Cultivation of Mycobacterium bovis BCG in bioreactors.

PubMed

Dietrich, Guido; Mollenkopf, Hans-Joachim; Weber, Heinz; Knapp, Bernhard; Diehl, Klaus-Dieter; Hess, Jürgen; Blackkolb, Friedrich; Bröker, Michael; Kaufmann, Stefan H E; Hundt, Erika

2002-07-03

The Mycobacterium bovis BCG vaccine for commercial use is classically produced as surface pellicles by culture on synthetic medium. Under these conditions, reproducibility of the cultures and quality assessment are hampered by slow growth of the bacilli, the formation of bacterial aggregates and a high proportion of dead bacilli after processing and final formulation of the vaccine. Here, we established dispersed cultures of M. bovis BCG in synthetic media in small-scale bioreactors. These cultures allow recording and adjusting of culture parameters and give rise to single bacilli with a high degree of live bacteria. In the murine model, bioreactor-grown M. bovis BCG exhibited slightly stronger replication and persistence than the vaccine produced under the classical conditions. The protective efficacy against challenge with M. tuberculosis was identical for both vaccine preparations.

Patterns and processes of Mycobacterium bovis evolution revealed by phylogenomic analyses

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis is an important animal pathogen worldwide that parasitizes wild and domesticated vertebrate livestock as well as humans. A comparison of the five M. bovis complete genomes from UK, South Korea, Brazil and USA revealed four novel large-scale structural variations of at least 2,000...

Genotyping did not evidence any contribution of Mycobacterium bovis to human tuberculosis in Brazil.

PubMed

Rocha, Adalgiza; Elias, Atina R; Sobral, Luciana F; Soares, Diego F; Santos, Alexandre C; Marsico, Ana-Grazia; Hacker, Mariana A; Caldas, Paulo C; Parente, Luiz C; Silva, Marcio R; Fonseca, Leila; Suffys, Philip; Boéchat, Neio

2011-01-01

The contribution of Mycobacterium bovis to the global burden of tuberculosis (TB) in man is likely to be underestimated due to its dysgonic growth characteristics and because of the absence of pyruvate in most used media is disadvantageous for its primary isolation. In Brazil Mycobacterium culture, identification and susceptibility tests are performed only in TB reference centers, usually for selected cases. Moreover, solid, egg-based, glycerol-containing (without pyruvate supplementation) Löwenstein-Jensen (L-J) or Ogawa media are routinely used, unfavouring M. bovis isolation. To determine the importance of M. bovis as a public health threat in Brazil we investigated 3046 suspected TB patients inoculating their clinical samples onto routine L-J and L-J pyruvate enriched media. A total of 1796 specimens were culture positive for Mycobacterium spp. and 702 TB cases were confirmed. Surprisingly we did not detect one single case of M. bovis in the resulting collection of 1674 isolates recovered from M. bovis favourable medium analyzed by conventional and molecular speciation methods. Also, bacillary DNA present on 454 sputum smears from 223 TB patients were OxyR genotyped and none was recognized as M. bovis. Our data indicate that M. bovis importance on the burden of human TB in Brazil is marginal. Copyright © 2010 Elsevier Ltd. All rights reserved.

Mycobacterium bovis infection in humans and cats in same household, Texas, USA, 2012

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis infection of cats is exceedingly rare in non-endemic regions for bovine tuberculosis. This case study describes the diagnosis and clinical management of pulmonary M. bovis infection in two indoor-housed cats and their association with at least one M. bovis-infected human in Texas...

Mycobacterium bovis infection at the interface between domestic and wild animals in Zambia

PubMed Central

2012-01-01

Background In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. Results A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units–Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. Conclusions These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host. PMID:23151267

The Effect of Mycobacterium avium Complex Infections on Routine Mycobacterium bovis Diagnostic Tests

PubMed Central

Barry, Claire; Corbett, David; Bakker, Douwe; Andersen, Peter; McNair, Jim; Strain, Sam

2011-01-01

Bovine tuberculosis (bTB) is diagnosed in naturally infected populations exposed to a wide variety of other pathogens. This study describes the cell-mediated immune responses of cattle exposed to Mycobacterium avium subspecies paratuberculosis (Map) and Mycobacterium avium subspecies avium with particular reference to routine antefmortem Mycobacterium bovis diagnostic tests. The IFN-γ released in response to stimulated blood was found to peak later in the Map-exposed group and was more sustained when compared to the Maa-exposed group. There was a very close correlation between the responses to the purified protein derivatives (PPD) used for stimulation (PPDa, PPDb, and PPDj) with PPDa and PPDj most closely correlated. On occasion, in the Map-infected cattle, PPDb-biased responses were seen compared to PPDa suggesting that some Map-infected cattle could be misclassified as M. bovis infected using this test with these reagents. This bias was not seen when PPDj was used. SICCT results were consistent with the respective infections and all calves would have been classed skin test negative. PMID:21772961

Structural definition of arabinomannans from Mycobacterium bovis BCG.

PubMed

Nigou, J; Gilleron, M; Brando, T; Vercellone, A; Puzo, G

1999-06-01

The structures of the hydrophilic parietal and cellular arabinomannans isolated from Mycobacterium bovis BCG cell wall [Nigou et al. (1997) J Biol Chem 272: 23094-103] were investigated. Their molecular mass as determined by MALDI-TOF mass spectrometry was around 16 kDa. Concerning cap structure, capillary electrophoresis analysis demonstrated that dimannoside (Manpalpha1-->2Manp) was the most abundant motif (65-75%). Using two-dimensional 1H-13C NMR spectroscopy, the mannan core was unambiguously demonstrated to be composed of -->6Manpalpha1--> backbone substituted at some O-2 by a single Manp unit. The branching degree was determined as 84%. Finally, arabinomannans were found to be devoid of the phosphatidyl-myo-inositol anchor and, by aminonaphthalene disulfonate tagging, the mannan core was shown to contain a reducing end. This constitutes the main difference between arabinomannans and lipoarabinomannans from Mycobacterium bovis BCG.

Loop-mediated isothermal amplification assay targeting the mpb70 gene for rapid differential detection of Mycobacterium bovis.

PubMed

Zhang, Hui; Wang, Zhen; Cao, Xudong; Wang, Zhengrong; Sheng, Jinliang; Wang, Yong; Zhang, Jing; Li, Zhiqiang; Gu, Xinli; Chen, Chuangfu

2016-11-01

Loop-mediated isothermal amplification (LAMP) is a highly sensitive, rapid, cost-effective nucleic acid amplification method. Tuberculosis (TB) is widely popular in the world and it is difficult to cure. The fundamental treatment is to clear the types of TB pathogens such as Mycobacterium bovis (M. bovis), Mycobacterium tuberculosis (M. tuberculosis). In order to detect and diagnose TB early, we constructed the differential diagnostic method of TB. In this study, we used LAMP for detection of M. bovis, based on amplification of the mpb70 gene which is a unique gene in M. bovis strain. The LAMP assay was able to detect only seven copies of the gene per reaction, whereas for the conventional PCR, it was 70 copies. The LAMP was evaluated for its specificity using six strains of five Mycobacterium species and 18 related non-Mycobacterium microorganism strains as controls. The target three Mycobacterium strains were all amplified, and no cross-reaction was found with 18 non-Mycobacterium microorganism strains. TB was detected by two methods, LAMP and conventional PCR (based on mpb70 gene); the positive rates of the two methods were 9.55 and 7.01 %, respectively. Our results indicate that the LAMP method should be a potential tool with high convenience, rapidity, sensitivity and specificity for the diagnosis of TB caused by M. bovis. Most importance is that the use of LAMP as diagnostic method in association with diagnostic tests based on mpb70 gene would allow the differentiation between M. bovis and other Mycobacterium in humans or animals. The LAMP method is actually in order to detect human TB, and it can be used for differential diagnosis in this paper.

