeted by the VirB T4SS effectors. Elucidating the functions of these effectors will help clarify the molecular role of T4SS during infection. Furthermore, studying the effectors secreted by Brucella spp. might provide insights into the mechanisms used by the bacteria to hijack the host signaling pathways and aid in the development of better vaccines and therapies against brucellosis.

Isolation of a novel 'atypical' Brucella strain from a bluespotted ribbontail ray (Taeniura lymma).

PubMed

Eisenberg, Tobias; Riße, Karin; Schauerte, Nicole; Geiger, Christina; Blom, Jochen; Scholz, Holger C

2017-02-01

A pleomorphic Gram-negative, motile coccobacillus was isolated from the gills of a wild-caught bluespotted ribbontail ray after its sudden death during quarantine. Strain 141012304 was observed to grow aerobically, to be clearly positive for cytochrome oxidase, catalase, urease and was initially identified as "Brucella melitensis" or "Ochrobactrum anthropi" by Matrix-assisted laser desorption/ionization-time of flight mass spectrometry and VITEK2-compact ® , respectively. Affiliation to the genus Brucella was confirmed by bcsp31 and IS711 PCR as well as by Brucella species-specific multiplex PCR, therein displaying a characteristic banding pattern recently described for Brucella strains obtained from amphibian hosts. Likewise, based on recA sequencing, strain 141012304 was found to form a separate lineage, within the so called 'atypical' Brucella, consisting of genetically more distantly related strains. The closest similarity was detected to brucellae, which have recently been isolated from edible bull frogs. Subsequent next generation genome sequencing and phylogenetic analysis confirmed that the ray strain represents a novel Brucella lineage within the atypical group of Brucella and in vicinity to Brucella inopinata and Brucella strain BO2, both isolated from human patients. This is the first report of a natural Brucella infection in a saltwater fish extending the host range of this medically important genus.

Analyses of Brucella pathogenesis, host immunity, and vaccine targets using systems biology and bioinformatics.

PubMed

He, Yongqun

2012-01-01

Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning.

Factors affecting Brucella spp. blood cultures positivity in children.

PubMed

Apa, Hurşit; Devrim, Ilker; Memur, Seyma; Günay, Ilker; Gülfidan, Gamze; Celegen, Mehmet; Bayram, Nuri; Karaarslan, Utku; Bağ, Ozlem; Işgüder, Rana; Oztürk, Aysel; Inan, Seyhan; Unal, Nurrettin

2013-03-01

Brucella infections have a wide spectrum of symptoms especially in children, making the diagnosis a complicated process. The gold standard for the final diagnosis for brucellosis is to identify the Brucella spp. isolated from blood or bone marrow cultures. The main purpose of this work was to evaluate the factors affecting the isolation of Brucella spp. from blood cultures. In our study, the ratio of fever, presence of hepatomegaly, and splenomegaly were found to be higher in the bacteremic group. In addition, C-reactive protein levels and liver function enzymes were found to be higher in the bacteremic group. In our opinion, while evaluating the febrile child with suspected Brucella infection, we highly recommend sampling blood cultures regardless of the history of previous antimicrobial therapy and duration of the symptoms.

Brucella infection inhibits macrophages apoptosis via Nedd4-dependent degradation of calpain2.

PubMed

Cui, Guimei; Wei, Pan; Zhao, Yuxi; Guan, Zhenhong; Yang, Li; Sun, Wanchun; Wang, Shuangxi; Peng, Qisheng

2014-11-07

The calcium-dependent protease calpain2 is involved in macrophages apoptosis. Brucella infection-induced up-regulation of intracellular calcium level is an essential factor for the intracellular survival of Brucella within macrophages. Here, we hypothesize that calcium-dependent E3 ubiquitin ligase Nedd4 ubiquitinates calpain2 and inhibits Brucella infection-induced macrophage apoptosis via degradation of calpain2.Our results reveal that Brucella infection induces increases in Nedd4 activity in an intracellular calcium dependent manner. Furthermore, Brucella infection-induced degradation of calpain2 is mediated by Nedd4 ubiquitination of calpain2. Brucella infection-induced calpain2 degradation inhibited macrophages apoptosis. Treatment of Brucella infected macrophages with calcium chelator BAPTA or Nedd4 knock-down decreased Nedd4 activity, prevented calpain2 degradation, and resulted in macrophages apoptosis. Copyright © 2014 Elsevier B.V. All rights reserved.

21 CFR 866.3085 - Brucella spp. serological reagents.

Code of Federal Regulations, 2010 CFR

2010-04-01

... 21 Food and Drugs 8 2010-04-01 2010-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp...

21 CFR 866.3085 - Brucella spp. serological reagents.

Code of Federal Regulations, 2011 CFR

2011-04-01

... 21 Food and Drugs 8 2011-04-01 2011-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp...

21 CFR 866.3085 - Brucella spp. serological reagents.

Code of Federal Regulations, 2013 CFR

2013-04-01

... 21 Food and Drugs 8 2013-04-01 2013-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp...

21 CFR 866.3085 - Brucella spp. serological reagents.

Code of Federal Regulations, 2012 CFR

2012-04-01

... 21 Food and Drugs 8 2012-04-01 2012-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp...

21 CFR 866.3085 - Brucella spp. serological reagents.

Code of Federal Regulations, 2014 CFR

2014-04-01

... 21 Food and Drugs 8 2014-04-01 2014-04-01 false Brucella spp. serological reagents. 866.3085 Section 866.3085 Food and Drugs FOOD AND DRUG ADMINISTRATION, DEPARTMENT OF HEALTH AND HUMAN SERVICES (CONTINUED) MEDICAL DEVICES IMMUNOLOGY AND MICROBIOLOGY DEVICES Serological Reagents § 866.3085 Brucella spp...

Establishment of Chronic Infection: Brucella's Stealth Strategy

PubMed Central

Ahmed, Waqas; Zheng, Ke; Liu, Zheng-Fei

2016-01-01

Brucella is a facultative intracellular pathogen that causes zoonotic infection known as brucellosis which results in abortion and infertility in natural host. Humans, especially in low income countries, can acquire infection by direct contact with infected animal or by consumption of animal products and show high morbidity, severe economic losses and public health problems. However for survival, host cells develop complex immune mechanisms to defeat and battle against attacking pathogens and maintain a balance between host resistance and Brucella virulence. On the other hand as a successful intracellular pathogen, Brucella has evolved multiple strategies to evade immune response mechanisms to establish persistent infection and replication within host. In this review, we mainly summarize the “Stealth” strategies employed by Brucella to modulate innate and the adaptive immune systems, autophagy, apoptosis and possible role of small noncoding RNA in the establishment of chronic infection. The purpose of this review is to give an overview for recent understanding how this pathogen evades immune response mechanisms of host, which will facilitate to understanding the pathogenesis of brucellosis and the development of novel, more effective therapeutic approaches to treat brucellosis. PMID:27014640

Analyses of Brucella Pathogenesis, Host Immunity, and Vaccine Targets using Systems Biology and Bioinformatics

PubMed Central

He, Yongqun

2011-01-01

Brucella is a Gram-negative, facultative intracellular bacterium that causes zoonotic brucellosis in humans and various animals. Out of 10 classified Brucella species, B. melitensis, B. abortus, B. suis, and B. canis are pathogenic to humans. In the past decade, the mechanisms of Brucella pathogenesis and host immunity have been extensively investigated using the cutting edge systems biology and bioinformatics approaches. This article provides a comprehensive review of the applications of Omics (including genomics, transcriptomics, and proteomics) and bioinformatics technologies for the analysis of Brucella pathogenesis, host immune responses, and vaccine targets. Based on more than 30 sequenced Brucella genomes, comparative genomics is able to identify gene variations among Brucella strains that help to explain host specificity and virulence differences among Brucella species. Diverse transcriptomics and proteomics gene expression studies have been conducted to analyze gene expression profiles of wild type Brucella strains and mutants under different laboratory conditions. High throughput Omics analyses of host responses to infections with virulent or attenuated Brucella strains have been focused on responses by mouse and cattle macrophages, bovine trophoblastic cells, mouse and boar splenocytes, and ram buffy coat. Differential serum responses in humans and rams to Brucella infections have been analyzed using high throughput serum antibody screening technology. The Vaxign reverse vaccinology has been used to predict many Brucella vaccine targets. More than 180 Brucella virulence factors and their gene interaction networks have been identified using advanced literature mining methods. The recent development of community-based Vaccine Ontology and Brucellosis Ontology provides an efficient way for Brucella data integration, exchange, and computer-assisted automated reasoning. PMID:22919594

Review of Detection of Brucella sp. by Polymerase Chain Reaction

PubMed Central

Yu, Wei Ling; Nielsen, Klaus

2010-01-01

Here we present a review of most of the currently used polymerase chain reaction (PCR)-based methods for identification of Brucella bacteria in biological samples. We focused in particular on methods using single-pair primers, multiplex primers, real-time PCRs, PCRs for marine Brucella, and PCRs for molecular biotyping. These methods are becoming very important tools for the identification of Brucella, at the species level and recently also at the biovar level. These techniques require minimum biological containment and can provide results in a very short time. In addition, genetic fingerprinting of isolates aid in epidemiological studies of the disease and its control. PCR-based methods are more useful and practical than conventional methods used to identify Brucella spp., and new methods for Brucella spp identification and typing are still being developed. However, the sensitivity, specificity, and issues of quality control and quality assurance using these methods must be fully validated on clinical samples before PCR can be used in routine laboratory testing for brucellosis. PMID:20718083

Phenotypic and molecular characterisation of Brucella isolates from marine mammals

PubMed Central

Dawson, Claire E; Stubberfield, Emma J; Perrett, Lorraine L; King, Amanda C; Whatmore, Adrian M; Bashiruddin, John B; Stack, Judy A; MacMillan, Alastair P

2008-01-01

Background Bacteria of the genus Brucella are the causative organisms of brucellosis in animals and man. Previous characterisation of Brucella strains originating from marine mammals showed them to be distinct from the terrestrial species and likely to comprise one or more new taxa. Recently two new species comprising Brucella isolates from marine mammals, B. pinnipedialis and B. ceti, were validly published. Here we report on an extensive study of the molecular and phenotypic characteristics of marine mammal Brucella isolates and on how these characteristics relate to the newly described species. Results In this study, 102 isolates of Brucella originating from eleven species of marine mammals were characterised. Results obtained by analysis using the Infrequent Restriction Site (IRS)-Derivative PCR, PCR-RFLP of outer membrane protein genes (omp) and IS711 fingerprint profiles showed good consistency with isolates originating from cetaceans, corresponding to B. ceti, falling into two clusters. These correspond to isolates with either dolphins or porpoises as their preferred host. Isolates originating predominantly from seals, and corresponding to B. pinnipedialis, cluster separately on the basis of IS711 fingerprinting and other molecular approaches and can be further subdivided, with isolates from hooded seals comprising a distinct group. There was little correlation between phenotypic characteristics used in classical Brucella biotyping and these groups. Conclusion Molecular approaches are clearly valuable in the division of marine mammal Brucella into subtypes that correlate with apparent ecological divisions, whereas conventional bioyping is of less value. The data presented here confirm that there are significant subtypes within the newly described marine mammal Brucella species and add to a body of evidence that could lead to the recognition of additional species or sub-species within this group. PMID:19091076

Evaluation of PCR methods for detection of Brucella strains from culture and tissues.

PubMed

Çiftci, Alper; İça, Tuba; Savaşan, Serap; Sareyyüpoğlu, Barış; Akan, Mehmet; Diker, Kadir Serdar

2017-04-01

The genus Brucella causes significant economic losses due to infertility, abortion, stillbirth or weak calves, and neonatal mortality in livestock. Brucellosis is still a zoonosis of public health importance worldwide. The study was aimed to optimize and evaluate PCR assays used for the diagnosis of Brucella infections. For this aim, several primers and PCR protocols were performed and compared with Brucella cultures and biological material inoculated with Brucella. In PCR assays, genus- or species-specific oligonucleotide primers derived from 16S rRNA sequences (F4/R2, Ba148/928, IS711, BruP6-P7) and OMPs (JPF/JPR, 31ter/sd) of Brucella were used. All primers except for BruP6-P7 detected the DNA from reference Brucella strains and field isolates. In spiked blood, milk, and semen samples, F4-R2 primer-oriented PCR assays detected minimal numbers of Brucella. In spiked serum and fetal stomach content, Ba148/928 primer-oriented PCR assays detected minimal numbers of Brucella. Field samples collected from sheep and cattle were examined by bacteriological methods and optimized PCR assays. Overall, sensitivity of PCR assays was found superior to conventional bacteriological isolation. Brucella DNA was detected in 35.1, 1.1, 24.8, 5.0, and 8.0% of aborted fetus, blood, milk, semen, and serum samples by PCR assays, respectively. In conclusion, PCR assay in optimized conditions was found to be valuable in sensitive and specific detection of Brucella infections of animals.

Prevalence of Brucella spp in humans1

PubMed Central

Soares, Catharina de Paula Oliveira Cavalcanti; Teles, José Andreey Almeida; dos Santos, Aldenir Feitosa; Silva, Stemberg Oliveira Firmino; Cruz, Maria Vilma Rocha Andrade; da Silva-Júnior, Francisco Feliciano

2015-01-01

Objective: to determine the seroprevalence of Brucella spp in humans. Method: this is an observational study, developed with 455 individuals between 18 and 64 years old, who use the Estratégia de Saúde da Família (Brazil's family health strategy). The serum samples of volunteers underwent buffered acid antigen tests, such as screening, agar gel immunodiffusion and slow seroagglutination test in tubes and 2-Mercaptoethanol. Results: among the samples, 1.98% has responded to buffered-acid antigen, 2.85% to agar gel immunodiffusion test and 1.54% to the slow seroagglutination tests on tubes/2-Mercaptoethanol. The prevalence of Brucella spp was 4.4%, represented by the last two tests. Conclusion: the results of this research suggest that the studied population is exposed to Brucella spp infection. PMID:26487143

Isolation of Brucella inopinata-Like Bacteria from White's and Denny's Tree Frogs.

PubMed

Kimura, Masanobu; Une, Yumi; Suzuki, Michio; Park, Eun-Sil; Imaoka, Koichi; Morikawa, Shigeru

2017-05-01

Brucella inopinata strain BO1 and B. sp. strain BO2 isolated from human patients, respectively, are genetically different from classical Brucella species. We isolated bacteria of the genus Brucella from two species of wild-caught tropical frogs kept in the facilities in Japan: White's tree frog, which inhabits Oceania, and Denny's tree frog, which inhabits Southeast Asia. Phylogenetic analyses based on 16S rRNA and recA gene sequences and multilocus sequence analysis showed that two isolates of Brucella spp. showed significant similarity to BO1, BO2, and the isolates from other wild-caught frogs. These results suggest that a variety of frog species are susceptible to a novel clade of Brucella bacteria, including B. inopinata.

A rare case of anterior mediastinal mass caused by Brucella infection.

PubMed

Sabzi, Feridoun; Faraji, Reza

2017-03-01

A previously healthy man, who had undergone coronary artery bypass 10 years earlier and had been diagnosed with brucellosis due to Brucella septicemia after Brucella arthritis, presented with chest pain and high fever. Anti- Brucella antibiotics were started, but after 4 weeks, his high fever remained. An infected mass was confirmed by computed tomography, and surgical intervention was performed via a median sternotomy. A large amount of thick pus gushed from an abscess in the upper mediastinum. The abscess cavity had a thick granulation wall, and cultured pus was positive for Brucella only. The patient responded well to antibiotic therapy.

Metal acquisition and virulence in Brucella

PubMed Central

Roop, R. Martin

2013-01-01

Similar to other bacteria, Brucella strains require several biologically essential metals for their survival in vitro and in vivo. Acquiring sufficient levels of some of these metals, particularly iron, manganese and zinc, is especially challenging in the mammalian host, where sequestration of these micronutrients is a well-documented component of both the innate and acquired immune responses. This review describes the Brucella metal transporters that have been shown to play critical roles in the virulence of these bacteria in experimental and natural hosts. PMID:22632611

Brucella Intracellular Life Relies on the Transmembrane Protein CD98 Heavy Chain.

PubMed

Keriel, Anne; Botella, Eric; Estrach, Soline; Bragagnolo, Gabriel; Vergunst, Annette C; Feral, Chloe C; O'Callaghan, David

2015-06-01

Brucella are intracellular bacterial pathogens that use a type IV secretion system (T4SS) to escape host defenses and create a niche in which they can multiply. Although the importance of Brucella T4SS is clear, little is known about its interactions with host cell structures. In this study, we identified the eukaryotic protein CD98hc as a partner for Brucella T4SS subunit VirB2. This transmembrane glycoprotein is involved in amino acid transport, modulation of integrin signaling, and cell-to-cell fusion. Knockdown of CD98hc expression in HeLa cells demonstrated that it is essential for Brucella infection. Using knockout dermal fibroblasts, we confirmed its role for Brucella but found that it is not required for Salmonella infection. CD98hc transiently accumulates around the bacteria during the early phases of infection and is required for both optimal bacterial uptake and intracellular multiplication of Brucella. These results provide new insights into the complex interplay between Brucella and its host. © The Author 2014. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

[Use of multiple locus variable number tandem repeats analysis for the Brucella systematization].

PubMed

Kulakov, Iu K; Kovalev, D A; Misetova, E N; Golovneva, S I; Liapustina, L V; Zheludkov, M M

2012-01-01

The methods of molecular-genetic differentiation to strain level acquire increasing significance in the current system of struggle with brucellosis. MLVA (multiple locus variable number tandem repeats analysis) was selected for molecular-genetic differentiation to strain level and simultaneous establishment of the genetic relationship of investigated Brucella strains. The goal of this work was MLVA typing of three pathogenic Brucella species strains with the analysis of stability of chosen loci, discrimination power and concordance to conventional phenotypic methods of the Brucella differentiation for use in systematization of brucellosis causing agents. Twenty six Brucella strains representing reference (n = 15), vaccine (n = 2) and field strains of three pathogenic Brucella species were tested: B. melitensis (n = 3), B. abortus (n = 2), B. suis (n = 2), and isolates (n = 2) with unidentified taxonomic position using MLVA with 9 pairs primers on known variable loci of Brucella genome. The analysis of the stability of chosen loci, discrimination power on Hunter-Gaston discrimination index (HGDI) and consistency to phenotypic methods of identification was performed. MLVA was confirmed for the results of phenotypic methods of identification, stability of the chosen loci in majority reference, and vaccine strains with a high index of variability HGDI 0.9969 for all loci. A dendrogram was plotted on the basis of MLVA data on distributed Brucella strains in related clusters according to its taxonomic species and biovar positions and construction of 25 genotypes. B. melitensis strains formed cluster related to the reference strain of B. melitensis 63/9 biovar 2. Australian isolates of Brucella 83-4 and Brucella 83-6 isolated from rodents formed a cluster distant from other strains of Brucella. MLVA is a promising method for differentiation of Brucella strains with known and unresolved taxonomic status for their systematization and creation of MLVA genotype catalogue that

Brucella discriminates between mouse dendritic cell subsets upon in vitro infection.

PubMed

Papadopoulos, Alexia; Gagnaire, Aurélie; Degos, Clara; de Chastellier, Chantal; Gorvel, Jean-Pierre

2016-01-01

Brucella is a Gram-negative bacterium responsible for brucellosis, a worldwide re-emerging zoonosis. Brucella has been shown to infect and replicate within Granulocyte macrophage colony-stimulating factor (GMCSF) in vitro grown bone marrow-derived dendritic cells (BMDC). In this cell model, Brucella can efficiently control BMDC maturation. However, it has been shown that Brucella infection in vivo induces spleen dendritic cells (DC) migration and maturation. As DCs form a complex network composed by several subpopulations, differences observed may be due to different interactions between Brucella and DC subsets. Here, we compare Brucella interaction with several in vitro BMDC models. The present study shows that Brucella is capable of replicating in all the BMDC models tested with a high infection rate at early time points in GMCSF-IL15 DCs and Flt3l DCs. GMCSF-IL15 DCs and Flt3l DCs are more activated than the other studied DC models and consequently intracellular bacteria are not efficiently targeted to the ER replicative niche. Interestingly, GMCSF-DC and GMCSF-Flt3l DC response to infection is comparable. However, the key difference between these 2 models concerns IL10 secretion by GMCSF DCs observed at 48 h post-infection. IL10 secretion can explain the weak secretion of IL12p70 and TNFα in the GMCSF-DC model and the low level of maturation observed when compared to GMCSF-IL15 DCs and Flt3l DCs. These models provide good tools to understand how Brucella induce DC maturation in vivo and may lead to new therapeutic design using DCs as cellular vaccines capable of enhancing immune response against pathogens.

Construction of pTM series plasmids for gene expression in Brucella species.

PubMed

Tian, Mingxing; Qu, Jing; Bao, Yanqing; Gao, Jianpeng; Liu, Jiameng; Wang, Shaohui; Sun, Yingjie; Ding, Chan; Yu, Shengqing

2016-04-01

Brucellosis, the most common widespread zoonotic disease, is caused by Brucella spp., which are facultative, intracellular, Gram-negative bacteria. With the development of molecular biology techniques, more and more virulence-associated factors have been identified in Brucella spp. A suitable plasmid system is an important tool to study virulence genes in Brucella. In this study, we constructed three constitutive replication plasmids (pTM1-Cm, pTM2-Amp, and pTM3-Km) using the replication origin (rep) region derived from the pBBR1-MCS vector. Also, a DNA fragment containing multiple cloning sites (MCSs) and a terminator sequence derived from the pCold vector were produced for complementation of the deleted genes. Besides pGH-6×His, a plasmid containing the groE promoter of Brucella spp. was constructed to express exogenous proteins in Brucella with high efficiency. Furthermore, we constructed the inducible expression plasmid pZT-6×His, containing the tetracycline-inducible promoter pzt1, which can induce expression by the addition of tetracycline in the Brucella culture medium. The constructed pTM series plasmids will play an important role in the functional investigation of Brucella spp. Copyright © 2016 Elsevier B.V. All rights reserved.

Detection and differentiation of the six Brucella species by polymerase chain reaction.

PubMed

Sifuentes-Rincón, A M; Revol, A; Barrera-Saldaña, H A

1997-11-01

Brucelosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Its current diagnosis is based on clinical observations that may be complemented by serology and microbiological culture tests; however, the former is limited in sensitivity and specificity, the latter is time consuming. To improve brucelosis diagnosis we developed a test which is specific and sensitive and is capable of differentiating the six species of Brucella. Four primers were designed from B. abortus sequences at the well-conserved Omp2 locus that are able to amplify the DNAs of all six species of Brucella. Our test detected all six species of Brucella. Their differentiation resulted directly from differences in the amplification patterns or was achieved indirectly using a RFLP present in one of the PCR products. The sensitivity and specificity of the new test were then determined; it was applied successfully in confirming the diagnosis of a patient whose clinical history and serology indicated infection with Brucella. The results make possible the use of a PCR test for Brucella detection and differentiation without relying on the measurement of the antibodies or microorganism culture. Our first results showed that the PCR test can confirm the presence of Brucella in blood samples of infected patients.

A history of the development of Brucella vaccines.

PubMed

Avila-Calderón, Eric Daniel; Lopez-Merino, Ahidé; Sriranganathan, Nammalwar; Boyle, Stephen M; Contreras-Rodríguez, Araceli

2013-01-01

Brucellosis is a worldwide zoonosis affecting animal and human health. In the last several decades, much research has been performed to develop safer Brucella vaccines to control the disease mainly in animals. Till now, no effective human vaccine is available. The aim of this paper is to review and discuss the importance of methodologies used to develop Brucella vaccines in pursuing this challenge.

Evaluation of a New and Rapid Serologic Test for Detecting Brucellosis: Brucella Coombs Gel Test.

PubMed

Hanci, Hayrunisa; Igan, Hakan; Uyanik, Muhammet Hamidullah

2017-01-01

Many serological tests have been used for the diagnosis of human brucellosis. A new serological method is identified as Brucella Coombs gel test based on the principle of centrifugation gel system similar to the gel system used in blood group determination. In this system, if Brucella antibodies were present in the serum, antigen and antibody would remain as a pink complex on the gel. Otherwise, the pink Brucella antigens would precipitate at the bottom of the gel card system. In this study, we aimed to compare the Brucella Coombs gel test, a new, rapid screen and titration method for detection of non-agglutinating IgG with the Brucella Coombs test. For this study, a total of 88 serum samples were obtained from 45 healthy persons and 43 individuals who had clinical signs and symptoms of brucellosis. For each specimen, Rose Bengal test, standard agglutination test, Coombs test and Brucella Coombs gel test were carried out. Sensitivity and specificity of Brucella Coombs gel test were found as 100.0 and 82.2%, respectively. Brucella Coombs gel test can be used as a screening test with high sensitivity. By the help of pink Brucella antigen precipitation, the tests' evaluation is simple and objective. In addition, determination of Brucella antibody by rapid titration offers another important advantage.

MULTIPLE-LOCUS VARIABLE-NUMBER TANDEM REPEAT ANALYSIS OF BRUCELLA ISOLATES FROM THAILAND.

PubMed

Kumkrong, Khurawan; Chankate, Phanita; Tonyoung, Wittawat; Intarapuk, Apiradee; Kerdsin, Anusak; Kalambaheti, Thareerat

2017-01-01

Brucellosis-induced abortion can result in significant economic loss to farm animals. Brucellosis can be transmitted to humans during slaughter of infected animals or via consumption of contaminated food products. Strain identification of Brucella isolates can reveal the route of transmission. Brucella strains were isolated from vaginal swabs of farm animal, cow milk and from human blood cultures. Multiplex PCR was used to identify Brucella species, and owing to high DNA homology among Brucella isolates, multiple-locus variable-number tandem repeat analysis (MLVA) based on the number of tandem repeats at 16 different genomic loci was used for strain identification. Multiplex PCR categorized the isolates into B. abortus (n = 7), B. melitensis (n = 37), B. suis (n = 3), and 5 of unknown Brucella spp. MLVA-16 clustering analysis differentiated the strains into various genotypes, with Brucella isolates from the same geographic region being closely related, and revealed that the Thai isolates were phylogenetically distinct from those in other countries, including within the Southeast Asian region. Thus, MLVA-16 typing has utility in epidemiological studies.

[Molecular typing of 12 Brucella strains isolated in Guizhou province in 2010-2013].

PubMed

Wang, Yue; Chen, Hong; Liu, Ying; Zhou, Jingzhu; Li, Shijun; Hang, Yan; Tang, Guangpeng; Wang, Dingming; Chen, Guichun

2015-09-01

To identify and characterize the Brucella strains from Guizhou province in 2010-2013. A total of 12 strains of Brucella suspicious bacteria were isolated in Guizhou province from 2010 to 2013. Four strains (GZLL3, GZLL4, GZLL11 and SH2) were isolated from goat blood samples and eight strains (SH4, GZZY, GZSQ, GZZA, BR13001, BR13004, BR13005 and BR13006) were isolated from blood samples of patient 12 Brucella suspicious strains were identified and characterized using conventional methods. Brucella genus specific gene BCSP31-based PCR (BCSP31-PCR) was used to identify the genus of Brucella and IS711 insert sequence-based PCR (AMOS-PCR) was applied to identify the species of Brucella strains. Goats and patients originated Brucella strains were comparatively analysed using Pulse-field Gel Electrophoresis (PFGE). Both of conventional methods and PCR identified the 12 Brucella suspicious strains as B. melitensis biotype 3. BCSP31-PCR identification results showed that a specific DNA bands (223 bp) were detected in all the 12 strains and positive control samples with no DNA band in negative samples. AMOS-PCR amplified a 731 bp-DNA bands in all the 12 strains, with 731 bp, 498 bp and 275 bp in M5, S2 and A19 strains, respectively, and no DNA band was detected in the negative control samples. PFGE analysis showed that 12 Brucella isolates from patients and goats showed consistent PFGE patterns with the digestion of restriction enzyme Xba I. The epidemic species/type of Brucella in both human and animal in Guizhou province was B. melitensis biotype 3 and goat was the main animal source of infection of brucellosis in Guizhou province.

Detection and differentiation of the six Brucella species by polymerase chain reaction.

PubMed Central

Sifuentes-Rincón, A. M.; Revol, A.; Barrera-Saldaña, H. A.

1997-01-01

BACKGROUND: Brucelosis is a severe acute febrile disease caused by bacteria of the genus Brucella. Its current diagnosis is based on clinical observations that may be complemented by serology and microbiological culture tests; however, the former is limited in sensitivity and specificity, the latter is time consuming. To improve brucelosis diagnosis we developed a test which is specific and sensitive and is capable of differentiating the six species of Brucella. MATERIALS AND METHODS: Four primers were designed from B. abortus sequences at the well-conserved Omp2 locus that are able to amplify the DNAs of all six species of Brucella. RESULTS: Our test detected all six species of Brucella. Their differentiation resulted directly from differences in the amplification patterns or was achieved indirectly using a RFLP present in one of the PCR products. The sensitivity and specificity of the new test were then determined; it was applied successfully in confirming the diagnosis of a patient whose clinical history and serology indicated infection with Brucella. CONCLUSIONS: The results make possible the use of a PCR test for Brucella detection and differentiation without relying on the measurement of the antibodies or microorganism culture. Our first results showed that the PCR test can confirm the presence of Brucella in blood samples of infected patients. Images FIG. 2 FIG. 3 FIG. 4 FIG. 5 PMID:9407549

Brucella ceti Infection in a Common Minke Whale ( Balaenoptera acutorostrata ) with Associated Pathology.

PubMed

Davison, Nicholas J; Perrett, Lorraine L; Dawson, Claire; Dagleish, Mark P; Haskins, Gary; Muchowski, Jakub; Whatmore, Adrian M

2017-07-01

There are three major lineages of marine mammal strains of Brucella spp.: Brucella ceti ST23, found predominantly in porpoises; B. ceti ST26, in pelagic delphinids and ziphiids; and Brucella pinnipedialis ST24/25, predominantly in seals. The isolation of Brucella spp. in mysticetes has been described only in common minke whales ( Balaenoptera acutorostrata ) in Norway and Scotland. We report a third case of Brucella infection and isolation in a minke whale associated with a large abscess. In contrast to the two previous reports that involved isolates of B. pinnipedialis ST24 or the porpoise-associated B. ceti complex ST23, this case was associated with the dolphin-associated B. ceti ST26. Thus, minke whales can be infected naturally with members of all the distinct major lineages of Brucella associated with marine mammals. This report is unique in that the B. ceti ST26 did not originate from a pelagic delphinid or a beaked whale.

Comparative Genomics of Early-Diverging Brucella Strains Reveals a Novel Lipopolysaccharide Biosynthesis Pathway

PubMed Central

Wattam, Alice R.; Inzana, Thomas J.; Williams, Kelly P.; Mane, Shrinivasrao P.; Shukla, Maulik; Almeida, Nalvo F.; Dickerman, Allan W.; Mason, Steven; Moriyón, Ignacio; O’Callaghan, David; Whatmore, Adrian M.; Sobral, Bruno W.; Tiller, Rebekah V.; Hoffmaster, Alex R.; Frace, Michael A.; De Castro, Cristina; Molinaro, Antonio; Boyle, Stephen M.; De, Barun K.; Setubal, João C.

2012-01-01

ABSTRACT Brucella species are Gram-negative bacteria that infect mammals. Recently, two unusual strains (Brucella inopinata BO1T and B. inopinata-like BO2) have been isolated from human patients, and their similarity to some atypical brucellae isolated from Australian native rodent species was noted. Here we present a phylogenomic analysis of the draft genome sequences of BO1T and BO2 and of the Australian rodent strains 83-13 and NF2653 that shows that they form two groups well separated from the other sequenced Brucella spp. Several important differences were noted. Both BO1T and BO2 did not agglutinate significantly when live or inactivated cells were exposed to monospecific A and M antisera against O-side chain sugars composed of N-formyl-perosamine. While BO1T maintained the genes required to synthesize a typical Brucella O-antigen, BO2 lacked many of these genes but still produced a smooth LPS (lipopolysaccharide). Most missing genes were found in the wbk region involved in O-antigen synthesis in classic smooth Brucella spp. In their place, BO2 carries four genes that other bacteria use for making a rhamnose-based O-antigen. Electrophoretic, immunoblot, and chemical analyses showed that BO2 carries an antigenically different O-antigen made of repeating hexose-rich oligosaccharide units that made the LPS water-soluble, which contrasts with the homopolymeric O-antigen of other smooth brucellae that have a phenol-soluble LPS. The results demonstrate the existence of a group of early-diverging brucellae with traits that depart significantly from those of the Brucella species described thus far. PMID:22930339

The role of 'atypical' Brucella in amphibians: are we facing novel emerging pathogens?

PubMed

Mühldorfer, K; Wibbelt, G; Szentiks, C A; Fischer, D; Scholz, H C; Zschöck, M; Eisenberg, T

2017-01-01

To discuss together the novel cases of Brucella infections in frogs with the results of published reports to extend our current knowledge on 'atypical' brucellae isolated from amphibians and to discuss the challenges we face on this extraordinary emerging group of pathogens. Since our first description, an additional 14 isolates from four different frog species were collected. Novel isolates and a subset of Brucella isolates previously cultured from African bullfrogs were characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), Fourier transform-infrared (FT-IR) spectroscopy and broth microdilution susceptibility testing. MALDI-TOF MS worked very efficiently for an accurate bacterial identification to the genus level. Within the cluster analysis, 'atypical' brucellae grouped distant from Brucella melitensis and were even more separated by FT-IR spectroscopy with respect to their geographical origin. Minimum inhibitory concentrations of 14 antimicrobial substances are provided as baseline data on antimicrobial susceptibility. The case history of Brucella infections in amphibians reveals a variety of pathologies ranging from localized manifestations to systemic infections. Some isolates seem to be capable of causing high mortality in zoological exhibitions putting higher demands on the management of endangered frog species. There is considerable risk in overlooking and misidentifying 'atypical' Brucella in routine diagnostics. Brucella have only recently been described in cold-blooded vertebrates. Their presence in frog species native to Africa, America and Australia indicates a more common occurrence in amphibians than previously thought. This study provides an extensive overview of amphibian brucellae by highlighting the main features of their clinical significance, diagnosis and zoonotic potential. © 2016 The Society for Applied Microbiology.

Isolation and identification of bovine Brucella isolates from Pakistan by biochemical tests and PCR.

PubMed

Ali, Shahzad; Ali, Qurban; Melzer, Falk; Khan, Iahtasham; Akhter, Shamim; Neubauer, Heinrich; Jamal, Syed M

2014-01-01

Brucellosis is endemic in bovines in Pakistan. The Brucella species and biovars involved, however, are unknown. The objectives of the present study were to isolate and characterize brucellae from seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes which had recently aborted. The seropositive milk samples, aborted fetuses, and vaginal swabs of cattle and buffaloes were collected from the Potohar Plateau, Pakistan. Isolation of brucellae was done on modified Farrell's serum dextrose agar. Isolates were characterized by conventional biotyping methods, while molecular typing was done by genus (B4/B5) and species-specific (Brucella abortus, Brucella melitensis, Brucella ovis, and Brucella suis) polymerase chain reaction (PCR). A total of 30 isolates were recovered from milk (n = 5), aborted fetuses (n = 13), and vaginal swabs (n = 12). Most isolates were from cattle (56.7 %). All of them were identified as B. abortus biovar 1 based on conventional biotyping methods and genus and species-specific PCR. This preliminary study provides the first report on the prevalence of B. abortus biovar 1 in cattle and buffaloes in Pakistan.

Nucleotide sequences specific to Brucella and methods for the detection of Brucella

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

McCready, Paula M; Radnedge, Lyndsay; Andersen, Gary L

Nucleotide sequences specific to Brucella that serves as a marker or signature for identification of this bacterium were identified. In addition, forward and reverse primers and hybridization probes derived from these nucleotide sequences that are used in nucleotide detection methods to detect the presence of the bacterium are disclosed.

Multiple Locus Variable-Number Tandem-Repeat and Single-Nucleotide Polymorphism-Based Brucella Typing Reveals Multiple Lineages in Brucella melitensis Currently Endemic in China.

PubMed

Sun, Mingjun; Jing, Zhigang; Di, Dongdong; Yan, Hao; Zhang, Zhicheng; Xu, Quangang; Zhang, Xiyue; Wang, Xun; Ni, Bo; Sun, Xiangxiang; Yan, Chengxu; Yang, Zhen; Tian, Lili; Li, Jinping; Fan, Weixing

2017-01-01

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. In China, brucellosis is recognized as a reemerging disease mainly caused by Brucella melitensis specie. To better understand the currently endemic B. melitensis strains in China, three Brucella genotyping methods were applied to 110 B. melitensis strains obtained in past several years. By MLVA genotyping, five MLVA-8 genotypes were identified, among which genotypes 42 (1-5-3-13-2-2-3-2) was recognized as the predominant genotype, while genotype 63 (1-5-3-13-2-3-3-2) and a novel genotype of 1-5-3-13-2-4-3-2 were second frequently observed. MLVA-16 discerned a total of 57 MLVA-16 genotypes among these Brucella strains, with 41 genotypes being firstly detected and the other 16 genotypes being previously reported. By BruMLSA21 typing, six sequence types (STs) were identified, among them ST8 is the most frequently seen in China while the other five STs were firstly detected and designated as ST137, ST138, ST139, ST140, and ST141 by international multilocus sequence typing database. Whole-genome sequence (WGS)-single-nucleotide polymorphism (SNP)-based typing and phylogenetic analysis resolved Chinese B. melitensis strains into five clusters, reflecting the existence of multiple lineages among these Chinese B. melitensis strains. In phylogeny, Chinese lineages are more closely related to strains collected from East Mediterranean and Middle East countries, such as Turkey, Kuwait, and Iraq. In the next few years, MLVA typing will certainly remain an important epidemiological tool for Brucella infection analysis, as it displays a high discriminatory ability and achieves result largely in agreement with WGS-SNP-based typing. However, WGS-SNP-based typing is found to be the most powerful and reliable method in discerning Brucella strains and will be popular used in the future.

Brucella antibody seroprevalence in Antarctic seals (Arctocephalus gazella, Leptonychotes weddellii and Mirounga leonina).

PubMed

Jensen, Silje-Kristin; Nymo, Ingebjørg Helena; Forcada, Jaume; Hall, Ailsa; Godfroid, Jacques

2013-09-03

Brucellosis is a worldwide infectious zoonotic disease caused by Gram-negative bacteria of the genus Brucella, and Brucella infections in marine mammals were first reported in 1994. A serosurvey investigating the presence of anti-Brucella antibodies in 3 Antarctic pinniped species was undertaken with a protein A/G indirect enzyme-linked immunosorbent assay (iELISA) and the Rose Bengal test (RBT). Serum samples from 33 Weddell seals Leptonychotes weddelli were analysed, and antibodies were detected in 8 individuals (24.2%) with the iELISA and in 21 (65.6%) with the RBT. We tested 48 southern elephant seal Mirounga leonina sera and detected antibodies in 2 animals (4.7%) with both the iELISA and the RBT. None of the 21 Antarctic fur seals Arctocephalus gazella was found positive. This is the first report of anti-Brucella antibodies in southern elephant seals. The potential impact of Brucella infection in pinnipeds in Antarctica is not known, but Brucella spp. are known to cause abortion in terrestrial species and cetaceans. Our findings suggest that Brucella infection in pinnipeds is present in the Antarctic, but to date B. pinnipedialis has not been isolated from any Antarctic pinniped species, leaving the confirmation of infection pending.

Identification and typing of Brucella spp. in stranded harbour porpoises (Phocoena phocoena) on the Dutch coast.

PubMed

Maio, Elisa; Begeman, Lineke; Bisselink, Yvette; van Tulden, Peter; Wiersma, Lidewij; Hiemstra, Sjoukje; Ruuls, Robin; Gröne, Andrea; Roest, Hendrik-Ido-Jan; Willemsen, Peter; van der Giessen, Joke

2014-09-17

The presence of Brucella (B.) spp. in harbour porpoises stranded between 2008 and 2011 along the Dutch coast was studied. A selection of 265 tissue samples from 112 animals was analysed using conventional and molecular methods. In total, 4.5% (5/112) of the animals corresponding with 2.3% (6/265) Brucella positive tissue samples were Brucella positive by culture and these were all confirmed by real-time polymerase chain reaction (real-time PCR) based on the insertion element 711 (IS711). In addition, two more Brucella-positive tissue samples from two animals collected in 2011 were identified using real-time PCR resulting in an overall Brucella prevalence of 6.3% (7/112 animals). Brucella spp. were obtained from lungs (n=3), pulmonary lymph node (n=3) and lungworms (n=2). Multi Locus Variable Number of Tandem Repeats (VNTR) Analysis (MLVA) typing based on the MLVA-16 showed that the Brucella isolates were B. ceti. Additional in silico Multi Locus Sequence typing (MLST) after whole genome sequencing of the 6 Brucella isolates confirmed B. ceti ST 23. According to the Brucella 2010 MLVA database, the isolated Brucella strains encountered were of five genotypes, in two distinct subclusters divided in two different time periods of harbour porpoises collection. This study is the first population based analyses for Brucella spp. infections in cetaceans stranded along the Dutch coast. Copyright © 2014 The Authors. Published by Elsevier B.V. All rights reserved.

Marine Mammal Brucella Reference Strains Are Attenuated in a BALB/c Mouse Model.

PubMed

Nymo, Ingebjørg H; Arias, Maykel A; Pardo, Julián; Álvarez, María Pilar; Alcaraz, Ana; Godfroid, Jacques; Jiménez de Bagüés, María Pilar

2016-01-01

Brucellosis is a zoonosis of worldwide distribution with numerous animal host species. Since the novel isolation of Brucella spp. from marine mammals in 1994 the bacteria have been isolated from various marine mammal hosts. The marine mammal reference strains Brucella pinnipedialis 12890 (harbour seal, Phoca vitulina) and Brucella ceti 12891 (harbour porpoise, Phocoena phocoena) were included in genus Brucella in 2007, however, their pathogenicity in the mouse model is pending. Herein this is evaluated in BALB/c mice with Brucella suis 1330 as a control. Both marine mammal strains were attenuated, however, B. ceti was present at higher levels than B. pinnipedialis in blood, spleen and liver throughout the infection, in addition B. suis and B. ceti were isolated from brains and faeces at times with high levels of bacteraemia. In B. suis-infected mice serum cytokines peaked at day 7. In B. pinnipedialis-infected mice, levels were similar, but peaked predominantly at day 3 and an earlier peak in spleen weight likewise implied an earlier response. The inflammatory response induced pathology in the spleen and liver. In B. ceti-infected mice, most serum cytokine levels were comparable to those in uninfected mice, consistent with a limited inflammatory response, which also was indicated by restricted spleen and liver pathology. Specific immune responses against all three strains were detected in vitro after stimulation of splenocytes from infected mice with the homologous heat-killed brucellae. Antibody responses in vivo were also induced by the three brucellae. The immunological pattern of B. ceti in combination with persistence in organs and limited pathology has heretofore not been described for other brucellae. These two marine mammal wildtype strains show an attenuated pattern in BALB/c mice only previously described for Brucella neotomea.

BrucellaCapt versus classical tests in the serological diagnosis and management of human brucellosis.

PubMed

Casanova, Aurora; Ariza, Javier; Rubio, Manuel; Masuet, Cristina; Díaz, Ramón

2009-06-01

The BrucellaCapt test is an immunocapture agglutination test suggested as a possible substitute for the Coombs test in the diagnosis of human brucellosis. Here it is compared with classical tests using 321 samples from 48 patients with brucellosis (6.9 +/- 1.7 samples per patient), including 20 patients with focal disease and 8 patients with a total of 9 relapse episodes (mean follow-up, 18 months). The BrucellaCapt test was used according to the manufacturer's instructions, and we also used a variant of the BrucellaCapt test in which the microtiter plates were not coated with antibodies against total human immunoglobulin (BCAPV). The correlation between the BrucellaCapt and BCAPV tests was 0.982 (P < 0.001), with 260 coincident pairs of titers (81%). The areas under the receiver operating characteristic curve for the BrucellaCapt and BCAPV tests with respect to the Coombs test were 0.969 and 0.960, respectively. Upon admission, the BrucellaCapt, BCAPV, and Coombs tests and the microagglutination test (MAT) were positive for all cases: titers were 1/2,560 by the BrucellaCapt test, 1/2,560 by the BCAPV test, 1/1,280 by the Coombs test, and 1/320 by the MAT. The decreases in the BrucellaCapt and BCAPV titers over time were pronounced in comparison with the Coombs titers. Cumulative probabilities of persistence 12 months after therapy were as follows: 80% by the BrucellaCapt test, 80% by the BCAPV test, 87% by the Coombs test, and 35% by the MAT. Serological changes during relapse were detected in seven cases (88%) by the Coombs test, in five cases by the BrucellaCapt and BCAPV tests, and in three cases by the MAT. The BrucellaCapt test is a sensitive, specific, and simple test for routine use in human brucellosis. Similar results were obtained with the BCAPV test. However, in some cases of relapse and chronic forms of the disease, the slight changes observed in low-affinity antibodies alone are better detected by the Coombs test.

Identification of Brucella spp. isolated from human brucellosis in Malaysia using high-resolution melt (HRM) analysis.

PubMed

Mohamed Zahidi, Jama'ayah; Bee Yong, Tay; Hashim, Rohaidah; Mohd Noor, Azura; Hamzah, Siti Hawa; Ahmad, Norazah

2015-04-01

Molecular approaches have been investigated to overcome difficulties in identification and differentiation of Brucella spp. using conventional phenotypic methods. In this study, high-resolution melt (HRM) analysis was used for rapid identification and differentiation of members of Brucella genus. A total of 41 Brucella spp. isolates from human brucellosis were subjected to HRM analysis using 4 sets of primers, which identified 40 isolates as Brucella melitensis and 1 as Brucella canis. The technique utilized low DNA concentration and was highly reproducible. The assay is shown to be a useful diagnostic tool, which can rapidly differentiate Brucella up to species level. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

Glutamate Decarboxylase-Dependent Acid Resistance in Brucella spp.: Distribution and Contribution to Fitness under Extremely Acidic Conditions

PubMed Central

Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel

2014-01-01

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. PMID:25381237

[Detection of Brucella with an automatic hemoculture system: Bact/Alert].

PubMed

Casas, J; Partal, Y; Llosá, J; Leiva, J; Navarro, J M; de la Rosa, M

1994-12-01

The ability of in vitro and in vivo detection of Brucella spp. with the Bact/Alert system was studied. Three strains of Brucella melitensis and two of Brucella abortus were used. Different dilutions of the five strains were performed in trypticase soy broth (TSB), achieving concentrations of 1 cfu/ml, 5 cfu/ml, 10 cfu/ml and 100 cfu/ml. Ten ml of each dilution and strain were inoculated into 5 aerobic bottles Bact/Alert and 5 biphasic Hemóline bottles. Furthermore, over a 9 month period, 8,216 bottles of Bact/Alert bottles from hospitalized patients and from the emergency department were processed in the authors' laboratory. The mean detection time for Brucella growth was from 2 to 3 days with the Bact/Alert system, and 14 days in the biphasic bottles. Former bottles processed in the authors' laboratory, 11 aerobic bottles belonged to 5 patients in whom brucelosis was confirmed by bloodculture. The Bact/Alert system detected Brucella melitensis in only on bottle at 2.9 days of incubation. In 7 bottles Bact/Alert detected B. melitensis by a blind pass of these bottles at 10 to 20 days of incubation. These results suggest that the Bact/Alert system does not totally solve the diagnosis of brucellosis. Blind passes of the bloodcultures are required.

Global analysis of Brucella melitensis proteomes.

PubMed

Mujer, Cesar V; Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy; Kraycer, Jo Ann; Redkar, Rajendra; Hagius, Sue; Elzer, Philip; Delvecchio, Vito G

2002-10-01

Brucella melitensis is a facultative, intracellular, gram-negative cocco-bacillus that causes Malta fever in humans and brucellosis in animals. There are at least six species in the genus, and the disease is classified as zoonotic because several species infect humans. Using 2-D gel electrophoresis and mass spectrometry, we have initiated (i) a comprehensive mapping and identification of all the expressed proteins of B. melitensis virulent strain 16M, and (ii) a comparative study of its proteome with the attentuated vaccinal strain Rev 1. Comprehensive proteome maps of all six Brucella species will be generated in order to obtain vital information for vaccine development, identification of pathogenicity islands, and establishment of host specificity and evolutionary relatedness.

Molecular characterization of Brucella abortus chromosome II recombination.

PubMed

Tsoktouridis, Georgios; Merz, Christian A; Manning, Simon P; Giovagnoli-Kurtz, Renée; Williams, Leanne E; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; Redkar, Rajendra J; Patra, Guy; DelVecchio, Vito G

2003-10-01

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed.

MLVA and MLST typing of Brucella from Qinghai, China.

PubMed

Ma, Jun-Ying; Wang, Hu; Zhang, Xue-Fei; Xu, Li-Qing; Hu, Gui-Ying; Jiang, Hai; Zhao, Fang; Zhao, Hong-Yan; Piao, Dong-Ri; Qin, Yu-Min; Cui, Bu-Yun; Lin, Gong-Hua

2016-04-13

The Qinghai-Tibet Plateau (QTP) of China is an extensive pastoral and semi-pastoral area, and because of poverty and bad hygiene conditions, Brucella is highly prevalent in this region. In order to adequately prevent this disease in the QTP region it is important to determine the identity of Brucella species that caused the infection. A total of 65 Brucella isolates were obtained from human, livestock and wild animals in Qinghai, a Chinese province in east of the QTP. Two molecular typing methods, MLVA (multi-locus variable-number tandem-repeat analysis) and MLST (multi locus sequence typing) were used to identify the species and genotypes of these isolates. Both MLVA and MLST typing methods classified the 65 isolates into three species, B. melitensis, B. abortus and B. suis, which included 60, 4 and 1 isolates respectively. The MLVA method uniquely detected 34 (Bm01 ~ Bm34), 3 (Ba01 ~ Ba03), and 1 (Bs01) MLVA-16 genotypes for B. melitensis, B. abortus and B. suis, respectively. However, none of these genotypes exactly matched any of the genotypes in the Brucella2012 MLVA database. The MLST method identified five known ST types: ST7 and ST8 (B. melitensis), ST2 and ST5 (B. abortus), and ST14 (B. suis). We also detected a strain with a mutant type (3-2-3-2-?-5-3-8-2) of ST8 (3-2-3-2-1-5-3-8-2). Extensive genotype-sharing events could be observed among isolates from different host species. There were at least three Brucella (B. melitensis, B. abortus and B. suis) species in Qinghai, of which B. melitensis was the predominant species in the area examined. The Brucella population in Qinghai was very different from other regions of the world, possibly owing to the unique geographical characteristics such as extremely high altitude in QTP. There were extensive genotype-sharing events between isolates obtained from humans and other animals. Yaks, sheep and blue sheep were important zoonotic reservoirs of brucellosis causing species found in humans.

Ontology-based Brucella vaccine literature indexing and systematic analysis of gene-vaccine association network.

PubMed

Hur, Junguk; Xiang, Zuoshuang; Feldman, Eva L; He, Yongqun

2011-08-26

Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals. The VO-based SciMiner (VO-SciMiner) was developed to incorporate a total of 67 Brucella vaccine terms. A set of rules for term expansion of VO terms were learned from training data, consisting of 90 biomedical articles related to Brucella vaccine terms. VO-SciMiner demonstrated high recall (91%) and precision (99%) from testing a separate set of 100 manually selected biomedical articles. VO-SciMiner indexing exhibited superior performance in retrieving Brucella vaccine-related papers over that obtained with MeSH-based PubMed literature search. For example, a VO-SciMiner search of "live attenuated Brucella vaccine" returned 922 hits as of April 20, 2011, while a PubMed search of the same query resulted in only 74 hits. Using the abstracts of 14,947 Brucella-related papers, VO-SciMiner identified 140 Brucella genes associated with Brucella vaccines. These genes included known protective antigens, virulence factors, and genes closely related to Brucella vaccines. These VO-interacting Brucella genes were significantly over-represented in biological functional categories, including metabolite transport and metabolism, replication and repair, cell wall biogenesis, intracellular trafficking and secretion, posttranslational modification, and chaperones. Furthermore, a comprehensive interaction network of Brucella vaccines and genes were identified. The asserted

Ontology-based Brucella vaccine literature indexing and systematic analysis of gene-vaccine association network

PubMed Central

2011-01-01

Background Vaccine literature indexing is poorly performed in PubMed due to limited hierarchy of Medical Subject Headings (MeSH) annotation in the vaccine field. Vaccine Ontology (VO) is a community-based biomedical ontology that represents various vaccines and their relations. SciMiner is an in-house literature mining system that supports literature indexing and gene name tagging. We hypothesize that application of VO in SciMiner will aid vaccine literature indexing and mining of vaccine-gene interaction networks. As a test case, we have examined vaccines for Brucella, the causative agent of brucellosis in humans and animals. Results The VO-based SciMiner (VO-SciMiner) was developed to incorporate a total of 67 Brucella vaccine terms. A set of rules for term expansion of VO terms were learned from training data, consisting of 90 biomedical articles related to Brucella vaccine terms. VO-SciMiner demonstrated high recall (91%) and precision (99%) from testing a separate set of 100 manually selected biomedical articles. VO-SciMiner indexing exhibited superior performance in retrieving Brucella vaccine-related papers over that obtained with MeSH-based PubMed literature search. For example, a VO-SciMiner search of "live attenuated Brucella vaccine" returned 922 hits as of April 20, 2011, while a PubMed search of the same query resulted in only 74 hits. Using the abstracts of 14,947 Brucella-related papers, VO-SciMiner identified 140 Brucella genes associated with Brucella vaccines. These genes included known protective antigens, virulence factors, and genes closely related to Brucella vaccines. These VO-interacting Brucella genes were significantly over-represented in biological functional categories, including metabolite transport and metabolism, replication and repair, cell wall biogenesis, intracellular trafficking and secretion, posttranslational modification, and chaperones. Furthermore, a comprehensive interaction network of Brucella vaccines and genes were

Physical map of the Brucella melitensis 16 M chromosome.

PubMed Central

Allardet-Servent, A; Carles-Nurit, M J; Bourg, G; Michaux, S; Ramuz, M

1991-01-01

We present the first restriction map of the Brucella melitensis 16 M chromosome obtained by Southern blot hybridization of SpeI, XhoI, and XbaI fragments separated by pulsed-field gel electrophoresis. All restriction fragments (a total of 113) were mapped into an open circle. The main difficulty in mapping involved the exceedingly high number of restriction fragments, as was expected considering the 59% G + C content of the Brucella genome. Several cloned genes were placed on this map, especially rRNA operons which are repeated three times. The size of the B. melitensis chromosome, estimated as 2,600 kb long in a previous study, appeared longer (3,130 kb) by restriction mapping. This restriction map is an initial approach to achieve a genetic map of the Brucella chromosome. Images PMID:2007548

Identification of Brucella spp. by using the polymerase chain reaction.

PubMed Central

Herman, L; De Ridder, H

1992-01-01

The application of two synthetic oligonucleotides as probes and as primers in the polymerase chain reaction is presented for a specific, sensitive, and quick identification of Brucella spp. The specific oligonucleotide sequences were chosen on the basis of a 16S rRNA sequence alignment between Brucella abortus and Agrobacterium tumefaciens. Images PMID:1377903

Glutamate decarboxylase-dependent acid resistance in Brucella spp.: distribution and contribution to fitness under extremely acidic conditions.

PubMed

Damiano, Maria Alessandra; Bastianelli, Daniela; Al Dahouk, Sascha; Köhler, Stephan; Cloeckaert, Axel; De Biase, Daniela; Occhialini, Alessandra

2015-01-01

Brucella is an expanding genus of major zoonotic pathogens, including at least 10 genetically very close species occupying a wide range of niches from soil to wildlife, livestock, and humans. Recently, we have shown that in the new species Brucella microti, the glutamate decarboxylase (Gad)-dependent system (GAD system) contributes to survival at a pH of 2.5 and also to infection in mice by the oral route. In order to study the functionality of the GAD system in the genus Brucella, 47 isolates, representative of all known species and strains of this genus, and 16 strains of the closest neighbor genus, Ochrobactrum, were studied using microbiological, biochemical, and genetic approaches. In agreement with the genome sequences, the GAD system of classical species was not functional, unlike that of most strains of Brucella ceti, Brucella pinnipedialis, and newly described species (B. microti, Brucella inopinata BO1, B. inopinata-like BO2, and Brucella sp. isolated from bullfrogs). In the presence of glutamate, these species were more acid resistant in vitro than classical terrestrial brucellae. Expression in trans of the gad locus from representative Brucella species in the Escherichia coli MG1655 mutant strain lacking the GAD system restored the acid-resistant phenotype. The highly conserved GAD system of the newly described or atypical Brucella species may play an important role in their adaptation to acidic external and host environments. Furthermore, the GAD phenotype was shown to be a useful diagnostic tool to distinguish these latter Brucella strains from Ochrobactrum and from classical terrestrial pathogenic Brucella species, which are GAD negative. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Brucella abortus Synthesizes Phosphatidylcholine from Choline Provided by the Host

PubMed Central

Comerci, Diego J.; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.

2006-01-01

The Brucella cell envelope is characterized by the presence of phosphatidylcholine (PC), a common phospholipid in eukaryotes that is rare in prokaryotes. Studies on the composition of Brucella abortus 2308 phospholipids revealed that the synthesis of PC depends on the presence of choline in the culture medium, suggesting that the methylation biosynthetic pathway is not functional. Phospholipid composition of pmtA and pcs mutants indicated that in Brucella, PC synthesis occurs exclusively via the phosphatidylcholine synthase pathway. Transformation of Escherichia coli with an expression vector containing the B. abortus pcs homologue was sufficient for PC synthesis upon induction with IPTG (isopropyl-β-d-thiogalactopyranoside), while no PC formation was detected when bacteria were transformed with a vector containing pmtA. These findings imply that Brucella depends on choline provided by the host cell to form PC. We could not detect any obvious associated phenotype in the PC-deficient strain under vegetative or intracellular growth conditions in macrophages. However, the pcs mutant strain displays a reproducible virulence defect in mice, which suggests that PC is necessary to sustain a chronic infection process. PMID:16484204

Differential phenotyping of Brucella species using a newly developed semi-automated metabolic system.

PubMed

Al Dahouk, Sascha; Scholz, Holger C; Tomaso, Herbert; Bahn, Peter; Göllner, Cornelia; Karges, Wolfram; Appel, Bernd; Hensel, Andreas; Neubauer, Heinrich; Nöckler, Karsten

2010-10-23

A commercial biotyping system (Taxa Profile™, Merlin Diagnostika) testing the metabolization of various substrates by bacteria was used to determine if a set of phenotypic features will allow the identification of members of the genus Brucella and their differentiation into species and biovars. A total of 191 different amines, amides, amino acids, other organic acids and heterocyclic and aromatic substrates (Taxa Profile™ A), 191 different mono-, di-, tri- and polysaccharides and sugar derivates (Taxa Profile™ C) and 95 amino peptidase- and protease-reactions, 76 glycosidase-, phosphatase- and other esterase-reactions, and 17 classic reactions (Taxa Profile™ E) were tested with the 23 reference strains representing the currently known species and biovars of Brucella and a collection of 60 field isolates. Based on specific and stable reactions a 96-well "Brucella identification and typing" plate (Micronaut™) was designed and re-tested in 113 Brucella isolates and a couple of closely related bacteria.Brucella species and biovars revealed characteristic metabolic profiles and each strain showed an individual pattern. Due to their typical metabolic profiles a differentiation of Brucella isolates to the species level could be achieved. The separation of B. canis from B. suis bv 3, however, failed. At the biovar level, B. abortus bv 4, 5, 7 and B. suis bv 1-5 could be discriminated with a specificity of 100%. B. melitensis isolates clustered in a very homogenous group and could not be resolved according to their assigned biovars. The comprehensive testing of metabolic activity allows cluster analysis within the genus Brucella. The biotyping system developed for the identification of Brucella and differentiation of its species and biovars may replace or at least complement time-consuming tube testing especially in case of atypical strains. An easy to handle identification software facilitates the applicability of the Micronaut™ system for microbiology

Brucella abortus is Prevalent in Both Humans and Animals in Bangladesh.

PubMed

Rahman, A K M A; Saegerman, C; Berkvens, D; Melzer, F; Neubauer, H; Fretin, D; Abatih, E; Dhand, N; Ward, M P

2017-08-01

To determine the role of different Brucella (B.) spp. in Bangladesh, 62 animal samples and 500 human sera were tested. Animal samples from cattle, goats and sheep (including milk, bull semen, vaginal swabs and placentas) were cultured for Brucella spp. Three test-positive human sera and all animal samples were screened by Brucella genus-specific real-time PCR (RT-PCR), and positive samples were then tested by IS711 RT-PCR to detect B. abortus and B. melitensis DNA. Only B. abortus DNA was amplified from 13 human and six animal samples. This is the first report describing B. abortus as the aetiological agent of brucellosis in occupationally exposed humans in Bangladesh. Of note is failure to detect B. melitensis DNA, the species most often associated with human brucellosis worldwide. Further studies are required to explore the occurrence of Brucella melitensis in Bangladesh. © 2017 Blackwell Verlag GmbH.

Molecular Characterization of Brucella abortus Chromosome II Recombination

PubMed Central

Tsoktouridis, Georgios; Merz, Christian A.; Manning, Simon P.; Giovagnoli-Kurtz, Renée; Williams, Leanne E.; Mujer, Cesar V.; Hagius, Sue; Elzer, Philip; Redkar, Rajendra J.; Patra, Guy; DelVecchio, Vito G.

2003-01-01

Large-scale genomic rearrangements including inversions, deletions, and duplications are significant in bacterial evolution. The recently completed Brucella melitensis 16M and Brucella suis 1330 genomes have facilitated the investigation of such events in the Brucella spp. Suppressive subtractive hybridization (SSH) was employed in identifying genomic differences between B. melitensis 16M and Brucella abortus 2308. Analysis of 45 SSH clones revealed several deletions on chromosomes of B. abortus and B. melitensis that encoded proteins of various metabolic pathways. A 640-kb inversion on chromosome II of B. abortus has been reported previously (S. Michaux Charachon, G. Bourg, E. Jumas Bilak, P. Guigue Talet, A. Allardet Servent, D. O'Callaghan, and M. Ramuz, J. Bacteriol. 179:3244-3249, 1997) and is further described in this study. One end of the inverted region is located on a deleted TATGC site between open reading frames BMEII0292 and BMEII0293. The other end inserted at a GTGTC site of the cyclic-di-GMP phosphodiesterase A (PDEA) gene (BMEII1009), dividing PDEA into two unequal DNA segments of 160 and 977 bp. As a consequence of inversion, the 160-bp segment that encodes the N-terminal region of PDEA was relocated at the opposite end of the inverted chromosomal region. The splitting of the PDEA gene most likely inactivated the function of this enzyme. A recombination mechanism responsible for this inversion is proposed. PMID:14526025

Whole-genome analyses of the speciation events in the pathogenic Brucellae

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

Chain, P; Comerci, D; Tolmasky, M

Despite their high DNA identity and a proposal to group classical Brucella species as biovars of B. melitensis, the commonly recognized Brucella species can be distinguished by distinct biochemical and fatty acid characters as well as by a marked host range (e.g. B. suis for swine, B. melitensis for sheep and goats, B. abortus for cattle). Here we present the genome of B. abortus 2308, the virulent prototype biovar 1 strain, and its comparison to the two other human pathogenic Brucellae species and to the B. abortus field isolate 9-941. The global distribution of pseudogenes, deletions and insertions support previousmore » indications that B. abortus and B. melitensis share a common ancestor that diverged from B. suis. With the exception of a dozen genes, the genetic complement of both B. abortus strains is identical, whereas the three species differ in gene content and pseudogenes. The pattern of species-specific gene inactivations affecting transcriptional regulators and outer membrane proteins suggest that these inactivations may play an important role in the establishment of host-specificity and may have been a primary driver of speciation in the Brucellae. Despite being non-motile, the Brucellae contain flagellum gene clusters and display species-specific flagellar gene inactivations, which lead to the putative generation of different versions of flagellum-derived structures, and may contribute to differences in host-specificity and virulence. Metabolic changes such as the lack of complete metabolic pathways for the synthesis of numerous compounds (e.g. glycogen, biotin, NAD, and choline) are consistent with adaptation of Brucellae to an intracellular lifestyle.« less

Analysis of pan-genome to identify the core genes and essential genes of Brucella spp.

PubMed

Yang, Xiaowen; Li, Yajie; Zang, Juan; Li, Yexia; Bie, Pengfei; Lu, Yanli; Wu, Qingmin

2016-04-01

Brucella spp. are facultative intracellular pathogens, that cause a contagious zoonotic disease, that can result in such outcomes as abortion or sterility in susceptible animal hosts and grave, debilitating illness in humans. For deciphering the survival mechanism of Brucella spp. in vivo, 42 Brucella complete genomes from NCBI were analyzed for the pan-genome and core genome by identification of their composition and function of Brucella genomes. The results showed that the total 132,143 protein-coding genes in these genomes were divided into 5369 clusters. Among these, 1710 clusters were associated with the core genome, 1182 clusters with strain-specific genes and 2477 clusters with dispensable genomes. COG analysis indicated that 44 % of the core genes were devoted to metabolism, which were mainly responsible for energy production and conversion (COG category C), and amino acid transport and metabolism (COG category E). Meanwhile, approximately 35 % of the core genes were in positive selection. In addition, 1252 potential essential genes were predicted in the core genome by comparison with a prokaryote database of essential genes. The results suggested that the core genes in Brucella genomes are relatively conservation, and the energy and amino acid metabolism play a more important role in the process of growth and reproduction in Brucella spp. This study might help us to better understand the mechanisms of Brucella persistent infection and provide some clues for further exploring the gene modules of the intracellular survival in Brucella spp.

The Change of a Medically Important Genus: Worldwide Occurrence of Genetically Diverse Novel Brucella Species in Exotic Frogs.

PubMed

Scholz, Holger C; Mühldorfer, Kristin; Shilton, Cathy; Benedict, Suresh; Whatmore, Adrian M; Blom, Jochen; Eisenberg, Tobias

2016-01-01

The genus Brucella comprises various species of both veterinary and human medical importance. All species are genetically highly related to each other, sharing intra-species average nucleotide identities (ANI) of > 99%. Infections occur among various warm-blooded animal species, marine mammals, and humans. Until recently, amphibians had not been recognized as a host for Brucella. In this study, however, we show that novel Brucella species are distributed among exotic frogs worldwide. Comparative recA gene analysis of 36 frog isolates from various continents and different frog species revealed an unexpected high genetic diversity, not observed among classical Brucella species. In phylogenetic reconstructions the isolates consequently formed various clusters and grouped together with atypical more distantly related brucellae, like B. inopinata, strain BO2, and Australian isolates from rodents, some of which were isolated as human pathogens. Of one frog isolate (10RB9215) the genome sequence was determined. Comparative genome analysis of this isolate and the classical Brucella species revealed additional genetic material, absent from classical Brucella species but present in Ochrobactrum, the closest genetic neighbor of Brucella, and in other soil associated genera of the Alphaproteobacteria. The presence of gene clusters encoding for additional metabolic functions, flanked by tRNAs and mobile genetic elements, as well as by bacteriophages is suggestive for a different ecology compared to classical Brucella species. Furthermore it suggests that amphibian isolates may represent a link between free living soil saprophytes and the pathogenic Brucella with a preferred intracellular habitat. We therefore assume that brucellae from frogs have a reservoir in soil and, in contrast to classical brucellae, undergo extensive horizontal gene transfer.

Phosphatidylethanolamine Synthesis Is Required for Optimal Virulence of Brucella abortus▿

PubMed Central

Bukata, Lucas; Altabe, Silvia; de Mendoza, Diego; Ugalde, Rodolfo A.; Comerci, Diego J.

2008-01-01

The Brucella cell envelope contains the zwitterionic phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Synthesis of PC occurs exclusively via the PC synthase pathway, implying that the pathogen depends on the choline synthesized by the host cell to form PC. Notably, PC is necessary to sustain a chronic infection process, which suggests that the membrane lipid content is relevant for Brucella virulence. In this study we investigated the first step of PE biosynthesis in B. abortus, which is catalyzed by phosphatidylserine synthase (PssA). Disruption of pssA abrogated the synthesis of PE without affecting the growth in rich complex medium. In minimal medium, however, the mutant required choline supplementation for growth, suggesting that at least PE or PC is necessary for Brucella viability. The absence of PE altered cell surface properties, but most importantly, it impaired several virulence traits of B. abortus, such as intracellular survival in both macrophages and HeLa cells, the maturation of the replicative Brucella-containing vacuole, and mouse colonization. These results suggest that membrane phospholipid composition is critical for the interaction of B. abortus with the host cell. PMID:18931122

Intracellularly Induced Cyclophilins Play an Important Role in Stress Adaptation and Virulence of Brucella abortus

PubMed Central

García Fernández, Lucía; DelVecchio, Vito G.; Briones, Gabriel

2013-01-01

Brucella is an intracellular bacterial pathogen that causes the worldwide zoonotic disease brucellosis. Brucella virulence relies on its ability to transition to an intracellular lifestyle within host cells. Thus, this pathogen must sense its intracellular localization and then reprogram gene expression for survival within the host cell. A comparative proteomic investigation was performed to identify differentially expressed proteins potentially relevant for Brucella intracellular adaptation. Two proteins identified as cyclophilins (CypA and CypB) were overexpressed in the intracellular environment of the host cell in comparison to laboratory-grown Brucella. To define the potential role of cyclophilins in Brucella virulence, a double-deletion mutant was constructed and its resulting phenotype was characterized. The Brucella abortus ΔcypAB mutant displayed increased sensitivity to environmental stressors, such as oxidative stress, pH, and detergents. In addition, the B. abortus ΔcypAB mutant strain had a reduced growth rate at lower temperature, a phenotype associated with defective expression of cyclophilins in other microorganisms. The B. abortus ΔcypAB mutant also displays reduced virulence in BALB/c mice and defective intracellular survival in HeLa cells. These findings suggest that cyclophilins are important for Brucella virulence and survival in the host cells. PMID:23230297

Brucella Antibodies in Alaskan True Seals and Eared Seals-Two Different Stories.