Identification of proteins from Mycobacterium tuberculosis missing in attenuated Mycobacterium bovis BCG strains.

PubMed

Mattow, J; Jungblut, P R; Schaible, U E; Mollenkopf, H J; Lamer, S; Zimny-Arndt, U; Hagens, K; Müller, E C; Kaufmann, S H

2001-08-01

A proteome approach, combining high-resolution two-dimensional electrophoresis (2-DE) with mass spectrometry, was used to compare the cellular protein composition of two virulent strains of Mycobacterium tuberculosis with two attenuated strains of Mycobacterium bovis Bacillus Calmette-Guerin (BCG), in order to identify unique proteins of these strains. Emphasis was given to the identification of M. tuberculosis specific proteins, because we consider these proteins to represent putative virulence factors and interesting candidates for vaccination and diagnosis of tuberculosis. The genome of M. tuberculosis strain H37Rv comprises nearly 4000 predicted open reading frames. In contrast, the separation of proteins from whole mycobacterial cells by 2-DE resulted in silver-stained patterns comprising about 1800 distinct protein spots. Amongst these, 96 spots were exclusively detected either in the virulent (56 spots) or in the attenuated (40 spots) mycobacterial strains. Fifty-three of these spots were analyzed by mass spectrometry, of which 41 were identified, including 32 M. tuberculosis specific spots. Twelve M. tuberculosis specific spots were identified as proteins, encoded by genes previously reported to be deleted in M. bovis BCG. The remaining 20 spots unique for M. tuberculosis were identified as proteins encoded by genes that are not known to be missing in M. bovis BCG.

Sensitivity of Mycobacterium bovis to common beef processing interventions

USDA-ARS?s Scientific Manuscript database

Introduction. Cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis and a relevant zoonosis to humans, may be sent to slaughter before diagnosis of infection because of slow multiplication of the pathogen. Purpose. This study evaluates multiple processing interventi...

Evaluation of tissue fixation methods to inactivate Mycobacterium bovis under routine laboratory conditions

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis (M. bovis) is the etiological agent of tuberculosis in mammals including humans. The seriousness of disease and low infective dose require that the agent be handled under biosafety level 3 conditions. Many experimental protocols include histological examination of infected tissue...

MicroRNA-155 Is Required for Mycobacterium bovis BCG-Mediated Apoptosis of Macrophages

PubMed Central

Ghorpade, Devram Sampat; Leyland, Rebecca; Kurowska-Stolarska, Mariola; Patil, Shripad A.

2012-01-01

Pathogenic mycobacteria, including Mycobacterium tuberculosis and Mycobacterium bovis, cause significant morbidity and mortality worldwide. However, the vaccine strain Mycobacterium bovis BCG, unlike virulent strains, triggers extensive apoptosis of infected macrophages, a step necessary for the elicitation of robust protective immunity. We here demonstrate that M. bovis BCG triggers Toll-like receptor 2 (TLR2)-dependent microRNA-155 (miR-155) expression, which involves signaling cross talk among phosphatidylinositol 3-kinase (PI3K), protein kinase Cδ (PKCδ), and mitogen-activated protein kinases (MAPKs) and recruitment of NF-κB and c-ETS to miR-155 promoter. Genetic and signaling perturbations presented the evidence that miR-155 regulates PKA signaling by directly targeting a negative regulator of PKA, protein kinase inhibitor alpha (PKI-α). Enhanced activation of PKA signaling resulted in the generation of PKA C-α; phosphorylation of MSK1, cyclic AMP response element binding protein (CREB), and histone H3; and recruitment of phospho-CREB to the apoptotic gene promoters. The miR-155-triggered activation of caspase-3, BAK1, and cytochrome c translocation involved signaling integration of MAPKs and epigenetic or posttranslational modification of histones or CREB. Importantly, M. bovis BCG infection-induced apoptosis was severely compromised in macrophages derived from miR-155 knockout mice. Gain-of-function and loss-of-function studies validated the requirement of miR-155 for M. bovis BCG's ability to trigger apoptosis. Overall, M. bovis BCG-driven miR-155 dictates cell fate decisions of infected macrophages, strongly implicating a novel role for miR-155 in orchestrating cellular reprogramming during immune responses to mycobacterial infection. PMID:22473996

Human tuberculosis due to Mycobacterium bovis in the United States, 1995-2005.

PubMed

Hlavsa, Michele C; Moonan, Patrick K; Cowan, Lauren S; Navin, Thomas R; Kammerer, J Steve; Morlock, Glenn P; Crawford, Jack T; Lobue, Philip A

2008-07-15

Understanding the epidemiology of human Mycobacterium bovis tuberculosis (TB) in the United States is imperative; this disease can be foodborne or airborne, and current US control strategies are focused on TB due to Mycobacterium tuberculosis and airborne transmission. The National TB Genotyping Service's work has allowed systematic identification of M. tuberculosis-complex isolates and enabled the first US-wide study of M. bovis TB. Results of spacer oligonucleotide and mycobacterial interspersed repetitive units typing were linked to corresponding national surveillance data for TB cases reported for the period 2004-2005 and select cases for the period 1995-2003. We also used National TB Genotyping Service data to evaluate the traditional antituberculous drug resistance-based case definition of M. bovis TB. Isolates from 165 (1.4%) of 11,860 linked cases were identified as M. bovis. Patients who were not born in the United States, Hispanic patients, patients <15 years of age, patients reported to be HIV infected, and patients with extrapulmonary disease each had increased adjusted odds of having M. bovis versus M. tuberculosis TB. Most US-born, Hispanic patients with TB due to M. bovis (29 [90.6%] of 32) had extrapulmonary disease, and their overall median age was 9.5 years. The National TB Genotyping Service's data indicated that the pyrazinamide-based case definition's sensitivity was 82.5% (95% confidence interval; 75.3%-87.9%) and that data identified 14 errors in pyrazinamide-susceptibility testing or reporting. The prevalence of extrapulmonary disease in the young, US-born Hispanic population suggests recent transmission of M. bovis, possibly related to foodborne exposure. Because of its significantly different epidemiologic profile, compared with that of M. tuberculosis TB, we recommend routine surveillance of M. bovis TB. Routine surveillance and an improved understanding of M. bovis TB transmission dynamics would help direct the development of additional

Absence of Mycobacterium bovis infection in dogs and cats residing on infected cattle farms: Michigan, 2002

PubMed Central

WILKINS, M. J.; BARTLETT, P. C.; BERRY, D. E.; PERRY, R. L.; FITZGERALD, S. D.; BERNARDO, T. M.; THOEN, C. O.; KANEENE, J. B.

2008-01-01

SUMMARY A cross-sectional field study was performed to evaluate infection in dogs and cats living on farms with Mycobacterium bovis-infected cattle. The purpose was to determine pet infection status and assess their risk to farm families and/or tuberculosis-free livestock. Data and specimens were collected from 18 cats and five dogs from nine participating farms. ELISA testing for M. bovis and M. avium was conducted. Fifty-one biological samples were cultured; all were negative for M. bovis, although other Mycobacterium species were recovered. No radiographic, serological or skin test evidence of mycobacterial infection was found. These negative results may be due to the low level of M. bovis infection in the cattle and the limited duration of exposure of pets to infected cattle residing on the same farm. No evidence was found to indicate that pets residing on M. bovis-infected Michigan cattle farms pose a risk to humans or M. bovis-free livestock; however, precautionary advice for farm owners was provided. PMID:18325127

Bovine Tuberculosis (Mycobacterium bovis) in Wildlife in Spain

PubMed Central

Aranaz, Alicia; de Juan, Lucía; Montero, Natalia; Sánchez, Celia; Galka, Margarita; Delso, Consuelo; Álvarez, Julio; Romero, Beatriz; Bezos, Javier; Vela, Ana I.; Briones, Victor; Mateos, Ana; Domínguez, Lucas

2004-01-01

Mycobacterium bovis infection in wildlife and feral species is a potential source of infection for livestock and a threat to protected and endangered species. The aim of this study was to identify Spanish wild animal species infected with M. bovis through bacteriological culture and spacer oligonucleotide typing (spoligotyping) of isolates for epidemiological purposes. This study included samples from red deer (Cervus elaphus), fallow deer (Dama dama), wild boar (Sus scrofa), Iberian lynx (Lynx pardina), hare (Lepus europaeus), and cattle (Bos taurus). They were collected in several geographical areas that were selected for their unique ecological value and/or known relationships between wildlife and livestock. In the areas included in this survey, M. bovis strains with the same spoligotyping pattern were found infecting several wild species and livestock, which indicates an epidemiological link. A locally predominant spoligotype was found in these areas. Better understanding of the transmission and distribution of disease in these populations will permit more precise targeting of control measures. PMID:15184440

Tuberculosis patients co-infected with Mycobacterium bovis and Mycobacterium tuberculosis in an urban area of Brazil.