PubMed

Nymo, Ingebjørg H; Rødven, Rolf; Beckmen, Kimberlee; Larsen, Anett K; Tryland, Morten; Quakenbush, Lori; Godfroid, Jacques

2018-01-01

Brucella pinnipedialis was first isolated from true seals in 1994 and from eared seals in 2008. Although few pathological findings have been associated with infection in true seals, reproductive pathology including abortions, and the isolation of the zoonotic strain type 27 have been documented in eared seals. In this study, a Brucella enzyme-linked immunosorbent assay (ELISA) and the Rose Bengal test (RBT) were initially compared for 206 serum samples and a discrepancy between the tests was found. Following removal of lipids from the serum samples, ELISA results were unaltered while the agreement between the tests was improved, indicating that serum lipids affected the initial RBT outcome. For the remaining screening, we used ELISA to investigate the presence of Brucella antibodies in sera of 231 eared and 1,412 true seals from Alaskan waters sampled between 1975 and 2011. In eared seals, Brucella antibodies were found in two Steller sea lions ( Eumetopias jubatus ) (2%) and none of the 107 Northern fur seals ( Callorhinus ursinus ). The low seroprevalence in eared seals indicate a low level of exposure or lack of susceptibility to infection. Alternatively, mortality due to the Brucella infection may remove seropositive animals from the population. Brucella antibodies were detected in all true seal species investigated; harbor seals ( Phoca vitulina ) (25%), spotted seals ( Phoca largha ) (19%), ribbon seals ( Histriophoca fasciata ) (16%), and ringed seals ( Pusa hispida hispida ) (14%). There was a low seroprevalence among pups, a higher seroprevalence among juveniles, and a subsequent decreasing probability of seropositivity with age in harbor seals. Similar patterns were present for the other true seal species; however, solid conclusions could not be made due to sample size. This pattern is in accordance with previous reports on B. pinnipedialis infections in true seals and may suggest environmental exposure to B. pinnipedialis at the juvenile stage, with a

DNA polymorphism analysis of Brucella lipopolysaccharide genes reveals marked differences in O-polysaccharide biosynthetic genes between smooth and rough Brucella species and novel species-specific markers

PubMed Central

2009-01-01

Background The lipopolysaccharide is a major antigen and virulence factor of Brucella, an important bacterial pathogen. In smooth brucellae, lipopolysaccharide is made of lipid A-core oligosaccharide and N-formylperosamine O-polysaccharide. B. ovis and B. canis (rough species) lack the O-polysaccharide. Results The polymorphism of O-polysaccharide genes wbkE, manAO-Ag, manBO-Ag, manCO-Ag, wbkF and wbkD) and wbo (wboA and wboB), and core genes manBcore and wa** was analyzed. Although most genes were highly conserved, species- and biovar-specific restriction patterns were found. There were no significant differences in putative N-formylperosamyl transferase genes, suggesting that Brucella A and M serotypes are not related to specific genes. In B. pinnipedialis and B. ceti (both smooth), manBO-Ag carried an IS711, confirming its dispensability for perosamine synthesis. Significant differences between smooth and rough species were found in wbkF and wbkD, two adjacent genes putatively related to bactoprenol priming for O-polysaccharide polymerization. B. ovis wbkF carried a frame-shift and B. canis had a long deletion partially encompassing both genes. In smooth brucellae, this region contains two direct repeats suggesting the deletion mechanism. Conclusion The results define species and biovar markers, confirm the dispensability of manBO-Ag for O-polysaccharide synthesis and contribute to explain the lipopolysaccharide structure of rough and smooth Brucella species. PMID:19439075

Molecular Survey on Brucellosis in Rodents and Shrews - Natural Reservoirs of Novel Brucella Species in Germany?

PubMed

Hammerl, J A; Ulrich, R G; Imholt, C; Scholz, H C; Jacob, J; Kratzmann, N; Nöckler, K; Al Dahouk, S

2017-04-01

Brucellosis is a widespread zoonotic disease introduced from animal reservoirs to humans. In Germany, bovine and ovine/caprine brucellosis were eradicated more than a decade ago and mandatory measures in livestock have been implemented to keep the officially brucellosis-free status. In contrast, surveillance of wildlife is still challenging, and reliable data on the prevalence of brucellae in small mammal populations do not exist. To assess the epidemiology of Brucella spp. in rodents and shrews, a molecular survey was carried out. A total of 537 rodents and shrews were trapped in four federal states located throughout Germany and investigated for the presence of Brucella. Using a two-step molecular assay based on the detection of the Brucella-specific bcsp31 and IS711 sequences in tissue samples, 14.2% (n = 76) of the tested animals were positive. These originated mainly from western and south-western Germany, where preliminary analyses indicate population density-dependent Brucella prevalence in voles (Myodes glareolus) and mice (Apodemus spp.). recA typing revealed a close relationship to a potentially novel Brucella species recently isolated from red foxes (Vulpes vulpes) in Austria. The molecular detection of brucellae in various rodent taxa and for the first time in shrew species shows that these animals may be naturally infected or at least have a history of exposure to Brucella spp. © 2015 Blackwell Verlag GmbH.

Brucella Modulates Secretory Trafficking via Multiple Type IV Secretion Effector Proteins

PubMed Central

Myeni, Sebenzile; Child, Robert; Ng, Tony W.; Kupko, John J.; Wehrly, Tara D.; Porcella, Stephen F.; Knodler, Leigh A.; Celli, Jean

2013-01-01

The intracellular pathogenic bacterium Brucella generates a replicative vacuole (rBCV) derived from the endoplasmic reticulum via subversion of the host cell secretory pathway. rBCV biogenesis requires the expression of the Type IV secretion system (T4SS) VirB, which is thought to translocate effector proteins that modulate membrane trafficking along the endocytic and secretory pathways. To date, only a few T4SS substrates have been identified, whose molecular functions remain unknown. Here, we used an in silico screen to identify putative T4SS effector candidate proteins using criteria such as limited homology in other bacterial genera, the presence of features similar to known VirB T4SS effectors, GC content and presence of eukaryotic-like motifs. Using β-lactamase and CyaA adenylate cyclase reporter assays, we identified eleven proteins translocated into host cells by Brucella, five in a VirB T4SS-dependent manner, namely BAB1_0678 (BspA), BAB1_0712 (BspB), BAB1_0847 (BspC), BAB1_1671 (BspE) and BAB1_1948 (BspF). A subset of the translocated proteins targeted secretory pathway compartments when ectopically expressed in HeLa cells, and the VirB effectors BspA, BspB and BspF inhibited protein secretion. Brucella infection also impaired host protein secretion in a process requiring BspA, BspB and BspF. Single or combined deletions of bspA, bspB and bspF affected Brucella ability to replicate in macrophages and persist in the liver of infected mice. Taken together, these findings demonstrate that Brucella modulates secretory trafficking via multiple T4SS effector proteins that likely act coordinately to promote Brucella pathogenesis. PMID:23950720

Progress in Brucella vaccine development

PubMed Central

YANG, Xinghong; SKYBERG, Jerod A.; CAO, Ling; CLAPP, Beata; THORNBURG, Theresa; PASCUAL, David W.

2012-01-01

Brucella spp. are zoonotic, facultative intracellular pathogens, which cause animal and human disease. Animal disease results in abortion of fetuses; in humans, it manifests flu-like symptoms with an undulant fever, with osteoarthritis as a common complication of infection. Antibiotic regimens for human brucellosis patients may last several months and are not always completely effective. While there are no vaccines for humans, several licensed live Brucella vaccines are available for use in livestock. The performance of these animal vaccines is dependent upon the host species, dose, and route of immunization. Newly engineered live vaccines, lacking well-defined virulence factors, retain low residual virulence, are highly protective, and may someday replace currently used animal vaccines. These also have possible human applications. Moreover, due to their enhanced safety and efficacy in animal models, subunit vaccines for brucellosis show great promise for their application in livestock and humans. This review summarizes the progress of brucellosis vaccine development and presents an overview of candidate vaccines. PMID:23730309

Brucella vulpis sp. nov., isolated from mandibular lymph nodes of red foxes (Vulpes vulpes).

PubMed

Scholz, Holger C; Revilla-Fernández, Sandra; Al Dahouk, Sascha; Hammerl, Jens A; Zygmunt, Michel S; Cloeckaert, Axel; Koylass, Mark; Whatmore, Adrian M; Blom, Jochen; Vergnaud, Gilles; Witte, Angela; Aistleitner, Karin; Hofer, Erwin

2016-05-01

Two slow-growing, Gram-negative, non-motile, non-spore-forming, coccoid bacteria (strains F60T and F965), isolated in Austria from mandibular lymph nodes of two red foxes (Vulpes vulpes), were subjected to a polyphasic taxonomic analysis. In a recent study, both isolates were assigned to the genus Brucella but could not be attributed to any of the existing species. Hence, we have analysed both strains in further detail to determine their exact taxonomic position and genetic relatedness to other members of the genus Brucella. The genome sizes of F60T and F965 were 3 236 779 and 3 237 765 bp, respectively. Each genome consisted of two chromosomes, with a DNA G+C content of 57.2 %. A genome-to-genome distance of >80 %, an average nucleotide identity (ANI) of 97 % and an average amino acid identity (AAI) of 98 % compared with the type species Brucella melitensis confirmed affiliation to the genus. Remarkably, 5 % of the entire genetic information of both strains was of non-Brucella origin, including as-yet uncharacterized bacteriophages and insertion sequences as well as ABC transporters and other genes of metabolic function from various soil-living bacteria. Core-genome-based phylogenetic reconstructions placed the novel species well separated from all hitherto-described species of the genus Brucella, forming a long-branched sister clade to the classical species of Brucella. In summary, based on phenotypic and molecular data, we conclude that strains F60T and F965 are members of a novel species of the genus Brucella, for which the name Brucella vulpis sp. nov. is proposed, with the type strain F60T ( = BCCN 09-2T = DSM 101715T).

Highly Sensitive Bacteriophage-Based Detection of Brucella abortus in Mixed Culture and Spiked Blood

PubMed Central

Sergueev, Kirill V.; Filippov, Andrey A.; Nikolich, Mikeljon P.

2017-01-01

For decades, bacteriophages (phages) have been used for Brucella species identification in the diagnosis and epidemiology of brucellosis. Traditional Brucella phage typing is a multi-day procedure including the isolation of a pure culture, a step that can take up to three weeks. In this study, we focused on the use of brucellaphages for sensitive detection of the pathogen in clinical and other complex samples, and developed an indirect method of Brucella detection using real-time quantitative PCR monitoring of brucellaphage DNA amplification via replication on live Brucella cells. This assay allowed the detection of single bacteria (down to 1 colony-forming unit per milliliter) within 72 h without DNA extraction and purification steps. The technique was equally efficient with Brucella abortus pure culture and with mixed cultures of B. abortus and α-proteobacterial near neighbors that can be misidentified as Brucella spp., Ochrobactrum anthropi and Afipia felis. The addition of a simple short sample preparation step enabled the indirect phage-based detection of B. abortus in spiked blood, with the same high sensitivity. This indirect phage-based detection assay enables the rapid and sensitive detection of live B. abortus in mixed cultures and in blood samples, and can potentially be applied for detection in other clinical samples and other complex sample types. PMID:28604602

Serological trail of Brucella infection in an urban slum population in Brazil

PubMed Central

Angel, Martha Olivera; Ristow, Paula; Ko, Albert I.; Di-Lorenzo, Cecilia

2013-01-01

Introduction Brucellosis is a re-emerging zoonosis with new cases reported each year in many Latin American countries, but it is mostly under-recognized. This study presents a serological investigation of infection with Brucella abortus and Brucella canis in a poor urban community in the city of Salvador, Brazil. Methodology Human sera (n = 180) were randomly selected from 3,171 samples taken from healthy individuals during 2003-2004 and tested with C-ELISA for B. abortus and I-ELISA for B. canis. Results Thirteen percent (24/180) of the individuals were positive for B. abortus and 4.6 % (8/174) were positive for B. canis. Among the variables studied only age (older than 45 years) appeared to be a risk factor for the detection of Brucella antibodies. Conclusion These results indicate the presence of Brucella infection in this settlement and highlight the need to understand the epidemiology of infection under these circumstances to establish the necessary measures for surveillance and control. PMID:23000868

Development of a Selective Culture Medium for Primary Isolation of the Main Brucella Species▿

PubMed Central

De Miguel, M. J.; Marín, C. M.; Muñoz, P. M.; Dieste, L.; Grilló, M. J.; Blasco, J. M.

2011-01-01

Bacteriological diagnosis of brucellosis is performed by culturing animal samples directly on both Farrell medium (FM) and modified Thayer-Martin medium (mTM). However, despite inhibiting most contaminating microorganisms, FM also inhibits the growth of Brucella ovis and some B. melitensis and B. abortus strains. In contrast, mTM is adequate for growth of all Brucella species but only partially inhibitory for contaminants. Moreover, the performance of both culture media for isolating B. suis has never been established properly. We first determined the performance of both media for B. suis isolation, proving that FM significantly inhibits B. suis growth. We also determined the susceptibility of B. suis to the antibiotics contained in both selective media, proving that nalidixic acid and bacitracin are highly inhibitory, thus explaining the reduced performance of FM for B. suis isolation. Based on these results, a new selective medium (CITA) containing vancomycin, colistin, nystatin, nitrofurantoin, and amphotericin B was tested for isolation of the main Brucella species, including B. suis. CITA's performance was evaluated using reference contaminant strains but also field samples taken from brucella-infected animals or animals suspected of infection. CITA inhibited most contaminant microorganisms but allowed the growth of all Brucella species, to levels similar to those for both the control medium without antibiotics and mTM. Moreover, CITA medium was more sensitive than both mTM and FM for isolating all Brucella species from field samples. Altogether, these results demonstrate the adequate performance of CITA medium for the primary isolation of the main Brucella species, including B. suis. PMID:21270216

Comparison of a New and Rapid Method: Brucella Coombs Gel Test With Other Diagnostic Tests.

PubMed

Kalem, Fatma; Ergün, Ayşe Gül; Durmaz, Süleyman; Doğan, Metin; Ertuğrul, Ömür; Gündem, Seval

2016-09-01

The aim of this study was to detect reliability of Brucella Coombs gel test (BCGT) by comparing with with ELISA (IgG + IgM), Standard agglutination test, and Brucella immunocapture agglutination methods in serological diagnosis of brucellosis. Brucella Coombs gel test (BCGT), Brucella ELISA (IgG + IgM), Standard agglutination test, and Brucella immunocapture agglutination tests of 78 patients with presumptive diagnosis of brucellosis which were sent to Microbiology Laboratory of Konya Numune Hospital from various regions of Konya were studied. Of 78 patients with ELISA IgG and IgM, STA, BICA and BCGT; 26, 21, 10, 12 and 12 were positive. When compared with BICA, the sensitivity and specifity of BCGT were 100% and 100%, respectively. According to results BCGT can be used as a diagnostic test in routine laboratories after more comprehensive studies in control groups and patients. © 2016 Wiley Periodicals, Inc.

Cloning, Expression, and Purification of Brucella suis Outer Membrane Proteins

DTIC Science & Technology

2005-01-01

13-09-20061 4. TITLE AND SUBTITLE 5a. CONTRACT NUMBER Cloning, expression and purification of Brucella suis outer membrane proteins 5b. GRANT NUMBER...attractive for this purpose. In this study, we cloned, expressed and purified seven predicted OMPs of Brucella suis . The recombinant proteins were...fused with 6-his and V5 epitope tags at their C termini to facilitate detection and purification. The B. suis surface genes were PCR synthesized based

ISOLATION AND CHARACTERIZATION OF A NOVEL MARINE BRUCELLA FROM A SOUTHERN SEA OTTER (ENHYDRA LUTRIS NEREIS), CALIFORNIA, USA.

PubMed

Miller, Melissa A; Burgess, Tristan L; Dodd, Erin M; Rhyan, Jack C; Jang, Spencer S; Byrne, Barbara A; Gulland, Frances M D; Murray, Michael J; Toy-Choutka, Sharon; Conrad, Patricia A; Field, Cara L; Sidor, Inga F; Smith, Woutrina A

2017-04-01

We characterize Brucella infection in a wild southern sea otter ( Enhydra lutris nereis) with osteolytic lesions similar to those reported in other marine mammals and humans. This otter stranded twice along the central California coast, US over a 1-yr period and was handled extensively at two wildlife rehabilitation facilities, undergoing multiple surgeries and months of postsurgical care. Ultimately the otter was euthanized due to severe, progressive neurologic disease. Necropsy and postmortem radiographs revealed chronic, severe osteoarthritis spanning the proximal interphalangeal joint of the left hind fifth digit. Numerous coccobacilli within the joint were strongly positive on Brucella immunohistochemical labelling, and Brucella sp. was isolated in pure culture from this lesion. Sparse Brucella-immunopositive bacteria were also observed in the cytoplasm of a pulmonary vascular monocyte, and multifocal granulomas were observed in the spinal cord and liver on histopathology. Findings from biochemical characterization, 16S ribosomal DNA, and bp26 gene sequencing of the bacterial isolate were identical to those from marine-origin brucellae isolated from cetaceans and phocids. Although omp2a gene sequencing revealed 100% homology with marine Brucella spp. infecting pinnipeds, whales, and humans, omp2b gene sequences were identical only to pinniped-origin isolates. Multilocus sequence typing classified the sea otter isolate as ST26, a sequence type previously associated only with cetaceans. Our data suggest that the sea otter Brucella strain represents a novel marine lineage that is distinct from both Brucella pinnipedialis and Brucella ceti. Prior reports document the zoonotic potential of the marine brucellae. Isolation of Brucella sp. from a stranded sea otter highlights the importance of wearing personal protective equipment when handling sea otters and other marine mammals as part of wildlife conservation and rehabilitation efforts.

Brucella and Coxiella; if you don't look, you don't find.

PubMed

Lambourne, Jonathan R; Brooks, Tim

2015-02-01

Brucella and Coxiella are similar; both are obligate intracellular, zoonotic pathogens with a broad geographic distribution. Infection in animals is usually asymptomatic, but causes fetal loss and therefore has significant economic impact. Human infection may be asymptomatic or give rise to either organ-specific or multi-system disease. Organism culture is challenging for Coxiella and can lack sensitivity for Brucella. Therefore, infection is most commonly diagnosed by serology, but this may be negative in early infection and serology results may be challenging to interpret. Both Brucella and Coxiella are typically susceptible to a wide range of antimicrobials, but long courses may be needed. © 2015 Royal College of Physicians.

Natural Brucella infection in Argentine wild foxes*

PubMed Central

Szyfres, B.; Tomé, J. González

1966-01-01

In the course of an investigation in 1962-64 into the natural occurrence of brucellosis among grey foxes in Argentina, agglutination tests were performed on 728 sera of the foxes Dusicyon gymnocercus antiquus and D. griseus griseus, captured in the provinces of Buenos Aires and Rio Negro. Agglutination titres of from 1:25 to 1:800 were found in 173 (23.8%) of the foxes tested, 11.3% having titres of 1:100 or more. In bacteriological testing, eight cultures of Brucella abortus, biotype 1, were obtained. Discussing their findings, the authors point out that it cannot be stated definitely whether Brucella is naturally shed by foxes or to what extent infected foxes contribute to the dissemination of brucellosis. PMID:5296540

Exposure of harbour seals Phoca vitulina to Brucella in declining populations across Scotland.

PubMed

Kershaw, Joanna L; Stubberfield, Emma J; Foster, Geoffrey; Brownlow, Andrew; Hall, Ailsa J; Perrett, Lorraine L

2017-09-20

Since 2000 there has been a major decline in the abundance of Scottish harbour seals Phoca vitulina. The causes of the decline remain uncertain. The aim of this study was to establish the extent to which the seals in the regions of greatest decline have been exposed to Brucella, a bacterial pathogen that causes reproductive failure in terrestrial mammalian hosts. Tissues from dead seals collected between 1992 and 2013 were cultured for Brucella (n = 150). Serum samples collected from live capture-released seals (n = 343) between 1997 and 2012 were tested for Brucella antibodies using the Rose Bengal plate agglutination test (RBT) and a competitive enzyme-linked immunosorbent assay (cELISA). In total, 16% of seals cultured had Brucella isolated from one or more tissues, but there were no pathological signs of infection. The cELISA results were more sensitive than the RBT results, showing that overall 25.4% of seals were seropositive, with the highest seroprevalence in juveniles. As there was no evidence of either a higher seroprevalence or higher circulating antibody levels in seropositive animals in the areas with the greatest declines, it was concluded that Brucella infection is likely not a major contributing factor to recent declines. However, the consistently high proportion of seals exposed to Brucella indicates possible endemicity in these populations, likely due to B. pinnipedialis, which has demonstrated a preference for pinniped hosts. Importantly, given the close proximity between seals, humans and livestock in many areas, there is the potential for cross-species infections.

Infection of California sea lions (Zalophus californianus) with terrestrial Brucella spp.

PubMed

Avalos-Téllez, Rosalía; Ramírez-Pfeiffer, Carlos; Hernández-Castro, Rigoberto; Díaz-Aparicio, Efrén; Sánchez-Domínguez, Carlos; Zavala-Norzagaray, Alan; Arellano-Reynoso, Beatriz; Suárez-Güemes, Francisco; Aguirre, A Alonso; Aurioles-Gamboa, David

2014-10-01

Infections with Brucella ceti and pinnipedialis are prevalent in marine mammals worldwide. A total of 22 California sea lions (Zalophus californianus) were examined to determine their exposure to Brucella spp. at San Esteban Island in the Gulf of California, Mexico, in June and July 2011. Although samples of blood, vaginal mucus and milk cultured negative for these bacteria, the application of rose Bengal, agar gel immunodiffusion, PCR and modified fluorescence polarization assays found that five animals (22.7%) had evidence of exposure to Brucella strains. The data also suggested that in two of these five sea lions the strains involved were of terrestrial origin, a novel finding in marine mammals. Further work will be required to validate and determine the epidemiological significance of this finding. Copyright © 2014 Elsevier Ltd. All rights reserved.

Diverse Genetic Regulon of the Virulence-Associated Transcriptional Regulator MucR in Brucella abortus 2308

PubMed Central

Caswell, Clayton C.; Elhassanny, Ahmed E. M.; Planchin, Emilie E.; Roux, Christelle M.; Weeks-Gorospe, Jenni N.; Ficht, Thomas A.; Dunman, Paul M.

2013-01-01

The Ros-type regulator MucR is one of the few transcriptional regulators that have been linked to virulence in Brucella. Here, we show that a Brucella abortus in-frame mucR deletion strain exhibits a pronounced growth defect during in vitro cultivation and, more importantly, that the mucR mutant is attenuated in cultured macrophages and in mice. The genetic basis for the attenuation of Brucella mucR mutants has not been defined previously, but in the present study the genes regulated by MucR in B. abortus have been elucidated using microarray analysis and real-time reverse transcription-PCR (RT-PCR). In B. abortus 2308, MucR regulates a wide variety of genes whose products may function in establishing and maintaining cell envelope integrity, polysaccharide biosynthesis, iron homeostasis, genome plasticity, and transcriptional regulation. Particularly notable among the MucR-regulated genes identified is arsR6 (nolR), which encodes a transcriptional regulator previously linked to virulence in Brucella melitensis 16 M. Importantly, electrophoretic mobility shift assays (EMSAs) determined that a recombinant MucR protein binds directly to the promoter regions of several genes repressed by MucR (including arsR6 [nolR]), and in Brucella, as in other alphaproteobacteria, MucR binds to its own promoter to repress expression of the gene that encodes it. Overall, these studies have uncovered the diverse genetic regulon of MucR in Brucella, and in doing so this work has begun to define the MucR-controlled genetic circuitry whose misregulation contributes to the virulence defect of Brucella mucR mutants. PMID:23319565

Brucella placentitis and seroprevalence in northern fur seals (Callorhinus ursinus) of the Pribilof Islands, Alaska

PubMed Central

Duncan, Colleen G.; Tiller, Rebekah; Mathis, Demetrius; Stoddard, Robyn; Kersh, Gilbert J.; Dickerson, Bobette; Gelatt, Tom

2017-01-01

Brucella species infect a wide range of hosts with a broad spectrum of clinical manifestations. In mammals, one of the most significant consequences of Brucella infection is reproductive failure. There is evidence of Brucella exposure in many species of marine mammals, but the outcome of infection is often challenging to determine. The eastern Pacific stock of northern fur seals (NFSs, Callorhinus ursinus) has declined significantly, spawning research into potential causes for this trend, including investigation into reproductive health. The objective of the current study was to determine if NFSs on St. Paul Island, Alaska have evidence of Brucella exposure or infection. Archived DNA extracted from placentas (n = 119) and serum (n = 40) samples were available for testing by insertion sequence (IS) 711 polymerase chain reaction (PCR) and the Brucella microagglutination test (BMAT), respectively. As well, placental tissue was available for histologic examination. Six (5%) placentas were positive by PCR, and a single animal had severe placentitis. Multilocus variable number tandem repeat analysis profiles were highly clustered and closely related to other Brucella pinnipedialis isolates. A single animal was positive on BMAT, and 12 animals had titers within the borderline range; 1 borderline animal was positive by PCR on serum. The findings suggest that NFSs on the Pribilof Islands are exposed to Brucella and that the organism has the ability to cause severe placental disease. Given the population trend of the NFS, and the zoonotic nature of this pathogen, further investigation into the epidemiology of this disease is recommended. PMID:24803576

Brucella placentitis and seroprevalence in northern fur seals ( Callorhinus ursinus) of the Pribilof Islands, Alaska.

PubMed

Duncan, Colleen G; Tiller, Rebekah; Mathis, Demetrius; Stoddard, Robyn; Kersh, Gilbert J; Dickerson, Bobette; Gelatt, Tom

2014-07-01

Brucella species infect a wide range of hosts with a broad spectrum of clinical manifestations. In mammals, one of the most significant consequences of Brucella infection is reproductive failure. There is evidence of Brucella exposure in many species of marine mammals, but the outcome of infection is often challenging to determine. The eastern Pacific stock of northern fur seals (NFSs, Callorhinus ursinus) has declined significantly, spawning research into potential causes for this trend, including investigation into reproductive health. The objective of the current study was to determine if NFSs on St. Paul Island, Alaska have evidence of Brucella exposure or infection. Archived DNA extracted from placentas ( n = 119) and serum ( n = 40) samples were available for testing by insertion sequence (IS) 711 polymerase chain reaction (PCR) and the Brucella microagglutination test (BMAT), respectively. As well, placental tissue was available for histologic examination. Six (5%) placentas were positive by PCR, and a single animal had severe placentitis. Multilocus variable number tandem repeat analysis profiles were highly clustered and closely related to other Brucella pinnipedialis isolates. A single animal was positive on BMAT, and 12 animals had titers within the borderline range; 1 borderline animal was positive by PCR on serum. The findings suggest that NFSs on the Pribilof Islands are exposed to Brucella and that the organism has the ability to cause severe placental disease. Given the population trend of the NFS, and the zoonotic nature of this pathogen, further investigation into the epidemiology of this disease is recommended.

High throughput MLVA-16 typing for Brucella based on the microfluidics technology

PubMed Central

2011-01-01

Background Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology. Results The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created. Conclusion In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other

Comparison and correlation of Simple Sequence Repeats distribution in genomes of Brucella species

PubMed Central

Kiran, Jangampalli Adi Pradeep; Chakravarthi, Veeraraghavulu Praveen; Kumar, Yellapu Nanda; Rekha, Somesula Swapna; Kruti, Srinivasan Shanthi; Bhaskar, Matcha

2011-01-01

Computational genomics is one of the important tools to understand the distribution of closely related genomes including simple sequence repeats (SSRs) in an organism, which gives valuable information regarding genetic variations. The central objective of the present study was to screen the SSRs distributed in coding and non-coding regions among different human Brucella species which are involved in a range of pathological disorders. Computational analysis of the SSRs in the Brucella indicates few deviations from expected random models. Statistical analysis also reveals that tri-nucleotide SSRs are overrepresented and tetranucleotide SSRs underrepresented in Brucella genomes. From the data, it can be suggested that over expressed tri-nucleotide SSRs in genomic and coding regions might be responsible in the generation of functional variation of proteins expressed which in turn may lead to different pathogenicity, virulence determinants, stress response genes, transcription regulators and host adaptation proteins of Brucella genomes. Abbreviations SSRs - Simple Sequence Repeats, ORFs - Open Reading Frames. PMID:21738309

Omics of Brucella: Species-Specific sRNA-Mediated Gene Ontology Regulatory Networks Identified by Computational Biology.

PubMed

Vishnu, Udayakumar S; Sankarasubramanian, Jagadesan; Gunasekaran, Paramasamy; Sridhar, Jayavel; Rajendhran, Jeyaprakash

2016-06-01

Brucella is an intracellular bacterium that causes the zoonotic infectious disease, brucellosis. Brucella species are currently intensively studied with a view to developing novel global health diagnostics and therapeutics. In this context, small RNAs (sRNAs) are one of the emerging topical areas; they play significant roles in regulating gene expression and cellular processes in bacteria. In the present study, we forecast sRNAs in three Brucella species that infect humans, namely Brucella melitensis, Brucella abortus, and Brucella suis, using a computational biology analysis. We combined two bioinformatic algorithms, SIPHT and sRNAscanner. In B. melitensis 16M, 21 sRNA candidates were identified, of which 14 were novel. Similarly, 14 sRNAs were identified in B. abortus, of which four were novel. In B. suis, 16 sRNAs were identified, and five of them were novel. TargetRNA2 software predicted the putative target genes that could be regulated by the identified sRNAs. The identified mRNA targets are involved in carbohydrate, amino acid, lipid, nucleotide, and coenzyme metabolism and transport, energy production and conversion, replication, recombination, repair, and transcription. Additionally, the Gene Ontology (GO) network analysis revealed the species-specific, sRNA-based regulatory networks in B. melitensis, B. abortus, and B. suis. Taken together, although sRNAs are veritable modulators of gene expression in prokaryotes, there are few reports on the significance of sRNAs in Brucella. This report begins to address this literature gap by offering a series of initial observations based on computational biology to pave the way for future experimental analysis of sRNAs and their targets to explain the complex pathogenesis of Brucella.

A case report of spontaneous abortion caused by Brucella melitensis biovar 3.

PubMed

Yang, Hong-Xia; Feng, Jun-Jun; Zhang, Qiu-Xiang; Hao, Rui-E; Yao, Su-Xia; Zhao, Rong; Piao, Dong-Ri; Cui, Bu-Yun; Jiang, Hai

2018-05-02

Brucellosis is a worldwide zoonotic disease caused by Brucella spp. Brucella invades the body through the skin mucosa, digestive tract, and respiratory tract. However, only a few studies on human spontaneous abortion attributable to Brucella have been reported. In this work, the patient living in Shanxi Province in China who had suffered a spontaneous abortion was underwent pathogen detection and Brucella melitensis biovar 3 was identified. The patient in this study was 22 years old. On July 16, 2015, she was admitted to Shanxi Grand Hospital, Shanxi Province, China because of one day of vaginal bleeding and three days of abdominal distension accompanied by fever after five months of amenorrhea. A serum tube agglutination test for brucellosis and blood culture were positive. At the time of discharge, she was prescribed oral doxycycline (100 mg/dose, twice a day) and rifampicin (600 mg/dose, once daily) for 6 weeks as recommended by the World Health Organization (WHO). No recurrence was observed during the six months of follow-up after the cessation of antibiotic treatment. This is the first reported case of miscarriage resulting from Brucella melitensis biovar 3 isolated from a pregnant woman who was infected through unpasteurized milk in China. Brucellosis infection was overlooked in the Maternity Hospital because of physician unawareness. Early recognition and prompt treatment of brucellosis infection are crucial for a successful outcome in pregnancy.

Brucella endocarditis: a report from Iran.

PubMed

Esmailpour, Negin; Borna, Sima; Nejad, Mehrnaz Rasooli; Badie, Sina Moradmand; Badie, Banafsheh Moradmand; Hadadi, Azar

2010-01-01

Endocarditis is a rare focal complication of brucellosis but the most common cause of mortality. The diagnosis of the complications of endemic diseases is therefore important. We evaluated Brucella endocarditis cases in a teaching hospital in Iran between April 1998 and March 2006. Nine patients with a median age of 38.11 years were recorded, of whom seven (77.7%) were male. Underlying cardiopathy was present in three patients (33.3%). The median duration of the symptoms prior to diagnosis was 5.33 months. Endocarditis involved the aortic valve in six cases (66.6%), the mitral valve in two cases (22.2%) and the aortic valve plus the mitral valve in one case (11.1%). Serologic tests were positive in eight (88.8%) and blood culture was positive in two (22.2%). Aortic valve replacement surgery was undertaken for five patients (55.5%). One patient died due to arrhythmia. A high degree of suspicion is therefore necessary in order to ameliorate the course of Brucella endocarditis.

Real-time PCR assays for detection of Brucella spp. and the identification of genotype ST27 in bottlenose dolphins (Tursiops truncatus).

PubMed

Wu, Qingzhong; McFee, Wayne E; Goldstein, Tracey; Tiller, Rebekah V; Schwacke, Lori

2014-05-01

Rapid detection of Brucella spp. in marine mammals is challenging. Microbiologic culture is used for definitive diagnosis of brucellosis, but is time consuming, has low sensitivity and can be hazardous to laboratory personnel. Serological methods can aid in diagnosis, but may not differentiate prior exposure versus current active infection and may cross-react with unrelated Gram-negative bacteria. This study reports a real-time PCR assay for the detection of Brucella spp. and application to screen clinical samples from bottlenose dolphins stranded along the coast of South Carolina, USA. The assay was found to be 100% sensitive for the Brucella strains tested, and the limit of detection was 0.27fg of genomic DNA from Brucella ceti B1/94 per PCR volume. No amplification was detected for the non-Brucella pathogens tested. Brucella DNA was detected in 31% (55/178) of clinical samples tested. These studies indicate that the real-time PCR assay is highly sensitive and specific for the detection of Brucella spp. in bottlenose dolphins. We also developed a second real-time PCR assay for rapid identification of Brucella ST27, a genotype that is associated with human zoonotic infection. Positive results were obtained for Brucella strains which had been identified as ST27 by multilocus sequence typing. No amplification was found for other Brucella strains included in this study. ST27 was identified in 33% (18/54) of Brucella spp. DNA-positive clinical samples. To our knowledge, this is the first report on the use of a real-time PCR assay for identification of Brucella genotype ST27 in marine mammals. Copyright © 2014 Elsevier B.V. All rights reserved.

Vector Development for the Expression of Foreign Proteins in the Vaccine Strain Brucella abortus S19

PubMed Central

Comerci, Diego J.; Pollevick, Guido D.; Vigliocco, Ana M.; Frasch, Alberto C. C.; Ugalde, Rodolfo A.

1998-01-01

A vector for the expression of foreign antigens in the vaccine strain Brucella abortus S19 was developed by using a DNA fragment containing the regulatory sequences and the signal peptide of the Brucella bcsp31 gene. This fragment was cloned in broad-host-range plasmid pBBR4MCS, resulting in plasmid pBEV. As a reporter protein, a repetitive antigen of Trypanosoma cruzi was used. The recombinant fusion protein is stably expressed and secreted into the Brucella periplasmic space, inducing a good antibody response against the T. cruzi antigen. The expression of the repetitive antigen in Brucella neither altered its growth pattern nor generated a toxic or lethal effect during experimental infection. The application of this strategy for the generation of live recombinant vaccines and the tagging of B. abortus S19 vaccine is discussed. This is the first time that a recombinant protein has been expressed in the periplasm of brucellae. PMID:9673273

Comparison of Brucella abortus and Brucella melitensis infections of mice and their effect on acquired cellular resistance.

PubMed Central

Young, E J; Gomez, C I; Yawn, D H; Musher, D M

1979-01-01

By using mice infected with strains of Brucella abortus and Brucella melitensis we examined the histological responses to infection, the relationship of histology to persistence of organisms, and the relation of persistence of organisms to the acquisition of acquired cellular resistance (ACR). Infection with B. abortus resulted in well-formed granulomas in the livers, which persisted for more than 30 days. In contrast, infection with B. melitensis produced microabscesses in the livers which resolved before 30 days. The clearance of organisms from the tissues was also different. A total of 30 days after infection, large numbers of viable bacteria were recovered from the tissues of B. abortus-infected mice whereas bacteria were no longer recoverable from B. melitensis-infected animals. ACR to Listeria monocytogenes, another intracellular pathogen, persisted for more than 30 days in B. abortus-infected mice but waned rapidly in B. melitensis-infected animals. This disappearance of ACR due to B. melitensis paralleled the clearance of bacteria from the tissues. Images PMID:121113

Host-Brucella interactions and the Brucella genome as tools for subunit antigen discovery and immunization against brucellosis

PubMed Central

Gomez, Gabriel; Adams, Leslie G.; Rice-Ficht, Allison; Ficht, Thomas A.

2013-01-01

Vaccination is the most important approach to counteract infectious diseases. Thus, the development of new and improved vaccines for existing, emerging, and re-emerging diseases is an area of great interest to the scientific community and general public. Traditional approaches to subunit antigen discovery and vaccine development lack consideration for the critical aspects of public safety and activation of relevant protective host immunity. The availability of genomic sequences for pathogenic Brucella spp. and their hosts have led to development of systems-wide analytical tools that have provided a better understanding of host and pathogen physiology while also beginning to unravel the intricacies at the host-pathogen interface. Advances in pathogen biology, host immunology, and host-agent interactions have the potential to serve as a platform for the design and implementation of better-targeted antigen discovery approaches. With emphasis on Brucella spp., we probe the biological aspects of host and pathogen that merit consideration in the targeted design of subunit antigen discovery and vaccine development. PMID:23720712

Serologic evidence of Brucella infection in pinnipeds along the coast of Hokkaido, the northernmost main island of Japan.

PubMed

Abe, Erika; Ohishi, Kazue; Ishinazaka, Tsuyoshi; Fujii, Kei; Maruyama, Tadashi

2017-04-01

Brucella infection in Hokkaido was serologically surveyed in four species of pinnipeds inhabiting Cape Erimo during 2008-2013 and the Shiretoko Peninsula in 1999 by ELISA using Brucella abortus and B. canis as antigens. Anti-Brucella positive sera showed higher absorbance to B. abortus than B. canis in almost all samples. Anti-B. abortus antibodies were detected in serum samples from 24% (n = 55) of Western Pacific harbor seals (Phoca vitulina stejnegeri) in Cape Erimo and from 66% (n = 41) of spotted seals (P. largha), 15% (n = 20) of ribbon seals (Histriophoca fasciata) and 18% (n = 17) of Western Steller's sea lions (Eumetopias jubatus jubatus) in the Shiretoko Peninsula. Anti-Brucella antibodies were detected at higher absorbance in 1- to 4-year-old harbor seals than in the pups and mature animals, suggesting either that Brucella infection mainly occurs after weaning or that it is maternally transmitted to pups with premature or suppressed immunity. Anti-Brucella antibodies were detected in both immature and mature spotted seals and ribbon seals, with higher absorbance in the former. The antibodies were detected only in mature Western Steller's sea lions. Western blot analysis of the serum samples showed some differences in band appearances, namely discrete versus smeary, and in the number of bands, indicating that multiple different Brucella may be prevalent in pinnipeds in Hokkaido. Alternatively, the Brucella of pinnipeds may have some intra-species diversity. © 2017 The Societies and John Wiley & Sons Australia, Ltd.

Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan.

PubMed

Lindahl-Rajala, Elisabeth; Hoffman, Tove; Fretin, David; Godfroid, Jacques; Sattorov, Nosirjon; Boqvist, Sofia; Lundkvist, Åke; Magnusson, Ulf

2017-03-01

Brucellosis is one of the most common zoonoses globally, and Central Asia remains a Brucella hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as Brucella. This study investigates the presence of Brucella DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. Brucella DNA was detected in 10.3% of 564 cow milk samples by IS711-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, Brucella DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of Brucella DNA. In addition, sequence analysis of the rpoB gene suggests that one cow was infected with B. abortus and another cow was most likely infected with B. melitensis. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in Brucella surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control Brucella infections in the livestock population but further research focusing on the isolation of Brucella is required to obtain a comprehensive understanding of the Brucella spp. circulating among the livestock in this region.

Detection and characterization of Brucella spp. in bovine milk in small-scale urban and peri-urban farming in Tajikistan

PubMed Central

Lindahl-Rajala, Elisabeth; Hoffman, Tove; Fretin, David; Godfroid, Jacques; Sattorov, Nosirjon; Boqvist, Sofia; Lundkvist, Åke; Magnusson, Ulf

2017-01-01

Brucellosis is one of the most common zoonoses globally, and Central Asia remains a Brucella hotspot. The World Health Organization classifies brucellosis as a neglected zoonotic disease that is rarely in the spotlight for research and mainly affects poor, marginalized people. Urban and peri-urban farming is a common practice in many low-income countries, and it increases the incomes of families that are often restrained by limited economic resources. However, there is a concern that the growing number of people and livestock living close together in these areas will increase the transmission of zoonotic pathogens such as Brucella. This study investigates the presence of Brucella DNA in bovine milk in the urban and peri-urban area of Dushanbe, Tajikistan. Brucella DNA was detected in 10.3% of 564 cow milk samples by IS711-based real-time PCR. This finding is concerning because consumption of unpasteurized dairy products is common in the region. Furthermore, Brucella DNA was detected in the milk of all seropositive cows, but 8.3% of the seronegative cows also showed the presence of Brucella DNA. In addition, sequence analysis of the rpoB gene suggests that one cow was infected with B. abortus and another cow was most likely infected with B. melitensis. The discrepancies between the serology and real-time PCR results highlight the need to further investigate whether there is a need for implementing complementary diagnostic strategies to detect false serological negative individuals in Brucella surveillance, control, and eradication programmes. Furthermore, vaccination of cattle with S19 in addition to vaccination of small ruminants with Rev 1 might be needed in order to control Brucella infections in the livestock population but further research focusing on the isolation of Brucella is required to obtain a comprehensive understanding of the Brucella spp. circulating among the livestock in this region. PMID:28296882

Persistence Testing of Brucella suis on Outdoor Materials ...

EPA Pesticide Factsheets

Report This report presents the results of an investigation to evaluate Brucella suis persistence on five materials (typically found in the outdoor environment) under various environmental conditions and exposure durations.

[Preparation and characterization of follwing the national standard anti-Brucella abortus serum, bovine].

PubMed

Li, Cui; Guan, Fushi; Dai, Zhihong; Jiang, Hui; Wen, Fang; Lu, Lianshou; Wang, Zaishi

2011-05-01

To prepare anti-Brucella abortus serum used for calibrate the agglutination test follwing the national standard, 4 anti-Brucella abortus sera were obtained from 4 cows infected with Brucella abortus naturally. By potency testing, the third serum was selected. Sterility, vaccum degree, residual moisture, uniformity and stability of this standard material were tested and proved to meet the national standard. Referring to the international standard, RBT (Rose-Bengal plate agglutination test), SAT (standard tube agglutination) and CFT (complement fixation test) titers of this standard material were measured to be 1:160 "+" 1:2 400 "++" and 1:800 "++", which are identical with the collaborative assay results. International unit of the standard material is 4 000 IU/mL.

Leptospira and Brucella antibodies in collared anteaters (Tamandua tetradactyla) in Brazilian zoos.

PubMed

Sales, Indiara dos Santos; Folly, Márcio Manhães; Garcia, Luize Néli Nunes; Ramos, Tatiane Mendes Varela; da Silva, Mariana Cristina; Pereira, Martha Maria

2012-12-01

The presence of Leptospira spp. and Brucella spp. antibodies was investigated in serum samples from 28 collared anteaters (Tamandua tetradactyla) kept in seven Brazilian zoos. Sera were tested against 19 Leptospira serovars using microscopic agglutination. Samples reacted to the following serovars: two (7.14%) to Patoc, three (10.71%) to Tarrasovi, three (10.71%) to both Patoc and Tarrasovi, two (7.14%) to Wolffi, and one (3.57%) to Australis. Two (7.14%) samples reacted to the buffered Brucella antigen test, but no confirmatory reaction occurred using the 2-mercaptoethanol slow slide agglutination test. No sample was reactive in the agar gel immunodiffusion test for rugose species of Brucella. The presence of anti-leptospira agglutinins in captive T. tetradactyla serum indicates that this species may be susceptible to infection by these bacteria.

Meta-analysis of variables affecting mouse protection efficacy of whole organism Brucella vaccines and vaccine candidates

PubMed Central

2013-01-01

Background Vaccine protection investigation includes three processes: vaccination, pathogen challenge, and vaccine protection efficacy assessment. Many variables can affect the results of vaccine protection. Brucella, a genus of facultative intracellular bacteria, is the etiologic agent of brucellosis in humans and multiple animal species. Extensive research has been conducted in developing effective live attenuated Brucella vaccines. We hypothesized that some variables play a more important role than others in determining vaccine protective efficacy. Using Brucella vaccines and vaccine candidates as study models, this hypothesis was tested by meta-analysis of Brucella vaccine studies reported in the literature. Results Nineteen variables related to vaccine-induced protection of mice against infection with virulent brucellae were selected based on modeling investigation of the vaccine protection processes. The variable "vaccine protection efficacy" was set as a dependent variable while the other eighteen were set as independent variables. Discrete or continuous values were collected from papers for each variable of each data set. In total, 401 experimental groups were manually annotated from 74 peer-reviewed publications containing mouse protection data for live attenuated Brucella vaccines or vaccine candidates. Our ANOVA analysis indicated that nine variables contributed significantly (P-value < 0.05) to Brucella vaccine protection efficacy: vaccine strain, vaccination host (mouse) strain, vaccination dose, vaccination route, challenge pathogen strain, challenge route, challenge-killing interval, colony forming units (CFUs) in mouse spleen, and CFU reduction compared to control group. The other 10 variables (e.g., mouse age, vaccination-challenge interval, and challenge dose) were not found to be statistically significant (P-value > 0.05). The protection level of RB51 was sacrificed when the values of several variables (e.g., vaccination route, vaccine viability

Epidemiology of Brucella infection in the human, livestock and wildlife interface in the Katavi-Rukwa ecosystem, Tanzania.

PubMed

Assenga, Justine A; Matemba, Lucas E; Muller, Shabani K; Malakalinga, Joseph J; Kazwala, Rudovick R

2015-08-08

Brucellosis is a zoonosis of public health importance worldwide. In Tanzania, the disease is underreported due to insufficient awareness, inadequate diagnostic protocols, including lack of appropriate reagents for diagnosis. Livestock and wildlife are considered potential sources of infection to humans; however, the role played by these carriers in the epidemiology of the disease in the ecosystems in Tanzania is not fully understood. The objective of this study was to establish the prevalence of anti-Brucella antibodies in humans, wildlife and livestock; and molecular prevalence of Brucella spp in cattle and goats in the Katavi- Rukwa ecosystem. Anti-Brucella antibodies were detected in humans at 0.6 % (95 % CI: 0.1, 2.1 %); cattle at 6.8 % (95 % CI: 5.4, 8.5 %), goats at 1.6 % (95 % CI: 0.4, 4.1 %) and buffaloes at 7.9 % (95 % CI: 1.7, 21.4 %). One of the two sampled lions tested positive. Cattle had a significantly higher prevalence of anti-Brucella antibodies as compared to goats (P < 0.05). A significantly higher seroprevalence was found in female than in male cattle and in adult than in young cattle (P < 0.05). There was an agreement of 95 and 89 % in cattle and goats, respectively, for the Rose Bengal plate Test (RBPT) and Competitive Enzyme Linked Immunosorbent Assay (c-ELISA) in detecting Brucella infection. Eight (3.5 %) out of 231 milk samples tested were positive for Brucella spp on Polymerase Chain Reaction (PCR), and Brucella abortus biovar 1 was detected in cattle milk. However, no Brucella spp were detected in goat milk. This study has shown the presence of anti- Brucella antibodies in humans, livestock, and wildlife in the Katavi- Rukwa ecosystem. Transmission of the infection between wildlife, livestock and humans is likely to continue due to increasing human activities in the human wildlife interface. This information is an important contribution to public health policy development in the human wildlife interface of the Katavi- Rukwa ecosystem.

The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response.

PubMed

Khan, Mike; Harms, Jerome S; Marim, Fernanda M; Armon, Leah; Hall, Cherisse L; Liu, Yi-Ping; Banai, Menachem; Oliveira, Sergio C; Splitter, Gary A; Smith, Judith A

2016-12-01

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host-Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a ΔbpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the ΔbpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase ΔcgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, ΔbpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas, USA

USDA-ARS?s Scientific Manuscript database

Various tissues, nasal swabs, urine, and blood samples were collected from 376 feral swine at two federally-inspected abattoirs in Texas during six separate sampling periods in 2015. Samples were tested for Brucella spp. by culture and serology. Brucella spp. were cultured from 13.0% of feral swin...

The Antibacterial Activity of Selected Labiatae (Lamiaceae) Essential Oils against Brucella melitensis.

PubMed

Al-Mariri, Ayman; Safi, Mazen

2013-03-01

Brucellosis, a zoonosis caused by four species of brucella, has a high morbidity. The major cause of brucellosis worldwide is brucella melitensis. Medicinal plants are considered as new antibacterial sources that could replace conventional antibiotics in the treatment of antibiotic-resistant bacteria. The aim of this study was to evaluate the efficacy of some native plants, alone and in combination with some antibiotics, in the treatment of brucellosis. The present experimental in vitro study was carried out to evaluate the anti-brucella activities of essential oils of Rosmarinus officinalis L., Origanum syriacum, Thymus syriacus, Salvia palaestina Benth, Mentha piperia, and Lavandula stoechas L., alone and in combination with some antibiotics. The activity against 16 tetracycline-resistant B. melitensis isolates was determined by disc diffusion method incorporating a concentration of 5%. Antibiotic discs were also used as a control. Microdilution brucella broth susceptibility assay was used in order to determine the MICs of essential oils and five antibiotics. Among all the herbs evaluated, only the essential oils of O. syriacum and T. syriacus plants demonstrated most effective anti-brucella activity, and were then chosen for MIC study. The minimal inhibitory concentrations (MIC50) of essential oils of O. syriacum and T. syriacus against tetracycline-resistant B. melitensis were 3.125 µl/ml and 6.25 µl/ml, respectively. Among the essential oils studied, those of O. syriacum and T. syriacus were most effective. Since a combination of levofloxacin and Thymus syriacus essential oil increased the efficacy of this antibiotic, O. syriacum and T. syriacus are recommended to be used as bactericidal agents against B. melitensis.

[Detection of a clonal complex with Brucella abortus biovar 2 genotype as founder in B. abortus isolates from Argentina].

PubMed

Hollender, Daiana; Conde, Sandra B; Salustio, Eduardo; Samartino, Luis E

2013-01-01

Brucella abortus is the causative agent of bovine brucellosis, a worldwide zoonosis. Up to date, eight biovars of B. abortus have been described. In Argentina, biovar 1 is the most frequently isolated. However, biovar 2, which is more pathogenic than biovar 1, is also found. Molecular methods for subtyping isolates are necessary for allowing epidemiological surveillance and control of eradication programs. Due to the genetic homogeneity of the genus Brucella, the development of molecular typing tools has been difficult. The publication of microorganism genomes facilitates the design of this approach. The aim of this work was to employ a Multiple Locus VNTR Analysis (MLVA) scheme for strains from Argentina isolated in our laboratory. From the 56 isolates analyzed, 47 different genotypic profiles were obtained. All the strains typed as biovar 2 showed the same profile. This scheme allowed assigning each isolate to the biovar it belongs to. All the genotypes were related using the goeBURST analysis and biovar 2 was proposed as founder. Copyright © 2013 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

Genotyping of Indian antigenic, vaccine, and field Brucella spp. using multilocus sequence typing.

PubMed

Shome, Rajeswari; Krithiga, Natesan; Shankaranarayana, Padmashree B; Jegadesan, Sankarasubramanian; Udayakumar S, Vishnu; Shome, Bibek Ranjan; Saikia, Girin Kumar; Sharma, Narendra Kumar; Chauhan, Harshad; Chandel, Bharat Singh; Jeyaprakash, Rajendhran; Rahman, Habibur

2016-03-31

Brucellosis is one of the most important zoonotic diseases that affects multiple livestock species and causes great economic losses. The highly conserved genomes of Brucella, with > 90% homology among species, makes it important to study the genetic diversity circulating in the country. A total of 26 Brucella spp. (4 reference strains and 22 field isolates) and 1 B. melitensis draft genome sequence from India (B. melitensis Bm IND1) were included for sequence typing. The field isolates were identified by biochemical tests and confirmed by both conventional and quantitative polymerase chain reaction (qPCR) targeting bcsp 31Brucella genus-specific marker. Brucella speciation and biotyping was done by Bruce ladder, probe qPCR, and AMOS PCRs, respectively, and genotyping was done by multilocus sequence typing (MLST). The MLST typing of 27 Brucella spp. revealed five distinct sequence types (STs); the B. abortus S99 reference strain and 21 B. abortus field isolates belonged to ST1. On the other hand, the vaccine strain B. abortus S19 was genotyped as ST5. Similarly, B. melitensis 16M reference strain and one B. melitensis field isolate were grouped into ST7. Another B. melitensis field isolate belonged to ST8 (draft genome sequence from India), and only B. suis 1330 reference strain was found to be ST14. The sequences revealed genetic similarity of the Indian strains to the global reference and field strains. The study highlights the usefulness of MLST for typing of field isolates and validation of reference strains used for diagnosis and vaccination against brucellosis.

Gene Discovery through Genomic Sequencing of Brucella abortus

PubMed Central

Sánchez, Daniel O.; Zandomeni, Ruben O.; Cravero, Silvio; Verdún, Ramiro E.; Pierrou, Ester; Faccio, Paula; Diaz, Gabriela; Lanzavecchia, Silvia; Agüero, Fernán; Frasch, Alberto C. C.; Andersson, Siv G. E.; Rossetti, Osvaldo L.; Grau, Oscar; Ugalde, Rodolfo A.

2001-01-01

Brucella abortus is the etiological agent of brucellosis, a disease that affects bovines and human. We generated DNA random sequences from the genome of B. abortus strain 2308 in order to characterize molecular targets that might be useful for developing immunological or chemotherapeutic strategies against this pathogen. The partial sequencing of 1,899 clones allowed the identification of 1,199 genomic sequence surveys (GSSs) with high homology (BLAST expect value < 10−5) to sequences deposited in the GenBank databases. Among them, 925 represent putative novel genes for the Brucella genus. Out of 925 nonredundant GSSs, 470 were classified in 15 categories based on cellular function. Seven hundred GSSs showed no significant database matches and remain available for further studies in order to identify their function. A high number of GSSs with homology to Agrobacterium tumefaciens and Rhizobium meliloti proteins were observed, thus confirming their close phylogenetic relationship. Among them, several GSSs showed high similarity with genes related to nodule nitrogen fixation, synthesis of nod factors, nodulation protein symbiotic plasmid, and nodule bacteroid differentiation. We have also identified several B. abortus homologs of virulence and pathogenesis genes from other pathogens, including a homolog to both the Shda gene from Salmonella enterica serovar Typhimurium and the AidA-1 gene from Escherichia coli. Other GSSs displayed significant homologies to genes encoding components of the type III and type IV secretion machineries, suggesting that Brucella might also have an active type III secretion machinery. PMID:11159979

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion

PubMed Central

Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant ΔrfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella Rough Mutant Induce Macrophage Death via Activating IRE1α Pathway of Endoplasmic Reticulum Stress by Enhanced T4SS Secretion.

PubMed

Li, Peng; Tian, Mingxing; Bao, Yanqing; Hu, Hai; Liu, Jiameng; Yin, Yi; Ding, Chan; Wang, Shaohui; Yu, Shengqing

2017-01-01

Brucella is a Gram-negative facultative intracellular pathogen that causes the worldwide zoonosis, known as brucellosis. Brucella virulence relies mostly on its ability to invade and replicate within phagocytic cells. The type IV secretion system (T4SS) and lipopolysaccharide are two major Brucella virulence factors. Brucella rough mutants reportedly induce the death of infected macrophages, which is T4SS dependent. However, the underlying molecular mechanism remains unclear. In this study, the T4SS secretion capacities of Brucella rough mutant and its smooth wild-type strain were comparatively investigated, by constructing the firefly luciferase fused T4SS effector, BPE123 and VceC. In addition, quantitative real-time PCR and western blotting were used to analyze the T4SS expression. The results showed that T4SS expression and secretion were enhanced significantly in the Brucella rough mutant. We also found that the activity of the T4SS virB operon promoter was notably increased in the Brucella rough mutant, which depends on quorum sensing-related regulators of VjbR upregulation. Cell infection and cell death assays revealed that deletion of vjbR in the Brucella rough mutant absolutely abolished cytotoxicity within macrophages by downregulating T4SS expression. This suggests that up-regulation of T4SS promoted by VjbR in rough mutant Δ rfbE contribute to macrophage death. In addition, we found that the Brucella rough mutant induce macrophage death via activating IRE1α pathway of endoplasmic reticulum stress. Taken together, our study provide evidence that in comparison to the Brucella smooth wild-type strain, VjbR upregulation in the Brucella rough mutant increases transcription of the virB operon, resulting in overexpression of the T4SS gene, accompanied by the over-secretion of effecter proteins, thereby causing the death of infected macrophages via activating IRE1α pathway of endoplasmic reticulum stress, suggesting novel insights into the molecular

Brucella neotomae Infection in Humans, Costa Rica.

PubMed

Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Jiménez-Rojas, César; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Víquez-Ruiz, Eunice; Rojas-Campos, Norman; Baker, Kate S; Oviedo-Sánchez, Gerardo; Amuy, Ernesto; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Guzmán-Verri, Caterina

2017-06-01

Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed.

Neospora caninum versus Brucella spp. exposure among dairy cattle in Ethiopia: a case control study.

PubMed

Asmare, Kassahun

2014-08-01

This case-control study aimed at assessing the relative association of Neospora caninum and Brucella species exposure with reproductive disorders. The study was carried out between October 2011 and June 2012 on 731 dairy cows sampled from 150 dairy farms in selected 17 conurbations of Ethiopia. Two hundred sixty-six of the cows were categorized as cases based on their history of abortion or stillbirth while the remaining 465 were controls. The presence of antibody to N. caninum was screened using indirect ELISA, while Brucella spp. exposure was assayed serially using Rose Bengal Plate Test and Complement Fixation Test. Exposure to N. caninum was more frequently observed among cases (23.8%) than controls (12.7%), while no significant difference (p > 0.05) was noted for Brucella exposure between the two groups. Moreover, the proportion of cows with disorders like retention of fetal membrane, endometritis and increased inter-calving period were significantly higher (p < 0.05) among Neospora seropositive cows. In conclusion, the finding discloses the strong association of N. caninum with reproductive disorders compared to Brucella spp. exposure. However, neither N. caninum nor Brucella spp. could explain the majority (73.2%) of the reported abortions and stillbirths in cattle. Hence, this observation underscores the need for more intensive investigation on the identification of causes of the aforementioned disorders in dairy cattle of Ethiopia.

Molecular strain typing of Brucella abortus isolates from Italy by two VNTR allele sizing technologies.

PubMed

De Santis, Riccardo; Ancora, Massimo; De Massis, Fabrizio; Ciammaruconi, Andrea; Zilli, Katiuscia; Di Giannatale, Elisabetta; Pittiglio, Valentina; Fillo, Silvia; Lista, Florigio

2013-10-01

Brucellosis, one of the most important re-emerging zoonoses in many countries, is caused by bacteria belonging to the genus Brucella. Furthermore these bacteria represent potential biological warfare agents and the identification of species and biovars of field strains may be crucial for tracing back source of infection, allowing to discriminate naturally occurring outbreaks instead of bioterrorist events. In the last years, multiple-locus variable-number tandem repeat analysis (MLVA) has been proposed as complement of the classical biotyping methods and it has been applied for genotyping large collections of Brucella spp. At present, the MLVA band profiles may be resolved by automated or manual procedures. The Lab on a chip technology represents a valid alternative to standard genotyping techniques (as agarose gel electrophoresis) and it has been previously used for Brucella genotyping. Recently, a new high-throughput genotyping analysis system based on capillary gel electrophoresis, the QIAxcel, has been described. The aim of the study was to evaluate the ability of two DNA sizing equipments, the QIAxcel System and the Lab chip GX, to correctly call alleles at the sixteen loci including one frequently used MLVA assay for Brucella genotyping. The results confirmed that these technologies represent a meaningful advancement in high-throughput Brucella genotyping. Considering the accuracy required to confidently resolve loci discrimination, QIAxcel shows a better ability to measure VNTR allele sizes compared to LabChip GX.

Brucella neotomae Infection in Humans, Costa Rica

PubMed Central

Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Jiménez-Rojas, César; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Víquez-Ruiz, Eunice; Rojas-Campos, Norman; Baker, Kate S.; Oviedo-Sánchez, Gerardo; Amuy, Ernesto; Chaves-Olarte, Esteban; Thomson, Nicholas R.; Moreno, Edgardo

2017-01-01

Several species of Brucella are known to be zoonotic, but B. neotomae infection has been thought to be limited to wood rats. In 2008 and 2011, however, B. neotomae was isolated from cerebrospinal fluid of 2 men with neurobrucellosis. The nonzoonotic status of B. neotomae should be reassessed. PMID:28518028

Promotion and Rescue of Intracellular Brucella neotomae Replication during Coinfection with Legionella pneumophila.

PubMed

Kang, Yoon-Suk; Kirby, James E

2017-05-01

We established a new Brucella neotomae in vitro model system for study of type IV secretion system-dependent (T4SS) pathogenesis in the Brucella genus. Importantly, B. neotomae is a rodent pathogen, and unlike B. abortus , B. melitensis , and B. suis , B. neotomae has not been observed to infect humans. It therefore can be handled more facilely using biosafety level 2 practices. More particularly, using a series of novel fluorescent protein and lux operon reporter systems to differentially label pathogens and track intracellular replication, we confirmed T4SS-dependent intracellular growth of B. neotomae in macrophage cell lines. Furthermore, B. neotomae exhibited early endosomal (LAMP-1) and late endoplasmic reticulum (calreticulin)-associated phagosome maturation. These findings recapitulate prior observations for human-pathogenic Brucella spp. In addition, during coinfection experiments with Legionella pneumophila , we found that defective intracellular replication of a B. neotomae T4SS virB4 mutant was rescued and baseline levels of intracellular replication of wild-type B. neotomae were significantly stimulated by coinfection with wild-type but not T4SS mutant L. pneumophila Using confocal microscopy, it was determined that intracellular colocalization of B. neotomae and L. pneumophila was required for rescue and that colocalization came at a cost to L. pneumophila fitness. These findings were not completely expected based on known temporal and qualitative differences in the intracellular life cycles of these two pathogens. Taken together, we have developed a new system for studying in vitro Brucella pathogenesis and found a remarkable T4SS-dependent interplay between Brucella and Legionella during macrophage coinfection. Copyright © 2017 American Society for Microbiology.

Development of a rapid recombinase polymerase amplification assay for detection of Brucella in blood samples.

PubMed

Ren, Hang; Yang, Mingjuan; Zhang, Guoxia; Liu, Shiwei; Wang, Xinhui; Ke, Yuehua; Du, Xinying; Wang, Zhoujia; Huang, Liuyu; Liu, Chao; Chen, Zeliang

2016-04-01

A rapid and sensitive recombinase polymerase amplification (RPA) assay, Bruce-RPA, was developed for detection of Brucella. The assay could detect as few as 3 copies of Brucella per reaction within 20 min. Bruce-RPA represents a candidate point-of-care diagnosis assay for human brucellosis. Copyright © 2016 Elsevier Ltd. All rights reserved.

Brucella β 1,2 Cyclic Glucan Is an Activator of Human and Mouse Dendritic Cells

PubMed Central

Martirosyan, Anna; Pérez-Gutierrez, Camino; Banchereau, Romain; Dutartre, Hélène; Lecine, Patrick; Dullaers, Melissa; Mello, Marielle; Pinto Salcedo, Suzana; Muller, Alexandre; Leserman, Lee; Levy, Yves; Zurawski, Gerard; Zurawski, Sandy; Moreno, Edgardo; Moriyón, Ignacio; Klechevsky, Eynav; Banchereau, Jacques; Oh, SangKon; Gorvel, Jean-Pierre

2012-01-01

Bacterial cyclic glucans are glucose polymers that concentrate within the periplasm of alpha-proteobacteria. These molecules are necessary to maintain the homeostasis of the cell envelope by contributing to the osmolarity of Gram negative bacteria. Here, we demonstrate that Brucella β 1,2 cyclic glucans are potent activators of human and mouse dendritic cells. Dendritic cells activation by Brucella β 1,2 cyclic glucans requires TLR4, MyD88 and TRIF, but not CD14. The Brucella cyclic glucans showed neither toxicity nor immunogenicity compared to LPS and triggered antigen-specific CD8+ T cell responses in vivo. These cyclic glucans also enhanced antigen-specific CD4+ and CD8+ T cell responses including cross-presentation by different human DC subsets. Brucella β 1,2 cyclic glucans increased the memory CD4+ T cell responses of blood mononuclear cells exposed to recombinant fusion proteins composed of anti-CD40 antibody and antigens from both hepatitis C virus and Mycobacterium tuberculosis. Thus cyclic glucans represent a new class of adjuvants, which might contribute to the development of effective antimicrobial therapies. PMID:23166489

Anti-Brucella Antibodies in Moose (Alces alces gigas), Muskoxen (Ovibos moschatus), and Plains Bison (Bison bison bison) in Alaska, USA.

PubMed

Nymo, Ingebjørg Helena; Beckmen, Kimberlee; Godfroid, Jacques

2016-01-01

We used an indirect enzyme-linked immunosorbent assay (iELISA) and the rose bengal test (RBT) to test for anti-Brucella antibodies in moose (Alces alces gigas), muskoxen (Ovibos moschatus), and plains bison (Bison bison bison) from various game management units (GMUs) in Alaska, US, sampled from 1982 to 2010. A portion of the sera had previously been tested with the standard plate test (SPT), the buffered Brucella antigen (BBA) card test, and the card test (CARD). No antibody-positive plains bison were identified. Anti-Brucella antibodies were detected in moose (iELISA, n=4/87; RBT, n=4/87; SPT, n=4/5; BBA, n=4/4) from GMU 23 captured in 1992, 1993, and 1995 and in muskoxen (iELISA, n=4/52; RBT, n=4/52; CARD, n=4/35) from GMUs 26A and 26B captured in 2004, 2006, and 2007. A negative effect of infection on the health of individuals of these species is probable. The presence of antibody-positive animals from 1992 to 2007 suggests presence of brucellae over time. The antibody-positive animals were found in northern Alaska, an area with a historically higher prevalence of Brucella-positive caribou, and a spillover of Brucella suis biovar 4 from caribou may have occurred. Brucella suis biovar 4 causes human brucellosis, and transmission from consumption of moose and muskoxen is possible.

Successful Management of Infected Facial Filler with Brucella.

PubMed

Alshaer, Zahra; Alsaadi, Yazeed; Mrad, Mohamed Amir

2018-06-11

The widespread desire to maintain youth and beauty with minimally invasive procedures made the use of soft tissue fillers an attractive option to correct numerous aesthetic problems. However, many complications have emerged recently especially with the use of non-FDA-approved permanent materials. In this case report, we are demonstrating the effective management of a patient with Brucella isolated from a facial abscess at the site of prior permanent filler injection done 17 years ago. A 56-year-old woman presented complaining of painful swelling of the right cheek after a failed trial of filler evacuation and intralesional corticosteroid injection. The patient was interviewed carefully, and physical examination was performed, followed by culture and imaging. The patient had a facial abscess that was complicated by parotid infiltration by Brucella. Eventually she was managed successfully by anti-Brucella antibiotics for 6 months with no further complaints. A review of causative organisms in the literature along with recommendations for management is discussed. Permanent fillers have shown many complications that can occur even years after injection. Therefore, physicians should be careful when using permanent fillers and should restrict their use to certain situations. Moreover, rare infections must be kept in mind and careful history, including travel history and animal contact, needs to be considered particularly in the unusual scenarios. This journal requires that authors assign a level of evidence to each article. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Brucella BioR regulator defines a complex regulatory mechanism for bacterial biotin metabolism.

PubMed

Feng, Youjun; Xu, Jie; Zhang, Huimin; Chen, Zeliang; Srinivas, Swaminath

2013-08-01

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of

Brucella BioR Regulator Defines a Complex Regulatory Mechanism for Bacterial Biotin Metabolism

PubMed Central

Xu, Jie; Zhang, Huimin; Srinivas, Swaminath

2013-01-01

The enzyme cofactor biotin (vitamin H or B7) is an energetically expensive molecule whose de novo biosynthesis requires 20 ATP equivalents. It seems quite likely that diverse mechanisms have evolved to tightly regulate its biosynthesis. Unlike the model regulator BirA, a bifunctional biotin protein ligase with the capability of repressing the biotin biosynthetic pathway, BioR has been recently reported by us as an alternative machinery and a new type of GntR family transcriptional factor that can repress the expression of the bioBFDAZ operon in the plant pathogen Agrobacterium tumefaciens. However, quite unusually, a closely related human pathogen, Brucella melitensis, has four putative BioR-binding sites (both bioR and bioY possess one site in the promoter region, whereas the bioBFDAZ [bio] operon contains two tandem BioR boxes). This raised the question of whether BioR mediates the complex regulatory network of biotin metabolism. Here, we report that this is the case. The B. melitensis BioR ortholog was overexpressed and purified to homogeneity, and its solution structure was found to be dimeric. Functional complementation in a bioR isogenic mutant of A. tumefaciens elucidated that Brucella BioR is a functional repressor. Electrophoretic mobility shift assays demonstrated that the four predicted BioR sites of Brucella plus the BioR site of A. tumefaciens can all interact with the Brucella BioR protein. In a reporter strain that we developed on the basis of a double mutant of A. tumefaciens (the ΔbioR ΔbioBFDA mutant), the β-galactosidase (β-Gal) activity of three plasmid-borne transcriptional fusions (bioBbme-lacZ, bioYbme-lacZ, and bioRbme-lacZ) was dramatically decreased upon overexpression of Brucella bioR. Real-time quantitative PCR analyses showed that the expression of bioBFDA and bioY is significantly elevated upon removal of bioR from B. melitensis. Together, we conclude that Brucella BioR is not only a negative autoregulator but also a repressor of

Modeling the survivability of brucella to exposure of Ultraviolet radiation and temperature

NASA Astrophysics Data System (ADS)

Howe, R.

Accumulated summation of daily Ultra Violet-B (UV-B = 290? to 320 ? ) data? from The USDA Ultraviolet Radiation Monitoring Program show good correlation (R^2 = 77%) with daily temperature data during the five month period from February through June, 1998. Exposure of disease organisms, such as brucella to the effects of accumulated UV-B radiation, can be modeled for a 5 month period from February through June, 1998. Estimates of a lethal dosage for brucell of UV-B in the environment is dependent on minimum/maximum temperature and Solar Zenith Angle for the time period. The accumulated increase in temperature over this period also effects the decomposition of an aborted fetus containing brucella. Decomposition begins at some minimum daily temperature at 27 to 30 degrees C and peaks at 39 to 40C. It is useful to view the summation of temperature as a threshold for other bacteria growth, so that accumulated temperature greater than some value causes decomposition through competition with other bacteria and brucella die from the accumulated effects of UV-B, temperature and organism competition. Results of a study (Cook 1998) to determine survivability of brucellosis in the environment through exposure of aborted bovine fetuses show no one cause can be attributed to death of the disease agent. The combination of daily increase in temperature and accumulated UV-B radiation reveal an inverse correlation to survivability data and can be modeled as an indicator of brucella survivability in the environment in arid regions.

Seroprevalence of Brucella antibodies in camels in Katsina State, Nigeria.

PubMed

Salisu, U S; Kudi, C A; Bale, J O O; Babashani, M; Kaltungo, B Y; Saidu, S N A; Asambe, A; Baba, A Y

2017-06-01

A cross-sectional study was carried out to determine the status of Brucella infection in one-humped (Dromedary) camels in the North and Central senatorial districts of Katsina State, Nigeria. Nine hundred and eighty serum samples from live and slaughtered camels were tested. Modified Rose Bengal plate test (RBPT) and serum agglutination test (SAT) with ethylenediaminetetraacetic acid, (EDTA) were used as screening and standard tests, respectively. The prevalence of Brucella antibodies were 110 (11.2%) and 103 (10.5%) for RBPT and SAT, respectively. Of the 472 and 508 serum samples tested from the herds and abattoirs, respectively, 63 (13.3%) and 47 (9.3%) were positive by RBPT while 62 (13.1%) and 41 (8.1%) were positive by SAT, respectively. Based on the results, it was concluded that Brucella antibodies were present in camels in the study area. Poor management practices and mixing of camels with other species of livestock as well as unrestricted movement of camels were proposed to be the reasons for the prevalence of the disease in the study area. In view of the public health importance of the disease, it is recommended that there is the need to develop a strategic plan to decrease spread of brucellosis in the study area.

Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis.

PubMed

De Santis, Riccardo; Ciammaruconi, Andrea; Faggioni, Giovanni; D'Amelio, Raffaele; Marianelli, Cinzia; Lista, Florigio

2009-04-07

Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.

Identification of Brucella ovis exclusive genes in field isolates from Argentina.

PubMed

Alvarez, Lucía Paula; García-Effrón, Guillermo; Robles, Carlos Alejandro

2016-03-01

Brucellosis caused by Brucella ovis is one of the most important infectious diseases of sheep. The aim of this study was to determine the presence of genes both inside and outside the specific B. ovis pathogenicity island 1 (BOPI-1) in a large collection of field isolates of B. ovis and other Brucella spp. from Argentina. The BOV_A0500 gene from B. ovis BOPI-1 was identified in all 104 B. ovis isolates studied. The BOPI-1 complete sequence was found to be conserved in 10 B. ovis strains from the collection, for which whole genome sequencing was performed. The BOV_0198 gene, which is outside BOPI-1 and considered exclusive to B. ovis, showed 90-100% identity with genomic regions of B. ovis, B. melitensis, B. abortus, B. canis, B. suis, B. microti, B. ceti and B. pinnipedialis. The results demonstrate that BOPI-1 is the only exclusive genetic region of B. ovis and marine Brucella spp. and that it is highly conserved in B. ovis field isolates from Argentina. Copyright © 2016 Elsevier Ltd. All rights reserved.

Structural Insights into the HWE Histidine Kinase Family: The Brucella Blue Light-Activated Histidine Kinase Domain.

PubMed

Rinaldi, Jimena; Arrar, Mehrnoosh; Sycz, Gabriela; Cerutti, María Laura; Berguer, Paula M; Paris, Gastón; Estrín, Darío Ariel; Martí, Marcelo Adrián; Klinke, Sebastián; Goldbaum, Fernando Alberto

2016-03-27

In response to light, as part of a two-component system, the Brucella blue light-activated histidine kinase (LOV-HK) increases its autophosphorylation, modulating the virulence of this microorganism. The Brucella histidine kinase (HK) domain belongs to the HWE family, for which there is no structural information. The HWE family is exclusively present in proteobacteria and usually coupled to a wide diversity of light sensor domains. This work reports the crystal structure of the Brucella HK domain, which presents two different dimeric assemblies in the asymmetric unit: one similar to the already described canonical parallel homodimers (C) and the other, an antiparallel non-canonical (NC) dimer, each with distinct relative subdomain orientations and dimerization interfaces. Contrary to these crystallographic structures and unlike other HKs, in solution, the Brucella HK domain is monomeric and still active, showing an astonishing instability of the dimeric interface. Despite this instability, using cross-linking experiments, we show that the C dimer is the functionally relevant species. Mutational analysis demonstrates that the autophosphorylation activity occurs in cis. The different relative subdomain orientations observed for the NC and C states highlight the large conformational flexibility of the HK domain. Through the analysis of these alternative conformations by means of molecular dynamics simulations, we also propose a catalytic mechanism for Brucella LOV-HK. Copyright © 2016 Elsevier Ltd. All rights reserved.

Public health consequences of a false-positive laboratory test result for Brucella--Florida, Georgia, and Michigan, 2005.

PubMed

2008-06-06

Human brucellosis, a nationally notifiable disease, is uncommon in the United States. Most human cases have occurred in returned travelers or immigrants from regions where brucellosis is endemic, or were acquired domestically from eating illegally imported, unpasteurized fresh cheeses. In January 2005, a woman aged 35 years who lived in Nassau County, Florida, received a diagnosis of brucellosis, based on results of a Brucella immunoglobulin M (IgM) enzyme immunoassay (EIA) performed in a commercial laboratory using analyte specific reagents (ASRs); this diagnosis prompted an investigation of dairy products in two other states. Subsequent confirmatory antibody testing by Brucella microagglutination test (BMAT) performed at CDC on the patient's serum was negative. The case did not meet the CDC/Council of State and Territorial Epidemiologists' (CSTE) definition for a probable or confirmed brucellosis case, and the initial EIA result was determined to be a false positive. This report summarizes the case history, laboratory findings, and public health investigations. CDC recommends that Brucella serology testing only be performed using tests cleared or approved by the Food and Drug Administration (FDA) or validated under the Clinical Laboratory Improvement Amendments (CLIA) and shown to reliably detect the presence of Brucella infection. Results from these tests should be considered supportive evidence for recent infection only and interpreted in the context of a clinically compatible illness and exposure history. EIA is not considered a confirmatory Brucella antibody test; positive screening test results should be confirmed by Brucella-specific agglutination (i.e., BMAT or standard tube agglutination test) methods.

Identification of lptA, lpxE, and lpxO, Three Genes Involved in the Remodeling of Brucella Cell Envelope.

PubMed

Conde-Álvarez, Raquel; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Salvador-Bescós, Miriam; Bárcena-Varela, Marina; Aragón-Aranda, Beatriz; Martínez-Gómez, Estrella; Zúñiga-Ripa, Amaia; de Miguel, María J; Bartholomew, Toby Leigh; Hanniffy, Sean; Grilló, María-Jesús; Vences-Guzmán, Miguel Ángel; Bengoechea, José A; Arce-Gorvel, Vilma; Gorvel, Jean-Pierre; Moriyón, Ignacio; Iriarte, Maite

2017-01-01

The brucellae are facultative intracellular bacteria that cause a worldwide extended zoonosis. One of the pathogenicity mechanisms of these bacteria is their ability to avoid rapid recognition by innate immunity because of a reduction of the pathogen-associated molecular pattern (PAMP) of the lipopolysaccharide (LPS), free-lipids, and other envelope molecules. We investigated the Brucella homologs of lptA, lpxE , and lpxO , three genes that in some pathogens encode enzymes that mask the LPS PAMP by upsetting the core-lipid A charge/hydrophobic balance. Brucella lptA , which encodes a putative ethanolamine transferase, carries a frame-shift in B. abortus but not in other Brucella spp. and phylogenetic neighbors like the opportunistic pathogen Ochrobactrum anthropi. Consistent with the genomic evidence, a B. melitensis lptA mutant lacked lipid A-linked ethanolamine and displayed increased sensitivity to polymyxin B (a surrogate of innate immunity bactericidal peptides), while B. abortus carrying B. melitensis lptA displayed increased resistance. Brucella lpxE encodes a putative phosphatase acting on lipid A or on a free-lipid that is highly conserved in all brucellae and O. anthropi. Although we found no evidence of lipid A dephosphorylation, a B. abortus lpxE mutant showed increased polymyxin B sensitivity, suggesting the existence of a hitherto unidentified free-lipid involved in bactericidal peptide resistance. Gene lpxO putatively encoding an acyl hydroxylase carries a frame-shift in all brucellae except B. microti and is intact in O. anthropi . Free-lipid analysis revealed that lpxO corresponded to olsC , the gene coding for the ornithine lipid (OL) acyl hydroxylase active in O. anthropi and B. microti , while B. abortus carrying the olsC of O. anthropi and B. microti synthesized hydroxylated OLs. Interestingly, mutants in lptA, lpxE , or olsC were not attenuated in dendritic cells or mice. This lack of an obvious effect on virulence together with the presence

An outbreak of Brucella melitensis infection by airborne transmission among laboratory workers.

PubMed Central

Ollé-Goig, J E; Canela-Soler, J

1987-01-01

An outbreak of acute brucellosis infection was detected among the employees of a biologicals manufacturing laboratory located in Girona, Spain. The first cases appeared six weeks after a vaccine with attenuated Brucella melitensis, Rev-1 had been produced for one week. A clinical and epidemiologic investigation conducted among the 164 employees found 22 patients with clinical symptoms and positive serology, and six patients detected by serology only (attack rate: 17.1 per cent). Blood cultures were obtained from two patients and Brucella melitensis was isolated. Employees working in areas with open windows above the laboratory air extracting system had an attack rate of 39.5 per cent, substantially higher than those working in other locations. When vaccine was manufactured again, an electric oven reaching 300 degrees C had been installed in the air extracting system just before its exit to the exterior. Appropriate culture medium plates were exposed to the laboratory air before and after passing through the oven. Brucellae were isolated from the plates exposed to the air before passing through the oven but not after doing so. PMID:3812841

ISOLATION AND IDENTIFICATION OF BRUCELLA SUIS IN PIGS AS ZOONOTIC DISEASE IN ENDEMIC AREAS OF EAST JAVA, INDONESIA.

PubMed

S, Emy Koestanti; Misaco, Wiwik; Chusniati, Sri; Maslachah, Lilik

2018-01-01

Brucellosis in pigs at East Java Indonesia has not only cause great economic losses due to a decrease in productivity of livestock but also are zoonotic. Infection on free brucelosis pigs were initially begun with the infected pigs both male and female, or the use of superior male pigs together. The elimination of the disease either on a group or population is considered as the most effective way to prevent the spread of the disease in pigs. Prevention efforts mainly addressed to vaccination, sanitary maintenace and government policy. The purpose of this study was to isolated and identified Brucella suis as the causative agent. The survey area were the pig farm owned by breeder farmers in the area of East Java Indonesia, at Kediri, Malang, Blitar and Probolinggo district. Blood samples obtained were tested with RBT. Pigs are suspected of being infected with Brucella if the RBT was positive that characterized with agglutination in the test results. If RBT was positive, bacteriological examination will be performed, with samples of visceral foetus organ, ie liver, spleen, placenta and amniotic fluid. Isolation and identification of Brucella suis were used Brucella Broth and Brucella Agar, and if the bacteri growthwill be continued with biochemical test ie H2S, urease, citrate, catalase and oxidase test. The positive results of Brucella suis showed positive urease, catalase andoxidase, but negative for citrate and H2S. RBT and bacteriolgical examination showed that 1 sample was positive Brucella suis , and 19 negative. The positive results showed positive urease, catalase and oxidase, but negative for citrate and H2S. Based on RBT test and bacteriological examination, there was 1 positive sample of brucellla suis, that is sample coming from Kediri district.

ISOLATION AND IDENTIFICATION OF BRUCELLA SUIS IN PIGS AS ZOONOTIC DISEASE IN ENDEMIC AREAS OF EAST JAVA, INDONESIA

PubMed Central

S, Emy Koestanti; Misaco, Wiwik; Chusniati, Sri; Maslachah, Lilik

2018-01-01

Background: Brucellosis in pigs at East Java Indonesia has not only cause great economic losses due to a decrease in productivity of livestock but also are zoonotic. Infection on free brucelosis pigs were initially begun with the infected pigs both male and female, or the use of superior male pigs together. The elimination of the disease either on a group or population is considered as the most effective way to prevent the spread of the disease in pigs. Prevention efforts mainly addressed to vaccination, sanitary maintenace and government policy. The purpose of this study was to isolated and identified Brucella suis as the causative agent. Material and Methods: The survey area were the pig farm owned by breeder farmers in the area of East Java Indonesia, at Kediri, Malang, Blitar and Probolinggo district. Blood samples obtained were tested with RBT. Pigs are suspected of being infected with Brucella if the RBT was positive that characterized with agglutination in the test results. If RBT was positive, bacteriological examination will be performed, with samples of visceral foetus organ, ie liver, spleen, placenta and amniotic fluid. Isolation and identification of Brucella suis were used Brucella Broth and Brucella Agar, and if the bacteri growthwill be continued with biochemical test ie H2S, urease, citrate, catalase and oxidase test. The positive results of Brucella suis showed positive urease, catalase andoxidase, but negative for citrate and H2S. Results: RBT and bacteriolgical examination showed that 1 sample was positive Brucella suis, and 19 negative. The positive results showed positive urease, catalase and oxidase, but negative for citrate and H2S. Conclusion: Based on RBT test and bacteriological examination, there was 1 positive sample of brucellla suis, that is sample coming from Kediri district. PMID:29619446

A Rapid Detection Method of Brucella with Quantum Dots and Magnetic Beads Conjugated with Different Polyclonal Antibodies

NASA Astrophysics Data System (ADS)

Song, Dandan; Qu, Xiaofeng; Liu, Yushen; Li, Li; Yin, Dehui; Li, Juan; Xu, Kun; Xie, Renguo; Zhai, Yue; Zhang, Huiwen; Bao, Hao; Zhao, Chao; Wang, Juan; Song, Xiuling; Song, Wenzhi

2017-03-01

Brucella spp. are facultative intracellular bacteria that cause zoonotic disease of brucellosis worldwide. Traditional methods for detection of Brucella spp. take 48-72 h that does not meet the need of rapid detection. Herein, a new rapid detection method of Brucella was developed based on polyclonal antibody-conjugating quantum dots and antibody-modified magnetic beads. First, polyclonal antibodies IgG and IgY were prepared and then the antibody conjugated with quantum dots (QDs) and immunomagnetic beads (IMB), respectively, which were activated by N-(3-dimethylaminopropyl)- N'-ethylcar-bodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) to form probes. We used the IMB probe to separate the Brucella and labeled by the QD probe, and then detected the fluorescence intensity with a fluorescence spectrometer. The detection method takes 105 min with a limit of detection of 103 CFU/mL and ranges from 10 to 105 CFU/mL ( R 2 = 0.9983), and it can be well used in real samples.

Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution

PubMed Central

Suárez-Esquivel, Marcela; Baker, Kate S.; Ruiz-Villalobos, Nazareth; Hernández-Mora, Gabriela; Barquero-Calvo, Elías; González-Barrientos, Rocío; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Chacón-Díaz, Carlos; Cloeckaert, Axel; Chaves-Olarte, Esteban; Thomson, Nicholas R.; Moreno, Edgardo

2017-01-01

Abstract Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97–99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication. PMID:28854602

Development of a bead-based Luminex assay using lipopolysaccharide specific monoclonal antibodies to detect biological threats from Brucella species.

PubMed

Silbereisen, Angelika; Tamborrini, Marco; Wittwer, Matthias; Schürch, Nadia; Pluschke, Gerd

2015-10-05

Brucella, a Gram-negative bacterium, is classified as a potential bioterrorism agent mainly due to the low dose needed to cause infection and the ability to transmit the bacteria via aerosols. Goats/sheep, cattle, pigs, dogs, sheep and rodents are infected by B. melitensis, B. abortus, B. suis, B. canis, B. ovis and B. neotomae, respectively, the six classical Brucella species. Most human cases are caused by B. melitensis and B. abortus. Our aim was to specifically detect Brucellae with 'smooth' lipopolysaccharide (LPS) using a highly sensitive monoclonal antibody (mAb) based immunological assay. To complement molecular detection systems for potential bioterror agents, as required by international biodefense regulations, sets of mAbs were generated by B cell hybridoma technology and used to develop immunological assays. The combination of mAbs most suitable for an antigen capture assay format was identified and an immunoassay using the Luminex xMAP technology was developed. MAbs specific for the LPS O-antigen of Brucella spp. were generated by immunising mice with inactivated B. melitensis or B. abortus cells. Most mAbs recognised both B. melitensis and B. abortus and antigen binding was not impeded by inactivation of the bacterial cells by γ irradiation, formalin or heat treatment, a step required to analyse the samples immunologically under biosafety level two conditions. The Luminex assay recognised all tested Brucella species with 'smooth' LPS with detection limits of 2×10(2) to 8×10(4) cells per mL, depending on the species tested. Milk samples spiked with Brucella spp. cells were identified successfully using the Luminex assay. In addition, the bead-based immunoassay was integrated into a multiplex format, allowing for simultaneous, rapid and specific detection of Brucella spp., Bacillus anthracis, Francisella tularensis and Yersinia pestis within a single sample. Overall, the robust Luminex assay should allow detection of Brucella spp. in both natural

9 CFR 113.32 - Detection of Brucella contamination.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 9 Animals and Animal Products 1 2010-01-01 2010-01-01 false Detection of Brucella contamination. 113.32 Section 113.32 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... applicable Standard Requirement or in a filed Outline of Production for the product. (a) One ml of the minced...

Design and implementation of a database for Brucella melitensis genome annotation.

PubMed

De Hertogh, Benoît; Lahlimi, Leïla; Lambert, Christophe; Letesson, Jean-Jacques; Depiereux, Eric

2008-03-18

The genome sequences of three Brucella biovars and of some species close to Brucella sp. have become available, leading to new relationship analysis. Moreover, the automatic genome annotation of the pathogenic bacteria Brucella melitensis has been manually corrected by a consortium of experts, leading to 899 modifications of start sites predictions among the 3198 open reading frames (ORFs) examined. This new annotation, coupled with the results of automatic annotation tools of the complete genome sequences of the B. melitensis genome (including BLASTs to 9 genomes close to Brucella), provides numerous data sets related to predicted functions, biochemical properties and phylogenic comparisons. To made these results available, alphaPAGe, a functional auto-updatable database of the corrected sequence genome of B. melitensis, has been built, using the entity-relationship (ER) approach and a multi-purpose database structure. A friendly graphical user interface has been designed, and users can carry out different kinds of information by three levels of queries: (1) the basic search use the classical keywords or sequence identifiers; (2) the original advanced search engine allows to combine (by using logical operators) numerous criteria: (a) keywords (textual comparison) related to the pCDS's function, family domains and cellular localization; (b) physico-chemical characteristics (numerical comparison) such as isoelectric point or molecular weight and structural criteria such as the nucleic length or the number of transmembrane helix (TMH); (c) similarity scores with Escherichia coli and 10 species phylogenetically close to B. melitensis; (3) complex queries can be performed by using a SQL field, which allows all queries respecting the database's structure. The database is publicly available through a Web server at the following url: http://www.fundp.ac.be/urbm/bioinfo/aPAGe.

Serological activity of white-tail deer against several species of Brucella.

PubMed

Salinas-Meléndez, J A; Martínez-Muñoz, A; Avalos-Ramírez, R; Cerutti-Pereyra, N; Riojas-Valdés, V M

1998-01-01

In Mexico, brucellosis is a widely distributed disease of domesticated ruminants, but its frequency in wild ruminants has not been documented. Since northeast Mexico is the main distribution area of white-tailed deer and has been reported as an area positive for brucellosis in domesticated species, the present study was conducted in order to determine serological activity against several species of the genus Brucella in white-tailed deer. A total of 208 sera of white-tailed deer were collected during the springs of 1994 and 1995 in the north part of the states of Nuevo León and Coahuila. Each serum was analyzed for the detection of antibodies against two smooth (B. abortus and B. melitensis) and one rough (B. ovis) species of the genus Brucella. The serological tests used for the determination of the presence of antibodies against Brucella were card and plate agglutination for B. abortus, plate agglutination and rivanol precipitation for B. melitensis, and agar gel immunodiffusion for B. ovis. Each assay had positive and negative controls. None of the analyzed samples was found to be positive, and only two sera showed partial plate agglutination against B. melitensis at a dilution of 1:25; however, at higher dilutions and to the rivanol precipitation test the same samples were negative. Therefore, the percentage of positive sera was estimated at 0% (0/208). This result makes evident the absence of positive white-tailed deer against Brucella in the sampled area, despite that this disease is considered present in domesticated species. Therefore, white-tailed deer does not have, at the present time, an important role for the dispersion of the disease. The same result has been reported in other countries.

First isolation and characterization of Brucella microti from wild boar.

PubMed

Rónai, Zsuzsanna; Kreizinger, Zsuzsa; Dán, Ádám; Drees, Kevin; Foster, Jeffrey T; Bányai, Krisztián; Marton, Szilvia; Szeredi, Levente; Jánosi, Szilárd; Gyuranecz, Miklós

2015-07-11

Brucella microti was first isolated from common vole (Microtus arvalis) in the Czech Republic in Central Europe in 2007. As B. microti is the only Brucella species known to live in soil, its distribution, ecology, zoonotic potential, and genomic organization is of particular interest. The present paper is the first to report the isolation of B. microti from a wild boar (Sus scrofa), which is also the first isolation of this bacterial species in Hungary. The B. microti isolate was cultured, after enrichment in Brucella-selective broth, from the submandibular lymph node of a female wild boar that was taken by hunters in Hungary near the Austrian border in September 2014. Histological and immunohistological examinations of the lymph node sections with B. abortus-, B. suis- and B. canis-specific sera gave negative results. The isolate did not require CO2 for growth, was oxidase, catalase, and urease positive, H2S negative, grew well in the presence of 20 μg/ml basic fuchsin and thionin, and had brownish pigmentation after three days of incubation. It gave strong positive agglutination with anti-A and anti-M but had a negative reaction with anti-R monospecific sera. The API 20 NE test identified it as Ochrobactrum anthropi with 99.9% identity, and it showed B. microti-specific banding pattern in the Bruce- and Suis-ladder multiplex PCR systems. Whole genome re-sequencing identified 30 SNPs in orthologous loci when compared to the B. microti reference genome available in GenBank, and the MLVA analysis yielded a unique profile. Given that the female wild boar did not develop any clinical disease, we hypothesize that this host species only harboured the bacterium, serving as a possible reservoir capable of maintaining and spreading this pathogen. The infectious source could have been either a rodent, a carcass that had been eaten or infection occurred via the boar rooting in soil. The low number of discovered SNPs suggests an unexpectedly high level of genetic homogeneity

9 CFR 113.32 - Detection of Brucella contamination.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 1 2013-01-01 2013-01-01 false Detection of Brucella contamination. 113.32 Section 113.32 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of...

9 CFR 113.32 - Detection of Brucella contamination.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 1 2014-01-01 2014-01-01 false Detection of Brucella contamination. 113.32 Section 113.32 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of...

9 CFR 113.32 - Detection of Brucella contamination.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 1 2012-01-01 2012-01-01 false Detection of Brucella contamination. 113.32 Section 113.32 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of...

9 CFR 113.32 - Detection of Brucella contamination.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 1 2011-01-01 2011-01-01 false Detection of Brucella contamination. 113.32 Section 113.32 Animals and Animal Products ANIMAL AND PLANT HEALTH INSPECTION SERVICE... tissue used as the source of cells or 1 ml of the extract of the tissue prior to the addition of...

The bovine immune response to Brucella abortus. III. Preparation of antisera against a Brucella component precipitated by sera of some infected cattle.

PubMed Central

Stemshorn, B; Nielsen, K; Samagh, B

1981-01-01

Two methods are described for the partial purification of a high molecular weight, heat-resistant component (CO1) of sonicates of smooth and rough Brucella abortus which is precipitated by sera of some infected cattle. Method 1, a combination of gel filtration chromatography and polyacrylamide gel electrophoresis, was used to prepare CO1 from sonicates of a smooth field strain of B. abortus. Method 2, a combination of gel filtration chromatography and heat treatment, was used to obtain CO1, from sonicates of rough B. abortus strain 45/20. Rabbit antisera produced against CO1 prepared by either method contained only CO1 precipitins but were negative in standard agglutination and complement fixation tests conducted with whole cell antigens. Evidence is presented that CO1 is identical to Brucella antigen A2, and it is proposed that in future the designation A2 be employed. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:6791797

[Determination of in vitro susceptibilities of Brucella spp. strains against 11 different antibacterial gents isolated from blood cultures].

PubMed

Keşli, Recep; Bilgin, Hüseyin; Yılmaz, Halim

2017-07-01

Brucellosis is a worldwide zoonotic disease and still continuous to be a major public health problem. In this study, it was aimed to identify the Brucella strains to the species level isolated from blood cultures, and to determine the rate of antimicrobial susceptibility against eleven antibacterial agents. A total of 106 Brucella spp. strains were included in the study, which were isolated from blood cultures in University of Health Sciences, Konya Training and Research Hospital, Medical Microbiology Laboratory between January 2011 and June 2013. Identification of the isolated strains were mainly based on conventional methods. In vitro antibacterial susceptibilities of azithromycin, ciprofloxacin, doxycycline, gentamicin, levofloxacin, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline, and trimethoprim/sulfamethoxazole, were evaluated by using the gradient (E-test, bioMerieux, France) strip method. The bacterial suspensions adjusted to 0.5 McFarland turbidity was inoculated to Mueller Hinton agar plates, supplemented with 5% sheep blood, and E-test strips of selected antibacterial were applied. The plates were incubated in ambient air 48 hours at 37ºC and Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 29213 were used as quality control strains for antimicrobial susceptibility testing. Minimum inhibitors concentration (MIC) values were interpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines for slow-growing bacteria such as Haemophilus spp. Of the 106 Brucella spp. strains included in to the study, 90 were identified as Brucella melitensis, and 16 were Brucella abortus. MIC90 values of azithromycin, ciprofloxacin, doxycycline, gentamicin, levofloxacin, moxifloxacin, rifampicin, streptomycin, tetracycline, tigecycline, and trimethoprim/sulfamethoxazole were determined as 1 µg/ml, 0.25 µg/ml, 0.19 µg/ml, 0.25 µg/ml, 0.19 µg/ml, 0.75 µg/ml, 0.25 µg/ml, 0.75 µg/ml, 0.38 µg/ml, 0.64 µg/ml, and 0

Computational prediction of secretion systems and secretomes of Brucella: identification of novel type IV effectors and their interaction with the host.

PubMed

Sankarasubramanian, Jagadesan; Vishnu, Udayakumar S; Dinakaran, Vasudevan; Sridhar, Jayavel; Gunasekaran, Paramasamy; Rajendhran, Jeyaprakash

2016-01-01

Brucella spp. are facultative intracellular pathogens that cause brucellosis in various mammals including humans. Brucella survive inside the host cells by forming vacuoles and subverting host defence systems. This study was aimed to predict the secretion systems and the secretomes of Brucella spp. from 39 complete genome sequences available in the databases. Furthermore, an attempt was made to identify the type IV secretion effectors and their interactions with host proteins. We predicted the secretion systems of Brucella by the KEGG pathway and SecReT4. Brucella secretomes and type IV effectors (T4SEs) were predicted through genome-wide screening using JVirGel and S4TE, respectively. Protein-protein interactions of Brucella T4SEs with their hosts were analyzed by HPIDB 2.0. Genes coding for Sec and Tat pathways of secretion and type I (T1SS), type IV (T4SS) and type V (T5SS) secretion systems were identified and they are conserved in all the species of Brucella. In addition to the well-known VirB operon coding for the type IV secretion system (T4SS), we have identified the presence of additional genes showing homology with T4SS of other organisms. On the whole, 10.26 to 14.94% of total proteomes were found to be either secreted (secretome) or membrane associated (membrane proteome). Approximately, 1.7 to 3.0% of total proteomes were identified as type IV secretion effectors (T4SEs). Prediction of protein-protein interactions showed 29 and 36 host-pathogen specific interactions between Bos taurus (cattle)-B. abortus and Ovis aries (sheep)-B. melitensis, respectively. Functional characterization of the predicted T4SEs and their interactions with their respective hosts may reveal the secrets of host specificity of Brucella.

Evaluation of serological tests for diagnosis of Brucella melitensis infection of goats.

PubMed Central

Díaz-Aparicio, E; Marín, C; Alonso-Urmeneta, B; Aragón, V; Pérez-Ortiz, S; Pardo, M; Blasco, J M; Díaz, R; Moriyón, I

1994-01-01

Five serological assays were evaluated for the diagnosis of brucellosis in goats: the rose bengal test (RBT), complement fixation test (CFT), radial immunodiffusion (RID) with Brucella and Yersinia enterocolitica O:9 polysaccharides, counterimmunoelectrophoresis (CIEP) with cytosol, and enzyme-linked immunosorbent assay (ELISA) with polyclonal and protein G conjugates and smooth lipopolysaccharide (S-LPS), native hapten polysaccharide (NH), or cytosol antigens. For optimal sensitivity, RBT had to be used with sera-antigen at a 3:1 dilution. In the RID test, Brucella melitensis biotype 1 NH could not be replaced by Brucella abortus biotype 1 or Y. enterocolitica 0:9 polysaccharides. In the ELISA, S-LPS and NH gave similar results and the protein G conjugate increased the specificity. With the sera from 55 B. melitensis culture-positive goats, the sensitivity was 100% for RBT, CFT (titer > or = 4), and ELISA with S-LPS or NH; 94% for RID; and 93% for CIEP. All tests were negative (100% specific) when testing the sera from 127 brucella-free goats. Larger discrepancies among the results of the serological tests were obtained with sera from goats of areas where brucellosis is endemic. When the sera of 20 young goats vaccinated subcutaneously (10(9) CFU of B. melitensis Rev 1) and bled 6 months later were examined, the specificities were as follows: NH ELISA, 60%; CFT and S-LPS ELISA, 75%; RBT, 80%; CIEP, 90%; and RID, 94%. With the sera from 10 young goats vaccinated conjunctivally (10(9) CFU of B. melitensis Rev 1) all tests were 100% specific 4 months after vaccination. The proportion of goats giving a positive reaction after vaccination decreased faster in RID than in other tests. PMID:8051240

Brucella Genetic Variability in Wildlife Marine Mammals Populations Relates to Host Preference and Ocean Distribution.

PubMed

Suárez-Esquivel, Marcela; Baker, Kate S; Ruiz-Villalobos, Nazareth; Hernández-Mora, Gabriela; Barquero-Calvo, Elías; González-Barrientos, Rocío; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Chacón-Díaz, Carlos; Cloeckaert, Axel; Chaves-Olarte, Esteban; Thomson, Nicholas R; Moreno, Edgardo; Guzmán-Verri, Caterina

2017-07-01

Intracellular bacterial pathogens probably arose when their ancestor adapted from a free-living environment to an intracellular one, leading to clonal bacteria with smaller genomes and less sources of genetic plasticity. Still, this plasticity is needed to respond to the challenges posed by the host. Members of the Brucella genus are facultative-extracellular intracellular bacteria responsible for causing brucellosis in a variety of mammals. The various species keep different host preferences, virulence, and zoonotic potential despite having 97-99% similarity at genome level. Here, we describe elements of genetic variation in Brucella ceti isolated from wildlife dolphins inhabiting the Pacific Ocean and the Mediterranean Sea. Comparison with isolates obtained from marine mammals from the Atlantic Ocean and the broader Brucella genus showed distinctive traits according to oceanic distribution and preferred host. Marine mammal isolates display genetic variability, represented by an important number of IS711 elements as well as specific IS711 and SNPs genomic distribution clustering patterns. Extensive pseudogenization was found among isolates from marine mammals as compared with terrestrial ones, causing degradation in pathways related to energy, transport of metabolites, and regulation/transcription. Brucella ceti isolates infecting particularly dolphin hosts, showed further degradation of metabolite transport pathways as well as pathways related to cell wall/membrane/envelope biogenesis and motility. Thus, gene loss through pseudogenization is a source of genetic variation in Brucella, which in turn, relates to adaptation to different hosts. This is relevant to understand the natural history of bacterial diseases, their zoonotic potential, and the impact of human interventions such as domestication. © The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Clinical and Diagnostic Aspects of Brucellosis and Antimicrobial Susceptibility of Brucella Isolates in Hamedan, Iran.

PubMed

Torkaman Asadi, Fatemeh; Hashemi, Seyyed Hamid; Alikhani, Mohammad Yousef; Moghimbeigi, Abbas; Naseri, Zahra

2017-05-24

Current drug regimens for brucellosis are associated with relatively high rates of therapeutic failure or relapse. Reduced antimicrobial susceptibility of Brucella spp. has been proposed recently as a potential cause of therapeutic failure. The aim of this study was to evaluate the antibiotic resistance pattern of Brucella melitensis clinical isolates by E-test method in Hamadan, west of Iran. In a 15-month period, all patients with suspected brucellosis were enrolled. Blood specimens were collected for diagnosis of brucellosis by BACTEC system and serological tests. Antimicrobial susceptibility of clinical isolates to 7 antibiotics was assessed by the E-test method. One hundred forty-nine patients with brucellosis were evaluated. 38.3% of cultures of clinical samples were positive for BACTEC system, of which 91.2% were associated with a positive serological test result. No significant associations were found between serology and the culture method. All Brucella isolates were susceptible to doxycycline, streptomycin, gentamicin, ciprofloxacin, and moxifloxacin. However, decreased sensitivity to rifampin and trimethoprim-sulfamethoxazole was found in 35.1% and 3.5% of isolates, respectively. Because of the high rates of intermediate sensitivity to rifampin among Brucella isolates, this drug should be prescribed with caution. We recommend restricting the use of rifampin for treatment of brucellosis except as an alternative drug for special situations.

Brucellosis caused by the wood rat pathogen Brucella neotomae: two case reports.

PubMed

Villalobos-Vindas, Juan M; Amuy, Ernesto; Barquero-Calvo, Elías; Rojas, Norman; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzman-Verri, Caterina; Moreno, Edgardo

2017-12-19

Brucellosis is a chronic bacterial disease caused by members of the genus Brucella. Among the classical species stands Brucella neotomae, until now, a pathogen limited to wood rats. However, we have identified two brucellosis human cases caused by B. neotomae, demonstrating that this species has zoonotic potential. Within almost 4 years of each other, a 64-year-old Costa Rican white Hispanic man and a 51-year-old Costa Rican white Hispanic man required medical care at public hospitals of Costa Rica. Their hematological and biochemical parameters were within normal limits. No adenopathies or visceral abnormalities were found. Both patients showed intermittent fever, disorientation, and general malaise and a positive Rose Bengal test compatible with Brucella infection. Blood and cerebrospinal fluid cultures rendered Gram-negative coccobacilli identified by genomic analysis as B. neotomae. After antibiotic treatment, the patients recovered with normal mental activities. This is the first report describing in detail the clinical disease caused by B. neotomae in two unrelated patients. In spite of previous claims, this bacterium keeps zoonotic potential. Proposals to generate vaccines by using B. neotomae as an immunogen must be reexamined and countries housing the natural reservoir must consider the zoonotic risk.

Brucella melitensis infection in sheep: present and future.

PubMed

Garin-Bastuji, B; Blasco, J M; Grayon, M; Verger, J M

1998-01-01

Sheep brucellosis, a zoonosis mainly due to B. melitensis (biovar 1, 2 or 3), remains widespread world-wide. Pathologically and epidemiologically, the disease is very similar to B. abortus infection in cattle. The live B. melitensis Rev 1 strain is currently considered as the best vaccine available for the control of sheep brucellosis, especially when used at the standard dose by the conjunctival route. Used exhaustively in whole-flock vaccination programmes, it induces a great decrease in the prevalence in both sheep and human populations. The expensive test-and-slaughter strategy should be restricted to the lowest infected areas. Whenever possible, Brucella spp. should be isolated by culture using adequate selective media from uterine discharges, aborted fetuses, udder secretions or selected tissues, such as lymph nodes, testes or epididymides. Species and biovar identification is routinely based on cultural criteria, on lysis by phages and on simple biochemical and serological tests. The recently developed polymerase chain reaction methods provide additional means of detection and identification. Despite the high degree of DNA homology within the genus Brucella, several methods, including PCR-RFLP and Southern blot, have been developed which allow, to a certain extent, the differentiation between Brucella species and some of their biovars. While several ELISA tests have been developed recently, the rose bengal plate agglutination and complement fixation tests, based on the detection of anti-S-LPS antibody, are still recommended for screening flocks and individuals. However, these tests sometimes lack specificity or sensitivity. For pooled samples, there are no useful tests such as the milk ring test in cattle. The brucellin allergic skin test can be used as a screening or complementary test in unvaccinated flocks, provided that a purified, lipopolysaccharide (LPS)-free and standardized antigen preparation is used.

Brucella and Osteoarticular Cell Activation: Partners in Crime

PubMed Central

Giambartolomei, Guillermo H.; Arriola Benitez, Paula C.; Delpino, M. Victoria

2017-01-01

Osteoarticular brucellosis is the most common presentation of human active disease although its prevalence varies widely. The three most common forms of osteoarticular involvement are sacroiliitis, spondylitis, and peripheral arthritis. The molecular mechanisms implicated in bone damage have been recently elucidated. B. abortus induces bone damage through diverse mechanisms in which TNF-α and the receptor activator of nuclear factor kappa-B ligand (RANKL)-the natural modulator of bone homeostasis are involved. These processes are driven by inflammatory cells, like monocytes/macrophages, neutrophils, Th17 CD4+ T, and B cells. In addition, Brucella abortus has a direct effect on osteoarticular cells and tilts homeostatic bone remodeling. These bacteria inhibit bone matrix deposition by osteoblasts (the only bone cells involved in bone deposition), and modify the phenotype of these cells to produce matrix metalloproteinases (MMPs) and cytokine secretion, contributing to bone matrix degradation. B. abortus also affects osteoclasts (cells naturally involved in bone resorption) by inducing an increase in osteoclastogenesis and osteoclast activation; thus, increasing mineral and organic bone matrix resorption, contributing to bone damage. Given that the pathology induced by Brucella species involved joint tissue, experiments conducted on synoviocytes revealed that besides inducing the activation of these cells to secrete chemokines, proinflammatory cytokines and MMPS, the infection also inhibits synoviocyte apoptosis. Brucella is an intracellular bacterium that replicates preferentially in the endoplasmic reticulum of macrophages. The analysis of B. abortus-infected synoviocytes indicated that bacteria also replicate in their reticulum suggesting that they could use this cell type for intracellular replication during the osteoarticular localization of the disease. Finally, the molecular mechanisms of osteoarticular brucellosis discovered recently shed light on how the

Entry and Elimination of Marine Mammal Brucella spp. by Hooded Seal (Cystophora cristata) Alveolar Macrophages In Vitro

PubMed Central

Larsen, Anett K.; Nymo, Ingebjørg H.; Boysen, Preben; Tryland, Morten; Godfroid, Jacques

2013-01-01

A high prevalence of Brucella pinnipedialis serology and bacteriology positive animals has been found in the Northeast Atlantic stock of hooded seal ( Cystophora cristata ); however no associated gross pathological changes have been identified. Marine mammal brucellae have previously displayed different infection patterns in human and murine macrophages. To investigate if marine mammal Brucella spp. are able to invade and multiply in cells originating from a presumed host species, we infected alveolar macrophages from hooded seal with a B . pinnipedialis hooded seal isolate. Hooded seal alveolar macrophages were also challenged with B . pinnipedialis reference strain (NCTC 12890) from harbor seal ( Phoca vitulina ), B . ceti reference strain (NCTC 12891) from harbor porpoise ( Phocoena phocoena ) and a B . ceti Atlantic white-sided dolphin ( Lagenorhynchus acutus ) isolate (M83/07/1), to evaluate possible species-specific differences. Brucella suis 1330 was included as a positive control. Alveolar macrophages were obtained by post mortem bronchoalveolar lavage of euthanized hooded seals. Phenotyping of cells in the lavage fluid was executed by flow cytometry using the surface markers CD14 and CD18. Cultured lavage cells were identified as alveolar macrophages based on morphology, expression of surface markers and phagocytic ability. Alveolar macrophages were challenged with Brucella spp. in a gentamicin protection assay. Following infection, cell lysates from different time points were plated and evaluated quantitatively for colony forming units. Intracellular presence of B . pinnipedialis hooded seal isolate was verified by immunocytochemistry. Our results show that the marine mammal brucellae were able to enter hooded seal alveolar macrophages; however, they did not multiply intracellularly and were eliminated within 48 hours, to the contrary of B. suis that showed the classical pattern of a pathogenic strain. In conclusion, none of the four marine mammal strains

Brucella pinnipedialis hooded seal (Cystophora cristata) strain in the mouse model with concurrent exposure to PCB 153.

PubMed

Nymo, Ingebjørg H; das Neves, Carlos G; Tryland, Morten; Bårdsen, Bård-Jørgen; Santos, Renato Lima; Turchetti, Andreia Pereira; Janczak, Andrew M; Djønne, Berit; Lie, Elisabeth; Berg, Vidar; Godfroid, Jacques

2014-05-01

Brucellosis, a worldwide zoonosis, is linked to reproductive problems in primary hosts. A high proportion of Brucella-positive hooded seals (Cystophora cristata) have been detected in the declined Northeast Atlantic stock. High concentrations of polychlorinated biphenyls (PCBs) have also been discovered in top predators in the Arctic, including the hooded seal, PCB 153 being most abundant. The aim of this study was to assess the pathogenicity of Brucella pinnipedialis hooded seal strain in the mouse model and to evaluate the outcome of Brucella spp. infection after exposure of mice to PCB 153. BALB/c mice were infected with B. pinnipedialis hooded seal strain or Brucella suis 1330, and half from each group was exposed to PCB 153 through the diet. B. pinnipedialis showed a reduced pathogenicity in the mouse model as compared to B. suis 1330. Exposure to PCB 153 affected neither the immunological parameters, nor the outcome of the infection. Altogether this indicates that it is unlikely that B. pinnipedialis contribute to the decline of hooded seals in the Northeast Atlantic. Copyright © 2014 Elsevier Ltd. All rights reserved.

The International Standard for Anti-Brucella abortus Serum

PubMed Central

Stableforth, A. W.

1954-01-01

In field trials on the eradication of brucellosis from dairy herds in Great Britain, which began in 1933, a serum standard of reference was used for the examination of agglutinating suspensions prepared in different laboratories. In 1937, the Office International des Epizooties (OIE) adopted this standard and made recommendations for its use internationally. These recommendations were revised by OIE in 1948, by the Third Inter-American Congress on Brucellosis and by the Joint FAO/WHO Expert Panel on Brucellosis in 1950, and again by the latter body in 1952. A new batch equivalent in potency to the original standard was established by the WHO Expert Committee on Biological Standardization in 1952 as the International Standard for Anti-Brucella abortus Serum. The International Standard, or a national standard of equivalent potency, ensures comparability of the titres obtained in different countries by different methods, and the results of such comparisons can be expressed in a simple manner by describing the titres in terms of International Units of Brucella antibody. PMID:13199656

Identification of Brucella spp. in feral swine (Sus scrofa) at abattoirs in Texas, USA.

PubMed

Pedersen, K; Bauer, N E; Olsen, S; Arenas-Gamboa, A M; Henry, A C; Sibley, T D; Gidlewski, T

2017-12-01

Various tissues, nasal swabs, urine and blood samples were collected from 376 feral swine at two federally inspected abattoirs in Texas during six separate sampling periods in 2015. Samples were tested for Brucella spp. by culture and serology. Brucella spp. were cultured from 13.0% of feral swine, and antibodies were detected in 9.8%. Only 32.7% of culture-positive feral swine were also antibody positive, and 43.2% of antibody-positive feral swine were culture positive. Approximately, the same number of males (14.0%) and females (12.1%) were culture positive, and slightly more males (10.5%) than females (8.7%) were antibody positive. Our results indicate that serology likely underestimates the prevalence of feral swine infected, and that those who come in contact with feral swine should be aware of the symptoms of infection with Brucella spp. to ensure prompt treatment. Published 2017. This article is a U.S. Government work and is in the public domain in the USA.

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats

PubMed Central

Mailybayeva, Aigerim; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Sansyzbay, Abylai; Renukaradhya, Gourapura J.; Petrovsky, Nikolai

2017-01-01

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18–0.34, infection index, P = 0.37–0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse

Improved influenza viral vector based Brucella abortus vaccine induces robust B and T-cell responses and protection against Brucella melitensis infection in pregnant sheep and goats.

PubMed

Mailybayeva, Aigerim; Yespembetov, Bolat; Ryskeldinova, Sholpan; Zinina, Nadezhda; Sansyzbay, Abylai; Renukaradhya, Gourapura J; Petrovsky, Nikolai; Tabynov, Kaissar

2017-01-01

We previously developed a potent candidate vaccine against bovine brucellosis caused by Brucella abortus using the influenza viral vector expressing Brucella Omp16 and L7/L12 proteins (Flu-BA). Our success in the Flu-BA vaccine trial in cattle and results of a pilot study in non-pregnant small ruminants prompted us in the current study to test its efficacy against B. melitensis infection in pregnant sheep and goats. In this study, we improved the Flu-BA vaccine formulation and immunization method to achieve maximum efficacy and safety. The Flu-BA vaccine formulation had two additional proteins Omp19 and SOD, and administered thrice with 20% Montanide Gel01 adjuvant, simultaneously by both subcutaneous and conjunctival routes at 21 days intervals in pregnant sheep and goats. At 42 days post-vaccination (DPV) we detected antigen-specific IgG antibodies predominantly of IgG2a isotype but also IgG1, and also detected a strong lymphocyte recall response with IFN-γ production. Importantly, our candidate vaccine prevented abortion in 66.7% and 77.8% of pregnant sheep and goats, respectively. Furthermore, complete protection (absence of live B. melitensis 16M) was observed in 55.6% and 66.7% of challenged sheep and goats, and 72.7% and 90.0% of their fetuses (lambs/yeanlings), respectively. The severity of B. melitensis 16M infection in vaccinated sheep and goats and their fetuses (index of infection and rates of Brucella colonization in tissues) was significantly lower than in control groups. None of the protection parameters after vaccination with Flu-BA vaccine were statistically inferior to protection seen with the commercial B. melitensis Rev.1 vaccine (protection against abortion and vaccination efficacy, alpha = 0.18-0.34, infection index, P = 0.37-0.77, Brucella colonization, P = 0.16 to P > 0.99). In conclusion, our improved Flu-BA vaccine formulation and delivery method were found safe and effective in protecting pregnant sheep and goats against adverse

The genome of Brucella melitensis.

PubMed

DelVecchio, Vito G; Kapatral, Vinayak; Elzer, Philip; Patra, Guy; Mujer, Cesar V

2002-12-20

The genome of Brucella melitensis strain 16M was sequenced and contained 3,294,931 bp distributed over two circular chromosomes. Chromosome I was composed of 2,117,144 bp and chromosome II has 1,177,787 bp. A total of 3,198 ORFs were predicted. The origins of replication of the chromosomes are similar to each other and to those of other alpha-proteobacteria. Housekeeping genes such as those that encode for DNA replication, protein synthesis, core metabolism, and cell-wall biosynthesis were found on both chromosomes. Genes encoding adhesins, invasins, and hemolysins were also identified.

Immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody.

PubMed

Zheng, W Y; Wang, Y; Zhang, Z C; Yan, F

2015-10-05

We examined the immunological characteristics of outer membrane protein omp31 of goat Brucella and its monoclonal antibody. Genomic DNA from the M5 strain of goat Brucella was amplified by polymerase chain reaction and cloned into the prokaryotic expression vector pGEX-4T-1. The expression and immunological characteristics of the fusion protein GST-omp31 were subjected to preliminary western blot detection with goat Brucella rabbit immune serum. The Brucella immunized BALB/c mouse serum was detected using purified protein. The high-potency mouse splenocytes and myeloma Sp2/0 cells were fused. Positive clones were screened by enzyme-linked immunosorbent assay to establish a hybridoma cell line. Mice were inoculated intraperitoneally with hybridoma cells to prepare ascites. The mAb was purified using the n-caprylic acid-ammonium sulfate method. The characteristics of mAb were examined using western blotting and enzyme-linked immunosorbent assay. A 680-base pair band was observed after polymerase chain reaction. Enzyme digestion identification and sequencing showed that the pGEX-4T-1-omp31 prokaryotic expression vector was successfully established; a target band of approximately 57 kDa with an apparent molecular weight consistent with the size of the target fusion protein. At 25°C, the expression of soluble expression increased significantly; the fusion protein GST-omp31 was detected by western blotting. Anti-omp31 protein mAb was obtained from 2 strains of Brucella. The antibody showed strong specificity and sensitivity and did not cross-react with Escherichia coli, Staphylococcus aureus, Bacillus subtilis, Mycobacterium tuberculosis, or Bacillus pyocyaneus. The pGEX-4T-1-omp31 prokaryotic expression vector was successfully established and showed good immunogenicity. The antibody also showed strong specificity and good sensitivity.

Rapid Quantitative Detection of Brucella melitensis by a Label-Free Impedance Immunosensor Based on a Gold Nanoparticle-Modified Screen-Printed Carbon Electrode

PubMed Central

Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

2013-01-01

A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 104 to 4 × 106 CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 104 and 4 × 105 CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis. PMID:23881126

Rapid quantitative detection of Brucella melitensis by a label-free impedance immunosensor based on a gold nanoparticle-modified screen-printed carbon electrode.

PubMed

Wu, Haiyun; Zuo, Yueming; Cui, Chuanjin; Yang, Wei; Ma, Haili; Wang, Xiaowen

2013-07-04

A rapid and simple method for quantitative monitoring of Brucella melitensis using electrochemical impedance spectroscopy (EIS) is reported for the first time. The label-free immunosensors were fabricated by immobilizing Brucella melitensis antibody on the surface of gold nanoparticle-modified screen-printed carbon electrodes (GNP-SPCEs). Cyclic voltammetry (CV) and EIS were used to characterize the Brucella melitensis antigen interaction on the surface of GNP-SPCEs with antibody. A general electronic equivalent model of an electrochemical cell was introduced for interpretation of the impedance components of the system. The results showed that the change in electron-transfer resistance (Rct) was significantly different due to the binding of Brucella melitensis cells. A linear relationship between the Rct variation and logarithmic value of the cell concentration was found from 4 × 10(4) to 4 × 10(6) CFU/mL in pure culture. The label-free impedance biosensor was able to detect as low as 1 × 10(4) and 4 × 10(5) CFU/mL of Brucella melitensis in pure culture and milk samples, respectively, in less than 1.5 h. Moreover, a good selectivity versus Escherichia coli O157:H7 and Staphylococcus aureus cells was obtained for our developed immunosensor demonstrating its specificity towards only Brucella melitensis.

Colony-level assessment of Brucella and Leptospira in the Guadalupe fur seal, Isla Guadalupe, Mexico.

PubMed

Ziehl-Quirós, E Carolina; García-Aguilar, María C; Mellink, Eric

2017-01-24

The relatively small population size and restricted distribution of the Guadalupe fur seal Arctocephalus townsendi could make it highly vulnerable to infectious diseases. We performed a colony-level assessment in this species of the prevalence and presence of Brucella spp. and Leptospira spp., pathogenic bacteria that have been reported in several pinniped species worldwide. Forty-six serum samples were collected in 2014 from pups at Isla Guadalupe, the only place where the species effectively reproduces. Samples were tested for Brucella using 3 consecutive serological tests, and for Leptospira using the microscopic agglutination test. For each bacterium, a Bayesian approach was used to estimate prevalence to exposure, and an epidemiological model was used to test the null hypothesis that the bacterium was present in the colony. No serum sample tested positive for Brucella, and the statistical analyses concluded that the colony was bacterium-free with a 96.3% confidence level. However, a Brucella surveillance program would be highly recommendable. Twelve samples were positive (titers 1:50) to 1 or more serovars of Leptospira. The prevalence was calculated at 27.1% (95% credible interval: 15.6-40.3%), and the posterior analyses indicated that the colony was not Leptospira-free with a 100% confidence level. Serovars Icterohaemorrhagiae, Canicola, and Bratislava were detected, but only further research can unveil whether they affect the fur seal population.

Lymphocyte proliferative responses of goats vaccinated with Brucella melitensis 16M or a delta purE201 strain.

PubMed Central

Olsen, S C; Cheville, N F; Stevens, M G; Houng, H H; Drazek, E S; Hadfield, T L; Warren, R L; Hoover, D L

1997-01-01

The response to a Brucella melitensis purEK deletion mutant, delta purE201 (referred to as strain 201), was compared with the response to its parental strain, 16M, in juvenile goats. Proliferative responses to gamma-irradiated bacteria were detected earlier in strain 201-infected goats. Lymphocytes from strain 16M- or 201-infected goats proliferated in response to one-dimensional polyacrylamide gel electrophoresis-separated proteins of similar mass isolated from strain 16M or Brucella abortus RB51. Data from this study suggest that some antigens stimulating cell-mediated responses are conserved among Brucella species, as 201- and 16M-infected goats recognized similar proteins expressed by RB51 and 16M. PMID:9199478

Bison PRNP genotyping and potential association with Brucella spp. seroprevalence

USGS Publications Warehouse

Seabury, C.M.; Halbert, N.D.; Gogan, P.J.P.; Templeton, J.W.; Derr, J.N.

2005-01-01

The implication that host cellular prion protein (PrPC) may function as a cell surface receptor and/or portal protein for Brucella abortus in mice prompted an evaluation of nucleotide and amino acid variation within exon 3 of the prion protein gene (PRNP) for six US bison populations. A non-synonymous single nucleotide polymorphism (T50C), resulting in the predicted amino acid replacement M17T (Met ??? Thr), was identified in each population. To date, no variation (T50: Met) has been detected at the corresponding exon 3 nucleotide and/or amino acid position for domestic cattle. Notably, 80% (20 of 25) of the Yellowstone National Park bison possessing the C/C genotype were Brucella spp. seropositive, representing a significant (P = 0.021) association between seropositivity and the C/C genotypic class. Moreover, significant differences in the distribution of PRNP exon 3 alleles and genotypes were detected between Yellowstone National Park bison and three bison populations that were either founded from seronegative stock or previously subjected to test-and-slaughter management to eradicate brucellosis. Unlike domestic cattle, no indel polymorphisms were detected within the corresponding regions of the putative bison PRNP promoter, intron 1, octapeptide repeat region or 3???-untranslated region for any population examined. This study provides the first evidence of a potential association between nucleotide variation within PRNP exon 3 and the presence of Brucella spp. antibodies in bison, implicating PrPC in the natural resistance of bison to brucellosis infection. ?? 2005 International Society for Animal Genetics.

Real-time PCR detection of Brucella spp. DNA in lesions and viscera of bovine carcasses.

PubMed

Sola, Marília Cristina; da Veiga Jardim, Eurione A G; de Freitas, Marcius Ribeiro; de Mesquita, Albenones José

2014-09-01

This study reports a real-time PCR assay for the detection of Brucella spp. associated with the FTA® Elute method in lesions observed during sanitary inspections in beef slaughter. Of the total 276 samples, 78 (28.3%) tested positive and 198 (71.7%) negative for Brucella spp. The real-time PCR technique associated with the FTA® Elute method proved to be an important tool for the diagnosis, judgment about and disposal of carcasses and viscera of slaughtered animals. Copyright © 2014 Elsevier B.V. All rights reserved.

Antimicrobial Susceptibility of Brucella melitensis Isolates in Peru

DTIC Science & Technology

2011-03-01

cipruftoxacin, and tl’imethoprim-sulfamethoxazole were determined by the Etest method. All isulates were sensitive to tested drugs during the periuds...rifampin (RIF), strepto- mycin, gentamicin (GE), and trimethoprim -sulfarnethoxazole (SXT); generally, two or three drugs arc used in combination for... trimethoprim -sulfamethoxazole in Kuwait (5) and Mcxicu (9) has similarly been reported. In this study, we sought to evaluate the susceptibility of Brucella

Temporal Role for MyD88 in a Model of Brucella-Induced Arthritis and Musculoskeletal Inflammation

PubMed Central

Lacey, Carolyn A.; Mitchell, William J.; Brown, Charles R.

2017-01-01

ABSTRACT Brucella spp. are facultative intracellular Gram-negative bacteria that cause the zoonotic disease brucellosis, one of the most common global zoonoses. Osteomyelitis, arthritis, and musculoskeletal inflammation are common focal complications of brucellosis in humans; however, wild-type (WT) mice infected systemically with conventional doses of Brucella do not develop these complications. Here we report C57BL/6 WT mice infected via the footpad with 103 to 106 CFU of Brucella spp. display neutrophil and monocyte infiltration of the joint space and surrounding musculoskeletal tissue. Joint inflammation is detectable as early as 1 day postinfection and peaks 1 to 2 weeks later, after which WT mice are able to slowly resolve inflammation. B and T cells were dispensable for the onset of swelling but required for resolution of joint inflammation and infection. At early time points, MyD88−/− mice display decreased joint inflammation, swelling, and proinflammatory cytokine levels relative to WT mice. Subsequently, swelling of MyD88−/− joints surpassed WT joint swelling, and resolution of joint inflammation was prolonged. Joint bacterial loads in MyD88−/− mice were significantly greater than those in WT mice by day 3 postinfection and at all time points thereafter. In addition, MyD88−/− joint inflammatory cytokine levels on day 3 and beyond were similar to WT levels. Collectively these data demonstrate MyD88 signaling mediates early inflammatory responses in the joint but also contributes to subsequent clearance of Brucella and resolution of inflammation. This work also establishes a mouse model for studying Brucella-induced arthritis, musculoskeletal complications, and systemic responses, which will lead to a better understanding of focal complications of brucellosis. PMID:28069819

Temporal Role for MyD88 in a Model of Brucella-Induced Arthritis and Musculoskeletal Inflammation.

PubMed

Lacey, Carolyn A; Mitchell, William J; Brown, Charles R; Skyberg, Jerod A

2017-03-01

Brucella spp. are facultative intracellular Gram-negative bacteria that cause the zoonotic disease brucellosis, one of the most common global zoonoses. Osteomyelitis, arthritis, and musculoskeletal inflammation are common focal complications of brucellosis in humans; however, wild-type (WT) mice infected systemically with conventional doses of Brucella do not develop these complications. Here we report C57BL/6 WT mice infected via the footpad with 10 3 to 10 6 CFU of Brucella spp. display neutrophil and monocyte infiltration of the joint space and surrounding musculoskeletal tissue. Joint inflammation is detectable as early as 1 day postinfection and peaks 1 to 2 weeks later, after which WT mice are able to slowly resolve inflammation. B and T cells were dispensable for the onset of swelling but required for resolution of joint inflammation and infection. At early time points, MyD88 -/- mice display decreased joint inflammation, swelling, and proinflammatory cytokine levels relative to WT mice. Subsequently, swelling of MyD88 -/- joints surpassed WT joint swelling, and resolution of joint inflammation was prolonged. Joint bacterial loads in MyD88 -/- mice were significantly greater than those in WT mice by day 3 postinfection and at all time points thereafter. In addition, MyD88 -/- joint inflammatory cytokine levels on day 3 and beyond were similar to WT levels. Collectively these data demonstrate MyD88 signaling mediates early inflammatory responses in the joint but also contributes to subsequent clearance of Brucella and resolution of inflammation. This work also establishes a mouse model for studying Brucella -induced arthritis, musculoskeletal complications, and systemic responses, which will lead to a better understanding of focal complications of brucellosis. Copyright © 2017 American Society for Microbiology.

Seroprevalence and risk factors associated with Brucella seropositivity in dairy and mixed cattle herds from Ecuador.

PubMed

Carbonero, A; Guzmán, L T; García-Bocanegra, I; Borge, C; Adaszek, L; Arenas, A; Saa, L R

2018-01-01

An extensive cross-sectional study to determine the seroprevalence of and associated risk factors for Brucella infection was performed in dairy and mixed (dairy-beef) cattle herds in Ecuador. A total of 2666 serum samples from 386 farms were analyzed using Rose Bengal test and a blocking ELISA test. In addition, a questionnaire with 57 variables related to management, feeding, facilities, biosecurity, and animal health was filled in every cattle farm. A Generalized Estimating Equations model was used to determine the factors associated with Brucella seropositivity. The true prevalence of Brucella seropositivity in dairy and mixed cattle from Ecuador reached 17.0% (CI95% 15.6-18.4%). The herd prevalence was 45.1% (174/386) (CI95% 40.1-50.1%), and the within-herd prevalence ranged from 10 to 100% (mean 38.9%; Q1 14.3%, Q2 26.8%, Q3 52.5%). Seven factors were included in the GEE model for Brucella seropositivity: the nominal variables sex (OR 2.03; CI95% 1.32-3.13), herd type (dairy) (OR 1.79; CI95% 1.11-2.87), closed facilities in the farm (OR 1.80; CI95% 1.19-2.74), and ad libitum feeding (OR: 0.32; CI95%: 0.19-0.54), and the quantitative variables age (OR 1.005; CI95% 1.001-1.009), average slope in the farm (%) (OR 1.013; CI95% 1.002-1.024), and annual abortion rate (OR 1.016; CI95% 1.002-1.031). This study remarks the high spread of Brucella infection in cattle farms from Ecuador. In addition, it reports the risk factors associated to this infection in the predominant extensive system existent in this country.

Seroprevalence of Brucella antibodies in harbor seals in Alaska, USA, with age, regional, and reproductive comparisons.

PubMed

Hoover-Miller, A; Dunn, J L; Field, C L; Blundell, G; Atkinson, S

2017-09-20

Populations of harbor seal Phoca vitulina in the Gulf of Alaska have dramatically declined during the past 4 decades. Numbers of seals in Glacier Bay, in southeast Alaska, USA, have also declined despite extensive protection. Causes of the declines and slow recovery are poorly understood. Brucellosis is a zoonotic disease that adversely affects reproduction in many domestic species. We measured the seroprevalence of Brucella antibodies in 554 harbor seals in 3 Alaska locations: Prince William Sound (PWS), Glacier Bay (GB), and Tracy Arm Fords Terror (TAFT) Wilderness Area. Objectives included testing for regional, sex, age, and female reproductive state differences in Brucella antibody seroprevalence, persistence in titers in recaptured seals, and differences in titers between mother seals and their pups. Overall, 52% of adults (AD), 53% of subadults (SA), 77% of yearlings (YRL), and 26% of <5 mo old pups were seropositive. Matched mother-pup samples were consistent with dependent pups acquiring maternal passive immunity to Brucella. Results show higher seroprevalence (64%) for AD and SA seals in the depressed and declining populations in PWS and GB than in TAFT (29%). Lactating females were less likely to be seropositive than other AD females, including pregnant females. Further research is needed to seek evidence of Brucella infection in Alaskan harbor seals, identify effects on neonatal viability, and assess zoonotic implications for Alaska Natives who rely on harbor seals for food.

Brucella TIR-like protein TcpB/Btp1 specifically targets the host adaptor protein MAL/TIRAP to promote infection.

PubMed

Li, Wenna; Ke, Yuehua; Wang, Yufei; Yang, Mingjuan; Gao, Junguang; Zhan, Shaoxia; Xinying, Du; Huang, Liuyu; Li, Wenfeng; Chen, Zeliang; Li, Juan

2016-08-26

Brucella spp. are known to avoid host immune recognition and weaken the immune response to infection. Brucella like accomplish this by employing two clever strategies, called the stealth strategy and hijacking strategy. The TIR domain-containing protein (TcpB/Btp1) of Brucella melitensis is thought to be involved in inhibiting host NF-κB activation by binding to adaptors downstream of Toll-like receptors. However, of the five TIR domain-containing adaptors conserved in mammals, whether MyD88 or MAL, even other three adaptors, are specifically targeted by TcpB has not been identified. Here, we confirmed the effect of TcpB on B.melitensis virulence in mice and found that TcpB selectively targets MAL. By using siRNA against MAL, we found that TcpB from B.melitensis is involved in intracellular survival and that MAL affects intracellular replication of B.melitensis. Our results confirm that TcpB specifically targets MAL/TIRAP to disrupt downstream signaling pathways and promote intra-host survival of Brucella spp. Copyright © 2016 Elsevier Inc. All rights reserved.

The effect of tigecycline and ertapenem against clinical isolates of Brucella melitensis detected by E-test on different media.

PubMed

Tanyel, E; Coban, A Y; Fisgin, N Tasdelen; Tulek, N

2010-01-01

In this study, in vitro activity of tigecycline (TIG) and ertapenem (ERT) against clinical isolates of Brucella melitensis and the effect of different media on in vitro test results were investigated. The in vitro effects of TIG and ERT to 38 B. melitensis isolates were comparatively investigated in brucella agar and 5% sheep blood agar. MIC value of ERT was 0.032 μg/mL in 23 of 38 and 20 of 38 isolates on blood and brucella agar, respectively. Minimum inhibitory concentration values of TIG were substantially different ranging between 0.064-0.25 μg/mL on blood agar. However, MIC values of TIG were similar on brucella agar with 0.25 μg/mL in 15 of 38 isolates and 0.5 μg/mL in 10 of 38 isolates. In conclusion, although ERT and TIG were effective against B. melitensis isolates in vitro, further studies are needed in order to determine the use of these novel drugs in treatment of brucellosis.

Virulence Effects and Signaling Partners Modulated by Brucella melitensis Light-sensing Histidine Kinase

NASA Astrophysics Data System (ADS)

Gourley, Christopher R.

The facultative intracellular pathogen Brucella melitensis utilizes diverse virulence factors. A Brucella light sensing histidine kinase can influence in vitro virulence of the bacteria during intracellular infection. First, we demonstrated that the B. melitensis light sensing kinase (BM-LOV-HK) affects virulence in an IRF-1-/- mouse model of infection. Infection with a Δ BM-LOV-HK strain resulted in less bacterial colonization of IRF-1-/- spleens and extended survivorship compared to mice infected with wild type B. melitensis 16M. Second, using PCR arrays, we observed less expression of innate and adaptive immune system activation markers in ΔBM-LOV-HK infected mouse spleens than wild type B. melitensis 16M infected mouse spleens 6 days after infection. Third, we demonstrated by microarray analysis of B. melitensis that deletion of BM-LOV-HK alters bacterial gene expression. Downregulation of genes involved in control of the general stress response system included the alternative sigma factor RpoE1 and its anti-anti sigma factor PhyR. Conversely, genes involved in flagella production, quorum sensing, and the type IV secretion system (VirB operon) were upregulated in the Δ BM-LOV-HK strain compared to the wild type B. melitensis 16M. Analysis of genes differentially regulated in Δ BM-LOV-HK versus the wild type strain indicated an overlap of 110 genes with data from previous quorum sensing regulator studies of Δ vjbR and/ΔblxR(babR) strains. Also, several predicted RpoE1 binding sites located upstream of genes were differentially regulated in the ΔBM-LOV-HK strain. Our results suggest BM-LOV-HK is important for in vivo Brucella virulence, and reveals that BM-LOV-HK directly or indirect regulates members of the Brucella quorum sensing, type IV secretion, and general stress systems.

Coinfection and vertical transmission of Brucella and Morbillivirus in a neonatal sperm whale (Physeter macrocephalus) in Hawaii, USA.

PubMed

West, Kristi L; Levine, Gregg; Jacob, Jessica; Jensen, Brenda; Sanchez, Susan; Colegrove, Kathleen; Rotstein, David

2015-01-01

The viral genus Morbillivirus and the bacterial genus Brucella have emerged as important groups of pathogens that are known to affect cetacean health on a global scale, but neither pathogen has previously been reported from endangered sperm whales (Physeter macrocephalus). A female neonate sperm whale stranded alive and died near Laie on the island of Oahu, Hawaii, US, in May of 2011. Congestion of the cerebrum and enlarged lymph nodes were noted on the gross necropsy. Microscopic findings included lymphoid depletion, chronic meningitis, and pneumonia, suggesting an in utero infection. Cerebrum, lung, umbilicus, and select lymph nodes (tracheobronchial and mediastinal) were positive for Brucella by PCR. Brucella sp. was also cultured from the cerebrum and from mediastinal and tracheobronchial lymph nodes. Twelve different tissues were screened for Morbillivirus by reverse-transcriptase (RT)-PCR and select tissues by immunohistochemistry, but only the tracheobronchial lymph node and spleen were positive by RT-PCR. Pathologic findings observed were likely a result of Brucella, but Morbillivirus may have played a key role in immune suppression of the mother and calf. The in utero infection in this individual strongly supports vertical transmission of both pathogens.

Observations on brucellosis due to Brucella melitensis*

PubMed Central

Spink, Wesley W.

1953-01-01

A special study was made of the problem of brucellosis due to Brucella melitensis in visits to Mexico City in 1948, to the FAO/WHO Brucellosis Centres at Montpellier (France), Florence (Italy), and Rijeka (Yugoslavia) in 1951, and to Spain in 1952. Br. melitensis infection in human beings causes more severe illness than Br. abortus infection. It develops primarily in rural communities living in close contact with goats and sheep; cattle and swine may also harbour the infection. In diagnosis, the agglutination test has proved the most satisfactory procedure; testing would be more uniformly reliable if a single antigen were used. Lack of funds and technical assistance have in many instances limited the bacteriological studies upon which a more definitive diagnosis of brucellosis depends. Antibiotics, Brucella vaccines, and colloidal preparations of gold and silver—used separately and in combination—have proved of varying therapeutic value, although response to antibiotics is less favourable than in cases of Br. abortus infection. While the drastic measures—involving the slaughter of about 10,000 sheep—taken in Slovenia, Yugoslavia, in the late 1940's, against an outbreak of brucellosis, is an inspiring example of how the disease can be eradicated, the removal of all diseased animals is rarely feasible economically. It is hoped that future research will reveal a practicable alternative in the immunization of sheep and goats against the disease. PMID:13106703

Nasal Vaccination Stimulates CD8+ T Cells for Potent Protection Against Mucosal Brucella melitensis Challenge

PubMed Central

Clapp, Beata; Yang, Xinghong; Thornburg, Theresa; Walters, Nancy; Pascual, David W.