PubMed

Silva, Marcio Roberto; Rocha, Adalgiza da Silva; da Costa, Ronaldo Rodrigues; de Alencar, Andrea Padilha; de Oliveira, Vania Maria; Fonseca Júnior, Antônio Augusto; Sales, Mariana Lázaro; Issa, Marina de Azevedo; Filho, Paulo Martins Soares; Pereira, Omara Tereza Vianello; dos Santos, Eduardo Calazans; Mendes, Rejane Silva; Ferreira, Angela Maria de Jesus; Mota, Pedro Moacyr Pinto Coelho; Suffys, Philip Noel; Guimarães, Mark Drew Crosland

2013-05-01

In this cross-sectional study, mycobacteria specimens from 189 tuberculosis (TB) patients living in an urban area in Brazil were characterised from 2008-2010 using phenotypic and molecular speciation methods (pncA gene and oxyR pseudogene analysis). Of these samples, 174 isolates simultaneously grew on Löwenstein-Jensen (LJ) and Stonebrink (SB)-containing media and presented phenotypic and molecular profiles of Mycobacterium tuberculosis, whereas 12 had molecular profiles of M. tuberculosis based on the DNA analysis of formalin-fixed paraffin wax-embedded tissue samples (paraffin blocks). One patient produced two sputum isolates, the first of which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, and the second of which only grew on SB media and presented phenotypic profiles of Mycobacterium bovis. One patient provided a bronchial lavage isolate, which simultaneously grew on LJ and SB media and presented phenotypic and molecular profiles of M. tuberculosis, but had molecular profiles of M. bovis from paraffin block DNA analysis, and one sample had molecular profiles of M. tuberculosis and M. bovis identified from two distinct paraffin blocks. Moreover, we found a low prevalence (1.6%) of M. bovis among these isolates, which suggests that local health service procedures likely underestimate its real frequency and that it deserves more attention from public health officials.

Molecular findings and approaches spotlighting Mycobacterium bovis persistence in cattle.

PubMed

Alvarez, Angel H; Estrada-Chávez, Ciro; Flores-Valdez, Mario Alberto

2009-01-01

Mycobacterium tuberculosis (Mtb) and Mycobacterium bovis (M. bovis) are the etiological agents of human and bovine tuberculosis (TB, bTB) respectively, and share genetic identity over 99% at the whole genome level. Progress has been made towards explaining how mycobacteria and their infected hosts remain in balance without producing clinical symptoms of disease, a phenomenon referred to as latency or persistence, which can be mimicked by certain in vitro conditions. Latency/persistence has mainly been studied using Mtb, where the two-component signalling system, dosRS, has been assigned an instrumental role, and even constitutes the current basis for development of new diagnostic methods and treatment addressing this particular stage of TB. M. bovis conserves homolog genes that in Mtb play a role in human latent TB infection and that, by analogy, would allow it to enter a persistent state in infected cattle; nevertheless, little attention has been paid to this stage in bovine hosts. We suggest that many of the advances acquired through the study of Mtb can and should be taken into consideration by research groups and veterinary professionals dealing with bTB. The study of the infection in bovines, paying particular attention to defining the molecular and cellular markers of a M. bovis persistent infection in cattle, presents great opportunities for the development and trial of new diagnostic tests and vaccines, tools that will surely help in promoting eradication of bTB in high-burden settings.

Poly-L-glutamate/glutamine synthesis in the cell wall of Mycobacterium bovis is regulated in response to nitrogen availability

PubMed Central

2013-01-01

Background The cell wall of pathogenic mycobacteria is known to possess poly-L-glutamine (PLG) layer. PLG synthesis has been directly linked to glutamine synthetase (GS) enzyme. glnA1 gene encodes for GS enzyme in mycobacteria. PLG layer is absent in cell wall of avirulent Mycobacterium smegmatis, although M. smegmatis strain expressing GS enzyme of pathogenic mycobacteria can synthesize PLG layer in the cell wall. The role of GS enzyme has been extensively studied in Mycobacterium tuberculosis, however, little is known about GS enzyme in other mycobacterial species. Mycobacterium bovis, as an intracellular pathogen encounters nitrogen stress inside macrophages, thus it has developed nitrogen assimilatory pathways to survive in adverse conditions. We have investigated the expression and activity of M. bovis GS in response to nitrogen availability and effect on synthesis of PLG layer in the cell wall. M. smegmatis was used as a model to study the behaviour of glnA1 locus of M. bovis. Results We observed that GS expression and activity decreased significantly in high nitrogen grown conditions. In high nitrogen conditions, the amount of PLG in cell wall was drastically reduced (below detectable limits) as compared to low nitrogen condition in M. bovis and in M. smegmatis strain complemented with M. bovis glnA1. Additionally, biofilm formation by M. smegmatis strain complemented with M. bovis glnA1 was increased than the wild type M. smegmatis strain. Conclusions The physiological regulation of GS in M. bovis was found to be similar to that reported in other mycobacteria but this data revealed that PLG synthesis in the cell wall of pathogenic mycobacteria occurs only in nitrogen limiting conditions and on the contrary high nitrogen conditions inhibit PLG synthesis. This indicates that PLG synthesis may be a form of nitrogen assimilatory pathway during ammonium starvation in virulent mycobacteria. Also, we have found that M. smegmatis complemented with M. bovis glnA1

Mycobacterium bovis infection in domestic pigs in Great Britain.

PubMed

Bailey, Suzanne S; Crawshaw, Timothy R; Smith, Noel H; Palgrave, Christopher J

2013-11-01

Mycobacterium bovis, the causative agent of bovine tuberculosis (TB), infects a wide range of wild and domestic mammals. Despite a control programme spanning decades, M. bovis infection levels in cattle in Great Britain (GB) have continued to rise over recent years. As the incidence of infection in cattle and wildlife may be linked to that in swine, data relating to infection of pigs identified at slaughter were examined in this study. Between 2007 and 2011, almost all M. bovis-infected pigs originated from farms in the South-West and West-Midland regions of England. The data suggest that pigs raised outdoors or on holdings with poor biosecurity may be more vulnerable to infection with M. bovis. In the majority of cases, the same strains of M. bovis were found in pigs and cattle, despite that fact that direct contact between these species was rarely observed. Genotyping and geographical mapping data indicated that some strains found in pigs may correlate better with those present in badgers, rather than cattle. In consequence, it is proposed that pigs may represent a useful sentinel for M. bovis infection in wildlife in GB. Given the potential implications of this infection for the pig industry, and for the on-going effort to control bovine TB, the importance of understanding the epidemiology and pathogenesis of M. bovis infection, as well as monitoring its prevalence, in pigs should not be underestimated. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Oral vaccination of white-tailed deer (Odocoileus virginianus) with Mycobacterium bovis Bacillus Calmette-Guerin (BCG)

USDA-ARS?s Scientific Manuscript database

Wildlife reservoirs of Mycobacterium bovis represent serious obstacles to the eradication of tuberculosis from livestock, particularly cattle. In Michigan, USA tuberculous white-tailed deer transmit M. bovis to other deer and cattle. One approach in dealing with this wildlife reservoir is to vaccina...

Single nucleotide polymorphisms in the Mycobacterium bovis genome resolve phylogenetic relationships

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis isolates carry restricted allelic variation yet exhibit a range of disease phenotypes and host preferences. Conventional genotyping methods target small hyper-variable regions of their genome and provide anonymous biallelic information insufficient to develop phylogeny. To resolv...

Multinucleated giant cell cytokine expression in pulmonary granulomas of cattle experimentally infected with Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

Pathogenic mycobacteria of the Mycobacterium tuberculosis complex such as Mycobacterium bovis, induce a characteristic lesion known as a granulomas. Granulomas represent a specific host response to chronic antigenic stimuli, such as foreign bodies, certain bacterial components, or persistent pathoge...

Draft Genome Sequences of Two Mycobacterium bovis Strains Isolated from Beef Cattle in Paraguay

PubMed Central

Sanabria, Lidia; Lagrave, Lorena; Nishibe, Christiane; Ribas, Augusto C. A.; Zumárraga, Martín J.; Araújo, Flábio R.

2017-01-01

ABSTRACT This work reports the draft genome sequences of the Mycobacterium bovis strains M1009 and M1010, isolated from the lymph nodes of two infected cows on a beef farm in Paraguay. Comparative genomics between these strains and other regional strains may provide more insights regarding M. bovis epidemiology in South America. PMID:28705977

Human Tuberculosis Caused by Mycobacterium bovis in the United States, 2006-2013.

PubMed

Scott, Colleen; Cavanaugh, Joseph S; Pratt, Robert; Silk, Benjamin J; LoBue, Philip; Moonan, Patrick K

2016-09-01

Using genotyping techniques that have differentiated Mycobacterium bovis from Mycobacterium tuberculosis since 2005, we review the epidemiology of human tuberculosis caused by M. bovis in the United States and validate previous findings nationally. All tuberculosis cases with a genotyped M. tuberculosis complex isolate reported during 2006-2013 in the United States were eligible for analysis. We used binomial regression to identify characteristics independently associated with M. bovis disease using adjusted prevalence ratios (aPRs) and corresponding 95% confidence intervals (CIs). During 2006-2013, the annual percentages of tuberculosis cases attributable to M. bovis remained consistent nationally (range, 1.3%-1.6%) among all tuberculosis cases (N = 59 273). Compared with adults 25-44 years of age, infants aged 0-4 years (aPR, 1.9 [95% CI, 1.4-2.8]) and children aged 5-14 years (aPR, 4.0 [95% CI, 3.1-5.3]) had higher prevalences of M. bovis disease. Patients who were foreign-born (aPR, 1.4 [95% CI, 1.2-1.7]), Hispanic (aPR, 3.9 [95% CI, 3.0-5.0]), female (aPR, 1.4 [95% CI, 1.3-1.6]), and resided in US-Mexico border counties (aPR, 2.0 [95% CI, 1.7-2.4]) also had higher M. bovis prevalences. Exclusively extrapulmonary disease (aPR, 3.7 [95% CI, 3.3-4.2]) or disease that was both pulmonary and extrapulmonary (aPR, 2.4 [95% CI, 2.1-2.9]) were associated with a higher prevalence of M. bovis disease. Children, foreign-born persons, Hispanics, and females are disproportionately affected by M. bovis, which was independently associated with extrapulmonary disease. Targeted prevention efforts aimed at Hispanic mothers and caregivers are warranted. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Molecular characterization of Mycobacterium bovis strains isolated from cattle slaughtered at two abattoirs in Algeria

PubMed Central

Sahraoui, Naima; Müller, Borna; Guetarni, Djamel; Boulahbal, Fadéla; Yala, Djamel; Ouzrout, Rachid; Berg, Stefan; Smith, Noel H; Zinsstag, Jakob

2009-01-01

Background Bovine Tuberculosis is prevalent in Algeria despite governmental attempts to control the disease. The objective of this study was to conduct, for the first time, molecular characterization of a population sample of Mycobacterium bovis strains isolated from slaughter cattle in Algeria. Between August and November 2007, 7250 animals were consecutively screened at the abattoirs of Algiers and Blida. In 260 animals, gross visible granulomatous lesions were detected and put into culture. Bacterial isolates were subsequently analysed by molecular methods. Results Altogether, 101 bacterial strains from 100 animals were subjected to molecular characterization. M. bovis was isolated from 88 animals. Other bacteria isolated included one strain of M. caprae, four Rhodococcus equi strains, three Non-tuberculous Mycobacteria (NTM) and five strains of other bacterial species. The M. bovis strains isolated showed 22 different spoligotype patterns; four of them had not been previously reported. The majority of M. bovis strains (89%) showed spoligotype patterns that were previously observed in strains from European cattle. Variable Number of Tandem Repeat (VNTR) typing supported a link between M. bovis strains from Algeria and France. One spoligotype pattern has also been shown to be frequent in M. bovis strains from Mali although the VNTR pattern of the Algerian strains differed from the Malian strains. Conclusion M. bovis infections account for a high amount of granulomatous lesions detected in Algerian slaughter cattle during standard meat inspection at Algiers and Blida abattoir. Molecular typing results suggested a link between Algerian and European strains of M. bovis. PMID:19173726

Epidemiology of Mycobacterium bovis Disease in Humans, the Netherlands, 1993–2007

PubMed Central

Magis-Escurra, Cecile; van Ingen, Jakko; Boeree, Martin J.; van Soolingen, Dick

2011-01-01

In the Netherlands, 1.4% of tuberculosis (TB) cases are caused by Mycobacterium bovis. After we admitted 3 patients with M. bovis infections to our reference hospital, we conducted a retrospective analysis of all M. bovis disease in the Netherlands during 1993–2007. We analyzed data from 231 patients for clinical, demographic, treatment, and outcome characteristics and for risk factors. Most patients were native Dutch (n = 138; 59.7%) or Moroccan (n = 54; 23.4%). Disease was mainly extrapulmonary (n = 136; 58.9%). Although 95 patients had pulmonary disease, person-to-person transmission did not occur, as shown by structural DNA fingerprinting analysis. Lymph node TB was more likely to develop in women (p<0.0001), whereas pulmonary M. bovis disease developed more frequently in men (p<0.0001). Diagnosis was accurate but delayed and led to inadequate treatment in 26% of the cases. Proportion of deaths from M. bovis disease was higher than that for M. tuberculosis disease. PMID:21392437

Mycobacterium bovis infection of cattle and white-tailed deer: Translational research of relevance to human tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances with the s...

Viral Booster Vaccines Improve Mycobacterium bovis BCG-Induced Protection Against Bovine Tuberculosis

USDA-ARS?s Scientific Manuscript database

Previous work in small animal laboratory models of tuberculosis have shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacille Calmette-Guerin (BCG) to prime and Modified Vaccinia Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad8...

Humoral Immune Responses of White-tailed Deer (Odocoileus virginianus) to Mycobacterium bovis BCG Vaccination and Experimental Challenge with M. bovis

USDA-ARS?s Scientific Manuscript database

Monitoring serum antibody production kinetics to multiple mycobacterial antigens can be useful as a diagnostic tool for the detection of Mycobacterium bovis infection as well as for the characterization of disease progression and efficacy of intervention strategies in several species. Humoral immun...

Survival kinetics of Mycobacterium bovis during manufacture and ripening of raw milk Cheddar and Caerphilly cheese produced on a laboratory-scale.