2016-01-01

Brucellosis remains a significant zoonotic threat worldwide. Humans and animals acquire infection via their oropharynx and upper respiratory tract following oral or aerosol exposure. After mucosal infection, brucellosis develops into a systemic disease. Mucosal vaccination could offer a viable alternative to conventional injection practices to deter disease. Using a nasal vaccination approach, the ΔznuA B. melitensis was found to confer potent protection against pulmonary Brucella challenge, and reduce colonization of spleens and lungs by more than 2500-fold, with more than 50% of vaccinated mice showing no detectable brucellae. Furthermore, tenfold more brucellae-specific, IFN-γ-producing CD8+ T cells than CD4+ T cells were induced in the spleen and respiratory lymph nodes. Evaluation of pulmonary and splenic CD8+ T cells from mice vaccinated with ΔznuA B. melitensis revealed that these expressed an activated effector memory (CD44hiCD62LloCCR7lo) T cells producing elevated levels of IFN-γ, TNF-α, perforin, and granzyme B. To assess the relative importance of these increased numbers of CD8+ T cells, CD8−/− mice were challenged with virulent B. melitensis, and they showed markedly increased bacterial loads in organs in contrast to similarly challenged CD4−/− mice. Only ΔznuA B. melitensis- and Rev-1-vaccinated CD4−/− and wild-type mice, not CD8−/− mice, were completely protected against Brucella challenge. Determination of cytokines responsible for conferring protection showed the relative importance of IFN-γ, but not IL-17. Unlike wild-type mice, IL-17 was greatly induced in IFN-γ−/− mice, but IL-17 could not substitute for IFN-γ’s protection, although an increase in brucellae dissemination was observed upon in vivo IL-17 neutralization. These results show that nasal ΔznuA B. melitensis vaccination represents an attractive means to stimulate systemic and mucosal immune protection via CD8+ T cell engagement. PMID:26752510

Polymorphisms at the 3' untranslated region of SLC11A1 gene are associated with protection to Brucella infection in goats.

PubMed

Iacoboni, Paola A; Hasenauer, Flavia C; Caffaro, M Eugenia; Gaido, Analia; Rossetto, Cristina; Neumann, Roberto D; Salatin, Antonio; Bertoni, Emiliano; Poli, Mario A; Rossetti, Carlos A

2014-08-15

Goats are susceptible to brucellosis and the detection of Brucella-infected animals is carried out by serological tests. In other ruminant species, polymorphisms in microsatellites (Ms) of 3' untranslated region (3'UTR) of the solute carrier family 11 member A1 (SLC11A1) gene were associated with resistance to Brucella abortus infection. Goats present two polymorphic Ms at the 3'UTR end of SLC11A1 gene, called regions A and B. Here, we evaluated if polymorphisms in regions A and/or B are associated with Brucella infection in goats. Serum (for the detection of Brucella-specific antibodies) and hair samples (for DNA isolation and structure analysis of the SLC11A1 gene) were randomly collected from 229 adult native goats from the northwest of Argentina. Serological status was evaluated by buffer plate antigen test (BPAT) complemented by the fluorescent polarization assay (FPA), and the genotype of the 3'UTR of the SLC11A1 gene was determined by capillary electrophoresis and confirmed by sequence analysis. Polymorphisms in regions A and B of the 3'UTR SLC11A1 gene were found statistically significant associated with protection to Brucella infection. Specifically, the association study indicates statistical significance of the allele A15 and B7/B7 genotype with absence of Brucella-specific antibodies (p=0.0003 and 0.0088, respectively). These data open a promising opportunity for limiting goat brucellosis through selective breeding of animals based on genetic markers associated with natural resistance to B. melitensis infection. Copyright © 2014 Elsevier B.V. All rights reserved.

Evidence of Brucella strain ST27 in bottlenose dolphin (Tursiops truncatus) in Europe.

PubMed

Cvetnić, Željko; Duvnjak, Sanja; Đuras, Martina; Gomerčić, Tomislav; Reil, Irena; Zdelar-Tuk, Maja; Špičić, Silvio

2016-11-30

Marine mammal brucellosis has been known for more than 20 years, but recent work suggests it is more widespread than originally thought. Brucella (B.) pinnipedialis has been isolated from pinnipeds, while B. ceti strains have been associated with cetaceans. Here we report a Brucella strain isolated from multiple lymph nodes of one bottlenose dolphin (Tursiops truncatus) during routine examination of dolphin carcasses found in the Croatian part of the northern Adriatic Sea during the summer of 2015. Classical bacteriological biotyping, PCR-based techniques (single, multiplex, PCR-RFLP) and 16S rRNA DNA sequencing were used to identify Brucella spp. Multiple-locus variable number tandem repeat analysis of 16 loci and multilocus sequence typing of 9 loci were used for genotyping and species determination. The combination of bacteriological, molecular and genotyping techniques identified our strain as ST27, previously identified as a human pathogen. This report provides, to our knowledge, the first evidence of ST27 in the Adriatic Sea in particular and in European waters in general. The zoonotic nature of the strain and its presence in the Adriatic, which is inhabited by bottlenose dolphins, suggest that the strain may pose a significant threat to human health. Copyright © 2016 Elsevier B.V. All rights reserved.

Epidemiological investigation of the first human brucellosis case in Spain due to Brucella suis biovar 1 strain 1330.

PubMed

Compés Dea, Cecilia; Guimbao Bescós, Joaquín; Alonso Pérez de Ágreda, Juan Pablo; Muñoz Álvaro, Pilar María; Blasco Martínez, José María; Villuendas Usón, María Cruz

2017-03-01

No cases of human brucellosis caused by Brucella suis has been reported in Spain. This study involved interviews with the case and his co-workers, inspection of their workplace, checking infection control measures, and typing the Brucella strain isolated in the blood culture. Brucella suis biovar 1 strain 1330 was isolated from a patient who worked in a waste treatment plant. Food borne transmission, contact with animals, and risk jobs were ruled out. An accidental inoculation with a contaminated needle from a research laboratory waste container was identified as the most probable mode of transmission. There should be controls to ensure that waste containers are sealed. Copyright © 2016 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.

Advancement of knowledge of Brucella over the past 50 years

USDA-ARS?s Scientific Manuscript database

Fifty years ago, bacteria in the genus Brucella were known to cause infertility and reproductive losses. The genus was considered to contain only three species, B. abortus, B. melitensis and B. suis. Since the early 1960’s, at least seven new species have been identified as belonging to the Brucell...

Rapid identification of Yersinia pestis and Brucella melitensis by chip-based continuous flow PCR

NASA Astrophysics Data System (ADS)

Dietzsch, Michael; Hlawatsch, Nadine; Melzer, Falk; Tomaso, Herbert; Gärtner, Claudia; Neubauer, Heinrich

2012-06-01

To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

MEDICAL vs. MEDICAL AND SURGICAL TREATMENT FOR BRUCELLA ENDOCARDITIS: A REVIEW OF THE LITERATURE

PubMed Central

Keshtkar-Jahromi, Maryam; Razavi, Seyed-Mostafa; Gholamin, Sharareh; Keshtkar-Jahromi, Marzieh; Hossain, Mian; Sajadi, Mohammad

2012-01-01

This review was undertaken to determine the role of surgery in the treatment of brucella endocarditis. All English and French articles reporting brucella endocarditis (1966–2011) in Pubmed, Google and Scopus were reviewed. 308 cases were identified and Linear and Logistic regression was performed. Surgery improved outcomes by decreasing mortality from 32.7% in the medical treatment only group to 6.7% in the combined surgical and medical treatment group (p<.001). This association was still significant while controlling for other contributing factors. In the absence of a controlled trial, we recommend the utmost vigilance and consideration of surgical management in treating such patients. PMID:23102495

Microscopy-based Assays for High-throughput Screening of Host Factors Involved in Brucella Infection of Hela Cells.

PubMed

Casanova, Alain; Low, Shyan H; Emmenlauer, Mario; Conde-Alvarez, Raquel; Salcedo, Suzana P; Gorvel, Jean-Pierre; Dehio, Christoph

2016-08-05

Brucella species are facultative intracellular pathogens that infect animals as their natural hosts. Transmission to humans is most commonly caused by direct contact with infected animals or by ingestion of contaminated food and can lead to severe chronic infections. Brucella can invade professional and non-professional phagocytic cells and replicates within endoplasmic reticulum (ER)-derived vacuoles. The host factors required for Brucella entry into host cells, avoidance of lysosomal degradation, and replication in the ER-like compartment remain largely unknown. Here we describe two assays to identify host factors involved in Brucella entry and replication in HeLa cells. The protocols describe the use of RNA interference, while alternative screening methods could be applied. The assays are based on the detection of fluorescently labeled bacteria in fluorescently labeled host cells using automated wide-field microscopy. The fluorescent images are analyzed using a standardized image analysis pipeline in CellProfiler which allows single cell-based infection scoring. In the endpoint assay, intracellular replication is measured two days after infection. This allows bacteria to traffic to their replicative niche where proliferation is initiated around 12 hr after bacterial entry. Brucella which have successfully established an intracellular niche will thus have strongly proliferated inside host cells. Since intracellular bacteria will greatly outnumber individual extracellular or intracellular non-replicative bacteria, a strain constitutively expressing GFP can be used. The strong GFP signal is then used to identify infected cells. In contrast, for the entry assay it is essential to differentiate between intracellular and extracellular bacteria. Here, a strain encoding for a tetracycline-inducible GFP is used. Induction of GFP with simultaneous inactivation of extracellular bacteria by gentamicin enables the differentiation between intracellular and extracellular

Assessment of genetic diversity of zoonotic Brucella spp. recovered from livestock in Egypt using multiple locus VNTR analysis.

PubMed

Menshawy, Ahmed M S; Perez-Sancho, Marta; Garcia-Seco, Teresa; Hosein, Hosein I; García, Nerea; Martinez, Irene; Sayour, Ashraf E; Goyache, Joaquín; Azzam, Ragab A A; Dominguez, Lucas; Alvarez, Julio

2014-01-01

Brucellosis is endemic in most parts of Egypt, where it is caused mainly by Brucella melitensis biovar 3, and affects cattle and small ruminants in spite of ongoing efforts devoted to its control. Knowledge of the predominant Brucella species/strains circulating in a region is a prerequisite of a brucellosis control strategy. For this reason a study aiming at the evaluation of the phenotypic and genetic heterogeneity of a panel of 17 Brucella spp. isolates recovered from domestic ruminants (cattle, buffalo, sheep, and goat) from four governorates during a period of five years (2002-2007) was carried out using microbiological tests and molecular biology techniques (PCR, MLVA-15, and sequencing). Thirteen strains were identified as B. melitensis biovar 3 while all phenotypic and genetic techniques classified the remaining isolates as B. abortus (n = 2) and B. suis biovar 1 (n = 2). MLVA-15 yielded a high discriminatory power (h = 0.801), indicating a high genetic diversity among the B. melitensis strains circulating among domestic ruminants in Egypt. This is the first report of the isolation of B. suis from cattle in Egypt which, coupled with the finding of B. abortus, suggests a potential role of livestock as reservoirs of several zoonotic Brucella species in the region.

Brucella abortus Choloylglycine Hydrolase Affects Cell Envelope Composition and Host Cell Internalization

PubMed Central

Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C.; Mujer, Cesar V.; DelVecchio, Vito G.; Comerci, Diego J.

2011-01-01

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization. PMID:22174816

Brucella abortus choloylglycine hydrolase affects cell envelope composition and host cell internalization.

PubMed

Marchesini, María Inés; Connolly, Joseph; Delpino, María Victoria; Baldi, Pablo C; Mujer, Cesar V; DelVecchio, Vito G; Comerci, Diego J

2011-01-01

Choloylglycine hydrolase (CGH, E.C. 3.5.1.24) is a conjugated bile salt hydrolase that catalyses the hydrolysis of the amide bond in conjugated bile acids. Bile salt hydrolases are expressed by gastrointestinal bacteria, and they presumably decrease the toxicity of host's conjugated bile salts. Brucella species are the causative agents of brucellosis, a disease affecting livestock and humans. CGH confers Brucella the ability to deconjugate and resist the antimicrobial action of bile salts, contributing to the establishment of a successful infection through the oral route in mice. Additionally, cgh-deletion mutant was also attenuated in intraperitoneally inoculated mice, which suggests that CGH may play a role during systemic infection other than hydrolyzing conjugated bile acids. To understand the role CGH plays in B. abortus virulence, we infected phagocytic and epithelial cells with a cgh-deletion mutant (Δcgh) and found that it is defective in the internalization process. This defect along with the increased resistance of Δcgh to the antimicrobial action of polymyxin B, prompted an analysis of the cell envelope of this mutant. Two-dimensional electrophoretic profiles of Δcgh cell envelope-associated proteins showed an altered expression of Omp2b and different members of the Omp25/31 family. These results were confirmed by Western blot analysis with monoclonal antibodies. Altogether, the results indicate that Brucella CGH not only participates in deconjugation of bile salts but also affects overall membrane composition and host cell internalization.

Shedding Rates and SeroPrevalence of Brucella melitensis in Lactating Goats of Shahrekord, Iran.

PubMed

Ebrahimi, Azizollah; Milan, Jalal Sheykh Kanluye; Mahzoonieh, Mohamad Reza; Khaksar, Khadijeh

2014-03-01

Brucellosis remains a major worldwide zoonosis. Caprine brucellosis is a significant problem for both public health and animal production. Brucella melitensis causes disease in goats, sheep, humans, and occasionally cattle. Transmission is by ingestion or contact with infected materials, vaginal discharge, or milk. The current study aimed to determine the rate of B. melitensis seropositives and its probable shedding in lactating goats from flocks in Shahrekord district, Iran. In the current study, 1080 samples of milk, blood and vaginal swabs of 360 lactating goats (three samples from each animal) were randomly collected from 12 flocks in Shahrekord district. Serums from blood samples were examined by Rose Bengal plate (RBT) test and the titre of positives determined by tube agglutination test (TAT). Vaginal swab and milk (cream and sediment) samples were cultured on Brucella agar. Brucella spp. suspected pure cultures were incubated in the same conditions and then examined by Modified Zeil-Nelson (MZN) staining, oxidase and catalase tests. Positive isolates were examined by PCR. Out of 360 serum samples, 50 (13.9%) were positive by RBT, and six (1/66%) were positive by TAT. Culturing of milk and vaginal samples lead to isolation of 12 (3.33%) and 10 (2.77%) Brucella spp. suspected colonies, respectively. The PCR examinations of these isolates showed that ten (2.77%) milk and 6 vaginal swab samples (1.66%) belonged to B. melitensis species. Eight goats (2.22%) had positive results in RBT, culture and PCR examinations, simultaneously. The regional distribution of caprine brucellosis and shedding of B. melitensis through vaginal secretions and milk secretions of lactating goats indicated that 50% and 83.33% of the goat flocks contained vaginal and milk shedders, respectively.

Isolation & characterization of Brucella melitensis isolated from patients suspected for human brucellosis in India

PubMed Central

Barua, Anita; Kumar, Ashu; Thavaselvam, Duraipandian; Mangalgi, Smita; Prakash, Archana; Tiwari, Sapana; Arora, Sonia; Sathyaseelan, Kannusamy

2016-01-01

Background & objectives: Brucellosis is endemic in the southern part of India. A combination of biochemical, serological and molecular methods is required for identification and biotyping of Brucella. The present study describes the isolation and biochemical, molecular characterization of Brucella melitensis from patients suspected for human brucellosis. Methods: The blood samples were collected from febrile patients suspected to have brucellosis. A total of 18 isolates were obtained from 102 blood samples subjected to culture. The characterization of these 18 isolates was done by growth on Brucella specific medium, biochemical reactions, CO2 requirement, H2S production, agglutination with A and M mono-specific antiserum, dye sensitivity to basic fuchsin and thionin. Further, molecular characterization of the isolates was done by amplification of B. melitensis species specific IS711 repetitive DNA fragment and 16S (rRNA) sequence analysis. PCR-restriction fragment length polymorphism (RFLP) analysis of omp2 locus and IS711 gene was also done for molecular characterization. Results: All 102 suspected samples were subjected to bacteria isolation and of these, 18 isolates could be recovered on blood culture. The biochemical, PCR and PCR-RFLP and 16s rRNA sequencing revealed that all isolates were of B. melitensis and matched exactly with reference strain B. melitensis 16M. Interpretation & conclusions: The present study showed an overall isolation rate of 17.64 per cent for B. melitensis. There is a need to establish facilities for isolation and characterization of Brucella species for effective clinical management of the disease among patients as well as surveillance and control of infection in domestic animals. Further studies are needed from different geographical areas of the country with different level of endemicity to plan and execute control strategies against human brucellosis. PMID:27488010

Effects of gamma radiation and azathioprine on Brucella abortus infection in BALB/c mice

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

Elzer, P.H.; Rowe, G.E.; Enright, F.M.

Sublethal irradiation of BALB/c mice 4 hours prior to inoculation with 5 {times} 10(4) virulent Brucella abortus, caused significant (P less than 0.01) reductions in bacterial numbers in comparison with numbers in unirradiated controls. Numbers of brucellae in the spleen were significantly lower by 5 days after inoculation and decreased thereafter, so that at 2 and 3 weeks after inoculation, there were up to 1,000-fold fewer organisms in the spleen of irradiated mice. The number of brucellae in the spleen increased in irradiated mice thereafter. The course of events in the liver was similar, but developed more slowly, and peakmore » differences in bacterial numbers were about 1 log less. These phenomena were not attributable to differences in implantation of brucellae in the liver or spleen, nor to an abnormal distribution of organisms in other organs of irradiated mice. Irradiation of mice during the plateau phase of infection also resulted in significant (P less than 0.05) reductions in bacterial counts in the spleen during the succeeding 4 weeks. Macrophage activation in the spleen, measured by a Listeria monocytogenes-killing assay, was significantly (P less than 0.01) increased by irradiation alone at 1 week after inoculation and at that time was significantly (P less than 0.01) greater in B abortus-infected, irradiated mice than in B abortus-infected controls. Histologic, cytologic, and immunologic studies revealed that the decrease in numbers of organisms between 1 and 2 weeks after inoculation in irradiated mice occurred at a time when their immune response to B abortus was suppressed and when numbers of neutrophils and monocytes infiltrating the spleen were significantly (P less than 0.01) diminished.« less

Typing and comparative genome analysis of Brucella melitensis isolated from Lebanon.

PubMed

Abou Zaki, Natalia; Salloum, Tamara; Osman, Marwan; Rafei, Rayane; Hamze, Monzer; Tokajian, Sima

2017-10-16

Brucella melitensis is the main causative agent of the zoonotic disease brucellosis. This study aimed at typing and characterizing genetic variation in 33 Brucella isolates recovered from patients in Lebanon. Bruce-ladder multiplex PCR and PCR-RFLP of omp31, omp2a and omp2b were performed. Sixteen representative isolates were chosen for draft-genome sequencing and analyzed to determine variations in virulence, resistance, genomic islands, prophages and insertion sequences. Comparative whole-genome single nucleotide polymorphism analysis was also performed. The isolates were confirmed to be B. melitensis. Genome analysis revealed multiple virulence determinants and efflux pumps. Genome comparisons and single nucleotide polymorphisms divided the isolates based on geographical distribution but revealed high levels of similarity between the strains. Sequence divergence in B. melitensis was mainly due to lateral gene transfer of mobile elements. This is the first report of an in-depth genomic characterization of B. melitensis in Lebanon. © FEMS 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Seroprevalence of Brucella spp. in Cattle, Molecular Characterization in Milk, and the Analysis of Associated Risk Factors with Seroprevalence in Humans, Egypt.

PubMed

El-Diasty, Mohamed M; Ahmed, Heba A; Sayour, Ashraf E; El Hofy, Fatma I; Tahoun, Asmaa B M B; Shafik, Saleh M

2016-12-01

The objective of the present study was to estimate the seroprevalence of Brucella spp. in humans and cattle at Sharkia Governorate, Egypt. In addition, identification of Brucella spp. in milk samples by PCR and culture with the evaluation of the risk factors associated with Brucella spp. seroprevalence in humans were carried out. Overall, the seroprevalence of Brucella antibodies in the examined cattle was 23.8%, while in human participants it was 21%. The examination of 205 milk samples using PCR revealed that 6.3% were positive for B. abortus biovar 1 and the results were confirmed by culture methods. Multivariate logistic regression revealed that consumption of unpasteurized dairy products, occupational contact with animals, and knowledge about the disease are risk factors associated with infection in humans. This study documented the endemic status of brucellosis in Egypt. Hygienic measures and increased awareness about the disease are recommended to minimize the spread of infection from animals to humans.

Vaccination of elk (Cervus canadensis) with Brucella abortus strain RB51 overexpressing superoxide dismutase and glycosyltransferase genes does not induce adequate protection against experimental brucella abortus challenge

USDA-ARS?s Scientific Manuscript database

In recent years, elk (Cervus canadensis) have been implicated as the source of Brucella abortus infection for numerous cattle herds in the Greater Yellowstone Area (GYA). In the face of environmental and ecological changes on the landscape, the range of infected elk is expanding. Consequently, the d...

Comparative Proteome Analysis of Brucella melitensis Vaccine Strain Rev 1 and a Virulent Strain, 16M

PubMed Central

Eschenbrenner, Michel; Wagner, Mary Ann; Horn, Troy A.; Kraycer, Jo Ann; Mujer, Cesar V.; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G.

2002-01-01

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1. PMID:12193611

Comparative proteome analysis of Brucella melitensis vaccine strain Rev 1 and a virulent strain, 16M.

PubMed

Eschenbrenner, Michel; Wagner, Mary Ann; Horn, Troy A; Kraycer, Jo Ann; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G

2002-09-01

The genus Brucella consists of bacterial pathogens that cause brucellosis, a major zoonotic disease characterized by undulant fever and neurological disorders in humans. Among the different Brucella species, Brucella melitensis is considered the most virulent. Despite successful use in animals, the vaccine strains remain infectious for humans. To understand the mechanism of virulence in B. melitensis, the proteome of vaccine strain Rev 1 was analyzed by two-dimensional gel electrophoresis and compared to that of virulent strain 16M. The two strains were grown under identical laboratory conditions. Computer-assisted analysis of the two B. melitensis proteomes revealed proteins expressed in either 16M or Rev 1, as well as up- or down-regulation of proteins specific for each of these strains. These proteins were identified by peptide mass fingerprinting. It was found that certain metabolic pathways may be deregulated in Rev 1. Expression of an immunogenic 31-kDa outer membrane protein, proteins utilized for iron acquisition, and those that play a role in sugar binding, lipid degradation, and amino acid binding was altered in Rev 1.

Nylon bead enzyme-linked immunosorbent assay for detection of sub-picogram quantities of Brucella antigens.

PubMed Central

Perera, V Y; Creasy, M T; Winter, A J

1983-01-01

An indirect sandwich enzyme-linked immunosorbent assay, using antibody covalently coupled to nylon beads, has been adapted for the detection of Brucella antigens. Optimum conditions were achieved by incubation of 1 ml of reaction mixture with a single bead, and by minimizing nonspecific interactions through the use of beads coated with purified bovine antibodies, preabsorption of third layer rabbit antibodies with normal bovine serum, and treatment of beads with normal goat serum before addition of the goat anti-rabbit enzyme conjugate. Beta-galactosidase was selected for use with clinical samples primarily because of low levels of endogenous enzyme in bovine leukocytes. Use of a fluorogenic substrate enhanced sensitivity 20-fold. Under these conditions, 100 fg of solubilized crude lipopolysaccharide or 8 to 10 Brucella cells was detectable in a fixed volume of 1 ml. A system was also devised for concentrating antigen which permitted ready detection of 2 pg of lipopolysaccharide in a volume of 50 ml (40 fg/ml). Attempts to detect lipopolysaccharide in the presence of concentrated serum or plasma were unsuccessful, but 10 brucellae added to a suspension of leukocytes from 100 ml of normal bovine blood were easily measured. PMID:6415094

Serological survey for Brucella antibodies in donkeys of north-eastern Nigeria.

PubMed

Tijjani, A O; Junaidu, A U; Salihu, M D; Farouq, A A; Faleke, O O; Adamu, S G; Musa, H I; Hambali, I U

2017-08-01

A cross-sectional epidemiological study was conducted to determine seroprevalence and risk factors influencing the presence of Brucella antibodies in donkeys of Borno State, north-eastern Nigeria. The study aimed at providing baseline information that may be used in planning a control policy against equine brucellosis. Blood samples were collected from 601 donkeys, comprised of 374 males and 227 females from the six agricultural zones of the state between March 2013 and September 2014. The sera obtained were tested for Brucella antibodies using Rose Bengal plate test (RBPT) and competitive enzyme-linked immunosorbent assay (cELISA). Of the 601 donkeys tested, 43 (7.2%) and 40 (6.7%) were seropositive by RBPT and cELISA, respectively. A seroprevalence of 8.6% was obtained in male and 3.5% in female donkeys. According to age, the highest seroprevalence of 9.6% was obtained from donkeys of age group 4-6 years. With respect to pregnancy status, a higher seroprevalence (6.8%) was obtained from pregnant donkeys compared to 3.8% obtained from the non-pregnant ones. There were statistically significant associations between the presence of antibodies and sex (p < 0.05) and the presence of antibodies and age (p < 0.05) of the studied donkeys. However, no statistically significant association (p > 0.05) was observed between the pregnancy status and presence of antibodies. The study concludes that Brucella infection is present in donkeys in all the agricultural zones of the state. The relatively high seroprevalence (7.2%) obtained is of public health concern because of the close interaction between donkeys, ruminants, and humans in the study area.

Species identification and molecular typing of human Brucella isolates from Kuwait.

PubMed

Mustafa, Abu S; Habibi, Nazima; Osman, Amr; Shaheed, Faraz; Khan, Mohd W

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90-99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region.

Species identification and molecular typing of human Brucella isolates from Kuwait

PubMed Central

Osman, Amr; Shaheed, Faraz; Khan, Mohd W.

2017-01-01

Brucellosis is a zoonotic disease of major concern in Kuwait and the Middle East. Human brucellosis can be caused by several Brucella species with varying degree of pathogenesis, and relapses are common after apparently successful therapy. The classical biochemical methods for identification of Brucella are time-consuming, cumbersome, and provide information limited to the species level only. In contrast, molecular methods are rapid and provide differentiation at intra-species level. In this study, four molecular methods [16S rRNA gene sequencing, real-time PCR, enterobacterial repetitive intergenic consensus (ERIC)-PCR and multilocus variable-number tandem-repeat analysis (MLVA)-8, MLVA-11 and MLVA-16 were evaluated for the identification and typing of 75 strains of Brucella isolated in Kuwait. 16S rRNA gene sequencing of all isolates showed 90–99% sequence identity with B. melitensis and real-time PCR with genus- and species- specific primers identified all isolates as B. melitensis. The results of ERIC-PCR suggested the existence of 75 ERIC genotypes of B. melitensis with a discriminatory index of 0.997. Cluster classification of these genotypes divided them into two clusters, A and B, diverging at ~25%. The maximum number of genotypes (n = 51) were found in cluster B5. MLVA-8 analysis identified all isolates as B. melitensis, and MLVA-8, MLVA-11 and MLVA-16 typing divided the isolates into 10, 32 and 71 MLVA types, respectively. Furthermore, the combined minimum spanning tree analysis demonstrated that, compared to MLVA types discovered all over the world, the Kuwaiti isolates were a distinct group of MLVA-11 and MLVA-16 types in the East Mediterranean Region. PMID:28800594

Brucella taxonomy and evolution

PubMed Central

Ficht, Thomas

2010-01-01

Taxonomy and nomenclature represent man-made systems designed to enhance understanding of the relationship between organisms by comparison of discrete sets of properties. Initial efforts at bacterial taxonomy were flawed as a result of the previous use of nonsystematic approaches including common names resulting in confusing and inaccurate nomenclature. A decision was made to start afresh with bacterial nomenclature and to avoid the hazards experienced in the taxonomic classification of higher organisms. This was achieved by developing new rules designed to simplify classification and avoid unnecessary and confusing changes. This article reviews the work of a number of scientists attempting to reconcile new molecular data describing the phylogenetic relationship between Brucella organisms and a broader family of organisms with widely variant phenotypes that include human virulence and host range against a backdrop of strict regulatory requirements that fail to recognize significant differences between organisms with similar nomenclature. PMID:20521932

LOV Histidine Kinase Modulates the General Stress Response System and Affects the virB Operon Expression in Brucella abortus

PubMed Central

Sycz, Gabriela; Carrica, Mariela Carmen; Tseng, Tong-Seung; Bogomolni, Roberto A.; Briggs, Winslow R.; Goldbaum, Fernando A.; Paris, Gastón

2015-01-01

Brucella is the causative agent of the zoonotic disease brucellosis, and its success as an intracellular pathogen relies on its ability to adapt to the harsh environmental conditions that it encounters inside the host. The Brucella genome encodes a sensor histidine kinase containing a LOV domain upstream from the kinase, LOVHK, which plays an important role in light-regulated Brucella virulence. In this report we study the intracellular signaling pathway initiated by the light sensor LOVHK using an integrated biochemical and genetic approach. From results of bacterial two-hybrid assays and phosphotransfer experiments we demonstrate that LOVHK functionally interacts with two response regulators: PhyR and LovR, constituting a functional two-component signal-transduction system. LOVHK contributes to the activation of the General Stress Response (GSR) system in Brucella via PhyR, while LovR is proposed to be a phosphate-sink for LOVHK, decreasing its phosphorylation state. We also show that in the absence of LOVHK the expression of the virB operon is down-regulated. In conclusion, our results suggest that LOVHK positively regulates the GSR system in vivo, and has an effect on the expression of the virB operon. The proposed regulatory network suggests a similar role for LOVHK in other microorganisms. PMID:25993430

Brucella pelvic tubo-ovarian abscess mimicking a pelvic malignancy.

PubMed

Seoud, Muhiedine A F; Kanj, Suha S; Habli, Munira; Araj, George F; Khalil, Ali M

2003-01-01

A 57-y-old woman presented with recurrent abdominal and pelvic pain of 6 months' duration with low-grade fever. A computed tomographic scan indicated an ovarian tumor. Laparotomy revealed a pelvic abscess. Her symptoms resolved following surgery and antibiotic therapy. Pathology revealed an extensive inflammatory process. Tissue culture grew Brucella sp. The diagnosis and management of this previously undescribed pelvic tubo-ovarian abscess present a particular challenge.

Development and assessment of multiplex high resolution melting assay as a tool for rapid single-tube identification of five Brucella species.

PubMed

Gopaul, Krishna K; Sells, Jessica; Lee, Robin; Beckstrom-Sternberg, Stephen M; Foster, Jeffrey T; Whatmore, Adrian M

2014-12-11

The zoonosis brucellosis causes economically significant reproductive problems in livestock and potentially debilitating disease of humans. Although the causative agent, organisms from the genus Brucella, can be differentiated into a number of species based on phenotypic characteristics, there are also significant differences in genotype that are concordant with individual species. This paper describes the development of a five target multiplex assay to identify five terrestrial Brucella species using real-time polymerase chain reaction (PCR) and subsequent high resolution melt curve analysis. This technology offers a robust and cost effective alternative to previously described hydrolysis-probe Single Nucleotide Polymorphism (SNP)-based species defining assays. Through the use of Brucella whole genome sequencing five species defining SNPs were identified. Individual HRM assays were developed to these target these changes and, following optimisation of primer concentrations, it was possible to multiplex all five assays in a single tube. In a validation exercise using a panel of 135 Brucella strains of terrestrial and marine origin, it was possible to distinguish the five target species from the other species within this panel. The HRM multiplex offers a number of diagnostic advantages over previously described SNP-based typing approaches. Further, and uniquely for HRM, the successful multiplexing of five assays in a single tube allowing differentiation of five Brucella species in the diagnostic laboratory in a cost-effective and timely manner is described. However there are possible limitations to using this platform on DNA extractions direct from clinical material.

The bovine immune response to Brucella abortus I. A water soluble antigen precipitated by sera of some naturally infected cattle.

PubMed Central

Stemshorn, B; Nielsen, K

1977-01-01

Selected sera from cattle naturally infected with Brucella abortus precipitate water soluble antigens extracted by sonication from B. abortus. One of these antigens resembles antigen E (Baughn and Freeman) as it is excluded from Sephadex G-200 gels, migrates anodally when electrophoresed at pH 8.6, resists heating at 100 degrees C for ten minutes and appears to be susceptible to papain digestion. Precipitins specific for this antigen remained in sera from which all detectable Brucella agglutinating antibody had been removed by adsorption with live or heat killed B. abortus. The antigen has been extracted from smooth and rough strains of B abortus. Precipitins specific for this antigen have been detected in antisera produced against Brucella canis. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. PMID:405088

Detection of Brucella sp. infection through serological, microbiological, and molecular methods applied to buffaloes in Maranhão State, Brazil.

PubMed

Dos Santos, Larissa Sarmento; Sá, Joicy Cortez; Dos Santos Ribeiro, Diego Luiz; Chaves, Nancyleni Pinto; da Silva Mol, Juliana Pinto; Santos, Renato Lima; da Paixão, Tatiane Alves; de Carvalho Neta, Alcina Vieira

2017-04-01

The aim of the current study is to diagnose Brucella spp. infection using methods such as serology, bacterial isolation, and molecular analysis in buffaloes bred in Maranhão State. In order to do so, 390 samples of buffalo serum were subjected to serological tests, to Rose Bengal Plate Test (RBPT) and to 2-mercaptoethanol (2-ME) combined with slow agglutination test (SAT). Vaginal swabs were collected from seropositive animals and subjected to bacterial isolation and to generic PCR. According to the serological test, 16 animals had a positive reaction to the confirmatory test (2-ME/SAT). As for bacterial isolation, three samples resulted in the isolation of Brucella spp.-characteristic colonies, which were confirmed through PCR. These results confirmed Brucella spp. infection in the buffalo herd from Maranhão State.

Brucella melitensis infection within warthin tumor of the parotid gland.

PubMed

Horasan, Elif Sahin; Vaysoğlu, Yusuf; Unal, Murat; Uğuz, Mustafa; Kaya, Ali

2011-09-01

Brucellosis is a zoonotic systemic infectious disease, and multiorgan involvement is commonly seen, but involvement of the neck is a rare presentation of brucellosis. Granulomatous infections of the parotid gland are extremely rare. Warthin tumor is a well-known benign neoplasm of the salivary glands. In this report, we describe a Warthin tumor associated with Brucella melitensis in the same parotid gland.

Biosafety concerns related to Brucella and its potential use as a bioweapon

USDA-ARS?s Scientific Manuscript database

Brucellosis is endemic throughout the world and causes approximately 500,000 new human infections worldwide annually. Brucella melitensis is the most virulent species in humans and is associated with the majority of human infections under field and laboratory conditions. In the U.S., billions have ...

Entrance and Survival of Brucella pinnipedialis Hooded Seal Strain in Human Macrophages and Epithelial Cells

PubMed Central

Briquemont, Benjamin; Sørensen, Karen K.; Godfroid, Jacques

2013-01-01

Marine mammal Brucella spp. have been isolated from pinnipeds (B. pinnipedialis) and cetaceans (B. ceti) from around the world. Although the zoonotic potential of marine mammal brucellae is largely unknown, reports of human disease exist. There are few studies of the mechanisms of bacterial intracellular invasion and multiplication involving the marine mammal Brucella spp. We examined the infective capacity of two genetically different B. pinnipedialis strains (reference strain; NTCT 12890 and a hooded seal isolate; B17) by measuring the ability of the bacteria to enter and replicate in cultured phagocytes and epithelial cells. Human macrophage-like cells (THP-1), two murine macrophage cell lines (RAW264.7 and J774A.1), and a human malignant epithelial cell line (HeLa S3) were challenged with bacteria in a gentamicin protection assay. Our results show that B. pinnipedialis is internalized, but is then gradually eliminated during the next 72 – 96 hours. Confocal microscopy revealed that intracellular B. pinnipedialis hooded seal strain colocalized with lysosomal compartments at 1.5 and 24 hours after infection. Intracellular presence of B. pinnipedialis hooded seal strain was verified by transmission electron microscopy. By using a cholesterol-scavenging lipid inhibitor, entrance of B. pinnipedialis hooded seal strain in human macrophages was significantly reduced by 65.8 % (± 17.3), suggesting involvement of lipid-rafts in intracellular entry. Murine macrophages invaded by B. pinnipedialis do not release nitric oxide (NO) and intracellular bacterial presence does not induce cell death. In summary, B. pinnipedialis hooded seal strain can enter human and murine macrophages, as well as human epithelial cells. Intracellular entry of B. pinnipedialis hooded seal strain involves, but seems not to be limited to, lipid-rafts in human macrophages. Brucella pinnipedialis does not multiply or survive for prolonged periods intracellulary. PMID:24376851

Seroprevalence for Coxiella burnetii, Francisella tularensis, Brucella abortus and Brucella melitensis in Austrian adults: a cross-sectional survey among military personnel and civilians.

PubMed

Tobudic, Selma; Nedomansky, Klara; Poeppl, Wolfgang; Müller, Maria; Faas, Angelus; Mooseder, Gerhard; Allerberger, Franz; Stanek, Gerold; Burgmann, Heinz

2014-04-01

The prevalence of Coxiella burnetii, Francisella tularensis, Brucella abortus, and Brucella melitensis infections in Austria and the exposure risk of military personnel were assessed in an exploratory nationwide cross-sectional seroprevalence survey in 526 healthy adult individuals, 222 of which were soldiers and 304 were civilians. Screening for IgA/IgG antibodies to C. burnetii (Phase I) and IgG/IgM antibodies to C. burnetii (Phase II), and to F. tularensis was done with commercial enzyme-linked immunosorbent assays. To detect antibodies against B. abortus and B. melitensis, an in-house complement fixation test was used. Overall, 11 individuals (2.0%) showed antibodies to C. burnetii, 3 individuals (0.5%) were seropositive for F. tularensis, and one (0.3%) individual was borderline positive. All individuals positive or borderline for F. tularensis tested negative for antibodies against C. burnetii. All individuals tested negative for antibodies against B. melitensis/B. abortus. There were no significant differences between the seroprevalence of C. burnetii and F. tularensis among military personnel and civilians. Our data demonstrate serological evidence of a low rate of exposure to C. burnetii and F. tularensis among the Austrian adult population and military personnel. Copyright © 2014 Elsevier GmbH. All rights reserved.

Brucella melitensis and Mycobacterium tuberculosis depict overlapping gene expression patterns induced in infected THP-1 macrophages.

PubMed

Masoudian, M; Derakhshandeh, A; Ghahramani Seno, M M

2015-01-01

Pathogens infecting mammalian cells have developed various strategies to suppress and evade their hosts' defensive mechanisms. In this line, the intracellular bacteria that are able to survive and propagate within their host cells must have developed strategies to avert their host's killing attitude. Studying the interface of host-pathogen confrontation can provide valuable information for defining therapeutic approaches. Brucellosis, caused by the Brucella strains, is a zoonotic bacterial disease that affects thousands of humans and animals around the world inflicting discomfort and huge economic losses. Similar to many other intracellular dwelling bacteria, infections caused by Brucella are difficult to treat, and hence any attempt at identifying new and common therapeutic targets would prove beneficial for the purpose of curing infections caused by the intracellular bacteria. In THP-1 macrophage infected with Brucella melitensis we studied the expression levels of four host's genes, i.e. EMP2, ST8SIA4, HCP5 and FRMD5 known to be involved in pathogenesis of Mycobacterium tuberculosis. Our data showed that at this molecular level, except for FRMD5 that was downregulated, the other three genes were upregulated by B. melitensis. Brucella melitensis and M. tuberculosis go through similar intracellular processes and interestingly two of the investigated genes, i.e. EMP2 and ST4SIA8 were upregulated in THP-1 cell infected with B. melitensis similar to that reported for THP-1 cells infected with M. tuberculosis. At the host-pathogen interaction interface, this study depicts overlapping changes for different bacteria with common survival strategies; a fact that implies designing therapeutic approaches based on common targets may be possible.

Enabling comparative modeling of closely related genomes: Example genus Brucella

DOE PAGES

Faria, José P.; Edirisinghe, Janaka N.; Davis, James J.; ...

2014-03-08

For many scientific applications, it is highly desirable to be able to compare metabolic models of closely related genomes. In this study, we attempt to raise awareness to the fact that taking annotated genomes from public repositories and using them for metabolic model reconstructions is far from being trivial due to annotation inconsistencies. We are proposing a protocol for comparative analysis of metabolic models on closely related genomes, using fifteen strains of genus Brucella, which contains pathogens of both humans and livestock. This study lead to the identification and subsequent correction of inconsistent annotations in the SEED database, as wellmore » as the identification of 31 biochemical reactions that are common to Brucella, which are not originally identified by automated metabolic reconstructions. We are currently implementing this protocol for improving automated annotations within the SEED database and these improvements have been propagated into PATRIC, Model-SEED, KBase and RAST. This method is an enabling step for the future creation of consistent annotation systems and high-quality model reconstructions that will support in predicting accurate phenotypes such as pathogenicity, media requirements or type of respiration.« less

Enabling comparative modeling of closely related genomes: Example genus Brucella

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

Faria, José P.; Edirisinghe, Janaka N.; Davis, James J.

For many scientific applications, it is highly desirable to be able to compare metabolic models of closely related genomes. In this study, we attempt to raise awareness to the fact that taking annotated genomes from public repositories and using them for metabolic model reconstructions is far from being trivial due to annotation inconsistencies. We are proposing a protocol for comparative analysis of metabolic models on closely related genomes, using fifteen strains of genus Brucella, which contains pathogens of both humans and livestock. This study lead to the identification and subsequent correction of inconsistent annotations in the SEED database, as wellmore » as the identification of 31 biochemical reactions that are common to Brucella, which are not originally identified by automated metabolic reconstructions. We are currently implementing this protocol for improving automated annotations within the SEED database and these improvements have been propagated into PATRIC, Model-SEED, KBase and RAST. This method is an enabling step for the future creation of consistent annotation systems and high-quality model reconstructions that will support in predicting accurate phenotypes such as pathogenicity, media requirements or type of respiration.« less

Brucella ceti infection in dolphins from the Western Mediterranean sea.

PubMed

Isidoro-Ayza, Marcos; Ruiz-Villalobos, Nazareth; Pérez, Lola; Guzmán-Verri, Caterina; Muñoz, Pilar M; Alegre, Fernando; Barberán, Montserrat; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; González-Barrientos, Rocio; Moreno, Edgardo; Blasco, José María; Domingo, Mariano

2014-09-17

Brucella ceti infections have been increasingly reported in cetaceans. Brucellosis in these animals is associated with meningoencephalitis, abortion, discospondylitis', subcutaneous abscesses, endometritis and other pathological conditions B. ceti infections have been frequently described in dolphins from both, the Atlantic and Pacific Oceans. In the Mediterranean Sea, only two reports have been made: one from the Italian Tyrrhenian Sea and the other from the Adriatic Sea. We describe the clinical and pathological features of three cases of B. ceti infections in three dolphins stranded in the Mediterranean Catalonian coast. One striped dolphin had neurobrucellosis, showing lethargy, incoordination and lateral swimming due to meningoencephalitis, A B. ceti infected bottlenose dolphin had discospondylitis, and another striped dolphin did not show clinical signs or lesions related to Brucella infection. A detailed characterization of the three B. ceti isolates was performed by bacteriological, molecular, protein and fatty acid analyses. All the B. ceti strains originating from Mediterranean dolphins cluster together in a distinct phylogenetic clade, close to that formed by B. ceti isolates from dolphins inhabiting the Atlantic Ocean. Our study confirms the severity of pathological signs in stranded dolphins and the relevance of B. ceti as a pathogen in the Mediterranean Sea.

Detection of Brucella spp. in milk from seronegative cows by real-time polymerase chain reaction in the region of Batna, Algeria

PubMed Central

Sabrina, Rabehi; Mossadak, Hamdi Taha; Bakir, Mamache; Asma, Meghezzi; Khaoula, Boushaba

2018-01-01

Aim: The aim of this study was to detect Brucella spp. DNA in milk samples collected from seronegative cows using the real-time polymerase chain reaction (PCR) assay for diagnosis of brucellosis in seronegative dairy cows to prevent transmission of disease to humans and to reduce economic losses in animal production. Materials and Methods: In this study, 65 milk samples were investigated for the detection of Brucella spp. The detection of the IS711 gene in all samples was done by real-time PCR assay by comparative cycle threshold method. Results: The results show that of the 65 DNA samples tested, 2 (3.08%) were positive for Brucella infection. The mean cyclic threshold values of IS711 real-time PCR test were 37.97 and 40.48, indicating a positive reaction. Conclusion: The results of the present study indicated that the real-time PCR appears to offer several advantages over serological tests. For this reason, the real-time PCR should be validated on representative numbers of Brucella-infected and free samples before being implemented in routine diagnosis in human and animal brucellosis for controlling this disease. PMID:29657430

GLUTAMATE METABOLISM IN BRUCELLA ABORTUS STRAINS OF LOW AND HIGH VIRULENCE

PubMed Central

Dasinger, B. L.; Wilson, J. B.

1962-01-01

Dasinger, B. L. (University of Wisconsin, Madison) and J. B. Wilson. Glutamate metabolism in Brucella abortus strains of low and high virulence. J. Bacteriol. 84:911–915. 1962.—Brucella abortus strains of low virulence oxidize glutamate at a high rate, whereas strains of high virulence oxidize glutamate at a relatively low rate. Results indicated that this observation was not related to differences in pathway of glutamate oxidation or to differences in total enzyme activity. Permeability studies showed that the maximal rates of glutamate accumulation were 2.8 μmoles per 2 min per g (wet wt) for a strain of high virulence and 6.2 μmoles per 2 min per g for a strain of low virulence, but equal intracellular steady-state concentrations were attained by both types of strains. Evidence is presented which suggests that the site of glutamate oxidation is separate from the pool of glutamate being measured. Unequal rates of permeability at these sites could be the reason for the differences in rate of glutamate oxidation. PMID:14028057

The Bactec FX Blood Culture System Detects Brucella melitensis Bacteremia in Adult Patients within the Routine 1-Week Incubation Period.

PubMed

Sagi, Moshe; Nesher, Lior; Yagupsky, Pablo

2017-03-01

The performance of the Bactec FX blood culture system for detecting Brucella bacteremia within the routine 1-week incubation period was assessed in a prospective study conducted in an area in southern Israel in which Brucella melitensis is endemic. Aerobic vials (BD Bactec Plus Aerobic/F medium) inoculated with blood specimens obtained from adult patients with positive Rose-Bengal screening test results were monitored for 4 consecutive weeks, and blind subcultures of negative vials were performed on solid media on days 7 and 28. During a 16-month period, a total of 31 (35.2%) of 88 cultures, obtained from 19 (38.0%) of 50 patients, were positive for Brucella melitensis The blood culture instrument identified 30 (96.8%) of 31 positive vials within 7 days of incubation; the single positive vial that was missed by the automated readings was detected only by the blind subculture performed on day 28. It is concluded that the Bactec FX system is able to detect the vast majority of episodes of Brucella bacteremia within the 1-week incubation protocol instituted in most clinical microbiology laboratories and without the need to perform blind subcultures of negative vials, enabling early diagnosis and saving labor and incubation time and space. Copyright © 2017 American Society for Microbiology.

MyD88 and STING Signaling Pathways Are Required for IRF3-Mediated IFN-β Induction in Response to Brucella abortus Infection

PubMed Central

de Almeida, Leonardo A.; Carvalho, Natalia B.; Oliveira, Fernanda S.; Lacerda, Thais L. S.; Vasconcelos, Anilton C.; Nogueira, Lucas; Bafica, Andre; Silva, Aristóbolo M.; Oliveira, Sergio C.

2011-01-01

Type I interferons (IFNs) are cytokines that orchestrate diverse immune responses to viral and bacterial infections. Although typically considered to be most important molecules in response to viruses, type I IFNs are also induced by most, if not all, bacterial pathogens. In this study, we addressed the role of type I IFN signaling during Brucella abortus infection, a facultative intracellular bacterial pathogen that causes abortion in domestic animals and undulant fever in humans. Herein, we have shown that B. abortus induced IFN-β in macrophages and splenocytes. Further, IFN-β induction by Brucella was mediated by IRF3 signaling pathway and activates IFN-stimulated genes via STAT1 phosphorylation. In addition, IFN-β expression induced by Brucella is independent of TLRs and TRIF signaling but MyD88-dependent, a pathway not yet described for Gram-negative bacteria. Furthermore, we have identified Brucella DNA as the major bacterial component to induce IFN-β and our study revealed that this molecule operates through a mechanism dependent on RNA polymerase III to be sensed probably by an unknown receptor via the adaptor molecule STING. Finally, we have demonstrated that IFN-αβR KO mice are more resistant to infection suggesting that type I IFN signaling is detrimental to host control of Brucella. This resistance phenotype is accompanied by increased IFN-γ and NO production by IFN-αβR KO spleen cells and reduced apoptosis. PMID:21829705

Innocuity and immune response to Brucella melitensis Rev.1 vaccine in camels (Camelus dromedarius)

PubMed Central

Benkirane, A.; Idrissi, A.H. El; Doumbia, A.; de Balogh, K.

2014-01-01

A field trial was conducted in a camel brucellosis-free herd to evaluate antibody response to the Brucella melitensis Rev.1 vaccine in camels and assess shedding of the vaccine strain in milk. Twenty eight camels were divided into four groups according to their age and vaccination route. Groups A (n=3) and B (n=3) consisted of non-pregnant lactating female camels, vaccinated through subcutaneous and conjunctival routes, respectively. Groups C (n=10) consisted of 8-11 months old calves vaccinated through conjunctival route. The rest of the herd (n=12) composed of female and young camels were not vaccinated and were considered as the control group. Each animal from groups A, B and C was given the recommended dose of 2 × 109 colony forming units of Rev.1 vaccine irrespective of age or route of vaccination. Blood samples were collected from all the animals at the time of vaccination and at weekly, bi-weekly and monthly interval until 32 weeks post vaccination and from controls at weeks 8 and 24. The serological tests used were modified Rose Bengal Test, sero-agglutination test, and an indirect Enzyme Linked Immunosorbent Assay. Milk samples were collected from all vaccinated female camels and tested for the presence of Rev.1 vaccine strain. Most vaccinated animals started to show an antibody response at week 2 and remained positive until week 16. By week 20 post-vaccination all animals in the three groups were tested negative for Brucella antibodies. Bacteriological analysis of milk samples did not allow any isolation of Brucella melitensis. All samples were found Brucella negative in PCR analysis. The results of this study indicate that the Rev.1 vaccine induces seroconversion in camels. Rev.1 vaccine strain is not excreted in the milk of camels. These findings are promising as to the safe use of the Rev.1 vaccine in camels. PMID:26623347

Novel vector vaccine against Brucella abortus based on influenza A viruses expressing Brucella L7/L12 or Omp16 proteins: evaluation of protection in pregnant heifers.

PubMed

Tabynov, Kaissar; Yespembetov, Bolat; Sansyzbay, Abylai

2014-10-14

The present study provides the first information about the protection of a novel influenza viral vector vaccine expressing the Brucella proteins ribosomal L7/L12 or Omp16 containing the adjuvant Montanide Gel01 in pregnant heifers. Immunization of pregnant heifers was conducted via the conjunctival (n=10) or subcutaneous (n=10) route using cross prime and booster vaccination schedules at an interval of 28 days. The vector vaccine was evaluated in comparison with positive control groups vaccinated with Brucella abortus S19 (n=10) or B. abortus RB51 (n=10) and a negative (PBS+Montanide Gel01; n=10) control group. Via both the conjunctival or subcutaneous route, evaluation of protectiveness against abortion, effectiveness of vaccination and index of infection (in heifers and their fetuses or calves) demonstrated the vector vaccine provided good protection against B. abortus 544 infection compared to the negative control group (PBS+Montanide Gel01) and comparable protection to commercial vaccines B. abortus S19 or B. abortus RB51. Copyright © 2014 Elsevier Ltd. All rights reserved.

Proteomic Analysis of Detergent Resistant Membrane Domains during Early Interaction of Macrophages with Rough and Smooth Brucella melitensis

PubMed Central

Lauer, Sabine A.; Iyer, Srinivas; Sanchez, Timothy; Forst, Christian V.; Bowden, Brent; Carlson, Kay; Sriranganathan, Nammalwar; Boyle, Stephen M.

2014-01-01

The plasma membrane contains discrete nanometer-sized domains that are resistant to non-ionic detergents, and which are called detergent resistant membrane domains (DRMDs) or lipid rafts. Exposure of host cells to pathogenic bacteria has been shown to induce the re-distribution of specific host proteins between DRMDs and detergent soluble membranes, which leads to the initiation of cell signaling that enable pathogens to access host cells. DRMDs have been shown to play a role in the invasion of Brucella into host macrophages and the formation of replicative phagosomes called Brucella-containing vacuoles (BCVs). In this study we sought to characterize changes to the protein expression profiles in DRMDs and to respective cellular pathways and networks of Mono Mac 6 cells in response to the adherence of rough VTRM1 and smooth 16 M B. melitensis strains. DRMDs were extracted from Mono Mac 6 cells exposed for 2 minutes at 4°C to Brucella (no infection occurs) and from unexposed control cells. Protein expression was determined using the non-gel based quantitative iTRAQ (Isobaric Tags for Relative and Absolute Quantitation) mass spectrometry technique. Using the identified iTRAQ proteins we performed enrichment analyses and probed constructed human biochemical networks for interactions and metabolic reactions. We identified 149 proteins, which either became enriched, depleted or whose amounts did not change in DRMDs upon Brucella exposure. Several of these proteins were distinctly enriched or depleted in DRMDs upon exposure to rough and smooth B. melitensis strains which results in the differential engagement of cellular pathways and networks immediately upon Brucella encounter. For some of the proteins such as myosin 9, small G protein signaling modulator 3, lysine-specific demethylase 5D, erlin-2, and voltage-dependent anion-selective channel protein 2, we observed extreme differential depletion or enrichment in DRMDs. The identified proteins and pathways could provide

Comparison of Abortion and Infection after Experimental Challenge of Pregnant Bison and Cattle with Brucella abortus Strain 2308▿

PubMed Central

Olsen, S. C.; Johnson, C.

2011-01-01

A comparative study was conducted using data from naive bison (n = 45) and cattle (n = 46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered during midgestation. The incidence of abortion, fetal infection, uterine or mammary infection, or infection in maternal tissues after experimental challenge was greater (P < 0.05) in bison than in cattle. In animals that did abort, the time between experimental challenge and abortion was shorter (P < 0.05) for bison than for cattle. Brucella colonization of four target tissues and serologic responses on the standard tube agglutination test at the time of abortion did not differ (P > 0.05) between cattle and bison. The results of our study suggest that naive bison and cattle have similarities and differences after experimental exposure to a virulent B. abortus strain. Although our data suggest that bison may be more susceptible to infection with Brucella, some pathogenic characteristics of brucellosis were similar between bison and cattle. PMID:21976222

Treatment of a subdural empyema complicated by intracerebral abscess due to Brucella infection

PubMed Central

Zhang, J.; Chen, Z.; Xie, L.; Zhao, C.; Zhao, H.; Fu, C.; Chen, G.; Hao, Z.; Wang, L.; Li, W.

2017-01-01

A 55-year-old male presented with fever, stupor, aphasia, and left hemiparesis. A history of head trauma 3 months before was also reported. Cranial magnetic resonance imaging revealed slight contrast enhancement of lesions under the right frontal skull plate and right frontal lobe. Because of deterioration in nutritional status and intracranial hypertension, the patient was prepared for burr hole surgery. A subdural empyema (SDE) recurred after simple drainage. After detection of Brucella species in SDE, craniotomy combined with antibiotic treatment was undertaken. The patient received antibiotic therapy for 6 months (two doses of 2 g ceftriaxone, two doses of 100 mg doxycycline, and 700 mg rifapentine for 6 months) that resulted in complete cure of the infection. Thus, it was speculated that the preexisting subdural hematoma was formed after head trauma, which was followed by a hematogenous infection caused by Brucella species. PMID:28380194

Evaluation of Brucella abortus strain RB51 and strain 19 in pronghorn antelope

USGS Publications Warehouse

Elzer, P.H.; Smith, J.; Roffe, T.; Kreeger, T.; Edwards, J.; Davis, D.

2002-01-01

Free-roaming elk and bison in the Greater Yellowstone Area remain the only wildlife reservoirs for Brucella abortus in the United States, and the large number of animals and a lack of holding facilities make it unreasonable to individually vaccinate each animal. Therefore, oral delivery is being proposed as a possible option to vaccinate these wild ungulates. One of the main problems associated with oral vaccination is the potential exposure of nontarget species to the vaccines. The purpose of this study was to determine the effects of two Brucella vaccines, strain 19 (S19) and the rough strain RB51 (SRB51), in pregnant pronghorn antelope. We conclude that S19 and SRB51 rarely colonize maternal and fetal tissues of pregnant pronghorn and were not associated with fetal death. Oral delivery of either vaccine at this dose appears to be nonhazardous to pregnant pronghorn.

Brucella abortus Strain 2308 Wisconsin Genome: Importance of the Definition of Reference Strains

PubMed Central

Suárez-Esquivel, Marcela; Ruiz-Villalobos, Nazareth; Castillo-Zeledón, Amanda; Jiménez-Rojas, César; Roop II, R. Martin; Comerci, Diego J.; Barquero-Calvo, Elías; Chacón-Díaz, Carlos; Caswell, Clayton C.; Baker, Kate S.; Chaves-Olarte, Esteban; Thomson, Nicholas R.; Moreno, Edgardo; Letesson, Jean J.; De Bolle, Xavier; Guzmán-Verri, Caterina

2016-01-01

Brucellosis is a bacterial infectious disease affecting a wide range of mammals and a neglected zoonosis caused by species of the genetically homogenous genus Brucella. As in most studies on bacterial diseases, research in brucellosis is carried out by using reference strains as canonical models to understand the mechanisms underlying host pathogen interactions. We performed whole genome sequencing analysis of the reference strain B. abortus 2308 routinely used in our laboratory, including manual curated annotation accessible as an editable version through a link at https://en.wikipedia.org/wiki/Brucella#Genomics. Comparison of this genome with two publically available 2308 genomes showed significant differences, particularly indels related to insertional elements, suggesting variability related to the transposition of these elements within the same strain. Considering the outcome of high resolution genomic techniques in the bacteriology field, the conventional concept of strain definition needs to be revised. PMID:27746773

A case of acute septic arthritis hip caused by Brucella melitensis in an adolescent child.

PubMed

Jalan, Divesh; Elhence, Abhay; Elhence, Poonam; Jain, Princi

2015-09-21

Brucella is among the most common zoonotic diseases affecting humans. Although musculoskeletal involvement is seen in a large proportion of patients, the disease is often diagnosed late or misdiagnosed due to its subtle nature and rarity, and lack of awareness among clinicians. In this report, a 12-year-old girl was diagnosed with acute septic arthritis of the hip based on clinico-radiological features, and managed with standard treatment, including arthrotomy. However, the child did not respond to the treatment. Based on the histopathology and local endemicity, Brucella was suspected, and confirmed after serological testing. The child subsequently responded to treatment and, at latest follow-up at 1 year, had a full painless range of motion, with no relapse. 2015 BMJ Publishing Group Ltd.

Multi-locus variable-number tandem repeat analysis of Chinese Brucella strains isolated from 1953 to 2013.

PubMed

Tian, Guo-Zhong; Cui, Bu-Yun; Piao, Dong-Ri; Zhao, Hong-Yan; Li, Lan-Yu; Liu, Xi; Xiao, Pei; Zhao, Zhong-Zhi; Xu, Li-Qing; Jiang, Hai; Li, Zhen-Jun

2017-05-02

Brucellosis was a common human and livestock disease caused by Brucella strains, the category B priority pathogens by the US Center for Disease Control (CDC). Identified as a priority disease in human and livestock populations, the increasing incidence in recent years in China needs urgent control measures for this disease but the molecular background important for monitoring the epidemiology of Brucella strains at the national level is still lacking. A total of 600 Brucella isolates collected during 60 years (from 1953 to 2013) in China were genotyped by multiple locus variable-number tandem repeat analysis (MLVA) and the variation degree of MLVA11 loci was calculated by the Hunter Gaston Diversity Index (HGDI) values. The charts and map were processed by Excel 2013, and cluster analysis and epidemiological distribution was performed using BioNumerics (version 5.1). The 600 representative Brucella isolates fell into 104 genotypes with 58 singleton genotypes by the MLVA11 assay, including B. melitensis biovars 2 and 3 (five main genotypes), B. abortus biovars 1 and 3 (two main genotypes), B. suis biovars 1 and 3 (three main genotypes), and B. canis (two main genotypes) respectively. While most B. suis biovar 1 and biovar 3 were respectively found in northern provinces and southern provinces, B. melitensis and B. abortus strains were dominant in China. Canine Brucellosis was only found in animals without any human cases reported. Eight Brucellosis epidemic peaks emerged during the 60 years between 1953 and 2013: 1955 - 1959, 1962 - 1969, 1971 - 1975, 1977 - 1983, 1985 - 1989, 1992 - 1997, 2000 - 2008 and 2010 - 2013 in China. Brucellosis has its unique molecular epidemiological patterns with specific spatial and temporal distribution according to MLVA. IDOP-D-16-00101.

Infections and risk factors for livestock with species of Anaplasma, Babesia and Brucella under semi-nomadic rearing in Karamoja Region, Uganda.

PubMed

Lolli, Chiara; Marenzoni, Maria Luisa; Strona, Paolo; Lappo, Pier Giorgio; Etiang, Patrick; Diverio, Silvana

2016-03-01

A survey was conducted to estimate the prevalence of Anaplasma, Babesia and Brucella spp. infections in cattle, goats and sheep in the Karamoja Region of Uganda and to identify possible risk factors existing in this semi-nomadic and pastoral area. Low cost laboratory tests were used to diagnose infections (Rose Bengal test for Brucella spp. antibodies and direct microscopic examination for Anaplasma and Babesia spp.). Multivariable logistic regression models were applied to identify possible risk factors linked to gender, animal species, age (only for cattle) and districts. A total of 3935 cattle, 729 goats and 306 sheep of five districts of the Karamoja Region were tested. Seroprevalence for Brucella was 9.2 % (CI, 95 %: 8.4-10), for Anaplasma 19.5 % (CI 95 %: 18.4-20.6) and for Babesia 16 % (CI 95 %: 15-17.1). Significant differences in infections prevalence were observed against risk factors associated with districts and species. Cattle were the species with higher risk of the infections. Female gender was identified as at risk only for Brucella spp. infection. Cattle more than one year old had greater likelihood to be Brucella seropositive. Co-infections of Anaplasma and Babesia spp. were statistically associated, especially in goats and sheep. Further studies to identify risk factors related to host species and geographical districts are needed. The influence on the semi-nomadic agro-pastoral system in Karamoja of animal raids and animal mixing should be further investigated. Findings were important to sensitize Karamojong undertaking measures on infection control, especially on cattle, which are their main source of food.

Brucella melitensis prosthetic joint infection in a traveller returning to the UK from Thailand: Case report and review of the literature.

PubMed

Lewis, Joseph M; Folb, Jonathan; Kalra, Sanjay; Squire, S Bertel; Taegtmeyer, Miriam; Beeching, Nick J

Brucella spp. prosthetic joint infections are infrequently reported in the literature, particularly in returning travellers, and optimal treatment is unknown. We describe a prosthetic joint infection (PJI) caused by Brucella melitensis in a traveller returning to the UK from Thailand, which we believe to be the first detailed report of brucellosis in a traveller returning from this area. The 23 patients with Brucella-related PJI reported in the literature are summarised, together with our case. The diagnosis of Brucella-related PJI is difficult to make; only 30% of blood cultures and 75% of joint aspiration cultures were positive in the reported cases. Culture of intraoperative samples provides the best diagnostic yield. In the absence of radiological evidence of joint loosening, combination antimicrobial therapy alone may be appropriate treatment in the first instance; this was successful in 6/7 [86%] of patients, though small numbers of patients and the likelihood of reporting bias warrant caution in drawing any firm conclusions about optimal treatment. Aerosolisation of synovial fluid during joint aspiration procedures and nosocomial infection has been described. Brucella-related PJI should be considered in the differential of travellers returning from endemic areas with PJI, including Thailand. Personal protective equipment including fit tested filtering face piece-3 (FFP3) mask or equivalent is recommended for personnel carrying out joint aspiration when brucellosis is suspected. Travellers can reduce the risk of brucellosis by avoiding unpasteurised dairy products and animal contact (particularly on farms and abattoirs) in endemic areas and should be counselled regarding these risks as part of their pre-travel assessment. Copyright © 2016 The Author(s). Published by Elsevier Ltd.. All rights reserved.

Experimental infection of chicken embryos with recently described Brucella microti: Pathogenicity and pathological findings.

PubMed

Wareth, Gamal; Böttcher, Denny; Melzer, Falk; Shehata, Awad Ali; Roesler, Uwe; Neubauer, Heinrich; Schoon, Heinz-Adolf

2015-08-01

Brucellae are facultative intracellular pathogens causing disease in a wide range of domestic and wild animals as well as in humans. Brucella (B.) microti is a recently recognized species and was isolated from common voles (Microtus arvalis), red foxes and soil in Austria and the Czech Republic. Its pathogenicity for livestock and its zoonotic potential has not been confirmed yet. In the present study 25 SPF chicken embryos were inoculated at day 11 of age with 1.6×10(3) and 1.6×10(5)B. microti by yolk sac and allantoic sac routes. Re-isolation of B. microti indicated rapid multiplication of bacteria (up to 1.7×10(12)CFU). B. microti provoked marked gross lesions, i.e. hemorrhages and necroses. All inoculated embryos were dead (100% mortality) in between 2nd and 4th day post inoculation. The predominant histopathological lesion was necroses in liver, kidneys, lungs, spleen, gastrointestinal tract, spinal meninges, yolk sac and chorioallantoic membrane. Immunohistochemical examination showed the presence of Brucella antigen in nearly all of these organs, with infection being mainly restricted to non-epithelial cells or tissues. This study provides the first results on the multiplication and pathogenicity of the mouse pathogenic B. microti in chicken embryos. These data suggest that, even though chicken are not mammals, they could provide a useful tool for understanding the pathogenesis of B. microti associated disease. Copyright © 2015 Elsevier Ltd. All rights reserved.

Purification and properties of Cu-Zn superoxide dismutase extracted from Brucella abortus strain 19

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

Tabatabai, L.B.

Recent work showed that a recombinant 20 kDa protein from Brucella abortus expressed in E. coli is a Cu-Zn superoxide dismutase (SOD). Western blot and ELISA results indicated that cattle with brucellosis have antibody to SOD. Here the authors report the purification and properties of the native B. abortus Cu-Zn SOD. SOD was extracted from methanol-killed Brucella abortus strain 19 with 0.1 M sodium citrate-1.0 M sodium chloride solution. The extract was dialyzed and protein precipitated by ammonium sulfate at 70-100% saturation was collected. The SOD was purified by HPLC anion exchange chromatography. SOD activity was assayed with a coupledmore » enzyme assay using xanthine oxidase-cytochrome C reduction assay. The authors determined that the Brucella SOD is present in two molecular forms both inhibitable with KCN with Ki's of 0.32 mM and 4.98 mM, respectively. No other form of SOD was identified in the extract. Polyclonal antibody to SOD and polyclonal antibody to SOD synthetic peptide residues 134-143 inhibited SOD activity by 50% and 13%, respectively. Both SOD and the synthetic peptide inhibited binding of anti-SOD antibody to SOD by 60% and 20%, respectively. Based on these results the SOD and its amphipathic peptide will be considered as candidates for the design of synthetic multiple peptide vaccines and diagnostic reagents for bovine brucellosis.« less

Attenuated Signature-Tagged Mutagenesis Mutants of Brucella melitensis Identified during the Acute Phase of Infection in Mice

PubMed Central

Lestrate, P.; Dricot, A.; Delrue, R.-M.; Lambert, C.; Martinelli, V.; De Bolle, X.; Letesson, J.-J.; Tibor, A.

2003-01-01

For this study, we screened 1,152 signature-tagged mutagenesis mutants of Brucella melitensis 16M in a mouse model of infection and found 36 of them to be attenuated in vivo. Molecular characterization of transposon insertion sites showed that for four mutants, the affected genes were only present in Rhizobiaceae. Another mutant contained a disruption in a gene homologous to mosA, which is involved in rhizopine biosynthesis in some strains of Rhizobium, suggesting that this sugar may be involved in Brucella pathogenicity. A mutant was disrupted in a gene homologous to fliF, a gene potentially coding for the MS ring, a basal component of the flagellar system. Surprisingly, a mutant was affected in the rpoA gene, coding for the essential α-subunit of the RNA polymerase. This disruption leaves a partially functional protein, impaired for the activation of virB transcription, as demonstrated by the absence of induction of the virB promoter in the Tn5::rpoA background. The results presented here highlight the fact that the ability of Brucella to induce pathogenesis shares similarities with the molecular mechanisms used by both Rhizobium and Agrobacterium to colonize their hosts. PMID:14638795

Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16

PubMed Central

2014-01-01

Background Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16. Findings MLVA16 genotyping identified thirteen distinct genotypes and a Hunter-Gaston diversity index of 0.989 among the fourteen B. ovis genotyped strains. All B. ovis MLVA16 genotypes observed in the present study represented non-previously described profiles. Analyses of the eight conserved loci included in panel 1 (MLVA8) showed three different genotypes, two new and one already described for B. ovis isolates. Among ten B. ovis isolates from same herd only two strains had identical pattern, whereas the four isolates with no epidemiologic information exhibited a single MLVA16 pattern each. Analysis of minimal spanning tree, constructed using the fourteen B. ovis strains typed in this study together with all nineteen B. ovis MLVA16 genotypes available in the MLVAbank 2014, revealed the existence of two clearly distinct major clonal complexes. Conclusions In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil, by MLVA16. PMID:25015223

Genetic diversity of Brucella ovis isolates from Rio Grande do Sul, Brazil, by MLVA16.