PubMed

Forgrave, R; Donaghy, J A; Fisher, A; Rowe, M T

2016-11-01

Persistence of Mycobacterium bovis was investigated in UK raw milk cheeses. Replicating traditional cheese production methods under stringent CL3 containment conditions, Cheddar and Caerphilly cheeses were produced with Myco. bovis inoculated raw milk. High-inoculum investigations used three Myco. bovis genotypes; later low-inoculum investigations used only Myco. bovis AF2122/97. High-inoculum Cheddar (n = 9) and Caerphilly (n = 9) were matured for a minimum of 12 and 4 months respectively; maturation of low-inoculum Cheddar (n = 3) and Caerphilly (n = 3) was up to 11 weeks. Survival of Myco. bovis was monitored by enumeration at different points throughout cheese manufacture and ripening. D values were calculated as follows: 57 and 59 days in high-inoculum Cheddar and Caerphilly, respectively, and 41 and 24 days in low-inoculum Cheddar and Caerphilly respectively. Mycobacterium bovis is concentrated in cheese curd and a proportion lost with the whey. Reduction in viability during manufacturing is limited, while significant Myco. bovis inactivation occurs during maturation. Inactivation was improved, during Caerphilly ripening, when acid development was enhanced by increasing the proportion of starter culture. Mycobacterium bovis inactivation data obtained could be used to inform assessment of the risk posed to consumers by raw milk dairy products. © 2016 The Society for Applied Microbiology.

Whole genome sequencing of the monomorphic pathogen Mycobacterium bovis reveals local differentiation of cattle clinical isolates.

PubMed

Lasserre, Moira; Fresia, Pablo; Greif, Gonzalo; Iraola, Gregorio; Castro-Ramos, Miguel; Juambeltz, Arturo; Nuñez, Álvaro; Naya, Hugo; Robello, Carlos; Berná, Luisa

2018-01-02

Bovine tuberculosis (bTB) poses serious risks to animal welfare and economy, as well as to public health as a zoonosis. Its etiological agent, Mycobacterium bovis, belongs to the Mycobacterium tuberculosis complex (MTBC), a group of genetically monomorphic organisms featured by a remarkably high overall nucleotide identity (99.9%). Indeed, this characteristic is of major concern for correct typing and determination of strain-specific traits based on sequence diversity. Due to its historical economic dependence on cattle production, Uruguay is deeply affected by the prevailing incidence of Mycobacterium bovis. With the world's highest number of cattle per human, and its intensive cattle production, Uruguay represents a particularly suited setting to evaluate genomic variability among isolates, and the diversity traits associated to this pathogen. We compared 186 genomes from MTBC strains isolated worldwide, and found a highly structured population in M. bovis. The analysis of 23 new M. bovis genomes, belonging to strains isolated in Uruguay evidenced three groups present in the country. Despite presenting an expected highly conserved genomic structure and sequence, these strains segregate into a clustered manner within the worldwide phylogeny. Analysis of the non-pe/ppe differential areas against a reference genome defined four main sources of variability, namely: regions of difference (RD), variable genes, duplications and novel genes. RDs and variant analysis segregated the strains into clusters that are concordant with their spoligotype identities. Due to its high homoplasy rate, spoligotyping failed to reflect the true genomic diversity among worldwide representative strains, however, it remains a good indicator for closely related populations. This study introduces a comprehensive population structure analysis of worldwide M. bovis isolates. The incorporation and analysis of 23 novel Uruguayan M. bovis genomes, sheds light onto the genomic diversity of this

A proof of concept study to assess the potential of PCR testing to detect natural Mycobacterium bovis infection in South American camelids.

PubMed

Crawshaw, Timothy R; Chanter, Jeremy I; McGoldrick, Adrian; Line, Kirsty

2014-02-07

Cases of Mycobacterium bovis infection South American camelids have been increasing in Great Britain. Current antemortem immunological tests have some limitations. Cases at post mortem examination frequently show extensive pathology. The feasibility of detecting Mycobacterium bovis DNA in clinical samples was investigated. A sensitive extraction methodology was developed and used on nasal swabs and faeces taken post-mortem to assess the potential for a PCR test to detect Mycobacterium bovis in clinical samples. The gross pathology of the studied South American camelids was scored and a significantly greater proportion of South American camelids with more severe pathology were positive in both the nasal swab and faecal PCR tests. A combination of the nasal swab and faecal PCR tests detected 63.9% of all the South American camelids with pathology that were tested. The results suggest that antemortem diagnosis of Mycobacterium bovis in South American camelids may be possible using a PCR test on clinical samples, however more work is required to determine sensitivity and specificity, and the practicalities of applying the test in the field.

Mycobacterium bovis infections in slaughter pigs in Mubende district, Uganda: a public health concern.

PubMed

Muwonge, Adrian; Johansen, Tone B; Vigdis, Edvardsen; Godfroid, Jacques; Olea-Popelka, Francisco; Biffa, Demelash; Skjerve, Eystein; Djønne, Berit

2012-09-21

Bovine tuberculosis (TB) caused by Mycobacterium bovis is primarily a disease of ruminants, particularly cattle (Bos primigenius) and buffalo (Syncerus caffer), and is endemic in most developing countries. To date, studies done in Uganda have documented the prevalence of M. bovis in cattle, humans and wild life, in addition to non-tuberculous mycobacteria in pigs. Pigs are increasingly becoming an important component of the livestock sector and share the human ecosystem in rural Uganda. It is therefore of public health interest that they are not a source of human infections. As a follow up to previously published findings on mycobacteria in pigs, this study was aimed at investigating the occurrence and molecular characteristics of M. bovis detected in slaughter pigs in Mubende district, Uganda. One hundred fifty mesenteric lymph nodes with lesions suggestive of mycobacterial infections were collected from approximately one thousand slaughtered pigs in Mubende district over a period of five months. The isolation and identification of M. bovis was done using conventional mycobacteriological methods. Mycobacteria belonging to the Mycobacterium tuberculosis complex (MTC) were identified to species level using deletion analysis. Molecular typing was done using Spoligotyping and MIRU-VNTR analysis. Molecular data were analysed and interpreted using MIRU-VNTR plus, SpolDB4.0 and the Mycobacterium bovis spoligo database. Of the examined animals, one boar and two sows from Madudu Sub County were infected with M. bovis which presented as lesions of a deep yellow colour and a grit-like texture in the mesenteric lymph nodes. This represents 2% (3/150) of the lymph nodes where lesions suggestive of mycobacterial infections were detected. Molecular analysis revealed that the isolates from the infected pigs showed identical MIRU-VNTR profile and spoligotype (SB1469). This is the first study documenting the occurrence of M. bovis in slaughter pigs in Uganda, revealing that one in

Genetic profiling of Mycobacterium bovis strains from slaughtered cattle in Eritrea

PubMed Central

Hlokwe, Tiny; Rutten, Victor P. M. G.; Allepuz, Alberto; Cadmus, Simeon; Muwonge, Adrian; Robbe-Austerman, Suelee; Michel, Anita L.

2018-01-01

Mycobacterium bovis (M.bovis) is the main causative agent for bovine tuberculosis (BTB) and can also be the cause of zoonotic tuberculosis in humans. In view of its zoonotic nature, slaughterhouse surveillance, potentially resulting in total or partial condemnation of the carcasses and organs, is conducted routinely. Spoligotyping, VNTR profiling, and whole genome sequencing (WGS) of M. bovis isolated from tissues with tuberculosis-like lesions collected from 14 cattle at Eritrea’s largest slaughterhouse in the capital Asmara, were conducted.The 14 M. bovis isolates were classified into three different spoligotype patterns (SB0120, SB0134 and SB0948) and six VNTR profiles. WGS results matched those of the conventional genotyping methods and further discriminated the six VNTR profiles into 14 strains. Furthermore, phylogenetic analysis of the M. bovis isolates suggests two independent introductions of BTB into Eritrea possibly evolving from a common ancestral strain in Europe.This molecular study revealed the most important strains of M. bovis in Eritrea and their (dis)similarities with the strains generally present in East Africa and Europe, as well as potential routes of introduction of M. bovis. Though the sample size is small, the current study provides important information as well as platform for future in-depth molecular studies on isolates from both the dairy and the traditional livestock sectors in Eritrea and the region. This study provides information onthe origin of some of the M. bovis strains in Eritrea, its genetic diversity, evolution and patterns of spread between dairy herds. Such information is essential in the development and implementation of future BTB control strategy for Eritrea. PMID:29664901

Current knowledge and pending challenges in zoonosis caused by Mycobacterium bovis: a review.