PubMed

Dorneles, Elaine M S; Freire, Guilherme N; Dasso, Maurício G; Poester, Fernando P; Lage, Andrey P

2014-07-12

Ovine epididymitis is predominantly associated with Brucella ovis infection. Molecular characterization of Brucella spp. achieved by multi-locus variable number of tandem repeats (VNTR) analyses (MLVA) have proved to be a powerful tool for epidemiological trace-back studies. Thus, the aim of this study was to evaluate the genetic diversity of Brucella ovis isolates from Rio Grande do Sul State, Brazil, by MLVA16. MLVA16 genotyping identified thirteen distinct genotypes and a Hunter-Gaston diversity index of 0.989 among the fourteen B. ovis genotyped strains. All B. ovis MLVA16 genotypes observed in the present study represented non-previously described profiles. Analyses of the eight conserved loci included in panel 1 (MLVA8) showed three different genotypes, two new and one already described for B. ovis isolates. Among ten B. ovis isolates from same herd only two strains had identical pattern, whereas the four isolates with no epidemiologic information exhibited a single MLVA16 pattern each. Analysis of minimal spanning tree, constructed using the fourteen B. ovis strains typed in this study together with all nineteen B. ovis MLVA16 genotypes available in the MLVAbank 2014, revealed the existence of two clearly distinct major clonal complexes. In conclusion, the results of the present study showed a high genetic diversity among B. ovis field isolates from Rio Grande do Sul State, Brazil, by MLVA16.

Molecular Detection of Brucella spp. from Milk of Seronegative Cows from Some Selected Area in Bangladesh

PubMed Central

Islam, Md. Ariful; Khatun, Mst. Minara; Saha, Sukumar; Basir, Md. Samiul; Hasan, Md- Mahmodul

2018-01-01

Brucellosis is endemic in Bangladesh both in humans and in animals. A number of reasons complicate the diagnosis, as bovine brucellosis can be diagnosed by various serological tests. But the tests have a limitation; when the organism remains intracellular, the disease goes into chronic stage and the antibody titres may decline. The present study was conducted for isolation and detection of Brucella spp. by polymerase chain reaction (PCR) from seronegative cows. A total of 360 dairy cows from three geographical regions were screened serologically by Rose Bengal Plate Test (RBPT) where 24 samples were serologically positive and the rest of the samples were serologically negative. Among the 24 seropositive individuals, 11 were culture positive and 6 were culture positive from serologically negative dairy cows. The overall seroprevalence of brucellosis in cattle was 6.6% and in disease condition a higher prevalence was recorded in abortion (28.07%) followed by infertility (13.33%). To confirm the Brucella spp. in seronegative dairy cattle, the isolates were extracted and PCR was conducted, which produced 905 bp amplicon size of 6 Brucella spp. from milk sample. So, for the detection or eradication of brucellosis, a bacteriological test and a PCR technique should be performed with the serological test of milk. PMID:29568653

Proteomic analysis of Brucella abortus cell envelope and identification of immunogenic candidate proteins for vaccine development.

PubMed

Connolly, Joseph P; Comerci, Diego; Alefantis, Timothy G; Walz, Alexander; Quan, Marian; Chafin, Ryan; Grewal, Paul; Mujer, Cesar V; Ugalde, Rodolfo A; DelVecchio, Vito G

2006-07-01

Brucella abortus is the etiologic agent of bovine brucellosis and causes a chronic disease in humans known as undulant fever. In livestock the disease is characterized by abortion and sterility. Live, attenuated vaccines such as S19 and RB51 have been used to control the spread of the disease in animals; however, they are considered unsafe for human use and they induce abortion in pregnant cattle. For the development of a safer and equally efficacious vaccine, immunoproteomics was utilized to identify novel candidate proteins from B. abortus cell envelope (CE). A total of 163 proteins were identified using 2-DE with MALDI-TOF MS and LC-MS/MS. Some of the major protein components include outer-membrane protein (OMP) 25, OMP31, Omp2b porin, and 60 kDa chaperonin GroEL. 2-DE Western blot analyses probed with antiserum from bovine and a human patient infected with Brucella identified several new immunogenic proteins such as fumarate reductase flavoprotein subunit, F0F1-type ATP synthase alpha subunit, and cysteine synthase A. The elucidation of the immunome of B. abortus CE identified a number of candidate proteins for developing vaccines against Brucella infection in bovine and humans.

A novel lumazine synthase molecule from Brucella significantly promotes the immune-stimulation effects of antigenic protein.

PubMed

Du, Z Q; Wang, J Y

2015-10-27

Brucella, an intracellular parasite that infects some livestock and humans, can damage or destroy the reproductive system of livestock. The syndrome is referred to as brucellosis and often occurs in pastoral areas; it is contagious from livestock to humans. In this study, the intact Brucella suis outer membrane protein 31 (omp31) gene was cloned, recombinantly expressed, and examined as a subunit vaccine candidate. The intact Brucella lumazine synthase (bls) gene was cloned and recombinantly expressed to study polymerization function in vitro. Non-reducing gel electrophoresis showed that rBs-BLS existed in different forms in vitro, including as a dimer and a pentamer. An enzyme-linked immunosorbent assay result showed that rOmp31 protein could induce production of an antibody in rabbits. However, the rOmp31-BLS fusion protein could elicit a much higher antibody titer in rabbits; this construct involved fusion of the Omp31 molecule with the BLS molecule. Our results indicate that Omp31 is involved in immune stimulation, while BLS has a polymerizing function based on rOmp31-BLS fusion protein immunogenicity. These data suggest that Omp31 is an ideal subunit vaccine candidate and that the BLS molecule is a favorable transport vector for antigenic proteins.

Acute Brucella melitensis M16 infection model in mice treated with tumor necrosis factor-alpha inhibitors.

PubMed

Kutlu, Murat; Ergin, Çağrı; Şen-Türk, Nilay; Sayin-Kutlu, Selda; Zorbozan, Orçun; Akalın, Şerife; Şahin, Barboros; Çobankara, Veli; Demirkan, Neşe

2015-02-19

There is limited data in the literature about brucellosis related to an intracellular pathogen and anti-tumor necrosis factor alpha (anti-TNFα) medication. The aim of this study was to evaluate acute Brucella infections in mice receiving anti-TNFα drug treatment. Anti-TNFα drugs were injected in mice on the first and fifth days of the study, after which the mice were infected with B. melitensis M16 strain. Mice were sacrificed on the fourteenth day after infection. Bacterial loads in the liver and spleen were defined, and histopathological changes were evaluated. Neither the liver nor the spleen showed an increased bacterial load in all anti-TNFα drug groups when compared to a non-treated, infected group. The most significant histopathological findings were neutrophil infiltrations in the red pulp of the spleen and apoptotic cells with hepatocellular pleomorphism in the liver. There was no significant difference among the groups in terms of previously reported histopathological findings, such as extramedullary hematopoiesis and granuloma formation. There were no differences in hepatic and splenic bacterial load and granuloma formation, which indicate worsening of the acute Brucella infection in mice; in other words, anti-TNFα treatment did not exacerbate the acute Brucella spp. infection in mice.

In Situ Microscopy Analysis Reveals Local Innate Immune Response Developed around Brucella Infected Cells in Resistant and Susceptible Mice

PubMed Central

Copin, Richard; Vitry, Marie-Alice; Hanot Mambres, Delphine; Machelart, Arnaud; De Trez, Carl; Vanderwinden, Jean-Marie; Magez, Stefan; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

2012-01-01

Brucella are facultative intracellular bacteria that chronically infect humans and animals causing brucellosis. Brucella are able to invade and replicate in a broad range of cell lines in vitro, however the cells supporting bacterial growth in vivo are largely unknown. In order to identify these, we used a Brucella melitensis strain stably expressing mCherry fluorescent protein to determine the phenotype of infected cells in spleen and liver, two major sites of B. melitensis growth in mice. In both tissues, the majority of primary infected cells expressed the F4/80 myeloid marker. The peak of infection correlated with granuloma development. These structures were mainly composed of CD11b+ F4/80+ MHC-II+ cells expressing iNOS/NOS2 enzyme. A fraction of these cells also expressed CD11c marker and appeared similar to inflammatory dendritic cells (DCs). Analysis of genetically deficient mice revealed that differentiation of iNOS+ inflammatory DC, granuloma formation and control of bacterial growth were deeply affected by the absence of MyD88, IL-12p35 and IFN-γ molecules. During chronic phase of infection in susceptible mice, we identified a particular subset of DC expressing both CD11c and CD205, serving as a reservoir for the bacteria. Taken together, our results describe the cellular nature of immune effectors involved during Brucella infection and reveal a previously unappreciated role for DC subsets, both as effectors and reservoir cells, in the pathogenesis of brucellosis. PMID:22479178

A multiplex real-time polymerase chain reaction assay with two internal controls for the detection of Brucella species in tissues, blood, and feces from marine mammals.

PubMed

Sidor, Inga F; Dunn, J Lawrence; Tsongalis, Gregory J; Carlson, Jolene; Frasca, Salvatore

2013-01-01

Brucellosis has emerged as a disease of concern in marine mammals in the last 2 decades. Molecular detection techniques have the potential to address limitations of other methods for detecting infection with Brucella in these species. Presented herein is a real-time polymerase chain reaction (PCR) method targeting the Brucella genus-specific bcsp31 gene. The method also includes a target to a conserved region of the eukaryotic mitochondrial 16S ribosomal RNA gene to assess suitability of extracted DNA and a plasmid-based internal control to detect failure of PCR due to inhibition. This method was optimized and validated to detect Brucella spp. in multiple sample matrices, including fresh or frozen tissue, blood, and feces. The analytical limit of detection was low, with 95% amplification at 24 fg, or an estimated 7 bacterial genomic copies. When Brucella spp. were experimentally added to tissue or fecal homogenates, the assay detected an estimated 1-5 bacteria/µl. An experiment simulating tissue autolysis showed relative persistence of bacterial DNA compared to host mitochondrial DNA. When used to screen 1,658 field-collected marine mammal tissues in comparison to microbial culture, diagnostic sensitivity and specificity were 70.4% and 98.3%, respectively. In addition to amplification in fresh and frozen tissues, Brucella spp. were detected in feces and formalin-fixed, paraffin-embedded tissues from culture-positive animals. Results indicate the utility of this real-time PCR for the detection of Brucella spp. in marine species, which may have applications in surveillance or epidemiologic investigations.

Brucella abortus Induces the Premature Death of Human Neutrophils through the Action of Its Lipopolysaccharide

PubMed Central

Barquero-Calvo, Elías; Mora-Cartín, Ricardo; Arce-Gorvel, Vilma; de Diego, Juana L.; Chacón-Díaz, Carlos; Chaves-Olarte, Esteban; Guzmán-Verri, Caterina; Buret, Andre G.; Gorvel, Jean-Pierre; Moreno, Edgardo

2015-01-01

Most bacterial infections induce the activation of polymorphonuclear neutrophils (PMNs), enhance their microbicidal function, and promote the survival of these leukocytes for protracted periods of time. Brucella abortus is a stealthy pathogen that evades innate immunity, barely activates PMNs, and resists the killing mechanisms of these phagocytes. Intriguing clinical signs observed during brucellosis are the low numbers of Brucella infected PMNs in the target organs and neutropenia in a proportion of the patients; features that deserve further attention. Here we demonstrate that B. abortus prematurely kills human PMNs in a dose-dependent and cell-specific manner. Death of PMNs is concomitant with the intracellular Brucella lipopolysaccharide (Br-LPS) release within vacuoles. This molecule and its lipid A reproduce the premature cell death of PMNs, a phenomenon associated to the low production of proinflammatory cytokines. Blocking of CD14 but not TLR4 prevents the Br-LPS-induced cell death. The PMNs cell death departs from necrosis, NETosis and classical apoptosis. The mechanism of PMN cell death is linked to the activation of NADPH-oxidase and a modest but steadily increase of ROS mediators. These effectors generate DNA damage, recruitments of check point kinase 1, caspases 5 and to minor extent of caspase 4, RIP1 and Ca++ release. The production of IL-1β by PMNs was barely stimulated by B. abortus infection or Br-LPS treatment. Likewise, inhibition of caspase 1 did not hamper the Br-LPS induced PMN cell death, suggesting that the inflammasome pathway was not involved. Although activation of caspases 8 and 9 was observed, they did not seem to participate in the initial triggering mechanisms, since inhibition of these caspases scarcely blocked PMN cell death. These findings suggest a mechanism for neutropenia in chronic brucellosis and reveal a novel Brucella-host cross-talk through which B. abortus is able to hinder the innate function of PMN. PMID:25946018

Brucella endocarditis in a non-endemic area presenting as pyrexia of unknown origin

PubMed Central

Manade, Vivek Vilas; Kakrani, Arjun; Gadage, Siddharth Narayan; Misra, Rabindra

2014-01-01

A 67-year-old man with type 2 diabetes mellitus and hypertension since 7 years presented with a 3-month history of low-grade fever and malaise. Cardiac auscultation revealed the presence of an ejection systolic murmur in the primary aortic area. Most of the investigations for febrile illness were reported normal. His two-dimensional (2D) echocardiogram revealed a calcified aortic valve with mild aortic stenosis. In view of the prolonged fever and calcified aortic valve with mild aortic stenosis, a transoesophageal echocardiogram was performed, which showed small vegetation noted on right coronary cusp about 2.2 mm with free independent mobility. Blood culture was positive for Brucella spp from all the three venepuncture sites. Medical therapy for brucellosis was given with ciprofloxacin, doxycycline, co-trimoxazole and streptomycin, resulting in complete recovery. Brucella endocarditis is a rare, mostly ignored and missed clinical infection. It requires a high index of clinical suspicion for prompt diagnosis and treatment. PMID:25239983

Cervical Lymph Nodes as a Selective Niche for Brucella during Oral Infections

PubMed Central

von Bargen, Kristine; Gagnaire, Aurélie; Arce-Gorvel, Vilma; de Bovis, Béatrice; Baudimont, Fannie; Chasson, Lionel; Bosilkovski, Mile; Papadopoulos, Alexia; Martirosyan, Anna; Henri, Sandrine; Mège, Jean-Louis; Malissen, Bernard; Gorvel, Jean-Pierre

2015-01-01

Cervical lymph nodes (CLN) are the first lymph nodes encountered by material taking the oral route. To study their role in orally acquired infections, we analyzed 307 patients of up to 14 years treated in the university clinic of Skopje, Macedonia, for brucellosis, a zoonotic bacterial disease frequently acquired by ingestion of contaminated dairy products. From these children, 36% had lymphadenopathy. Among orally infected children, lymphadenopathy with CLN being the only lymph nodes affected was significantly more frequent as compared to those infected by contact with animals (83% vs. 63%), suggesting a possible involvement of CLN during orally acquired human brucellosis. Using a murine model where bacteria are delivered into the oral cavity, we show that Brucella quickly and selectively colonize the CLN where they proliferate and persist over long periods of time for up to 50 days post-infection. A similar efficient though less specific drainage to CLN was found for Brucella, Salmonella typhimurium and fluorescent microspheres delivered by gavage, a pathway likely representing a mixed infection mode of intragastric and oral infection, suggesting a central pathway of drained material. Microspheres as well as bacteria drained to CLN predominately reside in cells expressing CD68 and no or low levels of CD11c. Even though no systemic response could be detected, Brucella induced a locally restricted inflammatory reaction with increased expression levels of interferon γ, interleukin (IL)-6, IL-12, granzyme B and a delayed induction of Nos2. Inflammation led to pronounced lymphadenopathy, infiltration of macrophages/monocytes expressing high levels of major histocompatibility complex II and to formation of epitheloid granulomas. Together, these results highlight the role of CLN in oral infections as both, an initial and efficient trap for bacterial invaders and as possible reservoir for chronic pathogens. They likewise cast a new light on the significance of oral

African Lineage Brucella melitensis Isolates from Omani Livestock.

PubMed

Foster, Jeffrey T; Walker, Faith M; Rannals, Brandy D; Hussain, M Hammad; Drees, Kevin P; Tiller, Rebekah V; Hoffmaster, Alex R; Al-Rawahi, Abdulmajeed; Keim, Paul; Saqib, Muhammad

2017-01-01

Brucellosis is a common livestock disease in the Middle East and North Africa, but remains poorly described in the region both genetically and epidemiologically. Traditionally found in goats and sheep, Brucella melitensis is increasingly recognized as infecting camels. Most studies of brucellosis in camels to date have focused on serological surveys, providing only limited understanding of the molecular epidemiology of circulating strains. We genotyped B. melitensis isolates from Omani camels using whole genome SNP assays and VNTRs to provide context for regional brucellosis cases. We identified a lineage of B. melitensis circulating in camels as well as in goats, sheep, and cattle in Oman. This lineage is genetically distinct from most genotypes from the Arabian Peninsula and from isolates from much of the rest of the Middle East. We then developed diagnostic assays that rapidly identify strains from this lineage. In analyses of genotypes from throughout the region, Omani isolates were genetically most closely related to strains from brucellosis cases in humans and livestock in North Africa. Our findings suggest an African origin for B. melitensis in Oman that has likely occurred through the trade of infected livestock. Moreover, African lineages of B. melitensis appear to be undersampled and consequently are underrepresented in genetic databases for Brucella . As we begin to more fully understand global genomic diversity of B. melitensis , finding and characterizing these unique but widespread lineages is essential. We predict that increased sampling of humans and livestock in Africa will reveal little known diversity in this important zoonotic pathogen.

Goat farm management and Brucella serological test among goat keepers and livestock officers, 2011-2012, Nakhon Si Thammarat Province, southern Thailand.

PubMed

Te-Chaniyom, Thanidtha; Geater, Alan F; Kongkaew, Wandee; Chethanond, Usa; Chongsuvivatwong, Virasakdi

2016-12-01

Brucellosis, a zoonotic disease particularly affecting goats, emerged in Thailand in 2003, resulting in both an occupational hazard for goat keepers and livestock officers, and production losses. Farm management practices have been identified as risk factors associated with Brucella sero-positivity in many studies. Our finding in this study should be considered in order to strengthen the system of biosecurity control in farm animals as one health approach. The objectives of the study were to describe the distribution of potential risk factors by types of goat farms and to document the prevalence of human Brucella sero-positivity among goat keepers and livestock officers in Nakhon Si Thammarat, Thailand. A cross-sectional study was conducted from September to December 2012. The study population included three types of goat farms: standard, community enterprise and private goat farms that were located in Nakhon Si Thammmarat Province in southern Thailand. Information on whether the farm had any Brucella sero-positivity goats since 2011 was retrieved from the local livestock office records. Information on farming management was also traced back to 2011. Field researchers collected information from goat keepers of the selected farms using a structured questionnaire. Goat keepers on all farms pre-identified (January to June 2012) as having had at least one positive goat were considered to have been exposed. Goat keepers on a random sample of farms having all goats with negative results were considered to be unexposed. Venous blood samples were collected from goat keepers exposed and unexposed and from livestock officers and the samples were tested by IgG ELISA. Statistical analysis was done under the complex survey design in R software. Fourteen standard farms, 66 community enterprise farms and 68 private farms participated in the study; 82.4% (122/148) used public pasture and 53.4% (79/148) shared breeder goats with other farms. Farm management practices corresponding

Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans.

PubMed

Ohishi, Kazue; Bando, Takeharu; Abe, Erika; Kawai, Yasushi; Fujise, Yoshihiro; Maruyama, Tadashi

2016-10-01

In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994-2010 offshore surveys (21%, 285/1353) and in the 2006-2010 Japanese coastal surveys (20%, 86/436), in Bryde's whales (B. edeni brydei) in the 2000-2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002-2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000-2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000-2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde's whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level.

Efficacies of gentamicin-loaded magnetite block ionomer complexes against chronic Brucella melitensis infection

NASA Astrophysics Data System (ADS)

Jain-Gupta, Neeta; Pothayee, Nipon; Pothayee, Nikorn; Tyler, Ronald; Caudell, David L.; Balasubramaniam, Sharavanan; Hu, Nan; Davis, Richey M.; Riffle, Judy S.; Sriranganathan, Nammalwar

2013-11-01

Anionic copolymers can enable intracellular delivery of cationic drugs which otherwise cannot cross cell membrane barriers. We tested the efficacy of gentamicin-loaded magnetite block ionomer complexes (MBICs) against intracellular Brucella melitensis. Anionic block copolymers were used to coat nanomagnetite through adsorption of a portion of anions on the particle surfaces, then the remaining anions were complexed with 30-32 weight percentage of gentamicin. The zeta potential changed from -39 to -13 mV after encapsulation of the drug with complementary charge. The gentamicin-loaded MBICs had intensity average hydrodynamic diameters of 62 nm, while the polymer-coated nanomagnetite particles without drug were 34 nm in size. No toxicity as measured by a MTS assay was observed upon incubation of the MBICs with J774A.1 murine macrophage-like cells. Confocal microscopic images showed that the MBICs were taken up by the macrophages and distributed in the cell cytoplasm and endosomal/lysosomal compartments. Upon treatment with gentamicin-loaded MBICs (3.5 Log10), B. melitensis-infected macrophages showed significantly higher clearance of Brucella compared to the treatment with free g (0.9 Log10). Compared to doxycycline alone, a combination of doxycycline and gentamicin (either free or encapsulated in MBICs) showed significantly higher clearance of B. melitensis from chronically infected mice. Histopathological examination of kidneys from the MBICs-treated mice revealed multifocal infiltration of macrophages containing intracytoplasmic iron (MBICs) in peri-renal adipose. Although MBICs showed similar efficacy as free gentamicin against Brucella in mice, our strategy presents an effective way to deliver higher loads of drugs intracellularly and ability to study the bio-distribution of drug carriers.

Recombinant Brucella abortus gene expressing immunogenic protein

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

Mayfield, J.E.; Tabatabai, L.B.

This patent describes a synthetic recombinant DNA molecule containing a DNA sequence. It comprises a gene of Brucella abortus encoding an immunogenic protein having a molecular weight of approximately 31,000 daltons as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis under denaturing conditions, the protein having an isoelectric point around 4.9, and containing a twenty-five amino acid sequence from its amino terminal end consisting of Gln-Ala-Pro-Thr-Phe-Phe-Arg-Ile-Gly-Thr-Gly-Gly-Thr-Ala-Gly-Thr-Tyr-Tyr-Pro-Ile-Gly-Gly-Leu-Ile-Ala, wherein Gln, Ala, Pro, Thr, Phe, Arg, Ile, Gly, Tyr, and Leu, respectively, represent glutamine, alanine, proline, threonine, phenylalanine, arginine, isolecuine, glycine, tyrosine, and leucine.

Brucella ovis PA mutants for outer membrane proteins Omp10, Omp19, SP41, and BepC are not altered in their virulence and outer membrane properties.

PubMed

Sidhu-Muñoz, Rebeca S; Sancho, Pilar; Vizcaíno, Nieves

2016-04-15

Mutants in several genes have been obtained on the genetic background of virulent rough (lacking O-polysaccharide) Brucella ovis PA. The target genes encode outer membrane proteins previously associated with the virulence of smooth (bearing O-polysaccharide chains in the lipopolysaccharide) Brucella strains. Multiple attempts to delete omp16, coding for a homologue to peptidoglycan-associated lipoproteins, were unsuccessful, which suggests that Omp16 is probably essential for in vitro survival of B. ovis PA. Single deletion of omp10 or omp19-that encode two other outer membrane lipoproteins--was achieved, but the simultaneous removal of both genes failed, suggesting an essential complementary function between both proteins. Two other deletion mutants, defective in the Tol-C-homologue BepC or in the SP41 adhesin, were also obtained. Surprisingly when compared to previous results obtained with smooth Brucella, none of the B. ovis mutants showed attenuation in the virulence, either in the mouse model or in cellular models of professional and non-professional phagocytes. Additionally, and in contrast to the observations reported with smooth Brucella strains, several properties related to the outer membrane remained almost unaltered. These results evidence new distinctive traits between naturally rough B. ovis and smooth brucellae. Copyright © 2016 Elsevier B.V. All rights reserved.

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East

PubMed Central

Georgi, Enrico; Walter, Mathias C.; Pfalzgraf, Marie-Theres; Northoff, Bernd H.; Holdt, Lesca M.; Scholz, Holger C.; Zoeller, Lothar

2017-01-01

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is

Whole genome sequencing of Brucella melitensis isolated from 57 patients in Germany reveals high diversity in strains from Middle East.

PubMed

Georgi, Enrico; Walter, Mathias C; Pfalzgraf, Marie-Theres; Northoff, Bernd H; Holdt, Lesca M; Scholz, Holger C; Zoeller, Lothar; Zange, Sabine; Antwerpen, Markus H

2017-01-01

Brucellosis, a worldwide common bacterial zoonotic disease, has become quite rare in Northern and Western Europe. However, since 2014 a significant increase of imported infections caused by Brucella (B.) melitensis has been noticed in Germany. Patients predominantly originated from Middle East including Turkey and Syria. These circumstances afforded an opportunity to gain insights into the population structure of Brucella strains. Brucella-isolates from 57 patients were recovered between January 2014 and June 2016 with culture confirmed brucellosis by the National Consultant Laboratory for Brucella. Their whole genome sequences were generated using the Illumina MiSeq platform. A whole genome-based SNP typing assay was developed in order to resolve geographically attributed genetic clusters. Results were compared to MLVA typing results, the current gold-standard of Brucella typing. In addition, sequences were examined for possible genetic variation within target regions of molecular diagnostic assays. Phylogenetic analyses revealed spatial clustering and distinguished strains from different patients in either case, whereas multiple isolates from a single patient or technical replicates showed identical SNP and MLVA profiles. By including WGS data from the NCBI database, five major genotypes were identified. Notably, strains originating from Turkey showed a high diversity and grouped into seven subclusters of genotype II. MLVA analysis congruently clustered all isolates and predominantly matched the East Mediterranean genetic clade. This study confirms whole-genome based SNP-analysis as a powerful tool for accurate typing of B. melitensis. Furthermore it allows special allocation and therefore provides useful information on the geographic origin for trace-back analysis. However, the lack of reliable metadata in public databases often prevents a resolution below geographic regions or country levels and corresponding precise trace-back analysis. Once this obstacle is

Sacroiliitis as a sole manifestation of Brucella melitensis infection in a child

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

Miron, D.; Garty, I.; Tal, I.

A case of a 12-year-old boy with sacroiliitis documented by positive Tc-99m MDP and Ga-67 scans is described. Isolation of brucella melitensis from the blood and bone marrow established the diagnosis. He responded promptly to docycycline therapy. Throughout the course of his disease this boy had neither fever nor other signs of brucellosis, and x-ray was normal.

Long-term and large-scale epidemiology of Brucella infection in baleen whales and sperm whales in the western North Pacific and Antarctic Oceans

PubMed Central

OHISHI, Kazue; BANDO, Takeharu; ABE, Erika; KAWAI, Yasushi; FUJISE, Yoshihiro; MARUYAMA, Tadashi

2016-01-01

In a long-term, large-scale serologic study in the western North Pacific Ocean, anti-Brucella antibodies were detected in common minke whales (Balaenoptera acutorostrata) in the 1994–2010 offshore surveys (21%, 285/1353) and in the 2006–2010 Japanese coastal surveys (20%, 86/436), in Bryde’s whales (B. edeni brydei) in the 2000–2010 offshore surveys (9%, 49/542), in sei whales (B. borealis) in the 2002–2010 offshore surveys (5%, 40/788) and in sperm whales (Physeter macrocephalus) in the 2000–2010 offshore surveys (8%, 4/50). Anti-Brucella antibodies were not detected in 739 Antarctic minke whales (B. bonaerensis) in the 2000–2010 Antarctic surveys. This suggests that Brucella was present in the four large whale populations inhabiting the western North Pacific, but not in the Antarctic minke whale population. By PCR targeting for genes of outer membrane protein 2, the Brucella infection was confirmed in tissue DNA samples from Bryde’s whales (14%, 2/14), sei whales (11%, 1/9) and sperm whales (50%, 2/4). A placental tissue and an apparently healthy fetus from a sperm whale were found to be PCR-positive, indicating that placental transmission might have occurred and the newborn could act as a bacterial reservoir. Marked granulomatous testes were observed only in mature animals of the three species of baleen whales in the western North Pacific offshore surveys, especially in common minke whales, and 29% (307/1064) of total mature males had abnormal testes. This study provides an insight into the status of marine Brucella infection at a global level. PMID:27320816

In vitro bactericidal activity of aminoglycosides, including the next-generation drug plazomicin, against Brucella spp.

USDA-ARS?s Scientific Manuscript database

Plazomicin is a next-generation aminoglycoside with a potentially improved safety profile compared to other aminoglycosides. This study assessed plazomicin MICs and MBCs in four Brucella spp. reference strains. Like other aminoglycosides and aminocyclitols, plazomicin MBC values equaled MIC values ...

Immunization with Brucella VirB Proteins Reduces Organ Colonization in Mice through a Th1-Type Immune Response and Elicits a Similar Immune Response in Dogs

PubMed Central

Pollak, Cora N.; Wanke, María Magdalena; Estein, Silvia M.; Delpino, M. Victoria; Monachesi, Norma E.; Comercio, Elida A.; Fossati, Carlos A.

2014-01-01

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs. PMID:25540276

Immunization with Brucella VirB proteins reduces organ colonization in mice through a Th1-type immune response and elicits a similar immune response in dogs.

PubMed

Pollak, Cora N; Wanke, María Magdalena; Estein, Silvia M; Delpino, M Victoria; Monachesi, Norma E; Comercio, Elida A; Fossati, Carlos A; Baldi, Pablo C

2015-03-01

VirB proteins from Brucella spp. constitute the type IV secretion system, a key virulence factor mediating the intracellular survival of these bacteria. Here, we assessed whether a Th1-type immune response against VirB proteins may protect mice from Brucella infection and whether this response can be induced in the dog, a natural host for Brucella. Splenocytes from mice immunized with VirB7 or VirB9 responded to their respective antigens with significant and specific production of gamma interferon (IFN-γ), whereas interleukin-4 (IL-4) was not detected. Thirty days after an intraperitoneal challenge with live Brucella abortus, the spleen load of bacteria was almost 1 log lower in mice immunized with VirB proteins than in unvaccinated animals. As colonization reduction seemed to correlate with a Th1-type immune response against VirB proteins, we decided to assess whether such a response could be elicited in the dog. Peripheral blood mononuclear cells (PBMCs) from dogs immunized with VirB proteins (three subcutaneous doses in QuilA adjuvant) produced significantly higher levels of IFN-γ than cells from control animals upon in vitro stimulation with VirB proteins. A skin test to assess specific delayed-type hypersensitivity was positive in 4 out of 5 dogs immunized with either VirB7 or VirB9. As both proteins are predicted to locate in the outer membrane of Brucella organisms, the ability of anti-VirB antibodies to mediate complement-dependent bacteriolysis of B. canis was assessed in vitro. Sera from dogs immunized with either VirB7 or VirB9, but not from those receiving phosphate-buffered saline (PBS), produced significant bacteriolysis. These results suggest that VirB-specific responses that reduce organ colonization by Brucella in mice can be also elicited in dogs. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

[Expression and identification of eukaryotic expression vectors of Brucella melitensis lipoprotein OMP19].

PubMed

He, Zuoping; Luo, Peifang; Hu, Feihuan; Weng, Yunceng; Wang, Wenjing; Li, Chengyao

2016-04-01

To construct eukaryotic expression vectors carrying Brucella melitensis outer membrane protein 19 (OMP19), express them in transfected Huh7.5.1 and JEG-3 cells, and analyze their role in cell apoptosis. Brucella melitensis lipidated OMP19 (L-OMP19) gene and unlipidated OMP19 (U-OMP19) gene were amplified by PCR and inserted into the vector pZeroBack/blunt. The correct L-OMP19 and U-OMP19 genes verified by XbaI and BamHI double digestion and sequencing were cloned into the lentivirus expression vector pHAGE-CMV-MCS-IZsGreen to construct vectors pHAGE-L-OMP19 and pHAGE-U-OMP19, which were separately transfected into 293FT cells, Huh7.5.1 and JEG-3 cells. L-OMP19 and U-OMP19 in the cells were detected by Western blotting and immunofluorescence technique. Flow cytometry combined with annexin V-PE/7-AAD staining was used to detect the cell apoptosis. The lentiviral vectors pHAGE-L-OMP19 and pHAGE-U-OMP19 were constructed correctly and the recombinant lipoproteins L-OMP19 and U-OMP19 expressed in the above cells were well recognized by the specific antibodies against L-OMP19 in Western blotting and immunofluorescence technique. L-OMP19 and U-OMP19 induced JEG-3 cell death, but did not induce the apoptosis of Huh7.5.1 cells. The eukaryotic expression vectors of L-OMP19 and U-OMP19 have been constructed successfully. Recombinant lipoproteins L-OMP19 and U-OMP19 expressed in cells have a good antigenicity, which could be used as experimental materials for the research on the relationship between host cells and lipoproteins in Brucella infection.

Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence.

PubMed

Mancilla, Marcos

2015-01-01

Dissociation encompasses changes in a series of phenotypes: colony and cell morphology, inmunological and biochemical reactions and virulence. The concept is generally associated to the in vitro transition between smooth (S) and rough (R) colonies, a phenotypic observation in Gram-negative bacteria commonly made since the beginning of microbiology as a science. It is also well known that the loss of the O-polysaccharide, the most external lipopolysaccharide (LPS) moiety, triggers the change in the colony phenotype. Although dissociation is related to one of the most basic features used to distinguish between species, i.e., colony morphology, and, in the case of pathogens, predict their virulence behavior, it has been considered a laboratory artifact and thus did not gain further attention. However, recent insights into genetics and pathogenesis of members of Brucella, causative agents of brucellosis, have brought a new outlook on this experimental fact, suggesting that it plays a role beyond the laboratory observations. In this perspective article, the current knowledge on Brucella LPS genetics and its connection with dissociation in the frame of evolution is discussed. Latest reports support the notion that, by means of a better understanding of genetic pathways linked to R phenotype and the biological impact of this intriguing "old" phenomenon, unexpected applications can be achieved.

Smooth to Rough Dissociation in Brucella: The Missing Link to Virulence

PubMed Central

Mancilla, Marcos

2016-01-01

Dissociation encompasses changes in a series of phenotypes: colony and cell morphology, inmunological and biochemical reactions and virulence. The concept is generally associated to the in vitro transition between smooth (S) and rough (R) colonies, a phenotypic observation in Gram-negative bacteria commonly made since the beginning of microbiology as a science. It is also well known that the loss of the O-polysaccharide, the most external lipopolysaccharide (LPS) moiety, triggers the change in the colony phenotype. Although dissociation is related to one of the most basic features used to distinguish between species, i.e., colony morphology, and, in the case of pathogens, predict their virulence behavior, it has been considered a laboratory artifact and thus did not gain further attention. However, recent insights into genetics and pathogenesis of members of Brucella, causative agents of brucellosis, have brought a new outlook on this experimental fact, suggesting that it plays a role beyond the laboratory observations. In this perspective article, the current knowledge on Brucella LPS genetics and its connection with dissociation in the frame of evolution is discussed. Latest reports support the notion that, by means of a better understanding of genetic pathways linked to R phenotype and the biological impact of this intriguing “old” phenomenon, unexpected applications can be achieved. PMID:26779449

Misinterpretation of Gram Stain from the Stationary Growth Phase of Positive Blood Cultures for Brucella and Acinetobacter Species.

PubMed

Bazzi, Ali M; Al-Tawfiq, Jaffar A; Rabaan, Ali A

2017-01-01

Acinetobacter baumannii and Brucella species are Gram-negative organisms that are vulnerable to misinterpretation as Gram-positive or Gram-variable in blood cultures. We assess the random errors in gram stain interpretation to reduce the likelihood of such errors and therefore patient harm. Aerobic and anaerobic blood cultures from two patients in an acute care facility in Saudi Arabia were subjected to preliminary Gram-staining. In case 1, VITEK-2 Anaerobe Identification, repeat Gram staining from a blood agar plate, Remel BactiDrop™ Oxidase test, Urea Agar urease test and real-time PCR were used to confirm presence of Brucella and absence of Coryneform species. In case 2, repeat Gram- staining from the plate and the vials, VITEK-2 Gram-Negative Identification, real-time PCR and subculture on to Columbia agar, blood agar, and MacConkey agar were carried out to identify A. baumannii . In case 1, initially pleomorphic Gram-positive bacteria were identified. Coryneform species were suspected. Tiny growth was observed after 24 h on blood agar plates, and good growth by 48 h. Presence of Brucella species was ultimately confirmed. In case 2, preliminary Gram-stain results suggested giant Gram-positive oval cocci. Further testing over 18-24 h identified A. baumannii . Oxidase test from the plate and urease test from the culture vial is recommended after apparent identification of pleomorphic Gram-positive bacilli from blood culture, once tiny growth is observed, to distinguish Brucella from Corynebacterium species. If giant Gram-positive oval cocci are indicated by preliminary Gram-staining, it is recommended that the Gram stain be repeated from the plate after 4-6 h, or culture should be tested in Triple Sugar Iron (TSI) medium and the Gram stain repeated after 2-4 h incubation.

New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide

NASA Astrophysics Data System (ADS)

Casabuono, Adriana C.; Czibener, Cecilia; Del Giudice, Mariela G.; Valguarnera, Ezequiel; Ugalde, Juan E.; Couto, Alicia S.

2017-12-01

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. [Figure not available: see fulltext.

New Features in the Lipid A Structure of Brucella suis and Brucella abortus Lipopolysaccharide.

PubMed

Casabuono, Adriana C; Czibener, Cecilia; Del Giudice, Mariela G; Valguarnera, Ezequiel; Ugalde, Juan E; Couto, Alicia S

2017-12-01

Brucellaceae are Gram-negative bacteria that cause brucellosis, one of the most distributed worldwide zoonosis, transmitted to humans by contact with either infected animals or their products. The lipopolysaccharide exposed on the cell surface has been intensively studied and is considered a major virulence factor of Brucella. In the last years, structural studies allowed the determination of new structures in the core oligosaccharide and the O-antigen of this lipopolysaccharide. In this work, we have reinvestigated the lipid A structure isolated from B. suis and B. abortus lipopolysaccharides. A detailed study by MALDI-TOF mass spectrometry in the positive and negative ion modes of the lipid A moieties purified from both species was performed. Interestingly, a new feature was detected: the presence of a pyrophosphorylethanolamine residue substituting the backbone. LID-MS/MS analysis of some of the detected ions allowed assurance that the Lipid A structure composed by the diGlcN3N disaccharide, mainly hexa-acylated and penta-acylated, bearing one phosphate and one pyrophosphorylethanolamine residue. Graphical abstract ᅟ.

Intracellular trafficking of Brucella abortus in J774 macrophages.

PubMed

Arenas, G N; Staskevich, A S; Aballay, A; Mayorga, L S

2000-07-01

Brucella abortus is a facultative intracellular bacterium capable of surviving inside professional and nonprofessional phagocytes. The microorganism remains in membrane-bound compartments that in several cell types resemble modified endoplasmic reticulum structures. To monitor the intracellular transport of B. abortus in macrophages, the kinetics of fusion of phagosomes with preformed lysosomes labeled with colloidal gold particles was observed by electron microscopy. The results indicated that phagosomes containing live B. abortus were reluctant to fuse with lysosomes. Furthermore, newly endocytosed material was not incorporated into these phagosomes. These observations indicate that the bacteria strongly affect the normal maturation process of macrophage phagosomes. However, after overnight incubation, a significant percentage of the microorganisms were found in large phagosomes containing gold particles, resembling phagolysosomes. Most of the Brucella bacteria present in phagolysosomes were not morphologically altered, suggesting that they can also resist the harsh conditions prevalent in this compartment. About 50% colocalization of B. abortus with LysoSensor, a weak base that accumulates in acidic compartments, was observed, indicating that the B. abortus bacteria do not prevent phagosome acidification. In contrast to what has been described for HeLa cells, only a minor percentage of the microorganisms were found in compartments labeled with monodansylcadaverine, a marker for autophagosomes, and with DiOC6 (3,3'-dihexyloxacarbocyanine iodide), a marker for the endoplasmic reticulum. These results indicate that B. abortus bacteria alter phagosome maturation in macrophages. However, acidification does occur in these phagosomes, and some of them can eventually mature to phagolysosomes.

[Development and comparative evaluation of up-converting phosphor technology based lateral flow assay for rapid detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp].

PubMed

Li, Chunfeng; Zhang, Pingping; Wang, Xiaoying; Liu, Xiao; Zhao, Yong; Sun, Chongyun; Wang, Chengbin; Yang, Ruifu; Zhou, Lei

2015-01-01

To develop an up-converting phosphor technology based lateral flow (UPT-LF) assay for rapid and quantitative detection of Yersinia pestis, Bacillus anthracis spore and Brucella spp.and make the comparison with BioThreat Alert (BTA) test strips (Tetracore Inc., USA). Using up-converting phosphor nano-particles (UCP-NPs) as the bio-marker, three double-antibody-sandwich model based UPT-LF strips including Plague-UPT-LF, Anthrax-UPT-LF, Brucella-UPT-LF were prepared and its sensitivity, accuracy, linearity and specificity were determined by detecting 10(10), 10(9), 10(8), 10(7), 10(6), 10(5) and 0 CFU/ml series of concentrations of Y.pestis, B.anthracis, Brucella standards and other 27 kinds of 10(9) CFU/ml series of contrations of bacteria strains.Furthermore, the speed, sensitivity and accuracy of bacteria standards and simulated sample detection were compared between UPT-LF and BTA system. The detection limit of Plague-UPT-LF, Anthrax-UPT-LF and Brucella-LF was 10(5) CFU/ml. The CV of series of bacteria concentrations was ≤ 15%, and the r between lg (T/C-cut-off) and lg (concentration) was 0.996,0.998 and 0.999 (F values were 1 647.57, 743.51 and 1 822.17. All the P values were <0.001), respectively. The specificity of Plague-UPT-LF and Brucella-LF were excellent, while that of Anthrax-UPT-LF was a little bit regretful because of non-specific reaction with two isolates of B. subtilis and one B.cereus. On-site evaluation showed the detection time of UPT-LF for all Y.pestis, B.anthracis spore and Brucella spp.was 33, 36 and 37 min, while BTA was 115, 115 and 111 min, which revealed the higher detection speed and sensitivity of UPT-LF comparing with BTA. The negative rate of two methods for blank standard was both 5/5, the sensitivity of UPT-LF for Y.pestis,B.anthracis spore and Brucella spp. was all 10(5) CFU/ml, then BTA was 10(6), 10(6) and 10(5) CFU/ml, respectively. The detection rate of UPT-LF for all three bacteria analog positive samples was 16/16, while BTA

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

PubMed Central

Freer, E; Moreno, E; Moriyón, I; Pizarro-Cerdá, J; Weintraub, A; Gorvel, J P

1996-01-01

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or

Brucella-Salmonella lipopolysaccharide chimeras are less permeable to hydrophobic probes and more sensitive to cationic peptides and EDTA than are their native Brucella sp. counterparts.

PubMed

Freer, E; Moreno, E; Moriyón, I; Pizarro-Cerdá, J; Weintraub, A; Gorvel, J P

1996-10-01

A rough (R) Brucella abortus 45/20 mutant was more sensitive to the bactericidal activity of polymyxin B and lactoferricin B than was its smooth (S) counterpart but considerably more resistant than Salmonella montevideo. The outer membrane (OM) and isolated lipopolysaccharide (LPS) of S. montevideo showed a higher affinity for these cationic peptides than did the corresponding B. abortus OM and LPS. We took advantage of the moderate sensitivity of R B. abortus to cationic peptides to construct live R B. abortus-S-LPS chimeras to test the activities of polymyxin B, lactoferricin B, and EDTA. Homogeneous and abundant peripheral distribution of the heterologous S-LPS was observed on the surface of the chimeras, and this coating had no effect on the viability or morphology of the cells. When the heterologous LPS corresponded to the less sensitive bacterium S B. abortus S19, the chimeras were more resistant to cationic peptides; in contrast, when the S-LPS was from the more sensitive bacterium S. montevideo, the chimeras were more susceptible to the action of peptides and EDTA. A direct correlation between the amount of heterologous S-LPS on the surface of chimeric Brucella cells and peptide sensitivity was observed. Whereas the damage produced by polymyxin B in S. montevideo and B. abortus-S. montevideo S-LPS chimeras was manifested mainly as OM blebbing and inner membrane rolling, lactoferricin B caused inner membrane detachment, vacuolization, and the formation of internal electron-dense granules in these cells. Native S and R B. abortus strains were permeable to the hydrophobic probe N-phenyl-1-naphthylamine (NPN). In contrast, only reduced amounts of NPN partitioned into the OMs of the S. montevideo and B. abortus-S. montevideo S-LPS chimeras. Following peptide exposure, accelerated NPN uptake similar to that observed for S. montevideo was detected for the B. abortus-S. montevideo LPS chimeras. The partition of NPN into native or EDTA-, polymyxin B-, or

The Dutch Brucella abortus monitoring programme for cattle: the impact of false-positive serological reactions and comparison of serological tests.

PubMed

Emmerzaal, A; de Wit, J J; Dijkstra, Th; Bakker, D; van Zijderveld, F G

2002-02-01

The Dutch national Brucella abortus eradication programme for cattle started in 1959. Sporadic cases occurred yearly until 1995; the last infected herd was culled in 1996. In August 1999 the Netherlands was declared officially free of bovine brucellosis by the European Union. Before 1999, the programme to monitor the official Brucella-free status of bovine herds was primarily based on periodical testing of dairy herds with the milk ring test (MRT) and serological testing of all animals older than 1 year of age from non-dairy herds, using the micro-agglutination test (MAT) as screening test. In addition, serum samples of cattle that aborted were tested with the MAT. The high number of false positive reactions in both tests and the serum agglutination test (SAT) and complement fixation test (CFT) used for confirmation seemed to result in unnecessary blockade of herds, subsequent testing and slaughter of animals. For this reason, a validation study was performed in which three indirect enzyme-linked immunosorbent assays (ELISAs), the CFT and the SAT were compared using a panel of sera from brucellosis-free cattle, sera from experimentally infected cattle, and sera from cattle experimentally infected with bacteria which are known to induce cross-reactive antibodies (Pasteurella, Salmonella, Yersinia, and Escherichia). Moreover, four ELISAs and the MRT were compared using a panel of 1000 bulk milk samples from Brucella-free herds and 12 milk samples from Brucella abortus- infected cattle. It is concluded that the ELISA obtained from ID-Lelystad is the most suitable test to monitor the brucelosis free status of herds because it gives rise to fewer false-positive reactions than the SAT.

Transcriptome Analysis of the Brucella abortus BvrR/BvrS Two-Component Regulatory System

PubMed Central

Viadas, Cristina; Rodríguez, María C.; Sangari, Felix J.; Gorvel, Jean-Pierre; García-Lobo, Juan M.; López-Goñi, Ignacio

2010-01-01

Background The two-component BvrR/BvrS system is essential for Brucella abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of lipid A acylation. To determine the genes regulated by BvrR/BvrS, we performed a whole-genome microarray analysis using B. abortus RNA obtained from wild type and bvrR mutant cells grown in the same conditions. Methodology/Principal Findings A total of 127 differentially expressed genes were found: 83 were over expressed and 44 were less expressed in the bvrR mutant. Two operons, the phosphotransferase system and the maltose transport system, were down-regulated. Several genes involved in cell envelope or outer membrane biogenesis were differentially expressed: genes for outer membrane proteins (omp25a, omp25d), lipoproteins, LPS and fatty acid biosynthesis, stress response proteins, chaperones, flagellar genes, and twelve genes encoding ABC transport systems. Ten genes related with carbon metabolism (pckA and fumB among others) were up-regulated in the bvrR mutant, and denitrification genes (nirK, norC and nosZ) were also regulated. Notably, seven transcriptional regulators were affected, including VjbR, ExoR and OmpR that were less expressed in the bvrR mutant. Finally, the expression of eleven genes which have been previously related with Brucella virulence was also altered. Conclusions/Significance All these data corroborate the impact of BvrR/BvrS on cell envelope modulation, confirm that this system controls the carbon and nitrogen metabolism, and suggest a cross-talk among some regulators to adjust the Brucella physiology to the shift expected to occur during the transit from the extracellular to the intracellular niche. PMID:20422049

Draft genome sequence of field isolate Brucella melitensis strain 2007BM/1 from India.

PubMed

Singh, D K; Kumar, Bablu; Shrinet, Garima; Singh, R P; Das, Aparajita; Mantur, B G; Abhishek; Pandey, Aruna; Mondal, Piyali; Sajjanar, B K; Doimari, Soni; Singh, Vijayata; Kumari, Reena; Tiwari, A K; Gandham, Ravi Kumar

2018-04-21

Brucellosis is among one of the most widespread important global zoonotic diseases that is endemic in many parts of India. Brucella melitensis is supposed to be the most pathogenic species for humans. Here we report the draft genome sequence of B. melitensis strain 2007BM/1 isolated from a human in India. Genomic DNA was extracted from Brucella culture and was sequenced using an Illumina MiSeq platform. The generated reads were assembled using three de novo assemblers and the draft genome was annotated. This monoisolate, with a genome length of 3268756bp, was found to be resistant to azithromycin and trimethoprim/sulfamethoxazole but susceptible to tetracycline, ofloxacin, rifampicin, ciprofloxacin and doxycycline. The presence of virulence genes in the strain was identified. The results obtained will help in understanding drug resistance mechanisms and virulence factors in highly zoonotic B. melitensis and suggest the need for judicious use of antibiotics in livestock health and management practices. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

"HOOF-Print" Genotyping and Haplotype Inference Discriminates among Brucella spp Isolates From a Small Spatial Scale

USDA-ARS?s Scientific Manuscript database

We demonstrate that the “HOOF-Print” assay provides high power to discriminate among Brucella isolates collected on a small spatial scale (within Portugal). Additionally, we illustrate how haplotype identification using non-random association among markers allows resolution of B. melitensis biovars ...

Brucella spp. of amphibians comprise genomically diverse motile strains competent for replication in macrophages and survival in mammalian hosts

PubMed Central

Al Dahouk, Sascha; Köhler, Stephan; Occhialini, Alessandra; Jiménez de Bagüés, María Pilar; Hammerl, Jens Andre; Eisenberg, Tobias; Vergnaud, Gilles; Cloeckaert, Axel; Zygmunt, Michel S.; Whatmore, Adrian M.; Melzer, Falk; Drees, Kevin P.; Foster, Jeffrey T.; Wattam, Alice R.; Scholz, Holger C.

2017-01-01

Twenty-one small Gram-negative motile coccobacilli were isolated from 15 systemically diseased African bullfrogs (Pyxicephalus edulis), and were initially identified as Ochrobactrum anthropi by standard microbiological identification systems. Phylogenetic reconstructions using combined molecular analyses and comparative whole genome analysis of the most diverse of the bullfrog strains verified affiliation with the genus Brucella and placed the isolates in a cluster containing B. inopinata and the other non-classical Brucella species but also revealed significant genetic differences within the group. Four representative but molecularly and phenotypically diverse strains were used for in vitro and in vivo infection experiments. All readily multiplied in macrophage-like murine J774-cells, and their overall intramacrophagic growth rate was comparable to that of B. inopinata BO1 and slightly higher than that of B. microti CCM 4915. In the BALB/c murine model of infection these strains replicated in both spleen and liver, but were less efficient than B. suis 1330. Some strains survived in the mammalian host for up to 12 weeks. The heterogeneity of these novel strains hampers a single species description but their phenotypic and genetic features suggest that they represent an evolutionary link between a soil-associated ancestor and the mammalian host-adapted pathogenic Brucella species. PMID:28300153

Detection of Brucella abortus DNA in aborted goats and sheep in Egypt by real-time PCR.

PubMed

Wareth, Gamal; Melzer, Falk; Tomaso, Herbert; Roesler, Uwe; Neubauer, Heinrich

2015-06-03

Brucellosis is a major zoonoses affects wide range of domesticated as well as wild animals. Despite the eradication program of brucellosis in Egypt, the disease is still endemic among cattle, buffaloes, sheep, goats, and camels. In the present study, abortion occurred naturally among 25 animals (10 cows, 5 buffaloes, 9 Egyptian Baladi goats and 1 ewe) shared the same pasture were investigated by real-time polymerase chain reaction (RT-PCR). DNA of Brucella (B.) abortus was detected in serum of goats and sheep which has aborted recently by species-specific RT-PCR. The results suggest cross-species infection of B. abortus from cattle to non-preferred hosts raised in close contact. This article will renew our knowledge about the Brucella agent causing abortion in small ruminants in Egypt. Information provided in this study is important for surveillance program, because eradication programs and vaccination strategies may have to be adapted accordingly.

The milk delivery chain and presence of Brucella spp. antibodies in bulk milk in Uganda.

PubMed

Rock, Kim Toeroek; Mugizi, Denis Rwabiita; Ståhl, Karl; Magnusson, Ulf; Boqvist, Sofia

2016-06-01

This study examined the influence of informal milk delivery chains on the risk of human exposure to Brucella spp. through milk consumption in two regions of Uganda (Gulu and Soroti Districts). The work involved describing milk delivery chains, investigating brucellosis awareness amongst milk deliverers and determining the presence of Brucella spp. antibodies in cattle milk on delivery to primary collection points (boiling points and dairies). Milk samples (n = 331) were collected from deliverers at primary collection points and from street vendors at point of sale and analysed using indirect enzyme-linked immunosorbent assay (I-ELISA). A written questionnaire was used to collect data from deliverers (n = 279) on their milk delivery chains and their brucellosis awareness. The most common delivery points in Gulu District were small dairies and in Soroti District boiling points. The presence of Brucella spp. antibodies in milk samples was higher in Soroti (40 %) than in Gulu (11 %) (P < 0.0001). There are possible public health risk consequences of this finding as 42 % of deliverers in Soroti District reported drinking raw milk, compared with 15 % in Gulu District (P < 0.0001). Awareness of brucellosis was low, with 70 % of all milk deliverers reporting not having heard of the disease or the bacterium. Application of quality controls for milk (colour and odour) along the delivery chain varied depending upon supply and demand. This study provides evidence of the diversity of informal milk markets in low-income countries and of the potential public health risks of consuming unpasteurised milk. These results can be useful to those planning interventions to reduce brucellosis.

Immunogenicity and efficacy of a rough Brucella suis vaccine delivered orally or parenterally to feral swine

USDA-ARS?s Scientific Manuscript database

Brucella suis strain 353-1 is a stable vaccine strain that is clinically safe, does not cause positive serologic responses on conventional brucellosis surveillance tests, and induces humoral and cellular immunity in swine after vaccination. In this study, we evaluated tissue clearance and immunologi...

Spatial distribution of Brucella antibodies with reference to indigenous cattle populations among contrasting agro-ecological zones of Uganda.

PubMed

Kabi, Fredrick; Muwanika, Vincent; Masembe, Charles

2015-09-01

Indigenous cattle populations exhibit various degrees of agro-ecological fitness and provide desirable opportunities for investments to improve sustainable production for better rural small-scale farmers' incomes globally. However, they could be a source of infection to their attendants and other susceptible livestock if their brucellosis status remains unknown. This study investigated the spatial distribution of Brucella antibodies among indigenous cattle populations in Uganda. Sera from a total of 925 indigenous cattle (410 Ankole Bos taurus indicus, 50 Nganda and 465 East African Shorthorn Zebu (EASZ) - B. indicus) obtained randomly from 209 herds spread throughout Uganda were sequentially analysed for Brucella antibodies using the indirect (I) and competitive (C) enzyme linked Immuno-sorbent assays (ELISA). Recent incidences of abortion within the previous 12 months and routine hygienic practices during parturition were explored for public health risks. Brucella antibodies occurred in approximately 8.64% (80/925) and 28.70% (95% CI: 22.52, 34.89) of the sampled individual cattle and herds, respectively. Findings have shown that Ankole and EASZ cattle had similar seroprevalences. Indigenous cattle from the different study agro-ecological zones (AEZs) exhibited varying seroprevalences ranging from approximately 1.78% (95% CI: 0, 5.29) to 19.67% (95% CI: 8.99, 30.35) in the Lake Victoria Crescent (LVC) and North Eastern Drylands (NED) respectively. Significantly higher odds for Brucella antibodies occurred in the NED (OR: 3.40, 95% CI: 1.34, 8.57, p=0.01) inhabited by EASZ cattle compared to the KP (reference category) AEZ. Recent incidences of abortions within the previous 12 months were significantly (p<0.001) associated with seropositive herds. These findings add critical evidence to existing information on the widespread occurrence of brucellosis among indigenous cattle populations in Uganda and could guide allocation of meagre resources for awareness creation

Herd-level risk factors for Campylobacter fetus infection, Brucella seropositivity and within-herd seroprevalence of brucellosis in cattle in northern Nigeria.

PubMed

Mai, H M; Irons, P C; Kabir, J; Thompson, P N

2013-09-01

Brucellosis and campylobacteriosis are economically important diseases affecting bovine reproductive efficiency in Nigeria. A questionnaire-based survey was conducted in 271 cattle herds in Adamawa, Kaduna and Kano states of northern Nigeria using multistage cluster sampling. Serum from 4745 mature animals was tested for Brucella antibodies using the Rose-Bengal plate test and positives were confirmed in series-testing protocol using competitive enzyme-linked immunosorbent assay. Preputial scrapings from 602 bulls were tested using culture and identification for Campylobacter fetus. For each disease, a herd was classified as positive if one or more animals tested positive. For each herd, information on potential managemental and environmental risk factors was collected through a questionnaire administered during an interview with the manager, owner or herdsman. Multiple logistic regression models were used to model the odds of herd infection for each disease. A zero-inflated Poisson model was used to model the count of Brucella-positive animals within herds, with the number tested as an exposure variable. The presence of small ruminants (sheep and/or goats) on the same farm, and buying-in of >3 new animals in the previous year or failure to practice quarantine were associated with increased odds of herd-level campylobacteriosis and brucellosis, as well as increased within-herd counts of Brucella-positive animals. In addition, high rainfall, initial acquisition of animals from markets, practice of gynaecological examination and failure to practice herd prophylactic measures were positively associated with the odds of C. fetus infection in the herd. Herd size of >15, pastoral management system and presence of handling facility on the farm were associated with increased odds, and gynaecological examination with reduced odds of herd-level Brucella seropositivity. Furthermore, the zero-inflated Poisson model showed that borrowing or sharing of bulls was associated with

Identification of the Quorum-Sensing Target DNA Sequence and N-Acyl Homoserine Lactone Responsiveness of the Brucella abortus virB promoter▿

PubMed Central

Arocena, Gastón M.; Sieira, Rodrigo; Comerci, Diego J.; Ugalde, Rodolfo A.

2010-01-01

VjbR is a LuxR-type quorum-sensing (QS) regulator that plays an essential role in the virulence of the intracellular facultative pathogen Brucella, the causative agent of brucellosis. It was previously described that VjbR regulates a diverse group of genes, including the virB operon. The latter codes for a type IV secretion system (T4SS) that is central for the pathogenesis of Brucella. Although the regulatory role of VjbR on the virB promoter (PvirB) was extensively studied by different groups, the VjbR-binding site had not been identified so far. Here, we identified the target DNA sequence of VjbR in PvirB by DNase I footprinting analyses. Surprisingly, we observed that VjbR specifically recognizes a sequence that is identical to a half-binding site of the QS-related regulator MrtR of Mesorhizobium tianshanense. As shown by DNase I footprinting and electrophoretic mobility shift assays, generation of a palindromic MrtR-like-binding site in PvirB increased both the affinity and the stability of the VjbR-DNA complex, which confirmed that the QS regulator of Brucella is highly related to that of M. tianshanense. The addition of N-dodecanoyl homoserine lactone dissociated VjbR from the promoter, which confirmed previous reports that indicated a negative effect of this signal on the VjbR-mediated activation of PvirB. Our results provide new molecular evidence for the structure of the virB promoter and reveal unusual features of the QS target DNA sequence of the main regulator of virulence in Brucella. PMID:20400542

Vaccination with recombinant L7/L12-truncated Omp31 protein induces protection against Brucella infection in BALB/c mice.

PubMed

Golshani, Maryam; Rafati, Sima; Dashti, Amir; Gholami, Elham; Siadat, Seyed Davar; Oloomi, Mana; Jafari, Anis; Bouzari, Saeid

2015-06-01

Brucellosis is the most common bacterial zoonotic disease worldwide and no vaccine is available for the prevention of human brucellosis. In humans, brucellosis is mostly caused by Brucella melitensis and Brucella abortus. The Outer membrane protein 31 (Omp31) and L7/L12 are immunodominant and protective antigens conserved in human Brucella pathogens. In the present study, we evaluated the humoral and cellular immune responses induced by a fusion protein designed based on the Truncated form of Omp31 (TOmp31) and L7-L12 antigens. Vaccination of BALB/c mice with the recombinant fusion protein (rL7/L12-TOmp31) provided the significant protection level against B. melitensis and B. abortus challenge. Moreover, rL7/L12-TOmp31 elicited a strong specific IgG response (higher IgG2a titers) and significant IFN-γ/IL2 production and T-cell proliferation was also observed. The T helper1 (Th1) oriented response persisted for 12 weeks after the first immunization. The rL7/L12-TOmp31 could be a new potential antigen candidate for the development of a subunit vaccine against B. melitensis and B. abortus. Copyright © 2015 Elsevier Ltd. All rights reserved.

Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells

PubMed Central

Czibener, Cecilia; Ugalde, Juan Esteban

2011-01-01

Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection. PMID:21911075

Identification of a unique gene cluster of Brucella spp. that mediates adhesion to host cells.

PubMed

Czibener, Cecilia; Ugalde, Juan Esteban

2012-01-01

Brucella, the causative agent of brucellosis, a major zoonotic disease affecting a broad range of mammals, is a gram-negative bacterium whose virulence is dependent on the capacity to attach and invade different cells of the host. The bacterium is able to infect through a diverse repertoire of epitheliums: skin, airways or gastric. Although much has been studied on the mechanisms Brucella uses to establish an intracellular replication niche, almost none is known on how the bacterium adheres and invades host cells. We report here the identification of a pathogenicity island that harbors a gene homologous to proteins with bacterial immunoglobulin-like domains present in other pathogens that play a role in attachment and invasion. Deletion of the entire island results in a mutant with a reduced attachment capacity measured by intracellular replication and adhesion assays. Intraperitoneal and oral experimental infection of mice strongly suggests that this island plays a role during the oral infection probably mediating attachment and trespassing of the gastric epithelium to establish a systemic infection. Copyright © 2011 Institut Pasteur. Published by Elsevier Masson SAS. All rights reserved.

Prepatellar bursitis due to Brucella abortus: case report and analysis of the local immune response.

PubMed

Wallach, Jorge C; Delpino, M Victoria; Scian, Romina; Deodato, Bettina; Fossati, Carlos A; Baldi, Pablo C

2010-12-01

A case of prepatellar bursitis in a man with chronic brucellosis is presented. Brucella abortus biotype 1 was isolated from the abundant yellowish fluid obtained from the bursa. Clinical and epidemiological data did not suggest a direct inoculation of the agent in the bursa. However, the patient mentioned occasional local trauma due to recreational sports, which may have constituted a predisposing factor. As determined by ELISA, there were higher levels of IgG against Brucella LPS and cytosolic proteins detected in the patient's bursal synovial fluid when compared with serum. Levels of proinflammatory cytokines (tumour necrosis factor alpha, interleukin 1 beta, gamma interferon, interleukin 8 and MCP-1) were higher than in synovial fluids obtained from patients with rheumatoid arthritis and a patient with septic arthritis, and a zymographic analysis revealed a gelatinase of about 92 kDa. These findings indicate that it may be possible to diagnose brucellar bursitis by measuring specific antibodies in the bursal synovial fluid. In addition, our findings suggest a role of increased local levels of proinflammatory cytokines and gelatinases in the inflammatory manifestations of brucellar bursitis.

Detection of Antibodies to Brucella Cytoplasmic Proteins in the Cerebrospinal Fluid of Patients with Neurobrucellosis

PubMed Central

Baldi, Pablo C.; Araj, George F.; Racaro, Graciela C.; Wallach, Jorge C.; Fossati, Carlos A.

1999-01-01

The diagnosis of human neurobrucellosis usually relies on the detection of antibodies to Brucella lipopolysaccharide (LPS) in cerebrospinal fluid (CSF) by agglutination tests or enzyme-linked immunosorbent assay (ELISA). Here we describe the detection of immunoglobulin G (IgG) to cytoplasmic proteins (CP) of Brucella spp. by ELISA and Western blotting in seven CSF samples from five patients with neurobrucellosis. While IgG to CP (titers of 200 to 12,800) and IgG to LPS (800 to 6,400) were found in the CSF of these patients, these antibodies were not detected in CSF samples from two patients who had systemic brucellosis without neurological involvement. The latter, however, had serum IgG and IgM to both LPS and CP. No reactivity to these antigens was found in CSF samples from 14 and 20 patients suffering from nonbrucellar meningitis and noninfectious diseases, respectively. These findings suggest that, in addition to its usefulness in the serological diagnosis of human systemic brucellosis, the ELISA with CP antigen can be used for the specific diagnosis of human neurobrucellosis. PMID:10473531

Interaction Network and Localization of Brucella abortus Membrane Proteins Involved in the Synthesis, Transport, and Succinylation of Cyclic β-1,2-Glucans

PubMed Central

Guidolin, Leticia S.; Morrone Seijo, Susana M.; Guaimas, Francisco F.

2015-01-01

ABSTRACT Cyclic β-1,2-glucans (CβG) are periplasmic homopolysaccharides that play an important role in the virulence and interaction of Brucella with the host. Once synthesized in the cytoplasm by the CβG synthase (Cgs), CβG are transported to the periplasm by the CβG transporter (Cgt) and succinylated by the CβG modifier enzyme (Cgm). Here, we used a bacterial two-hybrid system and coimmunoprecipitation techniques to study the interaction network between these three integral inner membrane proteins. Our results indicate that Cgs, Cgt, and Cgm can form both homotypic and heterotypic interactions. Analyses carried out with Cgs mutants revealed that the N-terminal region of the protein (Cgs region 1 to 418) is required to sustain the interactions with Cgt and Cgm as well as with itself. We demonstrated by single-cell fluorescence analysis that in Brucella, Cgs and Cgt are focally distributed in the membrane, particularly at the cell poles, whereas Cgm is mostly distributed throughout the membrane with a slight accumulation at the poles colocalizing with the other partners. In summary, our results demonstrate that Cgs, Cgt, and Cgm form a membrane-associated biosynthetic complex. We propose that the formation of a membrane complex could serve as a mechanism to ensure the fidelity of CβG biosynthesis by coordinating their synthesis with the transport and modification. IMPORTANCE In this study, we analyzed the interaction and localization of the proteins involved in the synthesis, transport, and modification of Brucella abortus cyclic β-1,2-glucans (CβG), which play an important role in the virulence and interaction of Brucella with the host. We demonstrate that these proteins interact, forming a complex located mainly at the cell poles; this is the first experimental evidence of the existence of a multienzymatic complex involved in the metabolism of osmoregulated periplasmic glucans in bacteria and argues for another example of pole differentiation in Brucella

A Live Vaccine from Brucella abortus Strain 82 for Control of Cattle Brucellosis in the Russian Federation

USDA-ARS?s Scientific Manuscript database

During the first half of the 20th century, widespread regulatory efforts to control cattle brucellosis (Brucella abortus) in the Union of Soviet Socialist Republics were essentially nonexistent, and control was limited to selective test and slaughter of serologic agglutination reactors. By the 1950...

Brucellae through the food chain: the role of sheep, goats and springbok (Antidorcus marsupialis) as sources of human infections in Namibia.

PubMed

Magwedere, K; Bishi, A; Tjipura-Zaire, G; Eberle, G; Hemberger, Y; Hoffman, L C; Dziva, F

2011-12-01

A confirmed case of human brucellosis motivated an investigation into the potential source of infection in Namibia. Since domestic animals are principal sources of Brucella infection in humans, 1692 serum samples were screened from sheep, goats and cattle from 4 presumably at-risk farms and 900 springbok (Antidorcas marsupialis) serum samples from 29 mixed farming units for Brucella antibodies by the Rose-Bengal test (RBT) and positive cases confirmed by complement fixation test (CFT). To assess the prevalence of human brucellosis, 137 abattoir employees were tested for Brucella antibodies using the standard tube agglutination test (STAT) and by enzyme linked immunosorbent assay (ELISA). Cattle and sheep from all 4 farms were negative by RBT and CFT but 2 of the 4 farms (Ba and C) had 26/42 and 12/285 seropositive goats, respectively. Post mortem examination of seropositive goats revealed no gross pathological lesions typical of brucellosis except enlarged mesenteric and iliac lymph nodes seen in a single buck. Culture for brucellae from organs of seropositive animals was negative. None of the wildlife sera tested positive by either RBT or CFT. Interviews revealed that besides the case that prompted the investigation, a family and another person from other farms with confirmed brucellosis shared a common history of consumption of unpasteurised goat milk, home-made goat cheese and coffee with raw milk and prior contact with goats, suggesting goats as the likely source of infection. All 137 abattoir employees tested negative by STAT, but 3 were positive by ELISA. The 3 abattoir workers were clinically normal and lacked historical connections with clinical cases. Although goats are often associated with B. melitensis, these studies could not explicitly implicate this species owing to cross-reactivity with B. abortus, which can also infect goats. Nevertheless, these data reinforce the need for a better National Control Programme for brucellosis in Namibia.

First report of a Brucella suis infection in roe deer (Capreolus capreolus).

PubMed

Sting, Reinhard; Schwabe, Ingo; Oehme, Rainer; Elschner, Mandy Carolina; Melzer, Falk

2014-01-01

In the present case report the detection of Brucella (B.) suis biovar 2 in roe deer (Capreolus capreolus) is described for the first time. The roe deer fawn was found emaciated and moribund in a hunting ground in the district Hohenlohe in Baden-Württemberg, Germany, in February 2013. A post-mortem examination revealed particularly a high-grade fibrinous pleurisy caused by the pathogen which could be multiplied in a dense growth on sheep blood agar and confirmed and differentiated subsequently by PCR.