PubMed

Pérez-Lago, Laura; Navarro, Yurena; García-de-Viedma, Darío

2014-10-01

Mycobacterium bovis is both the causative agent of bovine tuberculosis (TB) and a zoonotic pathogen. In humans, considerably fewer cases of TB are caused by M. bovis than M. tuberculosis; nevertheless, diagnostic limitations mean that currently available data on prevalence grossly underestimate the true dimension of the problem. The routes of transmission from animals to humans are well known and include direct exposure to infected animals or consumption of contaminated animal products. Application of fingerprinting tools facilitates analysis of the molecular epidemiology of M. bovis in animal-to-human and human-to-human transmission. Apart from cattle and M. bovis, other animal species and members within the M. tuberculosis complex can contribute to the zoonosis. Improvements in diagnostic techniques, application of more advanced discriminatory genotyping tools, and collaboration between veterinary and human health care researchers are key to our understanding of this zoonosis. Copyright © 2013 Elsevier Ltd. All rights reserved.

Mycobacterium bovis infection at the interface between domestic and wild animals in Zambia.

PubMed

Hang'ombe, Mudenda B; Munyeme, Musso; Nakajima, Chie; Fukushima, Yukari; Suzuki, Haruka; Matandiko, Wigganson; Ishii, Akihiro; Mweene, Aaron S; Suzuki, Yasuhiko

2012-11-14

In Zambia, the presence of bovine tuberculosis in both wild and domestic animals has long been acknowledged and mutual transmission between them has been predicted without any direct evidence. Elucidation of the circulating Mycobacterium bovis strains at wild and domestic animals interphase area in Zambia, where bovine tuberculosis was diagnosed in wildlife seemed to be important. A PCR identified 15 and 37 M. bovis isolates from lechwe and cattle, respectively. Spoligotype analysis revealed that M. bovis strains from lechwe and cattle in Kafue basin clustered into a major node SB0120, where isolates outside the Kafue basin clustered into different nodes of SB0131 and SB0948. The comparatively higher variety of strains in cattle compared to lechwe elucidated by Mycobacterial Interspersed Repetitive Units-Variable Number Tandem Repeats analyses are consistent with cattle being the probable source of M. bovis in wild and domestic animals interphase area in Zambia. These results provide strong evidence of M. bovis strains transfer between cattle and lechwe, with the latter having developed into a sylvatic reservoir host.

β-Glucans inhibit intracellular growth of Mycobacterium bovis BCG but not virulent Mycobacterium tuberculosis in human macrophages

PubMed Central

Morris, Jessica D.; Rajaram, Murugesan V.S.; Schlesinger, Larry S.

2014-01-01

The yeast polysaccharide, β-glucan, has been shown to promote both anti-microbial and anti-tumor activities through its interaction with macrophages. Here we analyzed the effects of an insoluble whole glucan particle (WGP), a 1,3/1,6-β-glucan from Saccharomyces cerevisiae, and a soluble poly-1-6-β-d-glucopyranosyl-1-3-β-d-glucopyranose (PGG), a hydrolytic product of WGP, on the anti-microbial response of human macrophages against mycobacterial infection. Treatment of macrophages with WGP and PGG significantly decreased cell association and intracellular growth of Mycobacterium bovis BCG, but not Mycobacterium tuberculosis (M.tb) when compared to untreated controls. We characterized the influence of β-glucans on the generation of macrophage oxidative products and pro-inflammatory cytokines, two important anti-microbial defense mechanisms. WGP but not PGG treatment enhanced the oxidative response of macrophages as determined by the 2′,7′-dichlorofluorescin (DCF) assay. WGP treatment also induced macrophages to produce pro-inflammatory cytokines. The β-glucan receptor, Dectin-1, was found to be involved in the WGP-induced macrophage oxidative burst and intracellular growth inhibition of M. bovis BCG. This report indicates that although some forms of β-glucan are able to stimulate the respiratory burst and cytokine production in human macrophages, and exhibit antimicrobial properties against M. bovis BCG, the β-glucans tested here did not inhibit growth of M.tb within human macrophages. PMID:21762773

Tuberculous pleuritis secondary to Mycobacterium bovis in a veterinarian.

PubMed

Corcoran, John P; Hallifax, Robert J; Bettinson, Henry V; Psallidas, Ioannis; Rahman, Najib M

2016-07-01

Mycobacterium bovis is a rare cause of tuberculosis in humans, but should be considered in individuals at risk secondary to medical comorbidities (notably immunocompromise) or occupational exposure. Most cases are secondary to reactivation of latent infection in elderly individuals although cases of primary infection still occur, usually involving animal-to-human transmission. Pleural fluid culture in the context of suspected tuberculous pleuritis is frequently negative and pleural biopsy significantly increases the likelihood of confirming the diagnosis histologically and microbiologically. Although thoracoscopic biopsies are the reference standard, closed pleural biopsies are an appropriate and more accessible alternative in the majority of cases - these should be done under direct ultrasound guidance to maximise diagnostic yield. Treatment for M. bovis infection is with prolonged combination anti-tuberculous therapy, using an alternative to pyrazinamide as the organism is inherently resistant to this drug. © 2014 John Wiley & Sons Ltd.

Analysis of cytokine mRNA expression using a novel chromogenic in situ hybridization method in pulmonary granulomas of cattle experimentally infected by aerosolized Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis is the cause of tuberculosis in most animal species, including cattle and is a serious zoonotic pathogen. In humans, M. bovis infection can result in disease clinically indistinguishable from that caused by Mycobacterium tuberculosis, the cause of most tuberculosis in humans. Reg...

Impact of temperature and soil type on Mycobacterium bovis survival in the environment

PubMed Central

Barbier, Elodie; Rochelet, Murielle; Gal, Laurent; Boschiroli, Maria Laura

2017-01-01

Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides direct transmission via contaminated aerosols, indirect transmission of the M. bovis between wildlife and livestock might occur by inhalation or ingestion of environmental substrates contaminated through infected animal shedding. We monitored the survival of M. bovis in two soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay content). The population of M. bovis spiked in sterile soils was enumerated by a culture-based method after 14, 30, 60, 90, 120 and 150 days of incubation at 4°C and 22°C. A qPCR based assay targeting the IS1561’ locus was also performed to monitor M. bovis in both sterile and biotic spiked soils. The analysis of survival profiles using culture-based method showed that M. bovis survived longer at lower temperature (4°C versus 22°C) whereas the impact of soil characteristics on M. bovis persistence was not obvious. Furthermore, qPCR-based assay detected M. bovis for a longer period of time than the culture based method with higher gene copy numbers observed in sterile soils than in biotic ones. Impact of soil type on M. bovis persistence need to be deepened in order to fill the gap of knowledge concerning indirect transmission of the disease. PMID:28448585

Impact of temperature and soil type on Mycobacterium bovis survival in the environment.