Brucella melitensis infection following military duty in Iraq.

PubMed

Bechtol, D; Carpenter, L R; Mosites, E; Smalley, D; Dunn, J R

2011-11-01

Brucellosis is a common zoonotic disease worldwide; however, few cases are reported in the US. Brucella melitensis infections are primarily acquired via consumption of high-risk foods or travel to endemic areas. We describe a case of B. melitensis infection in a Tennessee soldier following deployment in Iraq. Initial symptoms included knee and back pain. Culture of an aspirate of the left sacroiliac joint yielded B. melitensis. Genetic analysis indicated that this isolate came from the Middle East. Investigation of laboratory workers identified risky exposures and positive serology prompting post-exposure prophylaxis. Military personnel and other travellers should be advised to reduce risk regarding food consumption and animal contact in endemic areas. Additionally, medical providers should remain vigilant for non-endemic zoonoses among recent travellers. © 2011 Blackwell Verlag GmbH.

Immuogenicity and safety of a natural rough mutant of Brucella suis as a vaccine for swine

USDA-ARS?s Scientific Manuscript database

The objective of the current study was to evaluate the safety, immunogenicity and clearance of the natural rough mutant of Brucella suis strain 353-1 (353-1) as a vaccine in domestic swine. In three studies encompassing 155 animals, pigs were inoculated with 353-1 by conjunctival (5 x 10**7 CFU), p...

Brucella spp. infection in large ruminants in an endemic area of Egypt: cross-sectional study investigating seroprevalence, risk factors and livestock owner's knowledge, attitudes and practices (KAPs).

PubMed

Holt, Hannah R; Eltholth, Mahmoud M; Hegazy, Yamen M; El-Tras, Wael F; Tayel, Ahmed A; Guitian, Javier

2011-05-19

Brucellosis is regarded as one of the major zoonotic infections worldwide. It was first reported in Egypt in 1939 and is now endemic, the predominate species of Brucella in cattle and buffalo in Egypt is B. melitensis. The aim of the study was to estimate seroprevalence of Brucella spp. in cattle and buffalo reared in households in an Egyptian village, identify risk factors for animals testing seropositive and to assess the knowledge, attitudes and practices (KAPs) of livestock owners with regards to brucellosis. A cross-sectional study was carried out in a village in Menufiya Governorate of Egypt. In June and July 2009, 107 households were selected using systematic sample and all lactating cattle and buffalo present in the household were sampled and tested for antibodies against Brucella spp. In addition, a questionnaire collecting information on potential risk factors for Brucella spp. infection in cattle and buffalo was administered to the household member responsible for rearing the livestock. Between December 2009 and February 2010 households were revisited and a second questionnaire regarding KAPs associated with brucellosis was administered. True individual and household seroprevalence were estimated to be 11.0% (95% CI: 3.06% to 18.4%) and 15.5% (95% CI: 6.61% to 24.7%), respectively. Cattle and buffalo kept in a household with sheep and goats had 6.32 (95% CI: 1.44 to 27.9) times the odds of testing seropositive for Brucella spp., compared to cattle and buffalo that were not. Most participants in the study stated that livestock owners assist in the parturition of ruminants without wearing gloves and that some farmers sell animals which they suspect are Brucella infected to butchers or at market. Many participants made their livestock's milk into cheese and other dairy products without pasteurising it. Brucellosis was endemic at high levels, in the current study. Although livestock owners had good general knowledge of brucellosis, they still appeared to

Brucella spp. infection in large ruminants in an endemic area of Egypt: cross-sectional study investigating seroprevalence, risk factors and livestock owner's knowledge, attitudes and practices (KAPs)

PubMed Central

2011-01-01

Background Brucellosis is regarded as one of the major zoonotic infections worldwide. It was first reported in Egypt in 1939 and is now endemic, the predominate species of Brucella in cattle and buffalo in Egypt is B. melitensis. The aim of the study was to estimate seroprevalence of Brucella spp. in cattle and buffalo reared in households in an Egyptian village, identify risk factors for animals testing seropositive and to assess the knowledge, attitudes and practices (KAPs) of livestock owners with regards to brucellosis. Methods A cross-sectional study was carried out in a village in Menufiya Governorate of Egypt. In June and July 2009, 107 households were selected using systematic sample and all lactating cattle and buffalo present in the household were sampled and tested for antibodies against Brucella spp. In addition, a questionnaire collecting information on potential risk factors for Brucella spp. infection in cattle and buffalo was administered to the household member responsible for rearing the livestock. Between December 2009 and February 2010 households were revisited and a second questionnaire regarding KAPs associated with brucellosis was administered. Results True individual and household seroprevalence were estimated to be 11.0% (95% CI: 3.06% to 18.4%) and 15.5% (95% CI: 6.61% to 24.7%), respectively. Cattle and buffalo kept in a household with sheep and goats had 6.32 (95% CI: 1.44 to 27.9) times the odds of testing seropositive for Brucella spp., compared to cattle and buffalo that were not. Most participants in the study stated that livestock owners assist in the parturition of ruminants without wearing gloves and that some farmers sell animals which they suspect are Brucella infected to butchers or at market. Many participants made their livestock's milk into cheese and other dairy products without pasteurising it. Conclusions Brucellosis was endemic at high levels, in the current study. Although livestock owners had good general knowledge of

TLR9 is required for MAPK/NF-κB activation but does not cooperate with TLR2 or TLR6 to induce host resistance to Brucella abortus.

PubMed

Gomes, Marco Túlio; Campos, Priscila Carneiro; Pereira, Guilherme de Sousa; Bartholomeu, Daniella Castanheira; Splitter, Gary; Oliveira, Sergio Costa

2016-05-01

Brucella abortus is a Gram-negative intracellular bacterial pathogen that causes a zoonosis of worldwide occurrence, leading to undulant fever in humans and abortion in domestic animals. B. abortus is recognized by several pattern-recognition receptors triggering pathways during the host innate immune response. Therefore, here, we determined the cooperative role of TLR9 with TLR2 or TLR6 receptors in sensing Brucella Furthermore, we deciphered the host innate immune response against B. abortus or its DNA, emphasizing the role of TLR9-MAPK/NF-κB signaling pathways in the production of proinflammatory cytokines. TLR9 is required for the initial host control of B. abortus, but this TLR was dispensable after 6 wk of infection. The susceptibility of TLR9(-/-)-infected animals to Brucella paralleled with lower levels of IFN-γ produced by mouse splenocytes stimulated with this pathogen compared with wild-type cells. However, no apparent cooperative interplay was observed between TLR2-TLR9 or TLR6-TLR9 receptors to control infection. Moreover, B. abortus or its DNA induced activation of MAPK/NF-κB pathways and production of IL-12 and TNF-α by macrophages partially dependent on TLR9 but completely dependent on MyD88. In addition, B. abortus-derived CpG oligonucleotides required TLR9 to promote IL-12 and TNF-α production by macrophages. By confocal microscopy, we demonstrated that TLR9 redistributed and colocalized with lysosomal-associated membrane protein-1 upon Brucella infection. Thus, B. abortus induced TLR9 traffic, leading to cell signaling activation and IL-12 and TNF-α production. Although TLR9 recognized Brucella CpG motifs, our results suggest a new pathway of B. abortus DNA-activating macrophages independent of TLR9. © Society for Leukocyte Biology.

Structural, functional and immunogenic insights on Cu,Zn Superoxide Dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

USDA-ARS?s Scientific Manuscript database

Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and he...

Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil.

PubMed

Furtado, Mariana Malzoni; Gennari, Solange Maria; Ikuta, Cassia Yumi; Jácomo, Anah Tereza de Almeida; de Morais, Zenaide Maria; Pena, Hilda Fátima de Jesus; Porfírio, Grasiela Edith de Oliveira; Silveira, Leandro; Sollmann, Rahel; de Souza, Gisele Oliveira; Tôrres, Natália Mundim; Ferreira Neto, José Soares

2015-01-01

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature.

Toll-Like Receptors 2 and 4 Cooperate in the Control of the Emerging Pathogen Brucella microti.

PubMed

Arias, Maykel A; Santiago, Llipsy; Costas-Ramon, Santiago; Jaime-Sánchez, Paula; Freudenberg, Marina; Jiménez De Bagüés, Maria P; Pardo, Julián

2016-01-01

Toll-like receptors (TLRs) recognize pathogen-derived molecules and play a critical role during the host innate and adaptive immune response. Brucella spp. are intracellular gram-negative bacteria including several virulent species, which cause a chronic zoonotic infection in a wide range of mammalian hosts known as brucellosis. A new Brucella species, Brucella microti , was recently isolated from wild rodents and found to be highly pathogenic in mice. Using this species-specific model, it was previously found that CD8 + T cells are required to control this infection. In order to find out the role of TLR-mediated responses in the control of this pathogen, the course of infection of B. microti was analyzed over 3 weeks in wild-type (WT) and TLR knock out (KO) mice including TLR2 -/- , TLR4 -/- , TLR9 -/- , TLR2×4 -/- and TLR2×4×9 -/- . WT and single TLR2, TLR4 and TLR9 KO mice similarly control infection in liver and spleen. In contrast, bacterial clearance was delayed in TLR2×4 -/- and TLR2×4×9 -/- mice at 7 and 14 days post-infection. This defect correlated with impaired maturation and pro-inflammatory cytokine production in B. microti -infected dendritic cells from TLR2×4 -/- and TLR2×4×9 -/- mice. Finally, it was found that Tc cells from TLR2×4 -/- and TLR2×4×9 -/- mice showed reduced ability to inhibit growth of B. microti in macrophages, suggesting the involvement of TLR2 and 4 in the generation of specific Tc cells. Our findings indicate that TLR2 and TLR4 are required to control B. microti infection in mice and that this effect could be related to its participation in the maturation of dendritic cells and the generation of specific CD8 + Tc cells.

Utilization of a specific in vitro lymphocyte immunostimulation assay as an aid in detection of brucella-infected cattle not detected by serological tests.

PubMed Central

Kaneene, J M; Johnson, D W; Anderson, R K; Muscoplat, C C

1978-01-01

Studies using the in vitro lymphocyte stimulation test (LST) were conducted with cattle in a dairy herd with a high percentage of reactors to several serological tests for brucellosis. Lymphocytes were prepared from peripheral bovine blood by the Ficoll-diatrizoate technique. Lymphocytes were cultured using microtitration culture plates. Brucella abortus soluble antigen, at a concentration of 4.4 microgram/culture, was added to the appropriate wells of microtitration culture plates and incubated for 6 days. The lymphocyte stimulation responses were measured by assaying for [3H]thymidine incorporation into DNA. Seroagglutination tests were conducted simultaneously with the LST, and tissues were collected after slaughter of the cattle for bacteriological culture to isolate B. abortus. All 21 animals studied were serologically negative for anti-brucella antibodies. Two of the 21 animals were classified as infected with Brucella by the LST, and B. abortus biotype 1 was isolated from tissues of these same two animals. The LST exhibited significant sensitivity and specificity in this study, and more observations of this nature might strengthen the application of this assay as an aid in the diagnosis of brucellosis. PMID:103888

Immunological response to Brucella abortus strain 19 vaccination of cattle in a communal area in South Africa.

PubMed

Simpson, Gregory J G; Marcotty, Tanguy; Rouille, Elodie; Chilundo, Abel; Letteson, Jean-Jacques; Godfroid, Jacques

2018-03-29

Brucellosis is of worldwide economic and public health importance. Heifer vaccination with live attenuated Brucella abortus strain 19 (S19) is the cornerstone of control in low- and middle-income countries. Antibody persistence induced by S19 is directly correlated with the number of colony-forming units (CFU) per dose. There are two vaccination methods: a 'high' dose (5-8 × 1010 CFU) subcutaneously injected or one or two 'low' doses (5 × 109 CFU) through the conjunctival route. This study aimed to evaluate serological reactions to the 'high' dose and possible implications of the serological findings on disease control. This study included 58 female cases, vaccinated at Day 0, and 29 male controls. Serum was drawn repeatedly and tested for Brucella antibodies using the Rose Bengal Test (RBT) and an indirect enzyme-linked immunosorbent assay (iELISA). The cases showed a rapid antibody response with peak RBT positivity (98%) at 2 weeks and iELISA (95%) at 8 weeks, then decreased in an inverse logistic curve to 14% RBT and 32% iELISA positive at 59 weeks and at 4.5 years 57% (4/7 cases) demonstrated a persistent immune response (RBT, iELISA or Brucellin skin test) to Brucella spp. Our study is the first of its kind documenting the persistence of antibodies in an African communal farming setting for over a year to years after 'high' dose S19 vaccination, which can be difficult to differentiate from a response to infection with wild-type B. abortus. A recommendation could be using a 'low' dose or different route of vaccination.

Gamma Interferon Loaded onto Albumin Nanoparticles: In Vitro and In Vivo Activities against Brucella abortus▿

PubMed Central

Segura, S.; Gamazo, C.; Irache, J. M.; Espuelas, S.

2007-01-01

The aim of this study was to evaluate the activity of gamma interferon (IFN-γ) when it was either adsorbed onto or loaded into albumin nanoparticles. Brucella abortus-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-γ-activated macrophages for the control of Brucella sp. infections. Whereas the encapsulation of IFN-γ inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-γ increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-γ, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-γ adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-γ-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-γ to macrophages and, thus, potentiate their therapeutic activity. PMID:17220401

Isolation of brucella abortus from two dogs in contact with bovine brucellosis.

PubMed Central

Prior, M G

1976-01-01

On a farm where several cattle were serologically positive for bovine brucellosis, three dogs were found to have titres greater than 400 i.u. to Brucella abortus. The titres persisted until the dogs were killed over two months later. Two male dogs were necropsied. B. abortus was isolated from the spleen of both dogs. While farm dogs are not thought to be a major reservoir of bovine brucellosis they may be considered as possible carriers in imfected herds and should be considered during the investigation and eradication of bovine brucellosis. PMID:826308

Comparison of abortion and infection after experimental challenge of pregnant bison and cattle with Brucella abortus strain 2308

USDA-ARS?s Scientific Manuscript database

A comparative study was conducted using data from naive bison (n=45) and cattle (n=46) from 8 and 6 studies, respectively, in which a standardized Brucella abortus strain 2308 experimental challenge was administered. The incidence of abortion, fetal infection, uterine or mammary infection, or infec...

Efficacy of dart or booster vaccination with strain RB51 in protecting bison against experimental Brucella abortus challenge

USDA-ARS?s Scientific Manuscript database

Vaccination is an effective tool for reducing the prevalence of brucellosis in natural hosts. In this study, we characterized the efficacy of the Brucella abortus strain RB51 (RB51) vaccine in bison when delivered by single intramuscular vaccination (Hand RB51), single pneumatic dart delivery (Dart ...

Serosurvey of Smooth Brucella, Leptospira spp. and Toxoplasma gondii in Free-Ranging Jaguars (Panthera onca) and Domestic Animals from Brazil

PubMed Central

Furtado, Mariana Malzoni; Gennari, Solange Maria; Ikuta, Cassia Yumi; Jácomo, Anah Tereza de Almeida; de Morais, Zenaide Maria; Pena, Hilda Fátima de Jesus; Porfírio, Grasiela Edith de Oliveira; Silveira, Leandro; Sollmann, Rahel; de Souza, Gisele Oliveira; Tôrres, Natália Mundim; Ferreira Neto, José Soares

2015-01-01

This study investigated the exposure of jaguar populations and domestic animals to smooth Brucella, Leptospira spp. and Toxoplasma gondii in the Cerrado, Pantanal and Amazon biomes of Brazil. Between February 2000 and January 2010, serum samples from 31 jaguars (Panthera onca), 1,245 cattle (Bos taurus), 168 domestic dogs (Canis lupus familiaris) and 29 domestic cats (Felis catus) were collected and analysed by rose bengal test for smooth Brucella, microscopic agglutination test for Leptospira spp. and modified agglutination test for T. gondii. Cattle populations from all sites (9.88%) were exposed to smooth Brucella, but only one jaguar from Cerrado was exposed to this agent. Jaguars captured in the Cerrado (60.0%) and in the Pantanal (45.5%) were seropositive for different serovars of Leptospira spp., cattle (72.18%) and domestic dogs (13.1%) from the three sites and one domestic cat from Pantanal were also seropositive for the agent. The most prevalent serotype of Leptospira spp. identified in jaguars from the Cerrado (Grippotyphosa) and the Pantanal (Pomona) biomes were distinct from those found in the domestic animals sampled. Jaguars (100%), domestic dogs (38.28%) and domestic cats (82.76%) from the three areas were exposed to T. gondii. Our results show that brucellosis and leptospirosis could have been transmitted to jaguars by domestic animals; and jaguars probably play an important role in the maintenance of T. gondii in nature. PMID:26605787

Fed batch fermentation and purification strategy for high yield production of Brucella melitensis recombinant Omp 28 kDa protein and its application in disease diagnosis.

PubMed

Karothia, B S; Athmaram, T N; D, Thavaselvam; Ashu, Kumar; Tiwari, Sapna; Singh, Anil K; Sathyaseelan, K; Gopalan, N

2013-07-01

Brucellosis is a disease caused by bacteria belonging to the genus Brucella. It affects cattle, goat, sheep, dog and humans. The serodiagnosis of brucellosis involves detection of antibodies generated against the LPS or whole cell bacterial extracts, however these tests lack sensitivity and specificity. The present study was performed to optimize the culture condition for the production of recombinant Brucella melitensis outer membrane protein 28 kDa protein in E.coli via fed batch fermentation. Expression was induced with 1.5mM isopropyl β thiogalactoside and the expressed recombinant protein was purified using Ni-NTA affinity chromatography. After fed-batch fermentation the dry cell weight of 17.81 g/L and a purified protein yield of 210.10 mg/L was obtained. The purified Brucella melitensis recombinant Omp 28 kDa protein was analyzed through SDS- poly acrylamide gel electrophoresis and western blotting. The obtained recombinant protein was evaluated for its diagnostic application through Indirect ELISA using brucellosis suspected human sera samples. Our results clearly indicate that recombinant Omp28 produced via fed batch fermentation has immense potential as a diagnostic reagent that could be employed in sero monitoring of brucellosis.

Pheno- and genotyping of Brucella abortus biovar 5 isolated from a water buffalo (Bubalus bubalis) fetus: First case reported in the Americas.

PubMed

Martínez, Diana; Thompson, Carolina; Draghi, Graciela; Canavesio, Vilma; Jacobo, Roberto; Zimmer, Patricia; Elena, Sebastián; Nicola, Ana M; de Echaide, Susana Torioni

2014-09-17

An isolate of Brucella spp. from an aborted water buffalo (Bubalus bubalis) fetus was characterized based on its pheno- and genotype. The phenotype was defined by carbon dioxide requirement, hydrogen sulfide production, sensitivity to thionin and basic fuchsin and agglutination with Brucella A and M monospecific antisera. The genotype was based on the amplification of the following genes: bcsp31, omp2ab, and eri and the species-specific localization of the insertion sequence IS711 in the Brucella chromosome via B. abortus-B. melitensis-B. ovis-B. suis (AMOS)-PCR. Unexpectedly, the isolate showed a phenotype different from B. abortus bv 1, the most prevalent strain in cattle in Argentina, and from vaccine strain 19, currently used in bovines and water buffaloes. Genotyping supported the phenotypic results, as the analysis of the omp2ab gene sequence showed an identical pattern to either B. abortus bv 5 or B. melitensis. Finally, the AMOS PCR generated a 1700-bp fragment from the isolate, different than those amplified from B. abortus bv 1 (498bp) and B. melitensis (731bp), confirming the presence of B. abortus bv 5. The OIE/FAO Reference Laboratory for Brucellosis confirmed this typing. This is the first report of B. abortus bv 5 from a water buffalo in the Americas. Copyright © 2014 Elsevier B.V. All rights reserved.

Immune Responses and Protection against Experimental Brucella suis biovar 1 Challenge in Non-vaccinated or RB51-Vaccinated Cattle

USDA-ARS?s Scientific Manuscript database

Twenty Hereford heifers, approximately 9 months of age, were vaccinated with saline (control) or 2 x 10**10 CFU of Brucella abortus strain RB51 (RB51) vaccine. Immunologic responses after inoculation demonstrated significantly greater (P<0.05) antibody and proliferative responses to RB51 antigens i...

Diagnosis and successful treatment of a lung abscess associated with Brucella species infection in a bottlenose dolphin (Tursiops truncatus).

PubMed

Cassle, Stephen E; Jensen, Eric D; Smith, Cynthia R; Meegan, Jennifer M; Johnson, Shawn P; Lutmerding, Betsy; Ridgway, Sam H; Francis-Floyd, Ruth

2013-06-01

This brief communication describes the clinical presentation, antemortem diagnosis, and successful treatment of a pulmonary abscess associated with a Brucella sp. in a 27-yr-old female bottlenose dolphin (Tursiops truncatus). Ultrasound revealed a 3-cm diameter hypoechoic mass deep to the pleural lining in the left lung field. Multiple ultrasound-guided fine-needle aspirates were performed and tested for bacterial and fungal etiology. All cultures were negative, but the infectious agent was identified by MicroSEQ analysis in two samples and confirmed with real-time polymerase chain reaction (PCR) amplification using known Brucella sp. primers. Amikacin was infused into the abscess and was followed by an oral doxycycline and rifampin protocol. Follow-up diagnostic imaging, including radiographs and computed tomography, revealed a resolved lesion with minimal mineralization within the affected lung fields. Brucellosis should be considered for pulmonary disease in dolphins, and personnel who interact with marine animals should use caution to prevent zoonotic brucellosis.

Development and trial of vaccines against Brucella.

PubMed

Lalsiamthara, Jonathan; Lee, John Hwa

2017-08-31

The search for ideal brucellosis vaccines remains active today. Currently, no licensed human or canine anti-brucellosis vaccines are available. In bovines, the most successful vaccine (S19) is only used in calves, as adult vaccination results in orchitis in male, prolonged infection, and possible abortion complications in pregnant female cattle. Another widely deployed vaccine (RB51) has a low protective efficacy. An ideal vaccine should exhibit a safe profile as well as enhance protective efficacy. However, currently available vaccines exhibit one or more major drawbacks. Smooth live attenuated vaccines suffer shortcomings such as residual virulence and serodiagnostic interference. Inactivated vaccines, in general, confer relatively low levels of protection. Recent developments to improve brucellosis vaccines include generation of knockout mutants by targeting genes involved in metabolism, virulence, and the lipopolysaccharide synthesis pathway, as well as generation of DNA vaccines, mucosal vaccines, and live vectored vaccines, have all produced varying degrees of success. Herein, we briefly review the bacteriology, pathogenesis, immunological implications, candidate vaccines, vaccinations, and models related to Brucella .

Development and trial of vaccines against Brucella

PubMed Central

Lalsiamthara, Jonathan

2017-01-01

The search for ideal brucellosis vaccines remains active today. Currently, no licensed human or canine anti-brucellosis vaccines are available. In bovines, the most successful vaccine (S19) is only used in calves, as adult vaccination results in orchitis in male, prolonged infection, and possible abortion complications in pregnant female cattle. Another widely deployed vaccine (RB51) has a low protective efficacy. An ideal vaccine should exhibit a safe profile as well as enhance protective efficacy. However, currently available vaccines exhibit one or more major drawbacks. Smooth live attenuated vaccines suffer shortcomings such as residual virulence and serodiagnostic interference. Inactivated vaccines, in general, confer relatively low levels of protection. Recent developments to improve brucellosis vaccines include generation of knockout mutants by targeting genes involved in metabolism, virulence, and the lipopolysaccharide synthesis pathway, as well as generation of DNA vaccines, mucosal vaccines, and live vectored vaccines, have all produced varying degrees of success. Herein, we briefly review the bacteriology, pathogenesis, immunological implications, candidate vaccines, vaccinations, and models related to Brucella. PMID:28859268

Brucella melitensis in France: Persistence in Wildlife and Probable Spillover from Alpine Ibex to Domestic Animals

PubMed Central

Mick, Virginie; Le Carrou, Gilles; Corde, Yannick; Game, Yvette; Jay, Maryne; Garin-Bastuji, Bruno

2014-01-01

Bovine brucellosis is a major zoonosis, mainly caused by Brucella abortus, more rarely by Brucella melitensis. France has been bovine brucellosis officially-free since 2005 with no cases reported in domestic/wild ruminants since 2003. In 2012, bovine and autochthonous human cases due to B. melitensis biovar 3 (Bmel3) occurred in the French Alps. Epidemiological investigations implemented in wild and domestic ruminants evidenced a high seroprevalence (>45%) in Alpine ibex (Capra ibex); no cases were disclosed in other domestic or wild ruminants, except for one isolated case in a chamois (Rupicapra rupicapra). These results raised the question of a possible persistence/emergence of Brucella in wildlife. The purpose of this study was to assess genetic relationships among the Bmel3 strains historically isolated in humans, domestic and wild ruminants in Southeastern France, over two decades, by the MLVA-panel2B assay, and to propose a possible explanation for the origin of the recent bovine and human infections. Indeed, this genotyping strategy proved to be efficient for this microepidemiological investigation using an interpretation cut-off established for a fine-scale setting. The isolates, from the 2012 domestic/human outbreak harbored an identical genotype, confirming a recent and direct contamination from cattle to human. Interestingly, they clustered not only with isolates from wildlife in 2012, but also with local historical domestic isolates, in particular with the 1999 last bovine case in the same massif. Altogether, our results suggest that the recent bovine outbreak could have originated from the Alpine ibex population. This is the first report of a B. melitensis spillover from wildlife to domestic ruminants and the sustainability of the infection in Alpine ibex. However, this wild population, reintroduced in the 1970s in an almost closed massif, might be considered as a semi-domestic free-ranging herd. Anthropogenic factors could therefore account with the

Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

PubMed Central

Goldbaum, F A; Leoni, J; Wallach, J C; Fossati, C A

1993-01-01

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24. Images PMID:8370742

Characterization of an 18-kilodalton Brucella cytoplasmic protein which appears to be a serological marker of active infection of both human and bovine brucellosis.

PubMed

Goldbaum, F A; Leoni, J; Wallach, J C; Fossati, C A

1993-08-01

Some anticytoplasmic protein monoclonal antibodies (MAbs) from mice immunized by infection with Brucella ovis cells have been obtained. One of these MAbs, BI24, was used to purify by immunoaffinity a protein with a pI of 5.6 and a molecular mass of 18 kDa. This protein was present in all of the rough and smooth Brucella species studied, but it could not be detected in Yersinia enterocolitica 09. Three internal peptides of this protein were partially sequenced; no homology with other bacterial proteins was found. The immunogenicity of the 18-kDa protein was studied with both human and bovine sera by a capture enzyme-linked immunosorbent assay system with MAb BI24.

Identification of Brucella melitensis Rev.1 vaccine-strain genetic markers: Towards understanding the molecular mechanism behind virulence attenuation.

PubMed

Issa, Mohammad Nouh; Ashhab, Yaqoub

2016-09-22

Brucella melitensis Rev.1 is an avirulent strain that is widely used as a live vaccine to control brucellosis in small ruminants. Although an assembled draft version of Rev.1 genome has been available since 2009, this genome has not been investigated to characterize this important vaccine. In the present work, we used the draft genome of Rev.1 to perform a thorough genomic comparison and sequence analysis to identify and characterize the panel of its unique genetic markers. The draft genome of Rev.1 was compared with genome sequences of 36 different Brucella melitensis strains from the Brucella project of the Broad Institute of MIT and Harvard. The comparative analyses revealed 32 genetic alterations (30 SNPs, 1 single-bp insertion and 1 single-bp deletion) that are exclusively present in the Rev.1 genome. In silico analyses showed that 9 out of the 17 non-synonymous mutations are deleterious. Three ABC transporters are among the disrupted genes that can be linked to virulence attenuation. Out of the 32 mutations, 11 Rev.1 specific markers were selected to test their potential to discriminate Rev.1 using a bi-directional allele-specific PCR assay. Six markers were able to distinguish between Rev.1 and a set of control strains. We succeeded in identifying a panel of 32 genome-specific markers of the B. melitensis Rev.1 vaccine strain. Extensive in silico analysis showed that a considerable number of these mutations could severely affect the function of the associated genes. In addition, some of the discovered markers were able to discriminate Rev.1 strain from a group of control strains using practical PCR tests that can be applied in resource-limited settings. Copyright © 2016 Elsevier Ltd. All rights reserved.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams.

PubMed

Moustacas, Valéria S; Silva, Teane M A; Costa, Luciana F; Xavier, Mariana N; Carvalho, Custódio A; Costa, Érica A; Paixão, Tatiane A; Santos, Renato L

2013-03-21

Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis.

Effects of Opsonization and Gamma Interferon on Growth of Brucella Melitensis 16M in Mouse Peritoneal Macrophages In Vitro

DTIC Science & Technology

2000-01-01

bacterial CFU (Fig. 3). Macrophages cultured without brucel - lae or cultured with brucellae but without IFN-7 made ɘ.3 nmol of nitrite/200 JJLI well...receptors used for the uptake of nonopsonized brucel - lae are unknown. Synergy between the receptor for the Fc domain of immunoglobulin G (FcR) and

Immunogenic proteins of Brucella abortus to minimize cross reactions in brucellosis diagnosis.

PubMed

Ko, Kyung Yuk; Kim, Jong-Wan; Her, Moon; Kang, Sung-Il; Jung, Suk Chan; Cho, Dong Hee; Kim, Ji-Yeon

2012-05-04

To overcome the limitations of serological diagnosis, including false positive reactions caused by other pathogens, specific antigens for diagnosis of brucellosis other than LPS have been required. The present study was conducted to separate and identify immuno-dominant insoluble proteins of Brucella abortus against the antisera of cattle infected with B. abortus, or/and Yersinia enterocolitica, or the sera of non-infected cattle. After separating insoluble proteins of B. abortus by two dimensional electrophoresis (2-DE), their immuno-reactivity was determined by western blotting. A portion of the immunogenic spots against the positive antisera of B. abortus that have the potential for use as specific antigens were identified by MS/MS analysis. Overall, 18 immunogenic insoluble proteins of B. abortus 1119-3 showed immuno-reactivity against only the positive antisera of B. abortus, but failed to have immunogenicity toward both the positive sera of Y. enterocolitica and the negative sera of B. abortus. Identification of these proteins revealed the following: F0F1 ATP synthase subunit β, solute-binding family 5 protein, 28 kDa OMP, Leu/Ile/Val-binding family protein, Histidinol dehyddrogenase, Hypothetical protein, Twin-arginine translocation pathway signal sequence domain-containing protein, Dihydroorotase, Serine protease family protein, β-hydroxyacyl-(acyl-carrier-protein) dehydratase FabA, Short-chain dehydrogenase-/reductase carbonic anhydrase, Orinithine carbamoyltransferase, Leucyl aminopeptidase, Cold shock DNA-binding domain-containing protein, Cu/Zn superoxide dismutase, and Methionine aminopeptidase. The 18 immunogenic proteins separated in the present study can be considered candidate antigens to minimize cross reaction in the diagnosis of brucellosis and useful sources for Brucella vaccine development. Copyright © 2011 Elsevier B.V. All rights reserved.

Brucella abortus Inhibits Major Histocompatibility Complex Class II Expression and Antigen Processing through Interleukin-6 Secretion via Toll-Like Receptor 2▿

PubMed Central

Barrionuevo, Paula; Cassataro, Juliana; Delpino, M. Victoria; Zwerdling, Astrid; Pasquevich, Karina A.; Samartino, Clara García; Wallach, Jorge C.; Fossati, Carlos A.; Giambartolomei, Guillermo H.

2008-01-01

The strategies that allow Brucella abortus to survive inside macrophages for prolonged periods and to avoid the immunological surveillance of major histocompatibility complex class II (MHC-II)-restricted gamma interferon (IFN-γ)-producing CD4+ T lymphocytes are poorly understood. We report here that infection of THP-1 cells with B. abortus inhibited expression of MHC-II molecules and antigen (Ag) processing. Heat-killed B. abortus (HKBA) also induced both these phenomena, indicating the independence of bacterial viability and involvement of a structural component of the bacterium. Accordingly, outer membrane protein 19 (Omp19), a prototypical B. abortus lipoprotein, inhibited both MHC-II expression and Ag processing to the same extent as HKBA. Moreover, a synthetic lipohexapeptide that mimics the structure of the protein lipid moiety also inhibited MHC-II expression, indicating that any Brucella lipoprotein could down-modulate MHC-II expression and Ag processing. Inhibition of MHC-II expression and Ag processing by either HKBA or lipidated Omp19 (L-Omp19) depended on Toll-like receptor 2 and was mediated by interleukin-6. HKBA or L-Omp19 also inhibited MHC-II expression and Ag processing of human monocytes. In addition, exposure to the synthetic lipohexapeptide inhibited Ag-specific T-cell proliferation and IFN-γ production of peripheral blood mononuclear cells from Brucella-infected patients. Together, these results indicate that there is a mechanism by which B. abortus may prevent recognition by T cells to evade host immunity and establish a chronic infection. PMID:17984211

Proteomic Profile of Brucella abortus-Infected Bovine Chorioallantoic Membrane Explants

PubMed Central

Mol, Juliana P. S.; Pires, Simone F.; Chapeaurouge, Alexander D.; Perales, Jonas; Santos, Renato L.; Andrade, Hélida M.; Lage, Andrey P.

2016-01-01

Brucella abortus is the etiological agent of bovine brucellosis, a zoonotic disease that causes significant economic losses worldwide. The differential proteomic profile of bovine chorioallantoic membrane (CAM) explants at early stages of infection with B. abortus (0.5, 2, 4, and 8 h) was determined. Analysis of CAM explants at 0.5 and 4 h showed the highest differences between uninfected and infected CAM explants, and therefore were used for the Differential Gel Electrophoresis (DIGE). A total of 103 spots were present in only one experimental group and were selected for identification by mass spectrometry (MALDI/ToF-ToF). Proteins only identified in extracts of CAM explants infected with B. abortus were related to recognition of PAMPs by TLR, production of reactive oxygen species, intracellular trafficking, and inflammation. PMID:27104343

A multicopper oxidase contributes to the copper tolerance of Brucella melitensis 16M.

PubMed

Wu, Tonglei; Wang, Shaohua; Wang, Zhen; Peng, Xiaowei; Lu, Yanli; Wu, Qingmin

2015-06-01

Copper is a potent antimicrobial agent. Multiple mechanisms of copper tolerance are utilized by some pathogenic bacteria. BMEII0580, which is significantly similar to the multicopper oxidase from Escherichia coli, was predicted to be the probable blue copper protein YacK precursor in Brucella melitensis 16M, and was designated as Brucella multicopper oxidase (BmcO). A bioinformatics analysis indicated that the typical motifs of multicopper oxidases are present in BmcO. BmcO, the expression of which was up-regulated by copper, could catalyze the oxidation of 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), dimethoxyphenol (DMP) and para-phenylenediamine (pPD), which are widely used as substrates for multicopper oxidase. Additionally, BmcO exhibited ferroxidase activity, which indicated that it might play an important role in the Fe(2+) uptake of B. melitensis. Importantly, the mutant strain 16MΔbmcO was more sensitive to copper than the wild-type strain B. melitensis 16M as well as its complementation strain 16MΔbmcO(bmcO). The infection assays of cells showed that similar bacterial numbers of B. melitensis 16M, 16MΔbmcO and 16MΔbmcO(bmcO) strains were recovered from the infected macrophages. This result indicated that BmcO was not essential for B. melitensis intracellular growth. In conclusion, our results confirm that BmcO is a multicopper oxidase and contributes to the copper tolerance of B. melitensis 16M. © FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Identification and Characterization of a High-Affinity Choline Uptake System of Brucella abortus

PubMed Central

Herrmann, Claudia K.; Bukata, Lucas; Melli, Luciano; Marchesini, M. Ines; Caramelo, Julio J.

2013-01-01

Phosphatidylcholine (PC), a common phospholipid of the eukaryotic cell membrane, is present in the cell envelope of the intracellular pathogen Brucella abortus, the etiological agent of bovine brucellosis. In this pathogen, the biosynthesis of PC proceeds mainly through the phosphatidylcholine synthase pathway; hence, it relies on the presence of choline in the milieu. These observations imply that B. abortus encodes an as-yet-unknown choline uptake system. Taking advantage of the requirement of choline uptake for PC synthesis, we devised a method that allowed us to identify a homologue of ChoX, the high-affinity periplasmic binding protein of the ABC transporter ChoXWV. Disruption of the choX gene completely abrogated PC synthesis at low choline concentrations in the medium, thus indicating that it is a high-affinity transporter needed for PC synthesis via the PC synthase (PCS) pathway. However, the synthesis of PC was restored when the mutant was incubated in media with higher choline concentrations, suggesting the presence of an alternative low-affinity choline uptake activity. By means of a fluorescence-based equilibrium-binding assay and using the kinetics of radiolabeled choline uptake, we show that ChoX binds choline with an extremely high affinity, and we also demonstrate that its activity is inhibited by increasing choline concentrations. Cell infection assays indicate that ChoX activity is required during the first phase of B. abortus intracellular traffic, suggesting that choline concentrations in the early and intermediate Brucella-containing vacuoles are limited. Altogether, these results suggest that choline transport and PC synthesis are strictly regulated in B. abortus. PMID:23161032

A B lymphocyte mitogen is a Brucella abortus virulence factor required for persistent infection

PubMed Central

Spera, Juan Manuel; Ugalde, Juan Esteban; Mucci, Juan; Comerci, Diego J.; Ugalde, Rodolfo Augusto

2006-01-01

Microbial pathogens with the ability to establish chronic infections have evolved strategies to actively modulate the host immune response. Brucellosis is a disease caused by a Gram-negative intracellular pathogen that if not treated during the initial phase of the infection becomes chronic as the bacteria persist for the lifespan of the host. How this pathogen and others achieve this action is a largely unanswered question. We report here the identification of a Brucella abortus gene (prpA) directly involved in the immune modulation of the host. PrpA belongs to the proline-racemase family and elicits a B lymphocyte polyclonal activation that depends on the integrity of its proline-racemase catalytic site. Stimulation of splenocytes with PrpA also results in IL-10 secretion. Construction of a B. abortus-prpA mutant allowed us to assess the contribution of PrpA to the infection process. Mice infected with B. abortus induced an early and transient nonresponsive status of splenocytes to both Escherichia coli LPS and ConA. This phenomenon was not observed when mice were infected with a B. abortus-prpA mutant. Moreover, the B. abortus-prpA mutant had a reduced capacity to establish a chronic infection in mice. We propose that an early and transient nonresponsive immune condition of the host mediated by this B cell polyclonal activator is required for establishing a successful chronic infection by Brucella. PMID:17053080

Bactericidal Action of Fresh Rabbit Blood Against Brucella abortus

PubMed Central

Joos, Richard W.; Hall, Wendell H.

1968-01-01

A photometric method was used to measure the bactericidal kinetics for Brucella abortus of freshly drawn rabbit blood during the time before clotting. This antibrucellar activity varied between rabbits in different immunologic states. Nonimmunized rabbits had moderate bactericidal activity after a lag of about 2 min. The blood of some immunized rabbits gave an immediate and strong kill, but in certain other immunized rabbits, especially when hyperimmunized, the bactericidal activity was inhibited. It appeared that serum bactericidins and complement are sometimes as active in unclotted blood as they are in serum. However, this bactericidal activity can be either increased or neutralized by immunization. The prozone bactericidal inhibition phenomenon (Neisser-Wechsberg) found in immune serum may, in fact, reflect inhibition taking place in vivo. Inhibition of the bactericidal activity in blood can contribute to the persistence of chronic infections and individual variations in resistance. PMID:4971893

CD8+ T Cell Exhaustion, Suppressed Gamma Interferon Production, and Delayed Memory Response Induced by Chronic Brucella melitensis Infection

PubMed Central

Durward-Diioia, Marina; Harms, Jerome; Khan, Mike; Hall, Cherisse; Smith, Judith A.

2015-01-01

Brucella melitensis is a well-adapted zoonotic pathogen considered a scourge of mankind since recorded history. In some cases, initial infection leads to chronic and reactivating brucellosis, incurring significant morbidity and economic loss. The mechanism by which B. melitensis subverts adaptive immunological memory is poorly understood. Previous work has shown that Brucella-specific CD8+ T cells express gamma interferon (IFN-γ) and can transition to long-lived memory cells but are not polyfunctional. In this study, chronic infection of mice with B. melitensis led to CD8+ T cell exhaustion, manifested by programmed cell death 1 (PD-1) and lymphocyte activation gene 3 (LAG-3) expression and a lack of IFN-γ production. The B. melitensis-specific CD8+ T cells that produced IFN-γ expressed less IFN-γ per cell than did CD8+ cells from uninfected mice. Both memory precursor (CD8+ LFA1HI CD127HI KLRG1LO) and long-lived memory (CD8+ CD27HI CD127HI KLRG1LO) cells were identified during chronic infection. Interestingly, after adoptive transfer, mice receiving cells from chronically infected animals were able to contain infection more rapidly than recipients of cells from acutely infected or uninfected donors, although the proportions of exhausted CD8+ T cells increased after adoptive transfer in both challenged and unchallenged recipients. CD8+ T cells of challenged recipients initially retained the stunted IFN-γ production found prior to transfer, and cells from acutely infected mice were never seen to transition to either memory subset at all time points tested, up to 30 days post-primary infection, suggesting a delay in the generation of memory. Here we have identified defects in Brucella-responsive CD8+ T cells that allow chronic persistence of infection. PMID:26416901

Modeling, molecular docking, probing catalytic binding mode of acetyl-CoA malate synthase G in Brucella melitensis 16M.

PubMed

Adi, Pradeepkiran Jangampalli; Yellapu, Nanda Kumar; Matcha, Bhaskar

2016-12-01

There are enormous evidences and previous reports standpoint that the enzyme of glyoxylate pathway malate synthase G (MSG) is a potential virulence factor in several pathogenic organisms, including Brucella melitensis 16M. Where the lack of crystal structures for best candidate proteins like MSG of B. melitensis 16M creates big lacuna to understand the molecular pathogenesis of brucellosis. In the present study, we have constructed a 3-D structure of MSG of Brucella melitensis 16M in MODELLER with the help of crystal structure of Mycobacterium tuberculosis malate synthase (PDB ID: 2GQ3) as template. The stereo chemical quality of the restrained model was evaluated by SAVES server; remarkably we identified the catalytic functional core domain located at 4 th cleft with conserved catalytic amino acids, start at ILE 59 to VAL 586 manifest the function of the protein. Furthermore, virtual screening and docking results reveals that best leadmolecules binds at the core domain pocket of MSG catalytic residues and these ligand leads could be the best prospective inhibitors to treat brucellosis.

Epididymitis Caused by Brucella ovis in a Southern Ontario Sheep Flock

PubMed Central

Buckrell, Brian C.; McEwen, Scott A.; Johnson, Walter H.; Savage, Neale C.

1985-01-01

Epididymitis was diagnosed in three rams in a commercial sheep flock in southern Ontario. The affected rams had palpably enlarged epididymides and two rams had semen which contained inflammatory cells and was of poor quality. Serum compliment fixation titers for Brucella ovis were 1:20, 1:80 and 1:90. Five other rams in the flock were clinically normal and without titers. Two of the affected rams had lesions similar to those produced by experimental infection with B. ovis. The infection in the rams had no apparent affect on ewe performance. The source of the infection remains unknown, but the rams were purchased from a flock which had imported ewes from the western U.S.A. ImagesFigure 1.Figure 2. PMID:17422577

Detection of Fusobacterium nucleatum in two cases of empyema and lung abscess using paromomycin-vancomycin supplemented Brucella HK agar.

PubMed

Nagaoka, Kentaro; Yanagihara, Katsunori; Morinaga, Yoshitomo; Kohno, Shigeru

2017-02-01

Fusobacterium nucleatum was found in patients with empyema or pulmonary abscess, using paromomycin-vancomycin Brucella HK agar. In vitro examination revealed that growth of the strains differed significantly in different media. Clinicians should be aware that suboptimal F. nucleatum cultivation methods may result in an underestimation of its frequency. Copyright © 2016 Elsevier Ltd. All rights reserved.

Species-specific multiplex PCR for the diagnosis of Brucella ovis, Actinobacillus seminis, and Histophilus somni infection in rams

PubMed Central

2013-01-01

Background Infectious ovine epididymitis results in substantial economic losses worldwide due to reproductive failure and culling of breeders. The most common causative agents of these infections are Brucella ovis, Actinobacillus seminis, and Histophilus somni. The aim of this study was to develop a multiplex PCR assay for simultaneous detection of Brucella ovis, Actinobacillus seminis, and Histophilus somni with species-specific primers applied to biological samples for molecular diagnosis of these infections. Results The multiplex assay was capable of detecting B. ovis, A. seminis, and H. somni DNA simultaneously from genomic bacterial DNA samples and pool of semen samples from experimentally infected rams. The method was highly specific since it did not amplify DNA from other bacterial species that can potentially cause epididymitis in rams as well as species phylogenetically related to B. ovis. All negative control samples were negative in PCR multiplex assay. Urine can be used as an alternative to semen samples. Conclusions The species-specific multiplex PCR assay developed in this study can be successfully used for the detection of three of the most common bacterial causes of ovine epididymitis. PMID:23514236

Brucella melitensis survival during manufacture of ripened goat cheese at two temperatures.

PubMed

Méndez-González, Karla Y; Hernández-Castro, Rigoberto; Carrillo-Casas, Erika M; Monroy, Jorge F; López-Merino, Ahide; Suárez-Güemes, Francisco

2011-12-01

The aim of the current work was to assess the influence of two temperatures, 4°C and 24°C, on pH and water activity and their association with Brucella melitensis survival during the traditional manufacture of ripened goat cheese. Raw milk from a brucellosis-free goat herd was used for the manufacture of ripened cheese. The cheese was inoculated with 5×10(9) of the B. melitensis 16M strain during the tempering stage. The cheeses were matured for 5, 20, and 50 days at both temperatures. To assess Brucella survival, the pH and a(w) were recorded at each stage of the process (curd cutting, draining whey, immersion in brine, ripening I, ripening II, and ripening III). B. melitensis was detected at ripening stage III (1×10(3) colony-forming unit [CFU]/mL) from cheeses matured at 4°C with a pH of 5.0 and a(w) of 0.90, and at a ripening stage II (1×10(4) CFU/mL) from cheeses ripened at 24°C with a pH of 4.0 and a(w) of 0.89. The remaining stages were free from the inoculated pathogen. In addition, viable B. melitensis was recovered in significant amounts (1-2×10(6) CFU/mL) from the whey fractions of both types of cheese ripened at 24°C and 4°C. These results revealed the effects of high temperature (24°C vs. 4°C) on the low pH (4) and a(w) (0.89) that appeared to be associated with the suppression of B. melitensis at the early stages of cheese ripening. In the ripened goat cheeses, B. melitensis survived under a precise combination of temperature during maturation, ripening time, and a(w) in the manufacturing process.

Efficacy of single calfhood vaccination of elk with Brucella abortus strain 19

USGS Publications Warehouse

Roffe, T.J.; Jones, L.C.; Coffin, K.; Drew, M.L.; Sweeney, Steven J.; Hagius, S.D.; Elzer, P.H.; Davis, D.

2004-01-01

Brucellosis has been eradicated from cattle in the states of Wyoming, Montana, and Idaho, USA. However, free-ranging elk (Cervus elaphus) that use feedgrounds in the Greater Yellowstone Area (GYA) and bison (Bison bison) in Yellowstone and Grand Teton national parks still have high seroprevalence to the disease and have caused loss of brucellosis-free status in Wyoming. Management tools to control or eliminate the disease are limited; however, wildlife vaccination is among the methods currently used by wildlife managers in Wyoming. We conducted a controlled challenge study of single calfhood vaccination. Elk calves, caught in January and February of 1999 and 2000 and acclimated to captivity for 3 weeks, were randomly assigned to control or vaccinate groups. The vaccinate groups received Brucetta abortus vaccine strain 19 (S19) by hand-delivered intramuscular injection. Calves were raised to adulthood and bred at either 2.5 or 3.5 years of age for 2000 and 1999 captures, respectively. Eighty-nine (44 controls, 45 vaccinates) pregnant elk entered the challenge portion of the study. We challenged elk at mid-gestation with pathogenic B. abortus strain 2308 by intraconjunctival instillation. Abortion occurred in significantly more (P = 0.002) controls (42; 93%) than vaccinates (32; 71%), and vaccine protected 25% of the vaccinate group. We used Brucella culture of fetus/calf tissues to determine the efficacy of vaccination for preventing infection, and we found that the number of infected fetuses/calves did not differ between controls and vaccinates (P = 0.14). Based on these data, single calfhood vaccination with S19 has low efficacy, will likely have only little to moderate effect on Brucella prevalence in elk, and is unlikely to eradicate the disease in wildlife of the GYA.

Identification of Protective Brucella Antigens and their Expressions in Vaccinia Virus to Prevent Disease in Animals and Humans.

DTIC Science & Technology

1996-05-01

see figure appendix; B. abortus sequence in similarity arrangement with secD of E.coli, H. influenzae, M. leprae and S. coelicalor). Highly related...low similarity (E. coil and M. leprae approx 13.5% similarity). The Brucella and E. coil sequences were 25% similar (see figures appendix). In E...1987. Micobacterial growth inhibition by interferon-g activated bone marrow macrophages and differential susceptibility among strains of Mycobacterium

Differential composition of culture supernatants from wild-type Brucella abortus and its isogenic virB mutants.

PubMed

Delpino, M Victoria; Comerci, Diego J; Wagner, Mary Ann; Eschenbrenner, Michel; Mujer, Cesar V; Ugalde, Rodolfo A; Fossati, Carlos A; Baldi, Pablo C; Delvecchio, Vito G

2009-07-01

The virB genes coding type IV secretion system are necessary for the intracellular survival and replication of Brucella spp. In this study, extracellular proteins from B. abortus 2308 (wild type, WT) and its isogenic virB10 polar mutant were compared. Culture supernatants harvested in the early stationary phase were concentrated and subjected to 2D electrophoresis. Spots present in the WT strain but absent in the virB10 mutant (differential spots) were considered extracellular proteins released in a virB-related manner, and were identified by MALDI-TOF analysis and matching with Brucella genomes. Among the 11 differential proteins identified, DnaK chaperone (Hsp70), choloylglycine hydrolase (CGH) and a peptidyl-prolyl cis-trans isomerase (PPIase) were chosen for further investigation because of their homology with extracellular and/or virulence factors from other bacteria. The three proteins were obtained in recombinant form and specific monoclonal antibodies (mAbs) were prepared. By Western blot with these mAbs, the three proteins were detected in supernatants from the WT but not in those from the virB10 polar mutant or from strains carrying non-polar mutations in virB10 or virB11 genes. These results suggest that the expression of virB genes affects the extracellular release of DnaK, PPIase and CGH, and possibly other proteins from B. abortus.

Evolution and genome specialization of Brucella suis biovar 2 Iberian lineages.

PubMed

Ferreira, Ana Cristina; Tenreiro, Rogério; de Sá, Maria Inácia Corrêa; Dias, Ricardo

2017-09-12

Swine brucellosis caused by B. suis biovar 2 is an emergent disease in domestic pigs in Europe. The emergence of this pathogen has been linked to the increase of extensive pig farms and the high density of infected wild boars (Sus scrofa). In Portugal and Spain, the majority of strains share specific molecular characteristics, which allowed establishing an Iberian clonal lineage. However, several strains isolated from wild boars in the North-East region of Spain are similar to strains isolated in different Central European countries. Comparative analysis of five newly fully sequenced B. suis biovar 2 strains belonging to the main circulating clones in Iberian Peninsula, with publicly available Brucella spp. genomes, revealed that strains from Iberian clonal lineage share 74% similarity with those reference genomes. Besides the 210 kb translocation event present in all biovar 2 strains, an inversion with 944 kb was presented in chromosome I of strains from the Iberian clone. At left and right crossover points, the inversion disrupted a TRAP dicarboxylate transporter, DctM subunit, and an integral membrane protein TerC. The gene dctM is well conserved in Brucella spp. except in strains from the Iberian clonal lineage. Intraspecies comparative analysis also exposed a number of biovar-, haplotype- and strain-specific insertion-deletion (INDELs) events and single nucleotide polymorphisms (SNPs) that could explain differences in virulence and host specificities. Most discriminative mutations were associated to membrane related molecules (29%) and enzymes involved in catabolism processes (20%). Molecular identification of both B. suis biovar 2 clonal lineages could be easily achieved using the target-PCR procedures established in this work for the evaluated INDELs. Whole-genome analyses supports that the B. suis biovar 2 Iberian clonal lineage evolved from the Central-European lineage and suggests that the genomic specialization of this pathogen in the Iberian Peninsula

The bovine immune response to Brucella abortus. II. Elimination of some sporadic serological reactions by chelation of divalent cations.

PubMed Central

Nielsen, K; Samagh, B S; Speckmann, G; Stemshorn, B

1979-01-01

The standard agglutination tests for detecting antibody to Brucella abortus were modified by addition of chelating agents (EDTA and EGTA) to the antigens. Approximately 80% of "singleton" agglutination test reactions, negative on the diagnostic complement fixation test, obtained with cattle sera were eliminated while no decrease in titer was apparent when sera from B. abortus infected or vaccinated cattle were tested. PMID:121242

Survival of a bacterioferritin deletion mutant of Brucella melitensis 16M in human monocyte-derived macrophages.

PubMed Central

Denoel, P A; Crawford, R M; Zygmunt, M S; Tibor, A; Weynants, V E; Godfroid, F; Hoover, D L; Letesson, J J

1997-01-01

A bacterioferritin (BFR) deletion mutant of Brucella melitensis 16M was generated by gene replacement. The deletion was complemented with a broad-host-range vector carrying the wild-type bfr gene, pBBR-bfr. The survival and growth of the mutant, B. melitensis PAD 2-78, were similar to those of its parental strain in human monocyte-derived macrophages (MDM). These results suggest that BFR is not essential for the intracellular survival of B. melitensis in human MDM. PMID:9317046

Recovery of a Medieval Brucella melitensis Genome Using Shotgun Metagenomics

PubMed Central

Kay, Gemma L.; Sergeant, Martin J.; Giuffra, Valentina; Bandiera, Pasquale; Milanese, Marco; Bramanti, Barbara

2014-01-01

ABSTRACT Shotgun metagenomics provides a powerful assumption-free approach to the recovery of pathogen genomes from contemporary and historical material. We sequenced the metagenome of a calcified nodule from the skeleton of a 14th-century middle-aged male excavated from the medieval Sardinian settlement of Geridu. We obtained 6.5-fold coverage of a Brucella melitensis genome. Sequence reads from this genome showed signatures typical of ancient or aged DNA. Despite the relatively low coverage, we were able to use information from single-nucleotide polymorphisms to place the medieval pathogen genome within a clade of B. melitensis strains that included the well-studied Ether strain and two other recent Italian isolates. We confirmed this placement using information from deletions and IS711 insertions. We conclude that metagenomics stands ready to document past and present infections, shedding light on the emergence, evolution, and spread of microbial pathogens. PMID:25028426

First isolation, identification, phenotypic and genotypic characterization of Brucella abortus biovar 3 from dairy cattle in Tanzania.

PubMed

Mathew, C; Stokstad, M; Johansen, T B; Klevar, S; Mdegela, R H; Mwamengele, G; Michel, P; Escobar, L; Fretin, D; Godfroid, J

2015-07-21

Brucellosis is a disease of worldwide public health and economic importance. Successful control is based on knowledge of epidemiology and strains present in an area. In developing countries, most investigations are based on serological assays. This study aimed at investigating a dairy herd experiencing abortions in order to establish within-herd seroprevalence to Brucella spp., identify, characterize Brucella strains by Multiple Loci Variable Number of Tandem Repeats Analysis (MLVA-VNTR) and investigate possible spillover to other species. The within-herd seroprevalence in cattle (n = 200) was 48 % (95 % CI 41-55), using an indirect ELISA, while the Rose Bengal Test (RBT) yielded lower prevalence (21.5 %; 95 % CI 16-27). Two sheep (n = 35) and one goat (n = 50) were seropositive using ELISA while none of the dogs (n = 6) was positive with the RBT. Three Brucella were isolated from an aborted fetus and associated membranes. Real time PCR (IS711), Bruce-ladder and classical biotyping classified the isolates as B. abortus biovar 3. MLVA-VNTR revealed two different but closely related genotypes. The isolates showed unique profiles, providing the first genotypic data from Tanzania. These genotypes were not related to B. abortus biovar 3 reference strain Tulya originally isolated from a human patient in Uganda in 1958, unlike the genotypes isolated and characterized recently in Kenya. High within-herd prevalence, isolation of the pathogen and abortion confirm that B. abortus is circulating in this herd with cattle as reservoir hosts. A low seroprevalence in sheep and goats suggests a spillover of B. abortus from cattle to small ruminants in the herd. This is the first isolation and characterization of B. abortus biovar 3 from a dairy cow with abortion in Tanzania. The origin of the Tanzanian genotypes remain elusive, although they seem to be related to genotypes found in Europe, Turkey and China but not related to B. abortus biovar 3 reference strain or genotypes from

BvrR/BvrS-Controlled Outer Membrane Proteins Omp3a and Omp3b Are Not Essential for Brucella abortus Virulence▿

PubMed Central

Manterola, Lorea; Guzmán-Verri, Caterina; Chaves-Olarte, Esteban; Barquero-Calvo, Elías; de Miguel, María-Jesús; Moriyón, Ignacio; Grilló, María-Jesús; López-Goñi, Ignacio; Moreno, Edgardo

2007-01-01

The Brucella abortus two-component regulatory system BvrR/BvrS controls the expression of outer membrane proteins (Omp) Omp3a (Omp25) and Omp3b (Omp22). Disruption of bvrS or bvrR generates avirulent mutants with altered cell permeability, higher sensitivity to microbicidal peptides, and complement. Consequently, the role of Omp3a and Omp3b in virulence was examined. Similar to bvrS or bvrR mutants, omp3a and omp3b mutants displayed increased attachment to cells, indicating surface alterations. However, they showed unaltered permeability; normal expression of Omp10, Omp16, Omp19, Omp2b, and Omp1; native hapten polysaccharide; and lipopolysaccharide and were resistant to complement and polymyxin B at ranges similar to those of the wild-type (WT) counterpart. Likewise, omp3a and omp3b mutants were able to replicate in murine macrophages and in HeLa cells, were resistant to the killing action of human neutrophils, and persisted in mice, like the WT strain. Murine macrophages infected with the omp3a mutant generated slightly higher levels of tumor necrosis factor alpha than the WT, whereas the bvrS mutant induced lower levels of this cytokine. Since the absence of Omp3a or Omp3b does not result in attenuation, it can be concluded that BvrR/BvrS influences additional Brucella properties involved in virulence. Our results are discussed in the light of previous works suggesting that disruption of omp3a generates attenuated Brucella strains, and we speculate on the role of group 3 Omps. PMID:17664262

Studies of Antigens for Complement Fixation and Gel Diffusion Tests in the Diagnosis of Infections Caused by Brucella ovis and Other Brucella

PubMed Central

Myers, Donald M.; Jones, Lois M.; Varela-Diaz, Victor M.

1972-01-01

Sonically treated and saline-extracted antigens of Brucella ovis, B. canis, B. abortus, and B. melitensis were compared in gel diffusion, complement fixation, and serum absorption tests. All the sonically extracted antigens showed cross-reactions with sera from animals infected or immunized with these species, whereas the saline-extracted antigens were specific for the surface of the rough or smooth colonial phase of the species or strain. The saline-extracted antigens of B. ovis and B. melitensis were both eluted as a single peak in the void volume by Sephadex G-200 column chromatography, in gel diffusion had staining characteristics of lipoproteins, but in immunoelectrophoresis showed distinct mobility patterns. Serological activity for both gel diffusion and complement fixation tests was demonstrated in the immunoglobulin G-containing fraction of sera taken from sheep 12 to 412 days after infection with B. ovis. The gel diffusion test with saline extract of B. ovis is as sensitive as the complement fixation test for the diagnosis of ram epididymitis and is more practical. Images PMID:4624210

Analysis of the Kinetics and Regulation of Cytokine Gene Expression During the Primary In Vivo Immune Response to Killed Brucella Abortus

DTIC Science & Technology

1992-08-10

R. Beining, D. Hochstein, Y. L. Lee, R . D. Angus , and B. Golding. 1992. LPS from Brucella abonus is less toxic than that from Escherichia coli...abonus endotoxin and LPS. Ann. Inst. Pasteur Microbial. 138:98. 76. RlIi, N. B. K., S. C. Cheung, I. Rosztoczy, and P. M. Pitha. 1992. Mouse genotype

Modeling, molecular dynamics, and docking assessment of transcription factor rho: a potential drug target in Brucella melitensis 16M

PubMed Central

Pradeepkiran, Jangampalli Adi; Kumar, Konidala Kranthi; Kumar, Yellapu Nanda; Bhaskar, Matcha

2015-01-01

The zoonotic disease brucellosis, a chronic condition in humans affecting renal and cardiac systems and causing osteoarthritis, is caused by Brucella, a genus of Gram-negative, facultative, intracellular pathogens. The mode of transmission and the virulence of the pathogens are still enigmatic. Transcription regulatory elements, such as rho proteins, play an important role in the termination of transcription and/or the selection of genes in Brucella. Adverse effects of the transcription inhibitors play a key role in the non-successive transcription challenges faced by the pathogens. In the investigation presented here, we computationally predicted the transcription termination factor rho (TtFRho) inhibitors against Brucella melitensis 16M via a structure-based method. In view the unknown nature of its crystal structure, we constructed a robust three-dimensional homology model of TtFRho’s structure by comparative modeling with the crystal structure of the Escherichia coli TtFRho (Protein Data Bank ID: 1PVO) as a template in MODELLER (v 9.10). The modeled structure was optimized by applying a molecular dynamics simulation for 2 ns with the CHARMM (Chemistry at HARvard Macromolecular Mechanics) 27 force field in NAMD (NAnoscale Molecular Dynamics program; v 2.9) and then evaluated by calculating the stereochemical quality of the protein. The flexible docking for the interaction phenomenon of the template consists of ligand-related inhibitor molecules from the ZINC (ZINC Is Not Commercial) database using a structure-based virtual screening strategy against minimized TtFRho. Docking simulations revealed two inhibitors compounds – ZINC24934545 and ZINC72319544 – that showed high binding affinity among 2,829 drug analogs that bind with key active-site residues; these residues are considered for protein-ligand binding and unbinding pathways via steered molecular dynamics simulations. Arg215 in the model plays an important role in the stability of the protein

Human brucellosis in northwest Ecuador: typifying Brucella spp., seroprevalence, and associated risk factors.

PubMed

Ron-Román, Jorge; Ron-Garrido, Lenin; Abatih, Emmanuel; Celi-Erazo, Maritza; Vizcaíno-Ordóñez, Laura; Calva-Pacheco, Jaime; González-Andrade, Pablo; Berkvens, Dirk; Benítez-Ortíz, Washington; Brandt, Jef; Fretin, David; Saegerman, Claude

2014-02-01

Human brucellosis in Ecuador is underreported and based only on passive surveillance. Since 2008, brucellosis was removed from the list of communicable diseases in the country. Until now, the true human brucellosis picture has not yet been determined. The aim of this study was to determine the seroprevalence of the disease, identify risk factors associated with brucellosis seropositivity in humans, and isolate circulating strains of Brucella spp. in the northwestern part of Ecuador. Between 2006 and 2008, a large transect survey was conducted, based on blood sampling of people from the northwestern part of Ecuador (n=3733) together with an epidemiological inquiry. On the basis of three diagnostic tests used in parallel, the overall seroprevalence was estimated as 1.88% (95% confidence interval [CI] 1.48-2.38). Based on a multivariable random effects logistic regression analysis, the main risk factors associated with human brucellosis seropositivity were contact with livestock (odds ratio [OR]=3.0; CI 1.25-7.08), consumption of fetus and placenta (OR=2.5; CI 1.18-5.22), and involvement in activities at risk for brucellosis infection (OR=1.8; CI 1.00-3.35). Noticeable variation in brucellosis seropositivity among humans within cantons was observed. The circulating strain was Brucella abortus biotype 4. This study emphasized that contact with livestock, consumption of fetus and placenta, and occupational hazard group were all significant risk factors for the transmission of brucellosis among individuals in the northwestern part of Ecuador. Alongside encouraging the launching of educational campaigns against brucellosis, especially in rural areas where 36% of the population lives, controlling this zoonotic disease in animals will directly benefit its prevention in humans, especially because there is no safe and efficacious vaccine against brucellosis in humans.

Brucella ceti and Brucellosis in Cetaceans

PubMed Central

Guzmán-Verri, Caterina; González-Barrientos, Rocío; Hernández-Mora, Gabriela; Morales, Juan-Alberto; Baquero-Calvo, Elías; Chaves-Olarte, Esteban; Moreno, Edgardo

2012-01-01

Since the first case of brucellosis detected in a dolphin aborted fetus, an increasing number of Brucella ceti isolates has been reported in members of the two suborders of cetaceans: Mysticeti and Odontoceti. Serological surveys have shown that cetacean brucellosis may be distributed worldwide in the oceans. Although all B. ceti isolates have been included within the same species, three different groups have been recognized according to their preferred host, bacteriological properties, and distinct genetic traits: B. ceti dolphin type, B. ceti porpoise type, and B. ceti human type. It seems that B. ceti porpoise type is more closely related to B. ceti human isolates and B. pinnipedialis group, while B. ceti dolphin type seems ancestral to them. Based on comparative phylogenetic analysis, it is feasible that the B. ceti ancestor radiated in a terrestrial artiodactyl host close to the Raoellidae family about 58 million years ago. The more likely mode of transmission of B. ceti seems to be through sexual intercourse, maternal feeding, aborted fetuses, placental tissues, vertical transmission from mother to the fetus or through fish or helminth reservoirs. The B. ceti dolphin and porpoise types seem to display variable virulence in land animal models and low infectivity for humans. However, brucellosis in some dolphins and porpoises has been demonstrated to be a severe chronic disease, displaying significant clinical and pathological signs related to abortions, male infertility, neurobrucellosis, cardiopathies, bone and skin lesions, strandings, and death. PMID:22919595

Brucella sp. vertebral osteomyelitis with intercurrent fatal Staphylococcus aureus toxigenic enteritis in a bottlenose dolphin (Tursiops truncatus)

PubMed Central

Goertz, Caroline E. C.; Frasca, Salvatore; Bohach, Gregory A.; Cowan, Daniel F.; Buck, John D.; French, Richard A.; De Guise, Sylvain; Maratea, Jennifer; Hinckley, Lynn; Ewalt, Darla; Schlievert, Patrick M.; Karst, Sheila M.; Deobald, Claudia F.; St. Aubin, David J.; Dunn, J. Lawrence

2013-01-01

A previously beach-stranded, juvenile, male, bottlenose dolphin (Tursiops truncatus) was diagnosed with vertebral osteomyelitis of unknown etiology. Antemortem serological testing suggested past or current Brucella sp. infection; however, this could not be confirmed prior to death despite multiple isolation attempts from aspirates, blood, and biopsies. Systemic antibiotics were administered for over a year to control the suspected infection; however, the animal succumbed peracutely to a highly pathogenic, enterotoxin-secreting Staphylococcus sp. Gross necropsy findings included a fistulous tract leading to locally extensive osteomyelitis of a coccygeal vertebra with sequestra and osteophytes from which a Brucella species was isolated. Histopathological examination of intestine revealed pseudomembranous enteritis with a uniform population of intraluminal Gram-positive cocci. Staphylococcus aureus was isolated in pure culture from the intestine and tested positive for the staphylococcal enterotoxin A gene by polymerase chain reaction analysis. Serum taken shortly before death had endotoxin and elevated antibody titers to staphylococcal enterotoxin A when compared to samples collected during a period of apparent good health eighteen months earlier. The isolation of a pyrogenic toxin superantigen-producing staphylococcal isolate, clinical signs, and diagnostic findings in this animal resembled some of those noted in human toxic shock syndrome. The present case highlights the clinical challenges of treating chronic illnesses, complications of long-term antibiotic use, and promotion of pathogenic strains in cases of prolonged rehabilitation of marine mammals. PMID:21908337

Gamma radiation grafted polymers for immobilization of Brucella antigen in diagnostic test studies

NASA Astrophysics Data System (ADS)

Docters, E. H.; Smolko, E. E.; Suarez, C. E.

The radiation grafting process has a wide field of industrial applications, and in the recent years the immobilization of biocomponents in grafted polymeric materials obtained by means of ionizing radiations is a new and important contribution to biotechnology. In the present work, gamma preirradiation grafting method was employed to produce acrylics hydrogels onto polyethylene (PE), polyvinyl chloride (PVC) and polystyrene (PS). Two monomers were used to graft the previously mentioned polymers: methacrylic acid (MAAc) and acrylamide (AAm), and several working conditions were considered as influencing the degree of grafting. All this grafted polymers were used to study the possibility of a subsequent immobilization of Brucella antigen (BAg) in diagnostic test studies (ELISA).

In Vitro Antibacterial Effects of Five Volatile Oil Extracts Against Intramacrophage Brucella Abortus 544

PubMed Central

Al-Mariri, Ayman; Saour, George; Hamou, Razan

2012-01-01

Background: Brucella abortus is a gram-negative facultative intracellular bacterium that can cause a highly contagious disease in sheep, goats, cattle and one-humped camels. It is responsible for one of the most important zoonosis in human. The aim of this study was to evaluate the role of Mentha piperita, Origanum majorana, Citrus lemon, Cinnamomum verum and Myristica fragrans essential volatile oil extracts on human macrophages infected by B. abortus 544. Methods: Essential volatile oil extracts from M. piperita, O. majorana, C. lemon, C. verum and M. fragrans were extracted. Human macrophages were cultured at a density of 2×105 cells per well in sterile 96-well microtiter plates, and infected with B. abortus 544 at a ratio of 1:100 bacteria/cell. Then essential volatile oil extracts were added at a concentration of 1%. At specified times; cells were washed, lysed with 0.1% Triton, and plated on 2YT agar to determine the number of intracellular bacteria. Results: Cinnamomum verum volatile oil at a concentration of 1% had the highest antibacterial activity against B. abortus 544 inside human macrophages. Its inhibitory effect observed from 24 h and continued till 144 h after the infection. Moreover, C. verum (0.1%) in combination with 1% concentration of M. piperita, O. majorana, C. lemon or M. fragrans volatile oil extracts produced a synergistic inhibitory effect against B. abortus 544. Conclusion: The results indicate that, among the five selected oil extracts, C. verum volatile oil applied either separately or in combination with other oil extracts had the most effective antimicrobial activity against Brucella. PMID:23115441

An outbreak of Brucella abortus biovar 2 in Canadian cattle

PubMed Central

Forbes, Lorry B.; Steele, Thomas B.

1989-01-01

An outbreak of brucellosis caused by Brucella abortus biovar 2 was identified in cattle in Alberta in December 1986. This was the only clinical infection discovered since the national cattle herd was declared brucellosisfree in 1985. It was the first report of B. abortus biovar 2 in Canadian cattle. The outbreak, involving three herds containing purebred Hereford cattle, was spread by the private treaty sale of untested cattle, and was identified following investigation of an abortion. The source of infection for the outbreak was not established, but several possibilities were identified including infected herds present in the area during the mid-1970's, latent infection originating in a Saskatchewan herd during the early 1960's, American cattle imported during the early 1970's, and brucellosis-infected bison in Wood Buffalo National Park. The containment and elimination of this nidus of infection appears to have been successful, and the national cattle herd at the time of writing is free of the disease. PMID:17423457

The genome sequence of the facultative intracellular pathogen Brucella melitensis.

PubMed

DelVecchio, Vito G; Kapatral, Vinayak; Redkar, Rajendra J; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross

2002-01-08

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other alpha-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti.

The genome sequence of the facultative intracellular pathogen Brucella melitensis

PubMed Central

DelVecchio, Vito G.; Kapatral, Vinayak; Redkar, Rajendra J.; Patra, Guy; Mujer, Cesar; Los, Tamara; Ivanova, Natalia; Anderson, Iain; Bhattacharyya, Anamitra; Lykidis, Athanasios; Reznik, Gary; Jablonski, Lynn; Larsen, Niels; D'Souza, Mark; Bernal, Axel; Mazur, Mikhail; Goltsman, Eugene; Selkov, Eugene; Elzer, Philip H.; Hagius, Sue; O'Callaghan, David; Letesson, Jean-Jacques; Haselkorn, Robert; Kyrpides, Nikos; Overbeek, Ross

2002-01-01

Brucella melitensis is a facultative intracellular bacterial pathogen that causes abortion in goats and sheep and Malta fever in humans. The genome of B. melitensis strain 16M was sequenced and found to contain 3,294,935 bp distributed over two circular chromosomes of 2,117,144 bp and 1,177,787 bp encoding 3,197 ORFs. By using the bioinformatics suite ERGO, 2,487 (78%) ORFs were assigned functions. The origins of replication of the two chromosomes are similar to those of other α-proteobacteria. Housekeeping genes, including those involved in DNA replication, transcription, translation, core metabolism, and cell wall biosynthesis, are distributed on both chromosomes. Type I, II, and III secretion systems are absent, but genes encoding sec-dependent, sec-independent, and flagella-specific type III, type IV, and type V secretion systems as well as adhesins, invasins, and hemolysins were identified. Several features of the B. melitensis genome are similar to those of the symbiotic Sinorhizobium meliloti. PMID:11756688

Development of a dual vaccine for prevention of Brucella abortus infection and Escherichia coli O157:H7 intestinal colonization.

PubMed

Iannino, Florencia; Herrmann, Claudia K; Roset, Mara S; Briones, Gabriel

2015-05-05

Zoonoses that affect human and animal health have an important economic impact. In the study now presented, a bivalent vaccine has been developed that has the potential for preventing the transmission from cattle to humans of two bacterial pathogens: Brucella abortus and Shiga toxin-producing Escherichia coli (STEC). A 66kDa chimeric antigen, composed by EspA, Intimin, Tir, and H7 flagellin (EITH7) from STEC, was constructed and expressed in B. abortus Δpgm vaccine strain (BabΔpgm). Mice orally immunized with BabΔpgm(EITH7) elicited an immune response with the induction of anti-EITH7 antibodies (IgA) that clears an intestinal infection of E. coli O157:H7 three times faster (t=4 days) than mice immunized with BabΔpgm carrier strain (t=12 days). As expected, mice immunized with BabΔpgm(EITH7) strain also elicited a protective immune response against B. abortus infection. A Brucella-based vaccine platform is described capable of eliciting a combined protective immune response against two bacterial pathogens with diverse lifestyles-the intracellular pathogen B. abortus and the intestinal extracellular pathogen STEC. Copyright © 2015 Elsevier Ltd. All rights reserved.

Studies on the genetic control of murine humoral response to immunization with a peptidoglycan-containing fraction extracted from Brucella melitensis.

PubMed

Cannat, A; Feingold, N; Caffin, J C; Serre, A

1979-01-01

A peptidoglycan containing fraction (fraction "5") extracted from Brucella melitensis has been injected in low infra-vaccinating doses into inbred mice. The genetic control of the resulting anti-Brucella humoral response has been studied in the C57BL/6 "good responder" X DBA2 "low responder" model. The results observed in F1, F2 and reciprocal backcrosses show that the "good responder" character, although transmitted as a dominant trait, is under polygenic control and independent of H2 haplotype, Ig allotype, sexual chromosoms or the "d" coat color gene. On the other hand, the phenotypic expression of at least one of the genes involved is sex-limited and influenced by hormonal environmental factors. Moreover the expression in females of one of these sex-dependent genes is associated with the "b" coat color gene. These results are discussed in terms of their possible relevance in spontaneous or vaccinal resistance to experimental brucellosis, of the relative role of the peptidoglycan and lipoprotein moieties in fraction "5" and of the possible importance of sex-dependent and chromosome 4-linked genetic factors for B-cell functions.

Global Rsh-dependent transcription profile of Brucella suis during stringent response unravels adaptation to nutrient starvation and cross-talk with other stress responses

PubMed Central

2013-01-01

Background In the intracellular pathogen Brucella spp., the activation of the stringent response, a global regulatory network providing rapid adaptation to growth-affecting stress conditions such as nutrient deficiency, is essential for replication in the host. A single, bi-functional enzyme Rsh catalyzes synthesis and hydrolysis of the alarmone (p)ppGpp, responsible for differential gene expression under stringent conditions. Results cDNA microarray analysis allowed characterization of the transcriptional profiles of the B. suis 1330 wild-type and Δrsh mutant in a minimal medium, partially mimicking the nutrient-poor intramacrophagic environment. A total of 379 genes (11.6% of the genome) were differentially expressed in a rsh-dependent manner, of which 198 were up-, and 181 were down-regulated. The pleiotropic character of the response was confirmed, as the genes encoded an important number of transcriptional regulators, cell envelope proteins, stress factors, transport systems, and energy metabolism proteins. Virulence genes such as narG and sodC, respectively encoding respiratory nitrate reductase and superoxide dismutase, were under the positive control of (p)ppGpp, as well as expression of the cbb3-type cytochrome c oxidase, essential for chronic murine infection. Methionine was the only amino acid whose biosynthesis was absolutely dependent on stringent response in B. suis. Conclusions The study illustrated the complexity of the processes involved in adaptation to nutrient starvation, and contributed to a better understanding of the correlation between stringent response and Brucella virulence. Most interestingly, it clearly indicated (p)ppGpp-dependent cross-talk between at least three stress responses playing a central role in Brucella adaptation to the host: nutrient, oxidative, and low-oxygen stress. PMID:23834488

Molecular typing of isolates obtained from aborted foetuses in Brucella-free Holstein dairy cattle herd after immunisation with Brucella abortus RB51 vaccine in Egypt.

PubMed

Wareth, Gamal; Melzer, Falk; Böttcher, Denny; El-Diasty, Mohamed; El-Beskawy, Mohamed; Rasheed, Nesma; Schmoock, Gernot; Roesler, Uwe; Sprague, Lisa D; Neubauer, Heinrich

2016-12-01

Bovine brucellosis is endemic in Egypt in spite of application of surveillance and control measures. An increase of abortions was reported in a Holstein dairy cattle herd with 600 animals in Damietta governorate in Egypt after immunisation with Brucella (B.) abortus RB51 vaccine. Twenty one (10.6%) of 197 vaccinated cows aborted after 3 months. All aborted cows had been tested seronegative for brucellosis in the past 3 years. B. abortus was isolated from four foetuses. Conventional biochemical and bacteriological identification and polymerase chain reaction (PCR) confirmed two B. abortus biovar (bv.) 1 smooth and two B. abortus rough strains. None of the B. abortus isolates were identified as RB51. Genotyping analysis by multiple locus of variable number tandem repeats analysis based on 16 markers (MLVA-16) revealed two different profiles with low genetic diversity. B. abortus bv1 was introduced in the herd and caused abortions. Copyright © 2016 Elsevier B.V. All rights reserved.

The Lipopolysaccharide of Brucella abortus BvrS/BvrR Mutants Contains Lipid A Modifications and Has Higher Affinity for Bactericidal Cationic Peptides

PubMed Central

Manterola, Lorea; Moriyón, Ignacio; Moreno, Edgardo; Sola-Landa, Alberto; Weiss, David S.; Koch, Michel H. J.; Howe, Jörg; Brandenburg, Klaus; López-Goñi, Ignacio

2005-01-01

The two-component BvrS/BvrR system is essential for Brucella abortus virulence. It was shown previously that its dysfunction abrogates expression of some major outer membrane proteins and increases bactericidal peptide sensitivity. Here, we report that BvrS/BvrR mutants have increased surface hydrophobicity and susceptibility to killing by nonimmune serum. The bvrS and bvrR mutant lipopolysaccharides (LPSs) bound more polymyxin B, chimeras constructed with bvrS mutant cells and parental LPS showed augmented polymyxin B resistance, and, conversely, parental cells and bvrS mutant LPS chimeras were more sensitive and displayed polymyxin B-characteristic outer membrane lesions, implicating LPS as being responsible for the phenotype of the BvrS/BvrR mutants. No qualitative or quantitative changes were detected in other envelope and outer membrane components examined: periplasmic β(1-2) glucans, native hapten polysaccharide, and phospholipids. The LPS of the mutants was similar to parental LPS in O-polysaccharide polymerization and fine structure but showed both increased underacylated lipid A species and higher acyl-chain fluidity that correlated with polymyxin B binding. These lipid A changes did not alter LPS cytokine induction, showing that in contrast to other gram-negative pathogens, recognition by innate immune receptors is not decreased by these changes in LPS structure. Transcription of Brucella genes required for incorporating long acyl chains into lipid A (acpXL and lpxXL) or implicated in lipid A acylation control (bacA) was not affected. We propose that in Brucella the outer membrane homeostasis depends on the functioning of BvrS/BvrR. Accordingly, disruption of BvrS/BvrR damages the outer membrane, thus contributing to the severe attenuation manifested by bvrS and bvrR mutants. PMID:16077108

Evaluation of Th1/Th2-Related Immune Response against Recombinant Proteins of Brucella abortus Infection in Mice.

PubMed

Im, Young Bin; Park, Woo Bin; Jung, Myunghwan; Kim, Suk; Yoo, Han Sang

2016-06-28

Brucellosis is a zoonotic disease caused by Brucella, a genus of gram-negative bacteria. Cytokines have key roles in the activation of innate and acquired immunities. Despite several research attempts to reveal the immune responses, the mechanism of Brucella infection remains unclear. Therefore, immune responses were analyzed in mice immunized with nine recombinant proteins. Cytokine production profiles were analyzed in the RAW 264.7 cells and naive splenocytes after stimulation with three recombinant proteins, metal-dependent hydrolase (r0628), bacterioferritin (rBfr), and thiamine transporter substrate-binding protein (rTbpA). Immune responses were analyzed by ELISA and ELISpot assay after immunization with proteins in mice. The production levels of NO, TNF-α, and IL-6 were time-dependently increased after having been stimulated with proteins in the RAW 264.7 cells. In naive splenocytes, the production of IFN-γ and IL-2 was increased after stimulation with the proteins. It was concluded that two recombinant proteins, r0628 and rTbpA, showed strong immunogenicity that was induced with Th1-related cytokines IFN-γ, IL-2, and TNF-α more than Th2-related cytokines IL-6, IL-4, and IL-5 in vitro. Conversely, a humoral immune response was activated by increasing the number of antigen-secreting cells specifically. Furthermore, these could be candidate diagnosis antigens for better understanding of brucellosis.

Evaluation of Brucella abortus Phosphoglucomutase (pgm) Mutant as a New Live Rough-Phenotype Vaccine

PubMed Central

Ugalde, Juan Esteban; Comerci, Diego José; Leguizamón, M. Susana; Ugalde, Rodolfo Augusto

2003-01-01

Brucella abortus S19 is the vaccine most frequently used against bovine brucellosis. Although it induces good protection levels, it cannot be administered to pregnant cattle, revaccination is not advised due to interference in the discrimination between infected and vaccinated animals during immune-screening procedures, and the vaccine is virulent for humans. Due to these reasons, there is a continuous search for new bovine vaccine candidates that may confer protection levels comparable to those conferred by S19 but without its disadvantages. A previous study characterized the phenotype associated with the phosphoglucomutase (pgm) gene disruption in Brucella abortus S2308, as well as the possible role for the smooth lipopolysaccharide (LPS) in virulence and intracellular multiplication in HeLa cells (J. E. Ugalde, C. Czibener, M. F. Feldman, and R. A. Ugalde, Infect. Immun. 68:5716-5723, 2000). In this report, we analyze the protection, proliferative response, and cytokine production induced in BALB/c mice by a Δpgm deletion strain. We show that this strain synthesizes O antigen with a size of approximately 45 kDa but is rough. This is due to the fact that the Δpgm strain is unable to assemble the O side chain in the complete LPS. Vaccination with the Δpgm strain induced protection levels comparable to those induced by S19 and generated a proliferative splenocyte response and a cytokine profile typical of a Th1 response. On the other hand, we were unable to detect a specific anti-O-antigen antibody response by using the fluorescence polarization assay. In view of these results, the possibility that the Δpgm mutant could be used as a vaccination strain is discussed. PMID:14573645

Immune responses and protection against experimental challenge after vaccination of bison with Brucella abortus strains RB51 or RB51 overexpressing superoxide dismutase and Glycosyltransferase genes

USDA-ARS?s Scientific Manuscript database

Vaccination is a tool that could be beneficial in managing the high prevalence of brucellosis in free-ranging bison in Yellowstone National Park. In this study, we characterized immunologic responses and protection against experimental challenge after vaccination of bison with Brucella abortus stra...

Crucial role of gamma interferon-producing CD4+ Th1 cells but dispensable function of CD8+ T cell, B cell, Th2, and Th17 responses in the control of Brucella melitensis infection in mice.

PubMed

Vitry, Marie-Alice; De Trez, Carl; Goriely, Stanislas; Dumoutier, Laure; Akira, Shizuo; Ryffel, Bernhard; Carlier, Yves; Letesson, Jean-Jacques; Muraille, Eric

2012-12-01

Brucella spp. are facultative intracellular bacterial pathogens responsible for brucellosis, a worldwide zoonosis that causes abortion in domestic animals and chronic febrile disease associated with serious complications in humans. There is currently no approved vaccine against human brucellosis, and antibiotic therapy is long and costly. Development of a safe protective vaccine requires a better understanding of the roles played by components of adaptive immunity in the control of Brucella infection. The importance of lymphocyte subsets in the control of Brucella growth has been investigated separately by various research groups and remains unclear or controversial. Here, we used a large panel of genetically deficient mice to compare the importance of B cells, transporter associated with antigen processing (TAP-1), and major histocompatibility complex class II-dependent pathways of antigen presentation as well as T helper 1 (Th1), Th2, and Th17-mediated responses on the immune control of Brucella melitensis 16 M infection. We clearly confirmed the key function played by gamma interferon (IFN-γ)-producing Th1 CD4(+) T cells in the control of B. melitensis infection, whereas IFN-γ-producing CD8(+) T cells or B cell-mediated humoral immunity plays only a modest role in the clearance of bacteria during primary infection. In the presence of a Th1 response, Th2 or Th17 responses do not really develop or play a positive or negative role during the course of B. melitensis infection. On the whole, these results could improve our ability to develop protective vaccines or therapeutic treatments against brucellosis.

Metabolic Control of Virulence Genes in Brucella abortus: HutC Coordinates virB Expression and the Histidine Utilization Pathway by Direct Binding to Both Promoters ▿ †

PubMed Central

Sieira, Rodrigo; Arocena, Gastón M.; Bukata, Lucas; Comerci, Diego J.; Ugalde, Rodolfo A.

2010-01-01

Type IV secretion systems (T4SS) are multicomponent machineries involved in the translocation of effector molecules across the bacterial cell envelope. The virB operon of Brucella abortus codes for a T4SS that is essential for virulence and intracellular multiplication of the bacterium in the host. Previous studies showed that the virB operon of B. abortus is tightly regulated within the host cells. In order to identify factors implicated in the control of virB expression, we searched for proteins of Brucella that directly bind to the virB promoter (PvirB). Using different procedures, we isolated a 27-kDa protein that binds specifically to PvirB. This protein was identified as HutC, the transcriptional repressor of the histidine utilization (hut) genes. Analyses of virB and hut promoter activity revealed that HutC exerts two different roles: it acts as a coactivator of transcription of the virB operon, whereas it represses the hut genes. Such activities were observed both intracellularly and in bacteria incubated under conditions that resemble the intracellular environment. Electrophoresis mobility shift assays (EMSA) and DNase I footprinting experiments revealed the structure, affinity, and localization of the HutC-binding sites and supported the regulatory role of HutC in both hut and virB promoters. Taken together, these results indicate that Brucella coopted the function of HutC to coordinate the Hut pathway with transcriptional regulation of the virB genes, probably as a way to sense its own metabolic state and develop adaptive responses to overcome intracellular host defenses. PMID:19854911

Brucella abortus Depends on Pyruvate Phosphate Dikinase and Malic Enzyme but Not on Fbp and GlpX Fructose-1,6-Bisphosphatases for Full Virulence in Laboratory Models

PubMed Central

Zúñiga-Ripa, Amaia; Barbier, Thibault; Conde-Álvarez, Raquel; Martínez-Gómez, Estrella; Palacios-Chaves, Leyre; Gil-Ramírez, Yolanda; Grilló, María Jesús; Letesson, Jean-Jacques; Iriarte, Maite

2014-01-01

The brucellae are the etiological agents of brucellosis, a worldwide-distributed zoonosis. These bacteria are facultative intracellular parasites and thus are able to adjust their metabolism to the extra- and intracellular environments encountered during an infectious cycle. However, this aspect of Brucella biology is imperfectly understood, and the nutrients available in the intracellular niche are unknown. Here, we investigated the central pathways of C metabolism used by Brucella abortus by deleting the putative fructose-1,6-bisphosphatase (fbp and glpX), phosphoenolpyruvate carboxykinase (pckA), pyruvate phosphate dikinase (ppdK), and malic enzyme (mae) genes. In gluconeogenic but not in rich media, growth of ΔppdK and Δmae mutants was severely impaired and growth of the double Δfbp-ΔglpX mutant was reduced. In macrophages, only the ΔppdK and Δmae mutants showed reduced multiplication, and studies with the ΔppdK mutant confirmed that it reached the replicative niche. Similarly, only the ΔppdK and Δmae mutants were attenuated in mice, the former being cleared by week 10 and the latter persisting longer than 12 weeks. We also investigated the glyoxylate cycle. Although aceA (isocitrate lyase) promoter activity was enhanced in rich medium, aceA disruption had no effect in vitro or on multiplication in macrophages or mouse spleens. The results suggest that B. abortus grows intracellularly using a limited supply of 6-C (and 5-C) sugars that is compensated by glutamate and possibly other amino acids entering the Krebs cycle without a critical role of the glyoxylate shunt. PMID:24936050

Geographic pattern of serum antibody prevalence for Brucella spp. in caribou, grizzly bears, and wolves from Alaska, 1975-1998.

PubMed

Zarnke, Randall L; Ver Hoef, Jay M; DeLong, Robert A

2006-07-01

Blood samples were collected from 2,635 caribou (Rangifer tarandus), 1,238 grizzly bears (Ursus arctos), and 930 wolves (Canis lupus) from throughout mainland Alaska during 1975-98. Sera were tested for evidence of exposure to Brucella spp. Serum antibody prevalences were highest in the northwestern region of the state. In any specific area, antibody prevalences for caribou and wolves were of a similar magnitude, whereas antibody prevalence for bears in these same areas were two to three times higher.

[Immune response and reproductive consequences in experimentally infected ewes with Brucella ovis during late pregnancy].

PubMed

Paolicchi, Fernando A; Nuñez, Marta; Fiorentino, María A; Malena, Rosana C; Trangoni, Marcos; Cravero, Silvio; Estein, Silvia M

2013-01-01

Ovine brucellosis by Brucella ovis is a highly prevalent disease in Argentina. This study aimed to evaluate the pathogenicity of B. ovis and the serological response in ewes during late pregnancy and in their offspring. Six adult ewes were distributed in two groupsG1 (pregnant females, n = 4) and G2 (nonpregnant females, n = 2). Three pregnant ewes at 15 days prepartum and one nonpregnant eve were inoculated with B. ovis. Sera of sheep and their offspring were analyzed by different serological tests. Samples of cervicovaginal mucus, placenta and milk were studied by bacteriology. A Brucella genus-specific PCR assay was carried out in placenta and milk samples. Placenta samples were hystopathologically processed. g1 females gave birth to live lambs, but one died hours postpartum. Serological techniques employed detected antibodies in serum of inoculated pregnant animal 5 days postchallenge. sera of female controls G1 and G2 remained negative throughout the study. Cervicovaginal mucus of infected ewes in G1 and G2 yielded negative results to bacteriology, but B. ovis was isolated from milk. The PCR assay was positive for the placenta and milk from inoculated pregnant ewes. Histopathology revealed necrotic suppurative placentitis in one placenta. However, although results demonstrated that B. ovis can invade the placenta and mammary gland, this bacterium did not cause abortion when it was inoculated intravenously at 15 days prepartum. B. ovis infection induced an early humoral response in pregnant ewes, but their lambs remained seronegative, indicating that there was no transfer of antibodies in infancy. Placenta colonization and milk excretion of B. ovis involves a potential source of infection for lambs, which could play a role as latent carriers of infection.

Structural elucidation of the Brucella melitensis M antigen by high-resolution NMR at 500 MHz

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

Bundle, D.R.; Cherwonogrodzky, J.W.; Perry, M.B.

The Brucella M antigen from the species type strain Brucella melitensis 16M has been identified as a component of the cell wall lipopolysaccharide (LPS). O polysaccharide liberated from this LPS by mild acid hydrolysis exhibited M activity in serological tests and was shown to be a homopolymer of 4-formamido-4,6-dideoxy-..cap alpha..-D-mannopyranosyl residues arranged in an oligosaccharide repeating unit as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the native lipopolysaccharide. Structural analysis of the O polysaccharide by NMR methods was difficult due to apparent microheterogeneity of the repeating unit, which was in fact caused by the presence of rotational isomers ofmore » the N-formyl moiety. This problem was resolved by chemical modification of the polysaccharide to its amino and N-acetyl derivatives, the 500-MHz /sup 1/H and 125-MHz /sup 13/C NMR spectra of which could be analyzed in terms of a unique structure through application of pH-dependent ..beta..-shifts and two-dimensional techniques that included COSY, relayed COSY, and NOESY experiments together with heteronuclear C/H shift correlation spectroscopy. On the basis of these experiments and supported by methylation and periodate oxidation data, the structure of the M polysaccharide was determined as a linear polymer of unbranched pentasaccharide repeating units consisting of four 1,2-linked and one 1,3-lined 4,6-dideoxy-4-formamido-..cap alpha..-D-mannopyranosyl residues. The marked structural similarity of the M antigen and the A antigen, which is known to be a 1,2-linked homopolysaccharide of 4,6-dideoxy-4-formamido-..cap alpha..-D-mannopyranosyl units, accounts for cross-serological reactions of the two and the long-standing confusion surrounding the nature of their antigenic determinants.« less

[Serological detection of Brucella suis, influenza virus and Aujeszky's disease virus in backyard and small swine holders in Argentina].

PubMed

Dibarbora, Marina; Cappuccio, Javier A; Aznar, María N; Bessone, Fernando A; Piscitelli, Hernán; Pereda, Ariel J; Pérez, Daniel R

Farmers raising less than 100 sows represent more than 99% of swine producers in Argentina, although little is known about their sanitary status and productive characteristics in the country. Sanitary and productive information was obtained. Furthermore, samples for serological studies were taken to detect antibodies against Brucella suis (Bs), Aujeszky's disease virus (AV) and influenza virus (IV) in 68 backyard and small producers with less than 100 sows located in the north, central and south regions of Argentina. Antibodies against H1 pandemic were detected in 80% of the farms while 11%, 11.7% and 6.0% of the producers were positive to influenza H3 cluster 2, AV and Bs, respectively. None of the producers was aware of the risk factors concerning the transmission of diseases from pigs to humans. A percentage of 47% of them buy pigs for breeding from other farmers and markets. With regard to biosecurity measures, only 16% of the farms had perimeter fences. The results of this study demonstrate that productive characterization and disease surveys are important to improve productivity and to reduce the risk of disease transmission among animals and humans. The study of sanitary status and risk factors is necessary for better control and eradication of diseases in backyard or small producers. More representative studies at country level should be carried out to detect the pathogensthat circulate and, with this knowledge, to implement prevention and control measures. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Comparison of potential protection conferred by three immunization strategies (protein/protein, DNA/DNA, and DNA/protein) against Brucella infection using Omp2b in BALB/c Mice.

PubMed

Golshani, Maryam; Rafati, Sima; Nejati-Moheimani, Mehdi; Ghasemian, Melina; Bouzari, Saeid

2016-12-25

In the present study, immunogenicity and protective efficacy of the Brucella outer membrane protein 2b (Omp2b) was evaluated in BALB/c mice using Protein/Protein, DNA/DNA and DNA/Protein vaccine strategies. Immunization of mice with three vaccine regimens elicited a strong specific IgG response (higher IgG2a titers over IgG1 titers) and provided Th1-oriented immune response. Vaccination of BALB/c mice with the DNA/Pro regimen induced higher levels of IFN-γ/IL-2 and conferred more protection levels against B. melitenisis and B. abortus challenge than did the protein or DNA alone. In conclusion, Omp2b is able to stimulate specific immune responses and to confer cross protection against B. melitensis and B. abortus infection. Therefore, it could be introduced as a new potential candidate for the development of a subunit vaccine against Brucella infection. Copyright © 2016 Elsevier B.V. All rights reserved.

Epidemiology and genetic characterization of BVDV, BHV-1, BHV-4, BHV-5 and Brucella spp. infections in cattle in Turkey

PubMed Central

ASLAN, Muhammet Eren; AZKUR, Ahmet Kursat; GAZYAGCI, Serkal

2015-01-01

The aim of the study was to determine the epidemiological data of bovine viral diarrhea virus (BVDV), bovine herpesvirus-1 (BHV-1), bovine herpesvirus-4 (BHV-4), bovine herpesvirus-5 (BHV-5) and Brucella–associated cattle that were previously reported to have abortion and infertility problems in Ankara, Corum, Kirikkale and Yozgat provinces, Turkey. Whole blood and sera samples were obtained from 656 cattle, and antibodies against Brucella spp. were detected in 45 (6.86%) and 41 (6.25%) animals by Rose Bengal plate and serum tube agglutination tests, respectively. The seropositivity rates against BVDV, BHV-1 and BHV-4 were 70.89%, 41.3% and 28.78%, respectively. RT-PCR and PCR were performed to detect RNA and DNA viruses in blood samples, respectively. The BVDV 5′-untranslated region and BHV-1 gB gene detected in this study were phylogenetically analyzed. The BVDV strains analyzed in this study were closely related to those previously reported from Turkey. The nucleotide sequence from the BHV-1 strain detected in this study is the first nucleotide sequence of BHV-1 circulating in this area of Turkey deposited in the GenBank. The presence of Brucella spp. and prevalence of BHV-1, BHV-4 and BVDV in cattle should be further investigated throughout these regions. PMID:26096964

Anti-Brucella activity of Caryopteris mongolica Bunge root extract against Brucella melitensis infection in mice.

PubMed

N, Tsevelmaa; B, Narangerel; O, Odgerel; D, Dariimaa; J, Batkhuu

2018-05-03

and to prevent Brucella infection.

First Report of Orchitis in Man Caused by Brucella abortus Biovar 1 in Ecuador

PubMed Central

Ron-Román, Jorge; Saegerman, Claude; Minda-Aluisa, Elizabeth; Benítez-Ortíz, Washington; Brandt, Jef; Douce, Richard

2012-01-01

We present a 44-year-old man from a rural community in northern Ecuador who worked on a cattle farm where he was involved with primary veterinary care, including assistance during births (or calving) and placenta retention and artificial insemination, with minimal precautions. In September of 2009, quite abruptly, he developed asthenia and hypersomnia without any apparent cause or symptoms like fever, chills, or night sweats. On November 14, 2009, he suffered from pain and edema in the right testicle that coincided with pain in the abdomen. Clinical, serological, and bacteriological investigations confirmed the first case of unilateral orchitis in man in Ecuador caused by Brucella abortus biovar 1. Because brucellosis is a neglected disease, special attention should be given to it in the training of medical and veterinary students. PMID:22826490

Differentiation of the serological response to Yersinia enterocolitica and Brucella abortus in cattle

PubMed Central

Corbel, M. J.; Cullen, G. A.

1970-01-01

The serological responses of cattle to inoculation with Brucella abortus and Yersinia enterocolitica type IX were compared. Complete cross-reactions were found in serum agglutination, antiglobulin, complement fixation and Rose Bengal plate tests. The cross-reaction between Br. abortus and Y. enterocolitica IX was confirmed by immunodiffusion tests. Although antibodies specific for each organism could also be detected by immunodiffusion tests with high titre rabbit or bovine sera, these tests were insufficiently sensitive for routine diagnostic use. A quantitative Rose Bengal plate test, using Rose Bengal stained Br. abortus and Y. enterocolitica IX, was developed which enabled the antibody responses to the two organisms to be differentiated. The specificity of this test was confirmed by cross-absorption experiments and its sensitivity was sufficient to permit evaluation of all bovine sera giving positive reactions to the serum agglutination test. ImagesFig. 1Fig. 2 PMID:4992575

A Homologue of an Operon Required for DNA Transfer in Agrobacterium Is Required in Brucella abortus for Virulence and Intracellular Multiplication

PubMed Central

Sieira, Rodrigo; Comerci, Diego J.; Sánchez, Daniel O.; Ugalde, Rodolfo A.

2000-01-01

As part of a Brucella abortus 2308 genome project carried out in our laboratory, we identified, cloned, and sequenced a genomic DNA fragment containing a locus (virB) highly homologous to bacterial type IV secretion systems. The B. abortus virB locus is a collinear arrangement of 13 open reading frames (ORFs). Between virB1 and virB2 and downstream of ORF12, two degenerated, palindromic repeat sequences characteristic of Brucella intergenic regions were found. Gene reporter studies demonstrated that the B. abortus virB locus constitutes an operon transcribed from virB1 which is turned on during the stationary phase of growth. A B. abortus polar virB1 mutant failed to replicate in HeLa cells, indicating that the virB operon plays a critical role in intracellular multiplication. Mutants with polar and nonpolar mutations introduced in virB10 showed different behaviors in mice and in the HeLa cell infection assay, suggesting that virB10 per se is necessary for the correct function of this type IV secretion apparatus. Mouse infection assays demonstrated that the virB operon constitutes a major determinant of B. abortus virulence. It is suggested that putative effector molecules secreted by this type IV secretion system determine routing of B. abortus to an endoplasmic reticulum-related replication compartment. PMID:10940027

Seroprevalence and Potential Risk Factors for Brucella Spp. Infection in Traditional Cattle, Sheep and Goats Reared in Urban, Periurban and Rural Areas of Niger

PubMed Central

Boukary, Abdou Razac; Saegerman, Claude; Abatih, Emmanuel; Fretin, David; Alambédji Bada, Rianatou; De Deken, Reginald; Harouna, Halimatou Adamou; Yenikoye, Alhassane; Thys, Eric

2013-01-01

Introduction In Niamey, Niger, interactions within the interface between animals, humans and the environment induce a potential risk of brucellosis transmission between animals and from animals to humans. Currently, little is known about the transmission of Brucella in this context. Results 5,192 animals from 681 herds were included in the study. Serum samples and hygroma fluids were collected. A household survey enabled to identify the risk factors for transmission of brucellosis. The true adjusted herd-level prevalence of brucellosis ranged between 11.2% and 17.2% and the true adjusted animal-population level prevalence was 1.3% (95% CI: 0.9–1.8%) based on indirect ELISA test for Brucella antibodies. Animals aged of 1–4 years were found to be more susceptible than animals less than 1 year old (Odds ratio [OR] of 2.7; 95% CI: 1.43–5.28). For cattle, the odds of brucellosis seropositivity were higher in rural compared to the periurban areas (OR of 2.8; 95% CI: 1.48–5.17) whereas for small ruminants the risk of seropositivity appeared to be higher in urban compared to periurban areas (OR of 5.5; 95% CI: 1.48–20.38). At herd level, the risk of transmission was increased by transhumance (OR of 5.4; 95% CI: 2.84–10.41), the occurrence of abortions (OR of 3.0; 95% CI: 1.40–6.41), and for herds having more than 50 animals (OR of 11.0; 95% CI: 3.75–32.46). Brucella abortus biovar 3 was isolated from the hygromas. Conclusion brucellosis in Niger is a serious problem among cattle especially in the rural areas around Niamey and among sheep in the urban areas of Niamey. The seroprevalence varies across strata and animal species with important risk factors including herd size, abortion and transhumance at herd level and age at animal population level. For effective control of brucellosis, an integrated approach seems appropriate involving all stakeholders working in public and animal health. PMID:24358261

The Use of Case History Studies to Differentiate Potentially Infected from Potentially Noninfected Herds with Reactors to Brucella abortus Antigens

PubMed Central

Martin, S. W.; Gerrow, A. F.

1978-01-01

Data on farm characteristics and the results of the first herd test for brucellosis were collected for 74 reactor and 74 negative herds in Wellington County, Ontario. Each reactor herd was classified as either probably infected or probably not infected using the occurrence of abortions prior to the first herd test or during the testing period, the total number of cattle removed and/or the spread of reactors within the herd as criteria of infection. Statistical techniques were used to select variables which were good “discriminators” between probably infected and noninfected herds. In general, reactor herds were primarily dairy herds and were somewhat larger than negative herds. The presence of only single suspicious reactors on the first test appeared to be a good predictor of lack of infection with Brucella abortus. Among the 37 farms in this category the single reactor was removed from only eight farms and no evidence o fthe spread of infection was observed. The presence of one or more positive reactors on the first herd test appeared to be a good predictor of the presence of infection. Of the 24 farms in this category, evidence of the spread of infection was present in ten farms and seven of these ten farms were eventually depopulated. The brucella milk ring test appeared to be the most effective means of identifying infected herds under the conditions present in Wellington County. PMID:417777

Immune responses of bison and efficacy after booster vaccination with Brucella abortus strain RB51.

PubMed

Olsen, S C; McGill, J L; Sacco, R E; Hennager, S G

2015-04-01

Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 10(10) CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1β (IL-1β) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 10(7) CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against

Immune Responses of Bison and Efficacy after Booster Vaccination with Brucella abortus Strain RB51

PubMed Central

McGill, J. L.; Sacco, R. E.; Hennager, S. G.

2015-01-01

Thirty-one bison heifers were randomly assigned to receive saline or a single vaccination with 1010 CFU of Brucella abortus strain RB51. Some vaccinated bison were randomly selected for booster vaccination with RB51 at 11 months after the initial vaccination. Mean antibody responses to RB51 were greater (P < 0.05) in vaccinated bison after initial and booster vaccination than in nonvaccinated bison. The proliferative responses by peripheral blood mononuclear cells (PBMC) from the vaccinated bison were greater (P < 0.05) than those in the nonvaccinated bison at 16 and 24 weeks after the initial vaccination but not after the booster vaccination. The relative gene expression of gamma interferon (IFN-γ) was increased (P < 0.05) in the RB51-vaccinated bison at 8, 16, and 24 weeks after the initial vaccination and at 8 weeks after the booster vaccination. The vaccinated bison had greater (P < 0.05) in vitro production of IFN-γ at all sampling times, greater interleukin-1β (IL-1β) production in various samplings after the initial and booster vaccinations, and greater IL-6 production at one sampling time after the booster vaccination. Between 170 and 180 days of gestation, the bison were intraconjunctivally challenged with approximately 1 × 107 CFU of B. abortus strain 2308. The incidences of abortion and infection were greater (P < 0.05) in the nonvaccinated bison after experimental challenge than in the bison receiving either vaccination treatment. Booster-vaccinated, but not single-vaccinated bison, had a reduced (P < 0.05) incidence of infection in fetal tissues and maternal tissues compared to that in the controls. Compared to the nonvaccinated bison, both vaccination treatments lowered the colonization (measured as the CFU/g of tissue) of Brucella organisms in all tissues, except in retropharyngeal and supramammary lymph nodes. Our study suggests that RB51 booster vaccination is an effective vaccination strategy for enhancing herd immunity against brucellosis in

Immunogencity of HSA-L7/L12 (Brucella abortus ribosomal protein) in an animal model.

PubMed

Pakzad, Iraj; Rezaee, Abbas; Rasaee, Mohammad Javad; Tabbaraee, Bahman; Delpisheh, Ali

2009-03-01

The immunogenic Brucella abortus ribosomal protein L7/L12 is a promising candidate antigen for the development of subunit vaccines against brucellosis. This study was aimed to evaluate the protection of recombinant Human Serum Albumin (HAS)-L7/L12 fusion protein in Balb/c mice. The amplified L7/L12 gene was cloned in pYHSA5 vector, pYHSA5-L7/L12 construct was transformed in Saccharomyces cerevisiae and the expressed protein from supernatant was purified by affinity chromatography. Balb/c mice were immunized in five groups by tHSA-L7/L12 fusion protein (group 1), Brucella abortus S19 (group 2), HSA (group 3), recombinant L7/L12 (group 4), PBS (group 5). ELISA to detect antibody production, LTT test to assess antigen specific lymphocyte response were conducted prior to virulent B. abortus strain 544 challenge two weeks after the last injection. Bacterial counts from spleens of immunized mice were done four weeks after challenge. In ELISA tests, the specific antibodies exhibited a dominance of immunoglobulin IgG1 over IgG2a. In addition, the tHSA-L7/L12 fusion protein and L7/L12 elicited a strong T-cell proliferative response upon restimulation in vitro with recombinant tHSA-L7/L12 and L7/L12, suggesting the induction of a cellular immunity response in vivo. However, there was no significant difference in proliferative response of L7/L12 and tHSA-L7/L12 fusion protein (p>0.05). The L7/L12 and tHSA-L7/L12 fusion protein vaccines could also induce significant protection against challenge with the virulent strain B. abortus 544 in Balb/c mice (p< or =0.05). The tHSA-L7/L12 fusion protein, similar to L7/L12 has the ability to induce antigen specific lymphocyte proliferation, stimulate humoral immunity and engender protection.

The prevalence and distribution of Brucella melitensis in goats in Malaysia from 2000 to 2009.

PubMed

Bamaiyi, P H; Hassan, L; Khairani-Bejo, S; ZainalAbidin, M; Ramlan, M; Adzhar, A; Abdullah, N; Hamidah, N H M; Norsuhanna, M M; Hashim, S N

2015-05-01

A study was conducted to describe the prevalence and distribution of zoonotic Brucella melitensis in goats in Peninsular Malaysia. Using serosurveillance data of the last decade (2000-2009) involving 119,799 goats and 3555 farms, the seroprevalence of brucellosis among goats was 0.91% (95% CI=0.86-0.96) and among farms was 7.09% (95% CI=6.27-7.98). The odds of brucellosis was significantly (P<0.05) higher in the later part of the decade, in larger herd size and among the states located in the peninsula as compared to eastern Malaysia. The infection was detected throughout Malaysia but at generally low seroprevalences with states like Perlis that border neighbouring countries having higher seroprevalence of brucellosis than other non-border states. Copyright © 2015 Elsevier B.V. All rights reserved.

Molecular Cloning and Characterization of cgs, the Brucella abortus Cyclic β(1-2) Glucan Synthetase Gene: Genetic Complementation of Rhizobium meliloti ndvB and Agrobacterium tumefaciens chvB Mutants

PubMed Central

Iñón de Iannino, Nora; Briones, Gabriel; Tolmasky, Marcelo; Ugalde, Rodolfo A.

1998-01-01

The animal pathogen Brucella abortus contains a gene, cgs, that complemented a Rhizobium meliloti nodule development (ndvB) mutant and an Agrobacterium tumefaciens chromosomal virulence (chvB) mutant. The complemented strains recovered the synthesis of cyclic β(1-2) glucan, motility, virulence in A. tumefaciens, and nitrogen fixation in R. meliloti; all traits were strictly associated with the presence of an active cyclic β(1-2) glucan synthetase protein in the membranes. Nucleotide sequencing revealed the presence in B. abortus of an 8.49-kb open reading frame coding for a predicted membrane protein of 2,831 amino acids (316.2 kDa) and with 51% identity to R. meliloti NdvB. Four regions of the B. abortus protein spanning amino acids 520 to 800, 1025 to 1124, 1284 to 1526, and 2400 to 2660 displayed similarities of higher than 80% with R. meliloti NdvB. Tn3-HoHo1 mutagenesis showed that the C-terminal 825 amino acids of the Brucella protein, although highly conserved in Rhizobium, are not necessary for cyclic β(1-2) glucan synthesis. Confirmation of the identity of this protein as B. abortus cyclic β(1-2) glucan synthetase was done by the construction of a B. abortus Tn3-HoHo1 insertion mutant that does not form cyclic β(1-2) glucan and lacks the 316.2-kDa membrane protein. The recovery of this mutant from the spleens of inoculated mice was decreased by 3 orders of magnitude compared with that of the parental strain; this result suggests that cyclic β(1-2) glucan may be a virulence factor in Brucella infection. PMID:9721274

The bhuQ Gene Encodes a Heme Oxygenase That Contributes to the Ability of Brucella abortus 2308 To Use Heme as an Iron Source and Is Regulated by Irr

PubMed Central

Ojeda, Jenifer F.; Martinson, David A.; Menscher, Evan A.

2012-01-01

The Brucella BhuQ protein is a homolog of the Bradyrhizobium japonicum heme oxygenases HmuD and HmuQ. To determine if this protein plays a role in the ability of Brucella abortus 2308 to use heme as an iron source, an isogenic bhuQ mutant was constructed and its phenotype evaluated. Although the Brucella abortus bhuQ mutant DCO1 did not exhibit a defect in its capacity to use heme as an iron source or evidence of increased heme toxicity in vitro, this mutant produced increased levels of siderophore in response to iron deprivation compared to 2308. Introduction of a bhuQ mutation into the B. abortus dhbC mutant BHB2 (which cannot produce siderophores) resulted in a severe growth defect in the dhbC bhuQ double mutant JFO1 during cultivation under iron-restricted conditions, which could be rescued by the addition of FeCl3, but not heme, to the growth medium. The bhuQ gene is cotranscribed with the gene encoding the iron-responsive regulator RirA, and both of these genes are repressed by the other major iron-responsive regulator in the alphaproteobacteria, Irr. The results of these studies suggest that B. abortus 2308 has at least one other heme oxygenase that works in concert with BhuQ to allow this strain to efficiently use heme as an iron source. The genetic organization of the rirA-bhuQ operon also provides the basis for the proposition that BhuQ may perform a previously unrecognized function by allowing the transcriptional regulator RirA to recognize heme as an iron source. PMID:22636783

Role of the Omp25/Omp31 Family in Outer Membrane Properties and Virulence of Brucella ovis▿

PubMed Central

Caro-Hernández, Paola; Fernández-Lago, Luis; de Miguel, María-Jesús; Martín-Martín, Ana I.; Cloeckaert, Axel; Grilló, María-Jesús; Vizcaíno, Nieves

2007-01-01

The genes coding for the five outer membrane proteins (OMPs) of the Omp25/Omp31 family expected to be located in the outer membrane (OM) of rough virulent Brucella ovis PA were inactivated to evaluate their role in virulence and OM properties. The OM properties of the mutant strains and of the mutants complemented with the corresponding wild-type genes were analyzed, in comparison with the parental strain and rough B. abortus RB51, in several tests: (i) binding of anti-Omp25 and anti-Omp31 monoclonal antibodies, (ii) autoagglutination of bacterial suspensions, and (iii) assessment of susceptibility to polymyxin B, sodium deoxycholate, hydrogen peroxide, and nonimmune ram serum. A tight balance of the members of the Omp25/Omp31 family was seen to be essential for the stability of the B. ovis OM, and important differences between the OMs of B. ovis PA and B. abortus RB51 rough strains were observed. Regarding virulence, the absence of Omp25d and Omp22 from the OM of B. ovis PA led to a drastic reduction in spleen colonization in mice. While the greater susceptibility of the Δomp22 mutant to nonimmune serum and its difficulty in surviving in the stationary phase might be on the basis of its dramatic attenuation, no defects in the OM able to explain the attenuation of the Δomp25d mutant were found, especially considering that the fully virulent Δomp25c mutant displayed more important OM defects. Accordingly, Omp25d, and perhaps Omp22, could be directly involved in the penetration and/or survival of B. ovis inside host cells. This aspect, together with the role of Omp25d and Omp22 in the virulence both of B. ovis in rams and of other Brucella species, should be thoroughly evaluated in future studies. PMID:17562767

Serological study of brucellosis in Argentine Creole sheep.

PubMed

López, Gustavo E; Peña, Sabrina; Escobar, Gabriela I; Hasan, Déborah B; Lucero, Nidia E

2018-01-05

Ovine cattle was introduced into America during the Spanish conquest with the second journey of Columbus to the Antilles and was disseminated throughout the region. In 1587, sheep were introduced into Argentina, later developing into the "Creole" breed. We selected 486 animals from different Argentine provinces with the aim of determining the serological status of brucellosis caused by Brucella melitensis and Brucella ovis. For the detection of antibodies against smooth Brucella spp., the Rose Bengal test (RBT) was performed as screening test while the serum agglutination test (SAT) and 2 mercapto-ethanol (2ME) were run as a confirmatory technique. Moreover, for the detection of antibodies against rough Brucella spp., we used the rapid slide agglutination test (RSAT) for screening and an indirect ELISA (IELISA) as confirmatory assay. This study showed that the total positive percentage of brucellosis due to B. ovis was 2.9%. Excluding the animals mixed with the Suffolk breed; seropositivity would be 0.6%. All animals tested negative for brucellosis caused by B. melitensis. Copyright © 2017 Asociación Argentina de Microbiología. Publicado por Elsevier España, S.L.U. All rights reserved.

Molecular assessment of the transplacental transmission of Toxoplasma gondii, Neospora caninum, Brucella canis and Ehrlichia canis in dogs.

PubMed

Taques, Isis Indaiara Gonçalves Granjeiro; Barbosa, Tatiane Rodrigues; Martini, Andresa de Cássia; Pitchenin, Letícia Camara; Braga, Ísis Assis; de Melo, Andréia Lima Tomé; Nakazato, Luciano; Dutra, Valéria; de Aguiar, Daniel Moura

2016-12-01

Given the fact that numerous microbial species can be detected in pregnant female dogs, the objective of this study was to assess the transplacental transmission of Brucella canis, Ehrlichia canis, Neospora caninum and Toxoplasma gondii in stillborn puppies. This study involved 41 stillborn puppies, 78.6% of which were positive for T. gondii, 52.4% for N. caninum and 59.5% for B. canis. E. canis was not detected in any of the analyzed puppies. Pregnancy is an important physiological condition for the transmission of infectious agents to puppies and transplacental transmission may be epidemiologically relevant in the spread of these opportunistic agents. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular epidemiological investigation of Brucella melitensis circulating in Mongolia by MLVA16.

PubMed

Kang, Sung-Il; Her, Moon; Erdenebaataar, Janchivdorj; Vanaabaatar, Batbaatar; Cho, Hyorim; Sung, So-Ra; Lee, Jin Ju; Jung, Suk Chan; Park, Yong Ho; Kim, Ji-Yeon

2017-02-01

Mongolia has a high incidence of brucellosis in human and animals due to livestock husbandry. To investigate the genetic characteristics of Mongolian B. melitensis, an MLVA (multi-locus variable-number tandem-repeat analysis)-16 assay was performed with 94 B. melitensis isolates. They were identified as B. melitensis biovar (bv.) 1 (67), 3 (10) and Rev. 1 vaccine strains (17) using a classical biotyping and multiplex PCR. In genotyping, three human isolates were grouped at 2 genotypes with sheep isolates, and it implies that B. melitensis are cross-infected between human and livestock. In the parsimony analysis, Mongolian B. melitensis isolates had high genetic similarity with Chinese strains, likely due to the geographical proximity, clustered distinctively as compared with other foreign isolates. B. melitensis Rev. 1 vaccine strains were divided into 4 genotypes with 92% similarity. In the analysis of Rev.1 strains, the risk of mutation of vaccine strain might not be overlooked. Animal quarantines should be strengthened to prevent the spread of Brucella species among adjacent countries. Copyright © 2016 Elsevier Ltd. All rights reserved.

Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis

PubMed Central

Ismael, Alaa Bassuny; Swelum, Ayman Abdel-Aziz; Mostafa, Salama A-H; Alhumiany, Abdel-Rahman A

2016-01-01

Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity. PMID:27207442

Latex agglutination using the periplasmic proteins antigen of Brucella melitensis is a successful, rapid, and specific serodiagnostic test for ovine brucellosis.

PubMed

Ismael, Alaa Bassuny; Swelum, Ayman Abdel-Aziz; Mostafa, Salama A-H; Alhumiany, Abdel-Rahman A

2016-09-01

Brucellosis, especially caused by Brucella melitensis, is considered the most-widespread zoonosis in the world, particularly in developing countries. This study was planned to develop an accurate test for diagnosis of ovine brucellosis using a specific hot saline extracted soluble Brucella melitensis periplasmic proteins (SBPPs). The efficacy of the latex agglutination test (LAT) using SBPPs compared to the Rose Bengal test (RBT), buffered plate agglutination test (BPAT), serum agglutination test (SAT), and an indirect enzyme-linked immunosorbent assay (i-ELISA) was evaluated in the field diagnosis of ovine brucellosis. The test performance was evaluated by estimating sensitivity (Se), specificity (Sp), positive predictive value (PPV), negative predictive value (NPV), disease prevalence (DP), positive likelihood ratio (PLR), and negative likelihood ratio (NLR) using test agreement and bacteriological culture in 1777 samples. The false-positive result was significantly (P ⩽0.05) lower in LAT than RBT, BPAT, SAT, and i-ELISA. With reference to test agreement, the Se, Sp, PPV, and PLR were highest (P ⩽0.05) in LAT 99.33%, 99.88%, 98.68%, and 827.25%, respectively. With reference to bacteriological culture, the LAT and i-ELISA tests showed a significant difference in Se with SAT. However, no significant difference in specificity was detected. The DP was 8.44% in the five tests. In conclusion, LAT using SBPPs of B. melitensis could be a suitable serodiagnostic field test for ovine brucellosis, with high sensitivity and specificity. © The Author(s) 2016.

Brucella canis infection in dogs attended in veterinary clinics from patos, ParabaĪBA state, Brazil

PubMed Central

Fernandes, Annielle Regina Fonseca; de Azevedo, Sérgio Santos; Pinheiro, Eliana Scarcelli; Genovez, Margareth Élide; de Azevedo, Adílio Santos; de Sousa Américo Batista, Carolina; Alves, Clebert José

2011-01-01

To determine the frequency of anti-Brucella canis antibodies in dogs attended in veterinary clinics from Patos, Paraíba State, Brazil, as well as to identify risk factors and to isolate and identify the agent, 193 dogs were used. Agar gel immunodiffusion test (AGID) was used to detect B. canis antibodies in sera. Isolation of B. canis was carried out in blood and bone marrow from seropositive animals. Six animals tested seropositive in AGID, resulting in a frequency of 3.11%. B. canis was isolated from bone marrow of one seropositive animal, with confirmation by PCR. Lack of cleaning of the dog’s environment was identified as risk factor (odds ratio = 7.91). This is the first report of isolation of B. canis in dogs from the Northeast region of Brazil. PMID:24031770

Brucella melitensis MucR, an orthologue of Sinorhizobium meliloti MucR, is involved in resistance to oxidative, detergent, and saline stresses and cell envelope modifications.

PubMed

Mirabella, A; Terwagne, M; Zygmunt, M S; Cloeckaert, A; De Bolle, X; Letesson, J J

2013-02-01

Brucella spp. and Sinorhizobium meliloti are alphaproteobacteria that share not only an intracellular lifestyle in their respective hosts, but also a crucial requirement for cell envelope components and their timely regulation for a successful infectious cycle. Here, we report the characterization of Brucella melitensis mucR, which encodes a zinc finger transcriptional regulator that has previously been shown to be involved in cellular and mouse infections at early time points. MucR modulates the surface properties of the bacteria and their resistance to environmental stresses (i.e., oxidative stress, cationic peptide, and detergents). We show that B. melitensis mucR is a functional orthologue of S. meliloti mucR, because it was able to restore the production of succinoglycan in an S. meliloti mucR mutant, as detected by calcofluor staining. Similar to S. meliloti MucR, B. melitensis MucR also represses its own transcription and flagellar gene expression via the flagellar master regulator ftcR. More surprisingly, we demonstrate that MucR regulates a lipid A core modification in B. melitensis. These changes could account for the attenuated virulence of a mucR mutant. These data reinforce the idea that there is a common conserved circuitry between plant symbionts and animal pathogens that regulates the relationship they have with their hosts.

Isolation of Aureimonas altamirensis, a Brucella canis-like bacterium, from an edematous canine testicle.

PubMed

Reilly, Thomas J; Calcutt, Michael J; Wennerdahl, Laura A; Williams, Fred; Evans, Tim J; Ganjam, Irene K; Bowman, Jesse W; Fales, William H

2014-11-01

Microbiological and histological analysis of a sample from a swollen testicle of a 2-year-old Border Collie dog revealed a mixed infection of the fungus Blastomyces dermatitidis and the Gram-negative bacterium Aureimonas altamirensis. When subjected to an automated microbial identification system, the latter isolate was provisionally identified as Psychrobacter phenylpyruvicus, but the organism shared several biochemical features with Brucella canis and exhibited agglutination, albeit weakly, with anti-B. canis antiserum. Unequivocal identification of the organism was only achieved by 16S ribosomal RNA gene sequencing, ultimately establishing the identity as A. altamirensis. Since its first description in 2006, this organism has been isolated infrequently from human clinical samples, but, to the authors' knowledge, has not been reported from a veterinary clinical sample. While of unknown clinical significance with respect to the pathology observed for the polymicrobial infection described herein, it highlights the critical importance to unambiguously identify the microbe for diagnostic, epidemiological, infection control, and public health purposes. © 2014 The Author(s).

Presence of Leishmania and Brucella Species in the Golden Jackal Canis aureus in Serbia

PubMed Central

Ćirović, Duško; Chochlakis, Dimosthenis; Tomanović, Snežana; Sukara, Ratko; Penezić, Aleksandra; Tselentis, Yannis; Psaroulaki, Anna

2014-01-01

The golden jackal Canis aureus occurs in south-eastern Europe, Asia, the Middle East, the Caucasus, and Africa. In Serbia, jackals neared extinction; however, during the last 30 years, the species started to spread quickly and to increase in number. Few studies in the past have revealed their potential role as carriers of zoonotic diseases. Animal samples were collected over a three-year period (01/2010–02/2013) from 12 sites all over Serbia. Of the tissue samples collected, spleen was chosen as the tissue to proceed; all samples were tested for Leishmania species and Brucella species by real-time PCR. Of the 216 samples collected, 15 (6.9%) were positive for Leishmania species, while four (1.9%) were positive for B. canis. The potential epidemiologic role of the golden jackal in carrying and dispersing zoonotic diseases in Serbia should be taken under consideration when applying surveillance monitoring schemes. PMID:24967397

Seroepidemiology of Brucella abortus infection in bovids in Sri Lanka.

PubMed

Silva, I; Dangolla, A; Kulachelvy, K

2000-07-03

From 1992 to 1995, 0.15% (n=3916) of the bovids (cattle and buffalo) in Sri Lanka were sampled, using a multi-stage sampling procedure. Serum antibodies for Brucella abortus were detected using the indirect enzyme-linked immunosorbent assay (ELISA). The age, the agroecological zone and the management system practiced in the farms of the sampled bovids were studied as risk factors for seropositivity. The overall seroprevalence of brucellosis in cattle was 4.7% (n=3076) and 4.2% in buffaloes (n=840). Bovids that were over 3 years of age, from the dry zone (annual rainfall 20-35 in.), and reared under an extensive management system had higher odds of being seropositive. Bovids from the dry zone were at approximately six times higher odds of being seropositive even after controlling for the possible effects of age and management system. Approximately 75% of the seropositive males were from the dry zone. Most bovids (84%) from the dry zone in this study were reared under an extensive management system (free grazing) which allows unrestricted contact between animals. These factors may have contributed to the spread of brucellosis in bovids in the dry zone. This infection might be an important cause of abortion in bovids in Sri Lanka. However, there is also evidence of other causes for abortion, repeat breeding and retained placenta.

Visual detection of Brucella in bovine biological samples using DNA-activated gold nanoparticles

PubMed Central

Kumar, Satish; Kaur, Gurpreet; Ali, Syed Atif; Shrivastava, Sameer; Gupta, Praveen K.; Cooper, Jonathan M.; Chaudhuri, Pallab

2017-01-01

Brucellosis is a bacterial disease, which, although affecting cattle primarily, has been associated with human infections, making its detection an important challenge. The existing gold standard diagnosis relies on the culture of bacteria which is a lengthy and costly process, taking up to 45 days. New technologies based on molecular diagnosis have been proposed, either through dip-stick, immunological assays, which have limited specificity, or using nucleic acid tests, which enable to identify the pathogen, but are impractical for use in the field, where most of the reservoir cases are located. Here we demonstrate a new test based on hybridization assays with metal nanoparticles, which, upon detection of a specific pathogen-derived DNA sequence, yield a visual colour change. We characterise the components used in the assay with a range of analytical techniques and show sensitivities down to 1000 cfu/ml for the detection of Brucella. Finally, we demonstrate that the assay works in a range of bovine samples including semen, milk and urine, opening up the potential for its use in the field, in low-resource settings. PMID:28719613

Human infection by Brucella melitensis: an outbreak attributed to contact with infected goats.

PubMed

Wallach, J C; Samartino, L E; Efron, A; Baldi, P C

1997-12-01

Although several outbreaks of Brucella melitensis infection have been reported among laboratory workers or goat cheese consumers, outbreaks related to rural labour have been rarely studied. An outbreak of human brucellosis among farm workers of Argentina was studied and revealed a close relationship with an epidemic of caprine abortions which occurred shortly before on the same farm. High rates of B. melitensis infection were found among goats. Active brucellosis was diagnosed in 33 subjects (14 with positive blood culture for B. melitensis), while other 27 did not show evidence of illness. While 25 of the brucellosis active patients were rural workers, only 5 of the healthy subjects were engaged in rural labour. Active brucellosis was diagnosed in 91.3% of the subjects in continuous contact with goats and in 32% of those having an occasional contact with the animals. All the 60 subjects denied consumption of goat cheese or milk. As shown here, epidemic human infections by B. melitensis may develop among people frequently in contact with infected goat herds or goat manure.

PPARγ-mediated increase in glucose availability sustains chronic Brucella abortus infection in alternatively activated macrophages

PubMed Central

Xavier, Mariana N.; Winter, Maria G.; Spees, Alanna M.; den Hartigh, Andreas B.; Nguyen, Kim; Roux, Christelle M.; Silva, Teane M. A.; Atluri, Vidya L.; Kerrinnes, Tobias; Keestra, A. Marijke; Monack, Denise M.; Luciw, Paul A.; Eigenheer, Richard A.; Bäumler, Andreas J.; Santos, Renato L.; Tsolis, Renée M.

2013-01-01

SUMMARY Eradication of persistent intracellular bacterial pathogens with antibiotic therapy is often slow or incomplete. However, strategies to augment antibiotics are hampered by our poor understanding of the nutritional environment that sustains chronic infection. Here we show that the intracellular pathogen Brucella abortus survives and replicates preferentially in alternatively activated macrophages (AAM), which are more abundant during chronic infection. A metabolic shift induced by peroxisome proliferator activated receptor γ (PPARγ), which increases intracellular glucose availability, is identified as a causal mechanism promoting enhanced bacterial survival in AAM. Glucose uptake was crucial for increased replication of B. abortus in AAM, and chronic infection, as inactivation of the bacterial glucose transporter gluP reduced both intracellular survival in AAM and persistence in mice. Thus, a shift in intracellular nutrient availability induced by PPARγ promotes chronic persistence of B. abortus within AAM and targeting this pathway may aid in eradicating chronic infection. PMID:23954155

Infection of cattle with Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park.

PubMed Central

Forbes, L B; Tessaro, S V

1996-01-01

An experiment was conducted to determine if cattle could be infected with a strain of Brucella abortus biovar 1 isolated from a bison in Wood Buffalo National Park. Three pregnant cows inoculated conjunctivally with 5.7 x 10(8) cfu of the bacterium, and their subsequent calves, showed seroconversion on standard serological tests for bovine brucellosis, and large numbers of the bacterium were isolated from numerous tissues at necropsy. A 4th cow that was moved into the pen that previously contained the inoculated cows subsequently showed seroconversion, and the same strain of B. abortus biovar 1 was isolated from numerous tissues. Although this strain from bison in Wood Buffalo National Park has existed in isolation from cattle for over 60 years, it remains infectious and contagious for cattle. PMID:8809394

An ecological perspective on the changing face of Brucella abortus in the western United States

USGS Publications Warehouse

Cross, Paul C.; Maichak, Eric J.; Brennan, Angela; Scurlock, Brandon M.; Henningsen, John C.; Luikart, Gordon

2013-01-01

After a hiatus during the 1990s, outbreaks of Brucella abortus in cattle are occurring more frequently in some of the western states of the United States, namely, Montana, Wyoming and Idaho. This increase is coincident with increasing brucellosis seroprevalence in elk (Cervus elaphus), which is correlated with elk density. Vaccines are a seductive solution, but their use in wildlife systems remains limited by logistical, financial, and scientific constraints. Cattle vaccination is ongoing in the region. Livestock regulations, however, tend to be based on serological tests that test for previous exposure and available vaccines do not protect against seroconversion. The authors review recent ecological studies of brucellosis, with particular emphasis on the Greater Yellowstone Area, and highlight the management options and implications of this work, including the potential utility of habitat modifications and targeted hunts, as well as scavengers and predators. Finally, the authors discuss future research directions that will help us to understand and manage brucellosis in wildlife.

Isolation, purification, and partial characterization of Brucella abortus matrix protein.

PubMed Central

Moriyon, I; Berman, D T

1983-01-01

Peptidoglycan sacculi with peptidoglycan-associated proteins were prepared from cell envelopes of Brucella abortus by extraction with sodium dodecyl sulfate (SDS) at 50 degrees C. On extraction of these preparations with SDS at 100 degrees C, a protein was obtained whose removal from peptidoglycan was confirmed by electron microscopy. Incubation of the 50 degrees C SDS-extracted cell envelopes with 50 mM MgCl2 in SDS-2-beta-mercaptoethanol at 37 degrees C also extracted the protein, along with lipopolysaccharide. At temperatures below 60 degrees C, the protein did not bind SDS strongly and had an apparent molecular weight greater than 92,000 in SDS-polyacrylamide gel electrophoresis. At higher temperatures, SDS bound strongly, and the apparent molecular weight was 38,000. Urea at 5 M did not alter the electrophoretic mobility of this 38,000-molecular-weight form. Immunoelectrophoresis in detergents with antisera to cell envelopes, carbohydrate staining of SDS-polyacrylamide gels, and production of anti-lipopolysaccharide antibodies by mice immunized with the purified protein indicated that lipopolysaccharide was present in free and protein-bound forms. Sequential gel filtration in SDS-EDTA and SDS-NaCl removed most lipopolysaccharide. After further purification by preparative SDS-polyacrylamide gel electrophoresis, a gas-liquid chromatographic analysis showed residual lipid tightly associated with the protein. The results suggested that the interactions between matrix proteins and other outer membrane components are stronger in B. abortus than in Escherichia coli, which was used as a control throughout. Images PMID:6401696

A Closed-tube Loop-Mediated Isothermal Amplification Assay for the Visual Endpoint Detection of Brucella spp. and Mycobacterium avium subsp. paratuberculosis.

PubMed

Trangoni, Marcos D; Gioffré, Andrea K; Cravero, Silvio L

2017-01-01

LAMP (loop-mediated isothermal amplification) is an isothermal nucleic acid amplification technique that is characterized by its efficiency, rapidity, high yield of final product, robustness, sensitivity, and specificity, with the blueprint that it can be implemented in laboratories of low technological complexity. Despite the conceptual complexity underlying the mechanistic basis for the nucleic acid amplification, the technique is simple to use and the amplification and detection can be carried out in just one step. In this chapter, we present a protocol based on LAMP for the rapid identification of isolates of Brucella spp. and Mycobacterium avium subsp. paratuberculosis, two major bacterial pathogens in veterinary medicine.

Express immunochromatographic detection of antibodies against Brucella abortus in cattle sera based on quantitative photometric registration and modulated cut-off level.

PubMed

Sotnikov, Dmitriy V; Byzova, Nadezhda A; Zherdev, Anatoly V; Eskendirova, Saule Z; Baltin, Kairat K; Mukanov, Kasim K; Ramankulov, Erlan M; Sadykhov, Elchin G; Dzantiev, Boris B

2015-01-01

An immunochromatographic test system was developed for rapid detection of the levels of specific IgG antibodies to Brucella abortus lipopolysaccharide, as a tool for diagnosis of brucellosis in cattle. The pilot test strips were examined using blood sera from sick (78 samples) and healthy (35 samples) cows. The results obtained by immunochromatographic assay, using a portable optical densitometer for digital video detection, correlate well with the results obtained by immunoenzyme assay and are in agreement with the results of the disease diagnosis. The new test system allows detection of antibodies within 10 min and can be proposed as an alternative to the methods available for serodiagnosis of brucellosis.

Animal and human dose-response models for Brucella species.

PubMed

Teske, Sondra S; Huang, Yin; Tamrakar, Sushil B; Bartrand, Timothy A; Weir, Mark H; Haas, Charles N

2011-10-01

Human Brucellosis is one of the most common zoonotic diseases worldwide. Disease transmission often occurs through the handling of domestic livestock, as well as ingestion of unpasteurized milk and cheese, but can have enhanced infectivity if aerosolized. Because there is no human vaccine available, rising concerns about the threat of Brucellosis to human health and its inclusion in the Center for Disease Control's Category B Bioterrorism/Select Agent List make a better understanding of the dose-response relationship of this microbe necessary. Through an extensive peer-reviewed literature search, candidate dose-response data were appraised so as to surpass certain standards for quality. The statistical programming language, "R," was used to compute the maximum likelihood estimation to fit two models, the exponential and the approximate beta-Poisson (widely used for quantitative risk assessment) to dose-response data. Dose-response models were generated for prevalent species of Brucella: Br. suis, Br. melitensis, and Br. abortus. Dose-response models were created for aerosolized Br. suis exposure to guinea pigs from pooled studies. A parallel model for guinea pigs inoculated through both aerosol and subcutaneous routes with Br. melitensis showed that the median infectious dose corresponded to a 30 colony-forming units (CFU) dose of Br. suis, much less than the N(50) dose of about 94 CFU for Br. melitensis organisms. When Br. melitensis was tested subcutaneously on mice, the N(50) dose was higher, 1,840 CFU. A dose-response model was constructed from pooled data for mice, rhesus macaques, and humans inoculated through three routes (subcutaneously/aerosol/intradermally) with Br. melitensis. © 2011 Society for Risk Analysis.

Brucella abortus Cyclic β-1,2-Glucan Mutants Have Reduced Virulence in Mice and Are Defective in Intracellular Replication in HeLa Cells

PubMed Central

Briones, Gabriel; Iñón de Iannino, Nora; Roset, Mara; Vigliocco, Ana; Paulo, Patricia Silva; Ugalde, Rodolfo A.

2001-01-01

Null cyclic β-1,2-glucan synthetase mutants (cgs mutants) were obtained from Brucella abortus virulent strain 2308 and from B. abortus attenuated vaccinal strain S19. Both mutants show greater sensitivity to surfactants like deoxycholic acid, sodium dodecyl sulfate, and Zwittergent than the parental strains, suggesting cell surface alterations. Although not to the same extent, both mutants display reduced virulence in mice and defective intracellular multiplication in HeLa cells. The B. abortus S19 cgs mutant was completely cleared from the spleens of mice after 4 weeks, while the 2308 mutant showed a 1.5-log reduction of the number of brucellae isolated from the spleens after 12 weeks. These results suggest that cyclic β-1,2-glucan plays an important role in the residual virulence of the attenuated B. abortus S19 strain. Although the cgs mutant was cleared from the spleens earlier than the wild-type parental strain (B. abortus S19) and produced less inflammatory response, its ability to confer protection against the virulent strain B. abortus 2308 was fully retained. Equivalent levels of induction of spleen gamma interferon mRNA and anti-lipopolysaccharide (LPS) of immunoglobulin G2a (IgG2a) subtype antibodies were observed in mice injected with B. abortus S19 or the cgs mutant. However, the titer of anti-LPS antibodies of the IgG1 subtype induced by the cgs mutant was lower than that observed with the parental S19 strain, thus suggesting that the cgs mutant induces a relatively exclusive Th1 response. PMID:11401996

Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

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

Nelson, O.D.

1997-09-04

This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage.

Oral immunization of mice with gamma-irradiated Brucella neotomae induces protection against intraperitoneal and intranasal challenge with virulent B. abortus 2308.