PubMed

Barbier, Elodie; Rochelet, Murielle; Gal, Laurent; Boschiroli, Maria Laura; Hartmann, Alain

2017-01-01

Mycobacterium bovis, the causative agent of the bovine tuberculosis (bTB), mainly affects cattle, its natural reservoir, but also a wide range of domestic and wild mammals. Besides direct transmission via contaminated aerosols, indirect transmission of the M. bovis between wildlife and livestock might occur by inhalation or ingestion of environmental substrates contaminated through infected animal shedding. We monitored the survival of M. bovis in two soil samples chosen for their contrasted physical and-chemical properties (i.e. pH, clay content). The population of M. bovis spiked in sterile soils was enumerated by a culture-based method after 14, 30, 60, 90, 120 and 150 days of incubation at 4°C and 22°C. A qPCR based assay targeting the IS1561' locus was also performed to monitor M. bovis in both sterile and biotic spiked soils. The analysis of survival profiles using culture-based method showed that M. bovis survived longer at lower temperature (4°C versus 22°C) whereas the impact of soil characteristics on M. bovis persistence was not obvious. Furthermore, qPCR-based assay detected M. bovis for a longer period of time than the culture based method with higher gene copy numbers observed in sterile soils than in biotic ones. Impact of soil type on M. bovis persistence need to be deepened in order to fill the gap of knowledge concerning indirect transmission of the disease.

Molecular detection of Mycobacterium bovis in cattle herds of the state of Pernambuco, Brazil.

PubMed

Cezar, Renata Duarte da Silva; Lucena-Silva, Norma; Filho, Antônio Fernando Barbosa Batista; Borges, Jonas de Melo; de Oliveira, Pollyane Raysa Fernandes; Lúcio, Érica Chaves; Arruda-Lima, Maíra; Santana, Vania Lucia de Assis; Pinheiro Junior, José Wilton

2016-02-20

The present study aimed to direct detect Mycobacterium bovis in milk (n = 401) and blood (n = 401) samples collected from 401 dairy cows of 20 properties located in the state of Pernambuco, Brazil, by real-time quantitative PCR (qPCR) targeting the region of difference 4 (RD4). Risk factors possibly associated with bovine tuberculosis (BTB) were also evaluated. Of the 802 samples analyzed, one milk (0.25%) and eight blood (2%) samples were positive for M. bovis in the qPCR and their identities were confirmed by sequencing. Animals positive for M. bovis were found in six (30%) of the 20 properties visited. None of the risk factors evaluated were statistically associated with BTB. M. bovis DNA was detected in one milk sample what may pose a risk to public health because raw milk is commonly consumed in Brazil.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil.

PubMed

Carvalho, Ricardo César Tavares; Vasconcellos, Sidra Ezidio Gonçalves; Issa, Marina de Azevedo; Soares Filho, Paulo Martins; Mota, Pedro Moacyr Pinto Coelho; Araújo, Flábio Ribeiro de; Carvalho, Ana Carolina da Silva; Gomes, Harrison Magdinier; Suffys, Philip Noel; Figueiredo, Eduardo Eustáquio de Souza; Paschoalin, Vânia Margaret Flosi

2016-01-01

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil.

Molecular Typing of Mycobacterium bovis from Cattle Reared in Midwest Brazil

PubMed Central

Carvalho, Ricardo César Tavares; Vasconcellos, Sidra Ezidio Gonçalves; Issa, Marina de Azevedo; Soares Filho, Paulo Martins; Mota, Pedro Moacyr Pinto Coelho; de Araújo, Flábio Ribeiro; Carvalho, Ana Carolina da Silva; Gomes, Harrison Magdinier; Suffys, Philip Noel; Paschoalin, Vânia Margaret Flosi

2016-01-01

Mycobacterium bovis is the causative agent of bovine tuberculosis (BTB), the pathogen responsible for serious economic impact on the livestock sector. In order to obtain data on isolated M. bovis strains and assist in the control and eradication program for BTB, a cross sectional descriptive molecular epidemiology study in the Brazilian Midwest was conducted. Through spoligotyping and 24-loci MIRU-VNTR methods, 37 clinical isolates of M. bovis circulating in the region were analyzed, 10 isolated from the state of Mato Grosso, 12 from the state of Mato Grosso do Sul and 15 from the state of Goiás. The spoligotyping analysis identified 10 distinct M. bovis profiles (SB0121 n = 14, SB0295 n = 6, SB0140 n = 6, SB0881 n = 3, SB1144 n = 2, SB1145 n = 2, SB0134 n = 1, SB1050 n = 1, SB1055 n = 1, SB1136 n = 1) grouped in six clusters and four orphan patterns. The MIRU-VNTR 24-loci grouped the same isolates in six clusters and 22 unique orphan patterns, showing higher discriminatory power than spoligotyping. When associating the results of both techniques, the isolates were grouped in five clusters and 24 unique M. bovis profiles. Among the 24-loci MIRU-VNTR evaluated, two, ETR-A and QUB 11b loci, showed high discriminatory ability (h = ≥ 0.50), while MIRU 16, MIRU 27, ETR-B, ETR-C, Mtub21 and QUB 26 loci showed moderate ability (h = 0.33 or h = 0.49) and were the most effective in evaluating the genotypic similarities among the clinical M. bovis isolate samples. Herein, the 29 patterns found amongst the 37 isolates of M. bovis circulating in the Brazilian Midwest can be due to the animal movement between regions, municipalities and farms, thus causing the spread of various M. bovis strains in herds from Midwest Brazil. PMID:27631383

Isolation of Mycobacterium bovis from Free-Ranging Wildlife in South Korea.

PubMed

Jang, Yunho; Ryoo, Soyoon; Lee, Hyunkyoung; Kim, Narae; Lee, Hang; Park, So-Young; Song, Woong-Seog; Kim, Jong-Taek; Lee, Hee Soo; Myung Kim, Jae

2017-01-01

We demonstrate Mycobacterium bovis infection in wild boar ( Sus scrofa ) in South Korea. During 2012-15, we attempted to isolate M. bovis from 847 wild animals, mainly Korean water deer ( Hydropotes inermis argyropus), raccoon dogs ( Nyctereutes procyonoides ), and wild boar, from 11 regions in South Korea. We isolated M. bovis from three of 118 wild boar (2.5%) captured in Gyeonggi Province, where bovine tuberculosis (bTB) outbreaks have also occurred in livestock. Spoligotypes and mycobacterial interspersed repetitive units-variable number tandem repeats types of these M. bovis isolates (SB0140 and SB1040, 4-2-3-3-7-5-5-4-4-3-4-3 and 5-2-3-3-7-5-5-4-3-10-5-2; MIRU4, MIRU16, MIRU27, MIRU31, ETR-A, ETR-B, ETR-C, QUB11b, QUB26, QUB3336, VNTR2401, and VNTR3171) have also been identified from farmed livestock such as cattle ( Bos taurus coreanae), Formosan sika deer ( Cervus nippon taiouanus), and American elk ( Cervus canadensis ) in the country. In South Korea, bTB appears to be endemic in livestock, and there are numerous opportunities for contact between wild boar and livestock due to high population densities and broad activity ranges. Our results support the hypothesis that M. bovis is transmitted between domestic and wild animals.

Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

USDA-ARS?s Scientific Manuscript database

Bovine gamma delta T cells are amongst the first cells to accumulate at the site of Mycobacterium bovis infection; however, their role in the developing lesion remains unclear. We utilized transcriptomics analysis, in situ hybridization, and a macrophage/gamma delta T cell co-culture system to eluc...

The most common spoligotype of Mycobacterium bovis isolated in the world and the recommended loci for VNTR typing; A systematic review.

PubMed

Ghavidel, Mahdis; Mansury, Davood; Nourian, Kimiya; Ghazvini, Kiarash

2018-03-22

Mycobacterium bovis is a neglected zoonotic organism that epidemiological studies are of crucial importance in identifying its source, control it and prevent it from spreading. The aim of this study was to investigate the most common spoligotypes of Mycobacterium bovis circulating around the world and introduce the most and least strong determine powers of loci for VNTR. We have used different databases such as ISC, science direct, Embase (Elsevier), Web of Science, Scopus and Medline via PubMed. Searches were performed by key words including: Mycobacterium bovis, MIRU -VNTR, spoligotyping and discrimination power. Finally, thirty-one articles were selected after filtering out some titles, abstracts and full texts. Spoligotype SB0120 was the most common circulating type on several continents while SB0121 existed in Europe, Africa and America. SB0140 was also detected in Asia, Europe and America. QUB3232 and QUB11b were more appropriate loci among the loci with high discriminatory power. MIRU 10 and MIRU4 were among the loci with poor discriminatory power. Taking the published data into consideration, SB0120 and SB0121 are predominant spoligotypes of M. bovis circulating among animals around the world. Determining the most common spoligotype of M. bovis is the key to find source of infection, control and prevent the disease. Copyright © 2018 Elsevier Ltd. All rights reserved.