PubMed

Dabral, Neha; Martha-Moreno-Lafont; Sriranganathan, Nammalwar; Vemulapalli, Ramesh

2014-01-01

Brucella spp. are Gram-negative, facultative intracellular coccobacilli that cause one of the most frequently encountered zoonosis worldwide. Humans naturally acquire infection through consumption of contaminated dairy and meat products and through direct exposure to aborted animal tissues and fluids. No vaccine against brucellosis is available for use in humans. In this study, we tested the ability of orally inoculated gamma-irradiated B. neotomae and B. abortus RB51 in a prime-boost immunization approach to induce antigen-specific humoral and cell mediated immunity and protection against challenge with virulent B. abortus 2308. Heterologous prime-boost vaccination with B. abortus RB51 and B. neotomae and homologous prime-boost vaccination of mice with B. neotomae led to the production of serum and mucosal antibodies specific to the smooth LPS. The elicited serum antibodies included the isotypes of IgM, IgG1, IgG2a, IgG2b and IgG3. All oral vaccination regimens induced antigen-specific CD4(+) and CD8(+) T cells capable of secreting IFN-γ and TNF-α. Upon intra-peritoneal challenge, mice vaccinated with B. neotomae showed the highest level of resistance against virulent B. abortus 2308 colonization in spleen and liver. Experiments with different doses of B. neotomae showed that all tested doses of 10(9), 10(10) and 10(11) CFU-equivalent conferred significant protection against the intra-peritoneal challenge. However, a dose of 10(11) CFU-equivalent of B. neotomae was required for affording protection against intranasal challenge as shown by the reduced bacterial colonization in spleens and lungs. Taken together, these results demonstrate the feasibility of using gamma-irradiated B. neotomae as an effective and safe oral vaccine to induce protection against respiratory and systemic infections with virulent Brucella.

Zepto-molar electrochemical detection of Brucella genome based on gold nanoribbons covered by gold nanoblooms

NASA Astrophysics Data System (ADS)

Rahi, Amid; Sattarahmady, Naghmeh; Heli, Hossein

2015-12-01

Gold nanoribbons covered by gold nanoblooms were sonoelectrodeposited on a polycrystalline gold surface at -1800 mV (vs. AgCl) with the assistance of ultrasound and co-occurrence of the hydrogen evolution reaction. The nanostructure, as a transducer, was utilized to immobilize a Brucella-specific probe and fabrication of a genosensor, and the process of immobilization and hybridization was detected by electrochemical methods, using methylene blue as a redox marker. The proposed method for detection of the complementary sequence, sequences with base-mismatched (one-, two- and three-base mismatches), and the sequence of non-complementary sequence was assayed. The fabricated genosensor was evaluated for the assay of the bacteria in the cultured and human samples without polymerase chain reactions (PCR). The genosensor could detect the complementary sequence with a calibration sensitivity of 0.40 μA dm3 mol-1, a linear concentration range of 10 zmol dm-3 to 10 pmol dm-3, and a detection limit of 1.71 zmol dm-3.

Effectiveness of Brucella abortus lipopolysaccharide as an adjuvant for tuberculin PPD.

PubMed

Jamalan, Mostafa; Ardestani, Susan Kaboudanian; Zeinali, Majid; Mosaveri, Nader; Mohammad Taheri, Mohammad

2011-01-01

Bacterial lipopolysaccharide (LPS) has T-helper 1 (Th1) immunostimulatory activities but because of toxicity and pyrogenicity cannot be used as an adjuvant. Brucella abortus LPS has less toxicity and no pyrogenic properties in comparison to other bacterial LPS. In the current study, the immunostimulatory properties of B. abortus LPS were evaluated for its adjuvant activity. Tuberculin purified protein derivative (PPD) from Mycobacterium tuberculosis was extracted and after anion-exchange chromatography on Q-sepharose column, two fractions (17 and 23), which dominantly contained 30- and 70-kDa antigens, were collected for immunological studies. BALB/c mice were immunized with four different antigen preparations (BCG, PPD, 17th and 23rd PPD fractions) along with complete Freund's adjuvant or B. abortus LPS. The T-cell immune response of mice was assessed by measurement of Th1-type cytokine (IFN-γ) and Th2-type cytokines (IL-5 and IL-10) levels. Also, the humoral immunity was evaluated by measuring the specific IgG levels. Our results showed that immunization of mice with 17th PPD fraction along with B. abortus LPS can induce a Th1-type cytokine response characterized with a high IFN-γ/IL-5 ratio, while immunization with PPD or 23rd PPD fraction along with the same adjuvant resulted to a mixed Th1/Th2-type cytokine response. Copyright © 2010 The International Association for Biologicals. Published by Elsevier Ltd. All rights reserved.

Genotyping of Brucella melitensis strains from dromedary camels (Camelus dromedarius) from the United Arab Emirates with multiple-locus variable-number tandem repeat analysis.

PubMed

Gyuranecz, Miklós; Wernery, Ulli; Kreizinger, Zsuzsa; Juhász, Judit; Felde, Orsolya; Nagy, Péter

2016-04-15

Camel brucellosis is a widespread zoonotic disease in camel-rearing countries caused by Brucella melitensis and Brucella abortus. The aim of this study was the first genetic analysis of B. melitensis strains isolated from dromedary camels (Camelus dromedarius) using multiple-locus variable-number tandem repeat analysis (MLVA). MLVA 16 and its MLVA 8 and MLVA11 subsets were used to determine the genotypes of 15 B. melitensis isolates from dromedary camels (11 strains) and other host species (4 strains) from the United Arab Emirates and the results were then compared to B. melitensis MLVA genotypes from other parts of the world. Five, including two novel genotypes were identified with MLVA 8. MLVA 16 further discriminated these five genotypes to ten variants. The eleven camel isolates clustered into four main genetic groups within the East-Mediterranean and African clades and this clustering correlated with the geographic origin of the hosts (United Arab Emirates, Kingdom of Saudi Arabia and Sudan) and the date of their isolation. The camel strains were also genetically related to strains isolated from wild and domestic ruminants from their close habitat or from other parts of the world. Although limited number of strains were analysed, based on our data imported animals from foreign countries, local small ruminants and wildlife species are hypothesized to be the main sources of camel brucellosis in the United Arab Emirates. MLVA was successfully applied to determine the epidemiological links between the different camel B. melitensis infections in the United Arab Emirates and it can be a beneficial tool in future disease control programs. Copyright © 2016 Elsevier B.V. All rights reserved.

Comparison of four polymerase chain reaction assays for the detection of Brucella spp. in clinical samples from dogs

PubMed Central

Boeri, Eduardo J.; Wanke, María M.; Madariaga, María J.; Teijeiro, María L.; Elena, Sebastian A.; Trangoni, Marcos D.

2018-01-01

Aim: This study aimed to compare the sensitivity (S), specificity (Sp), and positive likelihood ratios (LR+) of four polymerase chain reaction (PCR) assays for the detection of Brucella spp. in dog’s clinical samples. Materials and Methods: A total of 595 samples of whole blood, urine, and genital fluids were evaluated between October 2014 and November 2016. To compare PCR assays, the gold standard was defined using a combination of different serological and microbiological test. Bacterial isolation from urine and blood cultures was carried out. Serological methods such as rapid slide agglutination test, indirect enzyme-linked immunosorbent assay, agar gel immunodiffusion test, and buffered plate antigen test were performed. Four genes were evaluated: (i) The gene coding for the BCSP31 protein, (ii) the ribosomal gene coding for the 16S-23S intergenic spacer region, (iii) the gene coding for porins omp2a/omp2b, and (iv) the gene coding for the insertion sequence IS711. Results: The results obtained were as follows: (1) For the primers that amplify the gene coding for the BCSP31 protein: S: 45.64% (confidence interval [CI] 39.81-51.46), Sp: 95.62% (CI 93.13-98.12), and LR+: 10.43 (CI 6.04-18); (2) for the primers that amplify the ribosomal gene of the 16S-23S rDNA intergenic spacer region: S: 69.80% (CI 64.42-75.18), Sp: 95.62 % (CI 93.13-98.12), and LR+: 11.52 (CI 7.31-18.13); (3) for the primers that amplify the omp2a and omp2b genes: S: 39.26% (CI 33.55-44.97), Sp: 97.31% (CI 95.30-99.32), and LR+ 14.58 (CI 7.25-29.29); and (4) for the primers that amplify the insertion sequence IS711: S: 22.82% (CI 17.89 - 27.75), Sp: 99.66% (CI 98.84-100), and LR+ 67.77 (CI 9.47-484.89). Conclusion: We concluded that the gene coding for the 16S-23S rDNA intergenic spacer region was the one that best detected Brucella spp. in canine clinical samples. PMID:29657404

Risk factors for contacts between wild boar and outdoor pigs in Switzerland and investigations on potential Brucella suis spill-over.

PubMed

Wu, Natacha; Abril, Carlos; Thomann, Andreas; Grosclaude, Eleonore; Doherr, Marcus G; Boujon, Patrick; Ryser-Degiorgis, Marie-Pierre

2012-07-20

Due to the parallel increase of the number of free-ranging wild boar and domestic pigs reared outdoor, the risk that they interact has become higher. Contacts with wild boar can be the origin of disease outbreaks in pigs, as it has been documented for brucellosis in some European countries. This study aimed at quantifying the occurrence of contacts between wild boar and outdoor domestic pigs in Switzerland, and identifying risk factors for these contacts. Furthermore, exposed pigs were tested for pathogen spill-over, taking Brucella suis as an example because B. suis is widespread in Swiss wild boar while domestic pigs are officially free of brucellosis. Thirty-one percent of the game-wardens and 25% of the pig owners participating to a country-wide questionnaire survey reported contacts, including approaches of wild boar outside the fence, intrusions, and mating. Seventeen piggeries (5%) reported the birth of cross-bred animals. Risk factors for contacts identified by a uni- and multivariable logistic regression approach were: distance between pig enclosure and buildings, proximity of a forest, electric fences, and fences ≤ 60 cm. Pigs of the Mangalitza breed were most at risk for mating with wild boar (births of cross-bred animals). Blood and tissues of 218 outdoor pigs from 13 piggeries were tested for an infection with Brucella suis, using rose bengal test, complement fixation test, and an IS711-based real-time PCR. One piggery with previous wild boar contacts was found infected with B. suis, however, epidemiological investigations failed to identify the direct source of infection. Results show that interactions between wild boar and outdoor pigs are not uncommon, pointing at the existing risk of pathogen spill-over. Provided data on risk factors for these interactions could help the risk-based implementation of protection measures for piggeries. The documentation of a brucellosis outbreak in pigs despite the freedom-of-disease status underlines the

Risk factors for contacts between wild boar and outdoor pigs in Switzerland and investigations on potential Brucella suis spill-over

PubMed Central

2012-01-01

Background Due to the parallel increase of the number of free-ranging wild boar and domestic pigs reared outdoor, the risk that they interact has become higher. Contacts with wild boar can be the origin of disease outbreaks in pigs, as it has been documented for brucellosis in some European countries. This study aimed at quantifying the occurrence of contacts between wild boar and outdoor domestic pigs in Switzerland, and identifying risk factors for these contacts. Furthermore, exposed pigs were tested for pathogen spill-over, taking Brucella suis as an example because B. suis is widespread in Swiss wild boar while domestic pigs are officially free of brucellosis. Results Thirty-one percent of the game-wardens and 25% of the pig owners participating to a country-wide questionnaire survey reported contacts, including approaches of wild boar outside the fence, intrusions, and mating. Seventeen piggeries (5%) reported the birth of cross-bred animals. Risk factors for contacts identified by a uni- and multivariable logistic regression approach were: distance between pigs enclosure and houses, proximity of a forest, electric fences, and fences ≤ 60 cm. Pigs of the Mangalitza breed were most at risk for mating with wild boar (births of cross-bred animals). Blood and tissues of 218 outdoor pigs from 13 piggeries were tested for an infection with Brucella suis, using rose bengal test, complement fixation test, and an IS711-based real-time PCR. One piggery with previous wild boar contacts was found infected with B. suis, however, epidemiological investigations failed to identify the direct source of infection. Conclusions Results show that interactions between wild boar and outdoor pigs are not uncommon, pointing at the existing risk of pathogen spill-over. Provided data on risk factors for these interactions could help the risk-based implementation of protection measures for piggeries. The documentation of a brucellosis outbreak in pigs despite the freedom

Biosafety of parenteral Brucella abortus RB51 vaccine in bison calves

USGS Publications Warehouse

Roffe, T.J.; Olsen, S.C.; Gidlewski, T.; Jensen, A.E.; Palmer, M.V.; Huber, R.

1999-01-01

Vaccination is considered among the primary management tools for reducing brucellosis prevalence in Greater Yellowstone Area (GYA) ungulates. Before their use, however, vaccine safety and efficacy must be demonstrated. Twenty-seven female bison (Bison bison) calves (approx 5 months old) were vaccinated with Brucella abortus Strain RB51 (1.5 x 1010 colony forming units [CFU], subcutaneously) as part of routine management. We assessed the persistence, pathology, shedding, and transmission associated with RB51 by serial necropsy, bacteriology, histopathology, and serology of 20 of these 27 vaccinated calves, and RB51 serology of 10 nonvaccinated, commingling adult females. With the exception of 1 calf, RB51 dot-blot titers at necropsy were <1:80. Strain RB51 was cultured from lymph nodes in 4 of 4 calves at 14 weeks postvaccination (PV), 4 of 4 calves at 18 weeks PV, 1 of 4 calves at 22 weeks PV, 3 of 4 at 26 weeks PV, and 0 of 4 calves at 30 weeks PV. No gross lesions were observed. Mild histologic changes occurred only in a few draining lymph nodes early in sampling. Adverse clinical effects were not observed in vaccinates. Swabs from nasopharynx, conjunctiva, rectum, and vagina were uniformly culture negative for RB51. Strain RB51 dot-blot assays of bison cows were negative at a 1:20 dilution at 26 weeks PV. Our results suggest that RB51 persists longer in bison calves than in domestic cattle and is systemically distributed within lymphatic tissues. However, bison apparently clear the RB51 vaccine strain without shedding, transmission, or significant adverse reactions.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro.

PubMed

Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

2014-09-01

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4(+) T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2(-/-) and TLR4(-/-) mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2(-/-) and TLR4(-/-) mice. In addition, CD4(+) T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4(+) T cells from TLR2(-/-) and TLR4(-/-) mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2(-/-) and TLR4(-/-) mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2(-/-) and TLR4(-/-) mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response.

Brucellosis vaccines based on the open reading frames from genomic island 3 of Brucella abortus.

PubMed

Gómez, Leonardo; Alvarez, Francisco; Betancur, Daniel; Oñate, Angel

2018-05-17

Brucella abortus is the etiological agent of brucellosis, a zoonotic disease affecting cattle and humans. This disease has been partially controlled in cattle by immunization with live attenuated B. abortus S19 and RB51 strains. However, use of these vaccine strains has been associated with safety issues in animals and humans. New vaccines have since emerged in the prevention of brucellosis, particularly DNA vaccines, which have shown effectiveness and a good safety profile. Their protection efficacy in mice is associated with the induction of Th1 type and cytotoxic T cell mediated immune response against structural antigens and virulence factors expressed during B. abortus infection. Some antigenic candidate for vaccine design against brucellosis (mainly DNA vaccines) have been obtained from genomic island 3 (GI-3) of B. abortus, which encodes several open reading frames (ORFs) involved in the intracellular survival and virulence of this pathogen. The immunogenicity and protection conferred by these DNA vaccines in a murine model is reviewed in this article, suggesting that some of them could be safe and effective vaccine candidates against to prevent B. abortus infection. Copyright © 2018 Elsevier Ltd. All rights reserved.

Improved immunogenicity of tetanus toxoid by Brucella abortus S19 LPS adjuvant.

PubMed

Mohammadi, Mohsen; Kianmehr, Zahra; Kaboudanian Ardestani, Sussan; Gharegozlou, Behnaz

2014-09-01

Adjuvants are used to increase the immunogenicity of new generation vaccines, especially those based on recombinant proteins. Despite immunostimulatory properties, the use of bacterial lipopolysaccharide (LPS) as an adjuvant has been hampered due to its toxicity and pyrogenicity. Brucella abortus LPS is less toxic and has no pyrogenic properties compared to LPS from other gram negative bacteria. To evaluate the adjuvant effect of B. abortus (vaccine strain, S19) LPS for tetanus toxoid antigen (TT) and to investigate the protective effect of different tetanus vaccine preparations. LPS was extracted and purified from B. abortus S19 and KDO, glycan, phosphate content, and protein contamination were measured. Adipic acid dihydrazide (ADH) was used as a linker for conjugation of TT to LPS. Different amounts of B. abortus LPS, TT, TT conjugated with LPS, and TT mixed with LPS or complete Freund's adjuvant (CFA) were injected into mice and antibody production against TT was measured. The protective effect of induced antibodies was determined by LD50. Immunization of mice with TT+LPS produced the highest anti-TT antibody titer in comparison to the group immunized with TT without any adjuvant or the groups immunized with TT-LPS or TT+CFA. Tetanus toxid-S19 LPS also produced a 100% protective effect against TT in immunized mice. These data indicate that B. abortus LPS enhances the immune responses to TT and suggest the possible use of B. abortus LPS as an adjuvant in vaccine preparations.

Variations in the prevalence of antibody to brucella infection in cattle by farm, area and district in Kenya.

PubMed Central

Kadohira, M.; McDermott, J. J.; Shoukri, M. M.; Kyule, M. N.

1997-01-01

Variations in the sero-prevalence of antibody to brucella infection by cow, farm and area factors were investigated for three contrasting districts in Kenya: Samburu, an arid and pastoral area: Kiambu, a tropical highland area; and Kilifi, a typical tropical coastal area. Cattle were selected by a two-stage cluster sampling procedure and visited once between August 1991 and 1992. Schall's algorithm, a statistical model suitable for multi-level analysis was used. Using this model, older age, free grazing and large herd size (> or = 31) were associated with higher seroprevalence. Also, significant farm-to-farm, area-to-area and district-to-district variations were estimated. The patterns of high risk districts and areas seen were consistent with known animal husbandry and movement risk factors, but the larger than expected farm-to-farm variation within high risk areas and districts could not be explained. Thus, a multi-level method provided additional information beyond conventional analyses of sero-prevalence data. PMID:9042033

The Bactericidal Effect of High Temperature Is an Essential Resistance Mechanism of Chicken Macrophage against Brucella abortus Infection.

PubMed

Arayan, Lauren Togonon; Reyes, Alisha Wehdnesday Bernardo; Hop, Huynh Tan; Xuan, Huy Tran; Baek, Eun Jin; Min, Wongi; Kim, Suk

2017-10-28

Knowledge of avian host responses to brucellosis is critical to understanding how birds resist this infection; however, this mechanism is not well established. On the other hand, temperature has a major involvement in the physiology of living organisms, and cell death induced by heat is attributed to protein denaturation. This study demonstrates the direct bactericidal effect of a high temperature (41ºC) on Brucella abortus that resulted in the gradual reduction of intracellular bacteria and inhibited bacterial growth within avian macrophage HD11 in an increasing period of time. On the other hand, this study also revealed that high temperature does not affect the rate of bacterial uptake, as confirmed by the bacterial adherence assay. No significant difference was observed in the expression of target genes between infected and uninfected cells for both temperatures. This study suggests the susceptibility of B. abortus to bacterial death under a high temperature with an increased period of incubation, leading to suppression of bacterial growth.

Portable exhauster POR-007/Skid E and POR-008/Skid F storage plan

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

Nelson, O.D.

1998-07-25

This document provides storage requirements for 1,000 CFM portable exhausters POR-O07/Skid E and POR-008/Skid F. These requirements are presented in three parts: preparation for storage, storage maintenance and testing, and retrieval from storage. The exhauster component identification numbers listed in this document contain the prefix POR-007 or POR-008 depending on which exhauster is being used.

Outer membrane proteins from rough strains of four Brucella species.

PubMed Central

Santos, J M; Verstreate, D R; Perera, V Y; Winter, A J

1984-01-01

Outer membrane proteins from 15 rough strains of Brucella abortus, B. ovis, B. canis, and B. melitensis were extracted with a dipolar detergent, and outer membrane proteins from selected strains were purified by anion exchange chromatography and gel filtration (Verstreate et al., Infect. Immun. 35:979-989, 1982). Outer membrane proteins produced two types of profiles on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. One type, demonstrated by B. abortus, B. ovis, and B. canis strains, contained the three predominant protein groups present in smooth B. abortus strains (Verstreate et al., Infect. Immun. 35:979-989, 1982): groups 1, 2 (porin [Douglas et al., Infect. Immun. 44:16-21]), and 3. B. melitensis strains demonstrated the second profile type, in which there was an additional band between groups 1 and 2. The relative proportion of porin was considerably lower in B. ovis, B. canis, and B. melitensis than in B. abortus. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profiles could be used to distinguish B. abortus and B. melitensis from each other and from B. canis and B. ovis. The amino acid compositions of groups 2 and 3 from rough strains of B. abortus, B. canis, and B. melitensis were similar to those of corresponding proteins from smooth B. abortus strains. Zwittergent-soluble fractions from most rough strains contained antigen [b], which cross-reacted with group 2 from smooth B. abortus strains, and antigens [c] and [d], which cross-reacted with group 3 from smooth B. abortus strains. Antigen [a], shared by groups 2 and 3 (D. R. Verstreate and A. J. Winter, Infect. Immun. 46:182-187, 1984), was detected in most rough strains. None of these antigens were related to either rough or smooth lipopolysaccharide. Images PMID:6480106

[Multiplication of Brucella abortus and production of nitric oxide in two macrophage cell lines of different origin].

PubMed

Serafino, J; Conde, S; Zabal, O; Samartino, L

2007-01-01

Brucella abortus is a bacterium which causes abortions and infertility in cattle and undulant fever in humans. It multiplies intracellularly, evading the mechanisms of cellular death. Nitric oxide (NO) is important in the regulation of the immune response. In the present work, we studied the ability of three B. abortus strains to survive intracellularly in two macrophage cell lines. The bacterial multiplication in both cell lines was determined at two different times in UFC/ ml units. Moreover the inoculated cells were also observed under light-field and fluorescence microscopy stained with Giemsa and acridine orange, respectively. The stain of both cellular lines showed similar results with respect to the UFC/ml determination. The presence of B. abortus was confirmed by electronic microscopy. In both macrophage cell lines inoculated with the rough strain RB51, the multiplication diminished and the level of NO was higher, compared with cells inoculated with smooth strains (S19 and 2308). These results suggest that the absence of O-chain of LPS probably affects the intracellular growth of B. abortus.

Multicenter prospective study of treatment of Brucella melitensis brucellosis with doxycycline for 6 weeks plus streptomycin for 2 weeks.

PubMed Central

Cisneros, J M; Viciana, P; Colmenero, J; Pachón, J; Martinez, C; Alarcón, A

1990-01-01

The effectiveness of treating human brucellosis caused by Brucella melitensis with a 6-week course of doxcycline plus streptomycin for 2 of those weeks was analyzed by a multicenter prospective study of 139 patients. Subjects with central nervous system involvement, endocarditis, or spondylitis were excluded from the study. All but 5 of the 139 patients completed the full treatment schedule and became afebrile in the first week of therapy. Four patients suffered relapses during the follow-up period. Of the five patients who did not complete the treatment, two left because of adverse secondary effects (1.4%), another two left for noncomplicance with the treatment (1.4%), and the remaining patient was considered a therapeutic failure because his symptoms persisted after the first week of therapy (0.7%). We concluded that the combination of doxycycline and streptomycin is an effective treatment for the types of brucellosis included in our study. PMID:2193624

Brucella melitensis VirB12 recombinant protein is a potential marker for serodiagnosis of human brucellosis.

PubMed

Mirkalantari, Shiva; Zarnani, Amir-Hassan; Nazari, Mahboobeh; Irajian, Gholam Reza; Amirmozafari, Nour

2017-03-03

The numerous drawbacks of current serological tests for diagnosis of brucellosis which mainly results from cross reactivity with LPS from other gram-negative bacteria have generated an increasing interest to find more specific non-LPS antigens. Previous studies had indicated that Brucella VirB12 protein, a cell surface protein and component of type IV secretion system, induces antibody response during animal infection. However, this protein has not yet been tested as a serological diagnostic marker in human brucellosis. Recombinant VirB12 protein was prepared and evaluated the efficacy of it in an indirect enzyme-linked immunosorbent assay (ELISA) for brucellosis with sera collected from different region of Iran and the results were compared with a commercial ELISA kit. Sera from human brucellosis patients strongly reacted to the purified recombinant VirB12. The sensitivity, specificity, accuracy, negative predictive value and positive predictive value of recombinant VirB12-based ELISA related to the commercial-ELISA method were 87.8, 94, 90, 80 and 96.6% respectively. We concluded that antigenic VirB12 have a property value that can be considered as a candidate for using in serodiagnostic tests for human brucellosis.

Evaluation of a new immunocapture test for the diagnosis of ovine brucellosis caused by Brucella melitensis.

PubMed

Durán-Ferrer, M; Mendoza, J; Osuna, A; Caporale, V; Lucas, A; León, L; Garrido, F

2002-11-23

A new immunocapture technique has been applied to the diagnosis of ovine brucellosis under experimental conditions. The tests were made on a serum bank derived from both young and adult ewes vaccinated conjunctivally with the Rev 1 strain at a dose of 10(8) to 10(9) colony-forming units. Adult ewes were infected experimentally two-and-a-half years after they had been vaccinated and the results were compared with an unvaccinated control group. The condition of each animal in terms of infection with Brucella melitensis was determined by clinical and bacteriological investigations. The development of the immune response was compared by the rose bengal test, the complement fixation test, the Coombs' test and the immunocapture technique for 180 days after the vaccination and for 410 days after the experimental infection, that is, the two following gestations. The results suggest that the new technique is more specific in animals vaccinated conjunctivally, regardless of their age when they were vaccinated. After the experimental infection, significantly (P < 0.05) fewer of the vaccinated sheep which were free of clinical signs and were not excreting B melitensis reacted positively to the test.

Tissue distribution and cell tropism of Brucella canis in naturally infected canine foetuses and neonates.

PubMed

de Souza, Tayse Domingues; de Carvalho, Tatiane Furtado; Mol, Juliana Pinto da Silva; Lopes, João Vítor Menezes; Silva, Monique Ferreira; da Paixão, Tatiane Alves; Santos, Renato Lima

2018-05-08

Brucella canis infection is an underdiagnosed zoonotic disease. Knowledge about perinatal brucellosis in dogs is extremely limited, although foetuses and neonates are under risk of infection due to vertical transmission. In this study, immunohistochemistry was used to determine tissue distribution and cell tropism of B. canis in canine foetuses and neonates. Diagnosis of B. canis in tissues of naturally infected pups was based on PCR and sequencing of amplicons, bacterial isolation, and immunohistochemistry, whose specificity was confirmed by laser capture microdissection. PCR positivity among 200 puppies was 21%, and nine isolates of B. canis were obtained. Tissues from 13 PCR-positive puppies (4 stillborn and 9 neonates) presented widespread immunolabeling. Stomach, intestines, kidney, nervous system, and umbilicus were positive in all animals tested. Other frequently infected organs included the liver (92%), lungs (85%), lymph nodes (69%), and spleen (62%). Immunolabeled coccobacilli occurred mostly in macrophages, but they were also observed in erythrocytes, epithelial cells of gastrointestinal mucosa, renal tubules, epidermis, adipocytes, choroid plexus, ependyma, neuroblasts, blood vessels endothelium, muscle cells, and in the intestinal lumen. These results largely expand our knowledge about perinatal brucellosis in the dog, clearly demonstrating a pantropic distribution of B. canis in naturally infected foetuses and neonates.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

PubMed

Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

2016-08-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System

PubMed Central

Gorasia, Dhana G.; Veith, Paul D.; Hanssen, Eric G.; Glew, Michelle D.; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C.

2016-01-01

The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32–36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component. PMID:27509186

Isolation and expression of recombinant antibody fragments to the biological warfare pathogen Brucella melitensis.

PubMed

Hayhurst, Andrew; Happe, Scott; Mabry, Robert; Koch, Zephyr; Iverson, Brent L; Georgiou, George

2003-05-01

Brucella melitensis is a highly infectious animal pathogen able to cause a recurring debilitating disease in humans and is therefore high on the list of biological warfare agents. Immunoglobulin genes from mice immunized with gamma-irradiated B. melitensis strain 16M were used to construct a library that was screened by phage display against similarly prepared bacteria. The selected phage particles afforded a strong enzyme-linked immunosorbent assay (ELISA) signal against gamma-irradiated B. melitensis cells. However, extensive efforts to express the respective single chain antibody variable region fragment (scFv) in soluble form failed due to: (i) poor solubility and (ii) in vivo degradation of the c-myc tag used for the detection of the recombinant antibodies. Both problems could be addressed by: (i) fusing a human kappa light chain constant domain (Ck) chain to the scFv to generate single chain antibody fragment (scAb) antibody fragments and (ii) by co-expression of the periplasmic chaperone Skp. While soluble, functional antibodies could be produced in this manner, phage-displaying scFvs or scAbs were still found to be superior ELISA reagents for immunoassays, due to the large signal amplification afforded by anti-phage antibodies. The isolated phage antibodies were shown to be highly specific to B. melitensis and did not recognize Yersinia pseudotuberculosis in contrast to the existing diagnostic monoclonal YST 9.2.1.

TLR2 and TLR4 signaling pathways are required for recombinant Brucella abortus BCSP31-induced cytokine production, functional upregulation of mouse macrophages, and the Th1 immune response in vivo and in vitro

PubMed Central

Li, Jia-Yun; Liu, Yuan; Gao, Xiao-Xue; Gao, Xiang; Cai, Hong

2014-01-01

Brucella abortus is a zoonotic Gram-negative pathogen that causes brucelosis in ruminants and humans. Toll-like receptors (TLRs) recognize Brucella abortus and initiate antigen-presenting cell activities that affect both innate and adaptive immunity. In this study, we focused on recombinant Brucella cell-surface protein 31 (rBCSP31) to determine its effects on mouse macrophages. Our results demonstrated that rBCSP31 induced TNF-α, IL-6 and IL-12p40 production, which depended on the activation of mitogen-activated protein kinases (MAPKs) by stimulating the rapid phosphorylation of p38 and JNK and the activation of transcription factor NF-κB in macrophages. In addition, continuous exposure (>24 h) of RAW264.7 cells to rBCSP31 significantly enhanced IFN-γ-induced expression of MHC-II and the ability to present rBCSP31 peptide to CD4+ T cells. Furthermore, we found that rBCSP31 could interact with both TLR2 and TLR4. The rBCSP31-induced cytokine production by macrophages from TLR2−/− and TLR4−/− mice was lower than that from C57BL/6 macrophages, and the activation of NF-κB and MAPKs was attenuated in macrophages from TLR2−/− and TLR4−/− mice. In addition, CD4+ T cells from C57BL/6 mice immunized with rBCSP31 produced higher levels of IFN-γ and IL-2 compared with CD4+ T cells from TLR2−/− and TLR4−/− mice. Macrophages from immunized C57BL/6 mice produced higher levels of IL-12p40 than those from TLR2−/− and TLR4−/− mice. Furthermore, immunization with rBCSP31 provided better protection in C57BL/6 mice than in TLR2−/− and TLR4−/− mice after B. abortus 2308 challenge. These results indicate that rBCSP31 is a TLR2 and TLR4 agonist that induces cytokine production, upregulates macrophage function and induces the Th1 immune response. PMID:24769793

The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

PubMed Central

Vincent, Maxence S.; Durand, Eric; Cascales, Eric

2016-01-01

The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

Epidemiology of Brucellosis and Genetic Diversity of Brucella abortus in Kazakhstan

PubMed Central

Shevtsova, Elena; Shevtsov, Alexandr; Mukanov, Kasim; Filipenko, Maxim; Kamalova, Dinara; Sytnik, Igor; Syzdykov, Marat; Kuznetsov, Andrey; Akhmetova, Assel; Zharova, Mira; Karibaev, Talgat; Tarlykov, Pavel; Ramanculov, Erlan

2016-01-01

Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan. PMID

Epidemiology of Brucellosis and Genetic Diversity of Brucella abortus in Kazakhstan.

PubMed

Shevtsova, Elena; Shevtsov, Alexandr; Mukanov, Kasim; Filipenko, Maxim; Kamalova, Dinara; Sytnik, Igor; Syzdykov, Marat; Kuznetsov, Andrey; Akhmetova, Assel; Zharova, Mira; Karibaev, Talgat; Tarlykov, Pavel; Ramanculov, Erlan

2016-01-01

Brucellosis is a major zoonotic infection in Kazakhstan. However, there is limited data on its incidence in humans and animals, and the genetic diversity of prevalent strains is virtually unstudied. Additionally, there is no detailed overview of Kazakhstan brucellosis control and eradication programs. Here, we analyzed brucellosis epidemiological data, and assessed the effectiveness of eradication strategies employed over the past 70 years to counteract this infection. We also conducted multiple loci variable-number tandem repeat analysis (MLVA) of Brucella abortus strains found in Kazakhstan. We analyzed official data on the incidence of animal brucellosis in Kazakhstan. The records span more than 70 years of anti-brucellosis campaigns, and contain a brief description of the applied control strategies, their effectiveness, and their impact on the incidence in humans. The MLVA-16 method was used to type 94 strains of B. abortus and serial passages of B. abortus 82, a strain used in vaccines. MLVA-8 and MLVA-11 analyses clustered strains into a total of four and seven genotypes, respectively; it is the first time that four of these genotypes have been described. MLVA-16 analysis divided strains into 28 distinct genotypes having genetic similarity coefficient that varies from 60 to100% and a Hunter & Gaston diversity index of 0.871. MST analysis reconstruction revealed clustering into "Kazakhstani-Chinese (Central Asian)", "European" and "American" lines. Detection of multiple genotypes in a single outbreak confirms that poorly controlled trade of livestock plays a crucial role in the spread of infection. Notably, the MLVA-16 profile of the B. abortus 82 strain was unique and did not change during 33 serial passages. MLVA genotyping may thus be useful for epidemiological monitoring of brucellosis, and for tracking the source(s) of infection. We suggest that countrywide application of MLVA genotyping would improve the control of brucellosis in Kazakhstan.

Structural, functional and immunogenic insights on Cu,Zn superoxide dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

DOE PAGES

Pratt, Ashley J.; DiDonato, Michael; Shin, David S.; ...

2015-10-12

Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomicmore » details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against reactive oxygen

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus

PubMed Central

Pratt, Ashley J.; DiDonato, Michael; Shin, David S.; Cabelli, Diane E.; Bruns, Cami K.; Belzer, Carol A.; Gorringe, Andrew R.; Langford, Paul R.; Tabatabai, Louisa B.; Kroll, J. Simon; Tainer, John A.

2015-01-01

ABSTRACT Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against

Structural, Functional, and Immunogenic Insights on Cu,Zn Superoxide Dismutase Pathogenic Virulence Factors from Neisseria meningitidis and Brucella abortus.

PubMed

Pratt, Ashley J; DiDonato, Michael; Shin, David S; Cabelli, Diane E; Bruns, Cami K; Belzer, Carol A; Gorringe, Andrew R; Langford, Paul R; Tabatabai, Louisa B; Kroll, J Simon; Tainer, John A; Getzoff, Elizabeth D

2015-12-01

Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen and general pathogenicity factors and are therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomic details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and of SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly, and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes, and suggest general targets for antibacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors of or vaccines against these harmful pathogens. By protecting microbes against reactive oxygen

Structural, functional and immunogenic insights on Cu,Zn superoxide dismutase pathogenic virulence factors from Neisseria meningitidis and Brucella abortus

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

Pratt, Ashley J.; DiDonato, Michael; Shin, David S.

Bacterial pathogens Neisseria meningitidis and Brucella abortus pose threats to human and animal health worldwide, causing meningococcal disease and brucellosis, respectively. Mortality from acute N. meningitidis infections remains high despite antibiotics, and brucellosis presents alimentary and health consequences. Superoxide dismutases are master regulators of reactive oxygen, general pathogenicity factors and therefore therapeutic targets. Cu,Zn superoxide dismutases (SODs) localized to the periplasm promote survival by detoxifying superoxide radicals generated by major host antimicrobial immune responses. We discovered that passive immunization with an antibody directed at N. meningitidis SOD (NmSOD) was protective in a mouse infection model. To define the relevant atomicmore » details and solution assembly states of this important virulence factor, we report high-resolution and X-ray scattering analyses of NmSOD and SOD from B. abortus (BaSOD). The NmSOD structures revealed an auxiliary tetrahedral Cu-binding site bridging the dimer interface; mutational analyses suggested that this metal site contributes to protein stability, with implications for bacterial defense mechanisms. Biochemical and structural analyses informed us about electrostatic substrate guidance, dimer assembly and an exposed C-terminal epitope in the NmSOD dimer. In contrast, the monomeric BaSOD structure provided insights for extending immunogenic peptide epitopes derived from the protein. These collective results reveal unique contributions of SOD to pathogenic virulence, refine predictive motifs for distinguishing SOD classes and suggest general targets for anti-bacterial immune responses. The identified functional contributions, motifs, and targets distinguishing bacterial and eukaryotic SOD assemblies presented here provide a foundation for efforts to develop SOD-specific inhibitors or vaccines against these harmful pathogens. IMPORTANCE By protecting microbes against reactive oxygen

Detection of Leptospira spp. and Brucella abortus antibodies in free-living jaguars (Panthera onca) in two protected areas of northern Pantanal, Brazil.

PubMed

Onuma, Selma Samiko Miyazaki; Kantek, Daniel Luis Zanella; Crawshaw Júnior, Peter Gransden; Morato, Ronaldo Gonçalves; May-Júnior, Joares Adenilson; Morais, Zenaide Maria de; Ferreira Neto, José Soares; Aguiar, Daniel Moura de

2015-01-01

This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection.

Real-time PCR Detection of Brucella Abortus: A Comparative Study of SYBR Green I, 5'-exonuclease, and Hybridization Probe Assays

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

Newby, Deborah Trishelle; Hadfield, Ted; Roberto, Francisco Figueroa

Real-time PCR provides a means of detecting and quantifying DNA targets by monitoring PCR product accumulation during cycling as indicated by increased fluorescence. A number of different approaches can be used to generate the fluorescence signal. Three approaches—SYBR Green I (a double-stranded DNA intercalating dye), 5'-exonuclease (enzymatically released fluors), and hybridization probes (fluorescence resonance energy transfer)—were evaluated for use in a real-time PCR assay to detect Brucella abortus. The three assays utilized the same amplification primers to produce an identical amplicon. This amplicon spans a region of the B. abortus genome that includes portions of the alkB gene and themore » IS711 insertion element. All three assays were of comparable sensitivity, providing a linear assay over 7 orders of magnitude (from 7.5 ng down to 7.5 fg). However, the greatest specificity was achieved with the hybridization probe assay.« less

Brucella melitensis T Cell Epitope Recognition in Humans with Brucellosis in Peru

PubMed Central

Cannella, Anthony P.; Arlehamn, Cecilia S. Lindestam; Sidney, John; Patra, Kailash P.; Torres, Katherine; Tsolis, Renee M.; Liang, Li; Felgner, Philip L.; Saito, Mayuko; Gotuzzo, Eduardo; Gilman, Robert H.; Sette, Alessandro

2014-01-01

Brucella melitensis, one of the causative agents of human brucellosis, causes acute, chronic, and relapsing infection. While T cell immunity in brucellosis has been extensively studied in mice, no recognized human T cell epitopes that might provide new approaches to classifying and prognosticating B. melitensis infection have ever been delineated. Twenty-seven pools of 500 major histocompatibility complex class II (MHC-II) restricted peptides were created by computational prediction of promiscuous MHC-II CD4+ T cell derived from the top 50 proteins recognized by IgG in human sera on a genome level B. melitensis protein microarray. Gamma interferon (IFN-γ) and interleukin-5 (IL-5) enzyme-linked immunospot (ELISPOT) analyses were used to quantify and compare Th1 and Th2 responses of leukapheresis-obtained peripheral blood mononuclear cells from Peruvian subjects cured after acute infection (n = 9) and from patients who relapsed (n = 5). Four peptide epitopes derived from 3 B. melitensis proteins (BMEI 1330, a DegP/HtrA protease; BMEII 0029, type IV secretion system component VirB5; and BMEII 0691, a predicted periplasmic binding protein of a peptide transport system) were found repeatedly to produce significant IFN-γ ELISPOT responses in both acute-infection and relapsing patients; none of the peptides distinguished the patient groups. IL-5 responses against the panel of peptides were insignificant. These experiments are the first to systematically identify B. melitensis MHC-II-restricted CD4+ T cell epitopes recognized by the human immune response, with the potential for new approaches to brucellosis diagnostics and understanding the immunopathogenesis related to this intracellular pathogen. PMID:24126518

Isolation of two biologically active cell surface proteins from Brucella abortus by chromatofocusing

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

Tabatabai, L.B.; Deyoe, B.L.

1983-01-01

Brucella abortus contains a group of immunogenic cell surface proteins which have potential value as a vaccine or as a diagnostic reagent for the prevention and diagnosis of bovine brucellosis. Under nondenaturing conditions, these proteins range in molecular weight from 10,000-124,000, as determined by high performance liquid chromatography (HPLC) on TSK 3000sw. By analytical isoelectrofocusing, 6 major protein bands could be distinguished with pI's ranging from 4.0 to 6.0 and 3 additional major proteins with pI's of 7.5, 9.5, and 10. By chromatofocusing on Polybuffer Exchanger 94 with a pH gradient from 6-4, two of the six proteins from pImore » 4-6 were separated, a pI 4.9 and a pI 4.7 protein; a third fraction contained the high pI proteins. The former two proteins were homogeneous by analytical isoelectrofocusing, and a molecular weight of 54,000 daltons was found for both protein species by HPLC on TSK 3000sw. The pI 4-6 and not the pI 9.5 and 10 proteins, could be radiolabeled when intact cells were radioiodinated with diazotized (/sup 125/I)-iodosulfanilic acid. Biological activity of the proteins as assessed in lemmings indicated that immunization with the pI 4.7 and 4.9 proteins afforded better protection against experimental brucellosis than immunization with the high pI proteins. These results support our view that a single surface protein may be sufficient for the prevention of experimental brucellosis.« less

Understanding Virulence in the Brucellae and Francisellae: Towards Efficacious Treatments for Two Potential Biothreat Agents

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

Rasley, A; Parsons, D A; El-Etr, S

2009-12-30

Francisella tularensis, Yersinia pestis and Brucellae species are highly infectious pathogens classified as select agents by the Centers for Disease Control and Prevention (CDC) with the potential for use in bioterrorism attacks. These organisms are known to be facultative intracellular pathogens that preferentially infect human monocytes. As such, understanding how the host responds to infection with these organisms is paramount in detecting and combating human disease. We have compared the ability of fully virulent strains of each pathogen and their non-pathogenic near neighbors to enter and survive inside the human monocytic cell line THP-1 and have quantified the cellular responsemore » to infection with the goal of identifying both unique and common host response patterns. We expanded the scope of these studies to include experiments with pathogenic and non-pathogenic strains of Y. pestis, the causative agent of plague. Nonpathogenic strains of each organism were impaired in their ability to survive intracellularly compared with their pathogenic counterparts. Furthermore, infection of THP-1 cells with pathogenic strains of Y. pestis and F. tularensis resulted in marked increases in the secretion of the inflammatory chemokines IL-8, RANTES, and MIP-1{beta}. In contrast, B. melitensis infection failed to elicit any significant increases in a panel of cytokines tested. These differences may underscore distinct strategies in pathogenic mechanisms employed by these pathogens.« less

Early Acidification of Phagosomes Containing Brucella suis Is Essential for Intracellular Survival in Murine Macrophages

PubMed Central

Porte, Françoise; Liautard, Jean-Pierre; Köhler, Stephan

1999-01-01

Brucella suis is a facultative intracellular pathogen of mammals, residing in macrophage vacuoles. In this work, we studied the phagosomal environment of these bacteria in order to better understand the mechanisms allowing survival and multiplication of B. suis. Intraphagosomal pH in murine J774 cells was determined by measuring the fluorescence intensity of opsonized, carboxyfluorescein-rhodamine- and Oregon Green 488-rhodamine-labeled bacteria. Compartments containing live B. suis acidified to a pH of about 4.0 to 4.5 within 60 min. Acidification of B. suis-containing phagosomes in the early phase of infection was abolished by treatment of host cells with 100 nM bafilomycin A1, a specific inhibitor of vacuolar proton-ATPases. This neutralization at 1 h postinfection resulted in a 2- to 34-fold reduction of opsonized and nonopsonized viable intracellular bacteria at 4 and 6 h postinfection, respectively. Ammonium chloride and monensin, other pH-neutralizing reagents, led to comparable loss of intracellular viability. Addition of ammonium chloride at 7 h after the beginning of infection, however, did not affect intracellular multiplication of B. suis, in contrast to treatment at 1 h postinfection, where bacteria were completely eradicated within 48 h. Thus, we conclude that phagosomes with B. suis acidify rapidly after infection, and that this early acidification is essential for replication of the bacteria within the macrophage. PMID:10417172

DETECTION OF Leptospira spp. AND Brucella abortus ANTIBODIES IN FREE-LIVING JAGUARS (Panthera onca) IN TWO PROTECTED AREAS OF NORTHERN PANTANAL, BRAZIL

PubMed Central

ONUMA, Selma Samiko Miyazaki; KANTEK, Daniel Luis Zanella; CRAWSHAW, Peter Gransden; MORATO, Ronaldo Gonçalves; MAY-JÚNIOR, Joares Adenilson; de MORAIS, Zenaide Maria; FERREIRA, José Soares; de AGUIAR, Daniel Moura

2015-01-01

 This study aimed to assess the exposure of free-living jaguars (Panthera onca) to Leptospira spp. and Brucella abortus in two conservation units in the Pantanal of Mato Grosso, Brazil. The presence of antibodies in blood samples of eleven jaguars was investigated using autochthonous antigens isolated in Brazil added to reference antigen collection applied to diagnosis of leptospirosis by Microscopic Agglutination Test (MAT). The Rose Bengal test was applied for B. abortus antibodies. Two (18.2%) jaguars were seroreactive for the Leptospira spp. antigen and the serovar considered as most infective in both animals was a Brazilian isolate of serovar Canicola (L01). All jaguars were seronegative for B. abortus. These data indicate that the inclusion of autochthonous antigens in serological studies can significantly increase the number of reactive animals, as well as modify the epidemiological profile of Leptospira spp. infection. PMID:25923900

Comparative analysis of the early transcriptome of Brucella abortus - infected monocyte-derived macrophages from cattle naturally resistant or susceptible to brucellosis

PubMed Central

Rossetti, C.A.; Galindo, C.L.; Everts, R.E.; Lewin, H.A.; Garner, H.R.; Adams, L.G.

2010-01-01

Brucellosis is a worldwide zoonotic infectious disease that has a significant economic impact on animal production and human public health. We characterized the gene expression profile of B. abortus-infected monocyte-derived macrophages (MDMs) from naïve cattle naturally resistant (R) or susceptible (S) to brucellosis using a cDNA microarray technology. Our data indicate that 1) B. abortus induced a slightly increased genome activation in R MDMs and a down-regulated transcriptome in S MDMs, during the onset of infection, 2) R MDMs had the ability to mount a type 1 immune response against B. abortus infection which was impaired in S cells, and 3) the host cell activity was not altered after 12h post-B. abortus infection in R MDMs while the cell cycle was largely arrested in infected S MDMs at 12h p.i. These results contribute to understand of how host responses may be manipulated to prevent infection by brucellae. PMID:20932540

Diagnosing pyogenic, brucella and tuberculous spondylitis using histopathology and MRI: A retrospective study.

PubMed

Li, Tao; Liu, Tao; Jiang, Zhensong; Cui, Xingang; Sun, Jianmin

2016-10-01

The present study examined the histopathological and magnetic resonance imaging (MRI) features of pyogenic, brucella and tuberculous spondylitis (PS, BS and TS, respectively). A total of 22 PS, 20 BS and 20 TS patients were included in the study. Histopathological examination was used to assess the lesion structure and composition, and the MRI observation identified the lesion location and signal features. The following histopathological and MRI features were identified significantly more in patients with PS than in patients with BS and TS: Predominant neutrophil infiltration, abnormal intervertebral disk signal, lesions on the ventral and lateral sides of the vertebral bodies, and thick and irregular abscess walls. The following histopathological and MRI features were identified significantly more in patients with BS than in patients with PS and TS: Predominant lymphocyte infiltration, new bone formation, epithelioid granuloma, lesions on the ventral sides of the vertebral bodies, no, or very mild, vertebral body deformation, no abnormal paraspinal soft tissue signal, no intraosseous or paraspinal abscesses, and thin and irregular abscess walls. The following histopathological and MRI features were identified significantly more in patients with TS than in patients with BS and PS: Sequestrum, Langerhans giant cells, caseous necrosis, lesions primarily in the thoracic region and on the lateral sides of the vertebral bodies, no obvious intervertebral disk damage, obvious vertebral body deformation, abnormal paraspinal soft tissue signal, intraosseous or paraspinal abscesses, and thin and smooth abscess walls. In conclusion, it can be suggested that these significant differences in histopathological and MRI features between the three different types of spondylitis may contribute towards the differential diagnosis of the diseases.

Occurrence of oxidative stress in dairy cows seropositives for Brucella abortus.

PubMed

Perin, Géssica; Fávero, Juscivete F; Severo, Diego R T; Silva, Anielen D; Machado, Gustavo; Araújo, Hugo L; Lilenbaum, Walter; Morsch, Vera M; Schetinger, Maria Rosa C; Jordão, Ricardo S; Stefani, Lenita M; Bottari, Nathieli B; Da Silva, Aleksandro S

2017-09-01

Bovine brucellosis is an important zoonotic disease caused by the bacterium Brucella abortus that leads to economic losses due to animal discard and commercial restrictions. Since positive animals for brucellosis are culled, little is known about the pathogenesis of this disease. Therefore, the aims of this study were to evaluate possible changes in the activity of deaminase adenosine (ADA) and the oxidative stress in cows seropositives for brucellosis (Experiment I), and to evaluate the seroprevalence of B. abortus in dairy cows from the Western state of Santa Catarina, Southern Brazil (Experiment II). The Experiment I evaluated 20 pregnant cows: ten seropositives for B. abortus and ten seronegatives that were used as controls. The ADA activity and markers of oxidative stress (TBARS, catalase (CAT) and superoxide dismutase (SOD)) were evaluated in these animals. A reduction in the activity of ADA and catalase enzymes in seropositive animals was observed (p < 0.001). Conversely, there was an increase in TBARS levels and superoxide dismutase activity in cows infected by B. abortus (p < 0.001). The presence of oxidative stress and a reduction of ADA might be related to the modulation of the inflammatory response. The experiment II was performed due to a high number of herds with restrictions imposed by cases of brucellosis in the state of Santa Catarina in the last two years, and thus, the seroprevalence for B. abortus was evaluated in 1242 serum samples of cows of 69 herds. The serodiagnosis was performed using two tests: buffered acidified antigen and 2-mercaptoethanol. However, none of the serum samples were positive for B. abortus. Although we did not find seropositive animals for brucellosis in our study, the disease still requires continued surveillance, due to its economic impact, and to the oxidative stress caused by it, which may have contributed to cases of abortion in three seropositive cows (Experiment I) in the final third of the gestation

[Development and evaluation of a serological protocol of fluorescence polarization for the preliminary study of Brucella spp antibodies in humans].

PubMed

Sánchez-Villalobos, Alfredo; Urdaneta-Fernández, Margelys; Rubio-Fuenmayor, Elí; Molero-Saras, Gladys; Luzardo-Charris, Carlos; Corona-Mengual, Carlos

2011-03-01

In order to show the development and scope of a serological analysis method based on fluorescence polarization assay (FPA) from a drop of blood obtained by the capillary technique, a Brucella antibody assay was performed on a group of 321 high-risk workers. The results were compared with data from the analysis of blood serum by FPA and a competitive enzyme immunoassay (ELISA-c). The number of concordance was 318 (99.06%), and discordant 3 (0.93%), which were negative in serum by fluorescence polarization (FPAs) and ELISA-c, but positive with capillary FPA (FPAc). The comparative results FPAc were: sensitivity 100%; specificity: 99.05%; positive predictive value 66.67%; negative predictive value 100.0%; false positive rate: 0.95%; false negative rate: 0%; accuracy: 98.0%; odds ratio: 203.00. The youden J for both FPA methods was 0.667. The identification was considered reliable and the correlation of both procedures, FPA and ELISA-c, was no statistically different (P > 0.05%), which allows to highly recommend the study implementation of human brucellosis with capillary blood as a preliminary method.

B cell-deficient mice display markedly enhanced resistance to the intracellular bacterium Brucella abortus.

PubMed

Goenka, Radhika; Parent, Michelle A; Elzer, Philip H; Baldwin, Cynthia L

2011-04-15

Brucella species are facultative intracellular bacteria that cause lifelong infections in humans and livestock. Here we evaluated the contribution of B cells in control of murine brucellosis in the more susceptible BALB/c and the more resistant C57BL/6 mice by infecting B cell-deficient mice. Strikingly, in the absence of B cells in both C57BL/6 and BALB/c mice, 99% and 99.5% of the infection found in wild type mice was cleared, respectively. This augmented clearance was not reversed in either strain by passive transfer of immune serum. In C57BL/6 mice, the clearance of infection coincided with an increase in interferon γ (IFN-γ)-producing CD4 and CD8 T cells and a reduction in interleukin 10 (IL-10)-producing cells. In BALB/c mice, this clearance was IFN-γ-dependent, as B cell/IFN-γ dual knockout mice were unable to clear the infection, and was inversely related to the levels of transforming growth factor β (TGF-β). Furthermore, B cells were found to produce TGF-β and IL-10 during early stages of infection in BALB/c wild-type and C57BL/6 wild-type mice, respectively. Thus, we demonstrate that the establishment of the high plateau phase of infection is dependent on non-antibody-mediated B cell effector mechanisms, including B regulatory functions, during murine brucellosis. © The Author 2011. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved.

In vitro antibacterial effects of five volatile oil extracts against intramacrophage Brucella abortus 544.

PubMed

Al-Mariri, Ayman; Saour, George; Hamou, Razan

2012-06-01

Brucellaabortus is a gram-negative facultative intracellular bacterium that can cause a highly contagious disease in sheep, goats, cattle and one-humped camels. It is responsible for one of the most important zoonosis in human. The aim of this study was to evaluate the role of Mentha piperita, Origanum majorana, Citrus lemon, Cinnamomum verum and Myristica fragrans essential volatile oil extracts on human macrophages infected by B. abortus 544. Essential volatile oil extracts from M. piperita, O. majorana, C. lemon, C. verum and M. fragrans were extracted. Human macrophages were cultured at a density of 2×10(5) cells per well in sterile 96-well microtiter plates, and infected with B. abortus 544 at a ratio of 1:100 bacteria/cell. Then essential volatile oil extracts were added at a concentration of 1%. At specified times; cells were washed, lysed with 0.1% Triton, and plated on 2YT agar to determine the number of intracellular bacteria. Cinnamomum verum volatile oil at a concentration of 1% had the highest antibacterial activity against B. abortus 544 inside human macrophages. Its inhibitory effect observed from 24 h and continued till 144 h after the infection. Moreover, C. verum (0.1%) in combination with 1% concentration of M. piperita, O. majorana, C. lemon or M. fragrans volatile oil extracts produced a synergistic inhibitory effect against B. abortus 544. The results indicate that, among the five selected oil extracts, C. verum volatile oil applied either separately or in combination with other oil extracts had the most effective antimicrobial activity against Brucella.

Global analysis of the Brucella melitensis proteome: Identification of proteins expressed in laboratory-grown culture.

PubMed

Wagner, Mary Ann; Eschenbrenner, Michel; Horn, Troy A; Kraycer, Jo Ann; Mujer, Cesar V; Hagius, Sue; Elzer, Philip; DelVecchio, Vito G

2002-08-01

Brucella melitensis is a facultative intracellular bacterial pathogen that causes brucellosis, a zoonotic disease primarily infecting sheep and goats, characterized by undulant fever, arthritic pain and other neurological disorders in humans. A comprehensive proteomic study of strain 16M was conducted to identify and characterize the proteins expressed in laboratory-grown culture. Using overlapping narrow range immobilized pH gradient strips for two-dimensional gel electrophoresis, 883 protein spots were detected between pH 3.5 and 11. The average isoelectric point and molecular weight values of the detected spots were 5.22 and 46.5 kDa, respectively. Of the 883 observed protein spots, 440 have been identified by matrix-assisted laser desorption/ionization-mass spectrometry. These proteins represent 187 discrete open reading frames (ORFs) or 6% of the predicted 3197 ORFs contained in the genome. The corresponding ORFs of the identified proteins are distributed evenly between each of the two circular B. melitensis chromosomes, indicating that both replicons are functionally active. The presented proteome map lists those protein spots identified to date in this study. This map may serve as a baseline reference for future proteomic studies aimed at the definition of biochemical pathways associated with stress responses, host specificity, pathogenicity and virulence. It will also assist in characterization of global proteomic effects in gene-knockout mutants. Ultimately, it may aid in our overall understanding of the cell biology of B. melitensis, an important bacterial pathogen.

RegA Plays a Key Role in Oxygen-Dependent Establishment of Persistence and in Isocitrate Lyase Activity, a Critical Determinant of In vivo Brucella suis Pathogenicity

PubMed Central

Abdou, Elias; Jiménez de Bagüés, María P.; Martínez-Abadía, Ignacio; Ouahrani-Bettache, Safia; Pantesco, Véronique; Occhialini, Alessandra; Al Dahouk, Sascha; Köhler, Stephan; Jubier-Maurin, Véronique

2017-01-01

For aerobic human pathogens, adaptation to hypoxia is a critical factor for the establishment of persistent infections, as oxygen availability is low inside the host. The two-component system RegB/A of Brucella suis plays a central role in the control of respiratory systems adapted to oxygen deficiency, and in persistence in vivo. Using an original “in vitro model of persistence” consisting in gradual oxygen depletion, we compared transcriptomes and proteomes of wild-type and ΔregA strains to identify the RegA-regulon potentially involved in the set-up of persistence. Consecutive to oxygen consumption resulting in growth arrest, 12% of the genes in B. suis were potentially controlled directly or indirectly by RegA, among which numerous transcriptional regulators were up-regulated. In contrast, genes or proteins involved in envelope biogenesis and in cellular division were repressed, suggesting a possible role for RegA in the set-up of a non-proliferative persistence state. Importantly, the greatest number of the RegA-repressed genes and proteins, including aceA encoding the functional IsoCitrate Lyase (ICL), were involved in energy production. A potential consequence of this RegA impact may be the slowing-down of the central metabolism as B. suis progressively enters into persistence. Moreover, ICL is an essential determinant of pathogenesis and long-term interactions with the host, as demonstrated by the strict dependence of B. suis on ICL activity for multiplication and persistence during in vivo infection. RegA regulates gene or protein expression of all functional groups, which is why RegA is a key regulator of B. suis in adaptation to oxygen depletion. This function may contribute to the constraint of bacterial growth, typical of chronic infection. Oxygen-dependent activation of two-component systems that control persistence regulons, shared by several aerobic human pathogens, has not been studied in Brucella sp. before. This work therefore contributes

Subcellular fractions of Brucella ovis distinctively induce the production of interleukin-2, interleukin-4, and interferon-γ in mice

PubMed Central

2005-01-01

Abstract The aim of this study was to evaluate the effect of 3 Brucella ovis subcellular protein fractions: Outer membrane (OMP), inner membrane (IMP), and cytoplasm (CP), on cellular immune response by in vitro production of interleukin (IL)-2, IL-4, and interferon (IFN)-γ. Each fraction was inoculated 3 times into Balb/c mice, primary cultures of mice spleen cells were done, and these were then stimulated with the fractions. Culture supernatants were collected at 24, 48, 72, 96, and 120 h postinoculation. Cytokine concentration was measured by Duoset-enzyme-linked immunosorbent assay (ELISA). The OMP fraction induced highest cellular immune response of 1000 pg/mL of IL-2 at 24 h, which decreased to < 100 pg/mL by 96 h. The IL-2 response for the IMP fraction was low at 24 h, but exceeded that of the OMP fraction at 72, 96, and 120 h. The CP showed a poor IL response. Regarding the IFN-γ production, OMP and IMP induced a high response at 120 h. These results open the possibility for the use of B. ovis outer and inner membrane proteins as a subcellular vaccine. PMID:15745223

Clinical management of Brucella suis infection in dogs and implications for public health.

PubMed

James, D R; Golovsky, G; Thornton, J M; Goodchild, L; Havlicek, M; Martin, P; Krockenberger, M B; Marriott, Dje; Ahuja, V; Malik, R; Mor, S M

2017-01-01

Brucellosis caused by Brucella suis is a notifiable disease that has recently emerged in dogs in New South Wales (NSW). Given the potential for zoonotic transmission, euthanasia of affected dogs is recommended, but this action is not mandatory. We report the clinical management of three dogs that underwent treatment at their owners' request. A 14-month-old spayed female crossbreed originally obtained from an urban animal shelter underwent extensive investigations in 2011-12 for lameness and back pain, culminating in decompressive laminectomy. Diagnosis of multifocal discospondylitis and spinal empyema was made, with B. suis cultured from surgical biopsy specimens. The dog responded to long-term treatment using rifampicin and doxycycline. A second case of B. suis infection was diagnosed in January 2016 in a 3-year-old crossbreed pig-hunting dog with unilateral testicular enlargement. Following serological diagnosis the dog was given preliminary therapy using rifampicin and doxycycline, the affected testis was resected and the patient given a further month of combination therapy. In March 2016 a 7-year-old crossbreed pig-hunting dog with brucellosis was handled similarly, although both testes were removed. Brucellosis should be considered in the differential diagnosis of back pain, discospondylitis, lameness, abortion, prostatic abscessation and testicular/epididymal enlargement in dogs, especially if there is exposure to feral pigs or consumption of uncooked feral pig meat. Euthanasia is the only guarantee of reducing the public health risk to zero. However, where treatment is desired by the owner, combination therapy using rifampicin and doxycycline appears to be effective, when combined with surgical resection of infected tissues. Further monitoring of dogs during and after treatment is required to document cure. © 2017 Australian Veterinary Association.

Doxycycline-rifampin versus doxycycline-streptomycin in treatment of human brucellosis due to Brucella melitensis. The GECMEI Group. Grupo de Estudio de Castilla-la Mancha de Enfermedades Infecciosas.

PubMed

Solera, J; Rodríguez-Zapata, M; Geijo, P; Largo, J; Paulino, J; Sáez, L; Martínez-Alfaro, E; Sánchez, L; Sepulveda, M A; Ruiz-Ribó, M D

1995-09-01

Brucellosis is a common zoonosis in many parts of the world; the best regimen for the treatment of brucellosis has not been clearly determined. We have carried out a multicenter, open, controlled trial in five general hospitals in Spain to compare the efficacy and safety of doxycycline and rifampin (DR) versus doxycycline and streptomycin (DS) for the treatment of human brucellosis. The study included 194 ambulatory or hospitalized patients with acute brucellosis, without endocarditis or neurobrucellosis. The diagnostic criterion was isolation of Brucella species from blood or other tissues (n = 120) or a standard tube agglutination titer of 1/160 or more for anti-Brucella antibodies with compatible clinical findings (n = 74). Patients were randomly assigned to receive either 100 mg of doxycycline twice daily plus rifampin, 900 mg/day, in a single morning dose for 45 days (DR group) or the same dose of doxycycline for 45 days plus streptomycin, 1 g/day, intramuscularly for 14 days (DS group). A lack of therapeutic efficacy developed in 8 of the 100 patients in the DR group (8%) and in 2 of the 94 patients in the DS group (2%)(P = 0.10). Relapses occurred in 16 of the 100 patients in the DR group (16%) but in only 5 of the 94 patients in the DS group (5.3%) (P = 0.02). When relapse was considered in combination with initial lack of efficacy, 26 patients in the DR group (24%) and 7 patients in the DS group (7.45%) failed to respond to therapy (P = 0.0016). In general, therapy was well tolerated and only four patients (4%) in the DR group and two (2%) in the DS group had episodes of adverse effects necessitating discontinuation of treatment (P> 0.2). We conclude that a doxycycline-and-rifampin regimen is less effective than the doxycycline-and-streptomycin regimen in patients with acute brucellosis.

Pseudorabies Virus and Brucella abortus from an Expanding Wild Pig ( Sus scrofa ) Population in Southern Oklahoma, USA.

PubMed

Gaskamp, Joshua A; Gee, Kenneth L; Campbell, Tyler A; Silvy, Nova J; Webb, Stephen L

2016-04-28

Wild pigs ( Sus scrofa ) are causing increasing ecologic and economic damage at a global scale. Because wild pigs can carry ≥65 diseases that affect livestock, their widespread expansion threatens native wildlife and livestock. We screened wild pigs from south-central Oklahoma, US for antibodies against Brucella abortus , pseudorabies virus (PRV), and porcine reproductive and respiratory syndrome virus (PRRS). These pathogens were chosen because they are part of eradication programs in the US and could have large economic impacts on domestic livestock if transmitted from wild animals. We tested 282 serum samples during spring 2010 (n=149) and 2011 (n=133) and found an overall exposure rate to PRV of 24.1% (n=68); PRV was detected at two of three study sites. Two wild pigs had detectable antibody to B. abortus , and one had detectable antibody to PRRS. On average, 27% of wild pigs within a sounder were positive for PRV antibody, with 44% of the sounders (16/36) having at least one positive individual. These data highlight that wild pigs could carry pathogens that affect domestic livestock. Because the US is free of these pathogens in commercial livestock operations, continued surveillance and vaccination of domestic livestock are needed. Commercial livestock producers at the wildlife-livestock interface may benefit from spatial prioritization of risk zones to facilitate strategic control efforts.

Early transcriptional responses of internalization defective Brucella abortus mutants in professional phagocytes, RAW 264.7.

PubMed

Cha, Seung Bin; Lee, Won Jung; Shin, Min Kyoung; Jung, Myung Hwan; Shin, Seung Won; Yoo, An Na; Kim, Jong Wan; Yoo, Han Sang

2013-06-27

Brucella abortus is an intracellular zoonotic pathogen which causes undulant fever, endocarditis, arthritis and osteomyelitis in human and abortion and infertility in cattle. This bacterium is able to invade and replicate in host macrophage instead of getting removed by this defense mechanism. Therefore, understanding the interaction between virulence of the bacteria and the host cell is important to control brucellosis. Previously, we generated internalization defective mutants and analyzed the envelope proteins. The present study was undertaken to evaluate the changes in early transcriptional responses between wild type and internalization defective mutants infected mouse macrophage, RAW 264.7. Both of the wild type and mutant infected macrophages showed increased expression levels in proinflammatory cytokines, chemokines, apoptosis and G-protein coupled receptors (Gpr84, Gpr109a and Adora2b) while the genes related with small GTPase which mediate intracellular trafficking was decreased. Moreover, cytohesin 1 interacting protein (Cytip) and genes related to ubiquitination (Arrdc3 and Fbxo21) were down-regulated, suggesting the survival strategy of this bacterium. However, we could not detect any significant changes in the mutant infected groups compared to the wild type infected group. In summary, it was very difficult to clarify the alterations in host cellular transcription in response to infection with internalization defective mutants. However, we found several novel gene changes related to the GPCR system, ubiquitin-proteosome system, and growth arrest and DNA damages in response to B. abortus infection. These findings may contribute to a better understanding of the molecular mechanisms underlying host-pathogen interactions and need to be studied further.

T cell regulation of the thymus-independent antibody response to trinitrophenylated-Brucella abortus (TNP-BA)

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

Tanay, A.; Strober, S.

The authors have previously observed a reduction of the T cell-dependent primary antibody response to dinitrophenylated keyhole limpet hemocyanin, and an enhancement of the T cell-independent response to trinitrophenylated Brucella abortus (TNP-BA) in BALB/c mice after treatment with total lymphoid irradiation (TLI). To elucidate the relative contribution of T and B cells to the enhanced T cell-independent antibody responses after TLI, a syngeneic primary adoptive transfer system was utilized whereby irradiated hosts were reconstituted with unfractionated spleen cells or a combination of purified T and B cells from TLI-treated and untreated control mice. Antibody responses of purified splenic B cellsmore » from TLI-treated BALB/c mice (TLI/B) to TNP-BA were enhanced 10-fold as compared with those of unfractionated (UF) spleen cells or B cells from normal (NL) BALB/c mice (NL/UF and NL/B, respectively). Splenic T cells from normal animals (NL/T) suppressed the anti-TNP-BA response of TLI/B by more than 100-fold. NL/T neither suppressed nor enhanced the response of NL/B. On the other hand, T cells from TLI-treated mice (TLI/T) enhanced by 100-fold the anti-TNP-BA response of NL/B, but neither suppressed nor enhanced the response of TLI/B. Thus, T cells can regulate the T cell-independent antibody response to TNP-BA. However, experimental manipulation of the T and B cell populations is needed to demonstrate the regulatory functions.« less

Whole-genome mapping reveals a large chromosomal inversion on Iberian Brucella suis biovar 2 strains.

PubMed

Ferreira, Ana Cristina; Dias, Ricardo; de Sá, Maria Inácia Corrêa; Tenreiro, Rogério

2016-08-30

Optical mapping is a technology able to quickly generate high resolution ordered whole-genome restriction maps of bacteria, being a proven approach to search for diversity among bacterial isolates. In this work, optical whole-genome maps were used to compare closely-related Brucella suis biovar 2 strains. This biovar is the unique isolated in domestic pigs and wild boars in Portugal and Spain and most of the strains share specific molecular characteristics establishing an Iberian clonal lineage that can be differentiated from another lineage mainly isolated in several Central European countries. We performed the BamHI whole-genome optical maps of five B. suis biovar 2 field strains, isolated from wild boars in Portugal and Spain (three from the Iberian lineage and two from the Central European one) as well as of the reference strain B. suis biovar 2 ATCC 23445 (Central European lineage, Denmark). Each strain showed a distinct, highly individual configuration of 228-231 BamHI fragments. Nevertheless, a low divergence was globally observed in chromosome II (1.6%) relatively to chromosome I (2.4%). Optical mapping also disclosed genomic events associated with B. suis strains in chromosome I, namely one indel (3.5kb) and one large inversion (944kb). By using targeted-PCR in a set of 176 B. suis strains, including all biovars and haplotypes, the indel was found to be specific of the reference strain ATCC 23445 and the large inversion was shown to be an exclusive genomic marker of the Iberian clonal lineage of biovar 2. Copyright © 2016 Elsevier B.V. All rights reserved.