Comparative 'omics analyses differentiate Mycobacterium tuberculosis and Mycobacterium bovis and reveal distinct macrophage responses to infection with the human and bovine tubercle bacilli

PubMed Central

Malone, Kerri M.; Rue-Albrecht, Kévin; Magee, David A.; Conlon, Kevin; Schubert, Olga T.; Nalpas, Nicolas C.; Browne, John A.; Smyth, Alicia; Gormley, Eamonn; Aebersold, Ruedi; MacHugh, David E.; Gordon, Stephen V.

2018-01-01

Members of the Mycobacterium tuberculosis complex (MTBC) are the causative agents of tuberculosis in a range of mammals, including humans. A key feature of MTBC pathogens is their high degree of genetic identity yet distinct host tropism. Notably, while Mycobacterium bovis is highly virulent and pathogenic for cattle, the human pathogen M. tuberculosis is attenuated in cattle. Previous research also suggests that host preference amongst MTBC members has a basis in host innate immune responses. To explore MTBC host tropism, we present in-depth profiling of the MTBC reference strains M. bovis AF2122/97 and M. tuberculosis H37Rv at both the global transcriptional and the translational level via RNA-sequencing and SWATH MS. Furthermore, a bovine alveolar macrophage infection time course model was used to investigate the shared and divergent host transcriptomic response to infection with M. tuberculosis H37Rv or M. bovis AF2122/97. Significant differential expression of virulence-associated pathways between the two bacilli was revealed, including the ESX-1 secretion system. A divergent transcriptional response was observed between M. tuberculosis H37Rv and M. bovis AF2122/97 infection of bovine alveolar macrophages, in particular cytosolic DNA-sensing pathways at 48 h post-infection, and highlights a distinct engagement of M. bovis with the bovine innate immune system. The work presented here therefore provides a basis for the identification of host innate immune mechanisms subverted by virulent host-adapted mycobacteria to promote their survival during the early stages of infection. PMID:29557774

Surveillance for Mycobacterium bovis transmission from domestic cattle to wild ruminants in a Mexican wildlife-livestock interface area.

PubMed

Cisneros, Luis F; Valdivia, Arturo G; Waldrup, Kenneth; Díaz-Aparicio, Efrén; Martínez-de-Anda, Armando; Cruz-Vázquez, Carlos R; Ortiz, Raúl

2012-10-01

To assess the prevalence of Mycobacterium bovis infection in cattle and wild ruminants (WRs) in a wildlife-livestock interface area (WLIA) of the Mexican highland plateau. 24,400 cattle from 793 herds (including 17,351 commercially slaughtered cattle) and 142 WRs (110 white-tailed deer [Odocoileus virginianus], 20 red deer [Cervus elaphus], and 12 North American elk [Cervus canadensis]) harvested via controlled hunting. Cattle were serially tested for M bovis infection via caudal fold tuberculin and comparative cervical tuberculin tests during field surveillance. Carcasses of cattle and WRs were inspected for gross lesions; samples suggestive of tuberculosis were analyzed via histologic evaluation and mycobacterial culture (HMC). A PCR assay to detect Mycobacterium tuberculosis complex organisms was performed to confirm positive results of HMC. WRs had inflammatory lesions in lungs and lymph nodes, although HMC results did not indicate M bovis infection. Eight cattle had positive results for both tuberculin tests, and 31 had positive results for HMC of grossly detected lesions; all were from 7 herds, and ≥ 1 cow in each herd had positive PCR assay results. These 7 herds were depopulated; adjacent herds and herds related via commerce were quarantined. Calculated true prevalence of M bovis infection was 0.86% (95% confidence interval, 0.24% to 1.49%) in cattle; M bovis was not detected in any WRs. M bovis infection was present in cattle. Although transmission to WRs in this WLIA was not detected, diagnosis and prevention activities should be implemented and consolidated to prevent potential M bovis transmission between cattle and WRs.

Measuring bovine gamma delta T cell function at the site of Mycobacterium bovis infection

USDA-ARS?s Scientific Manuscript database

The causative agent of tuberculosis (TB) in cattle is Mycobacterium bovis. The characteristic lesions of bovine TB are well-organized pulmonary granulomas. Gamma delta T cells are a unique subset of nonconventional T cells that play major roles in both the innate and adaptive arms of the immune sys...

Epizootiologic survey of Mycobacterium bovis in wildlife and farm environments in northern Michigan.

PubMed

Witmer, Gary; Fine, Amanda E; Gionfriddo, James; Pipas, Michael; Shively, Kirk; Piccolo, Kim; Burke, Patrick

2010-04-01

Bovine tuberculosis (bovine TB), caused by Mycobacterium bovis, has reemerged in northern Michigan, USA, with detections in white-tailed deer (Odocoileus virginianus) in 1994 and in cattle in 1998. Since then, significant efforts have been directed toward reducing deer densities in the area in the hopes of reducing the bovine TB prevalence rate in deer and eliminating spillover of the disease into cattle. Despite the success of the efforts to reduce deer densities, additional cattle herds have become infected. Other mammals can be infected with M. bovis, and some carnivores and omnivores had been found to be infected with the disease in northern Michigan, USA. We conducted a multiyear surveillance effort to detect bovine TB in wild species of mammals in the Michigan, USA, outbreak area. From 2002 to 2004, tissue samples from 1,031 individual animals of 32 species were collected, processed, and cultured for M. bovis. Only 10 (1.0%) were culture-positive for M. bovis (five raccoons [Procyon lotor], four opossums [Didelphis virginiana], and one grey fox [Urocyon cinereoargenteus]). We also found two raccoons and four opossums to be positive for Mycobacterium avium. We collected 503 environmental samples from cattle farms recently identified as bovine TB positive; none yielded positive M. bovis culture results. Finally, we used infrared cameras to document wildlife use of four barns in the area. Many avian and mammalian species of wildlife were observed, with raccoons being the most commonly observed species. This surveillance study identified no new wildlife species that should be considered significant reservoirs of bovine TB in the outbreak area in northern Michigan, USA. However, the relatively high, apparent bovine TB prevalence rates in some carnivorous and omnivorous species, their relatively long life spans, and their frequent use of barns, suggests that removal of raccoons, opossums, foxes, and coyotes (Canis latrans) should be considered when a newly infected

Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance

PubMed Central

Bobadilla-del Valle, Miriam; Torres-González, Pedro; Cervera-Hernández, Miguel Enrique; Martínez-Gamboa, Areli; Crabtree-Ramirez, Brenda; Chávez-Mazari, Bárbara; Ortiz-Conchi, Narciso; Rodríguez-Cruz, Luis; Cervantes-Sánchez, Axel; Gudiño-Enríquez, Tomasa; Cinta-Severo, Carmen; Sifuentes-Osornio, José; Ponce de León, Alfredo

2015-01-01

Background Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Methodology/Principal Findings Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory’s database for the 2000–2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X 2 trend, p<0.001). Primary STR resistance was higher among M. bovis compared with M. tuberculosis isolates (10.9% vs.3.4%, p<0.001). Secondary multidrug resistance (MDR) rates were 38.5% and 34.4% for M. bovis and M. tuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000–2004 vs. 7.6% in 2010–2014; p = 0.02). Conclusions/Significance There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance. PMID:26421930

Specific recognition of mycobacterial protein and peptide antigens by gamma-delta T cell subsets following infection with virulent Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

Promoting effective immunity to Mycobacterium tuberculosis complex pathogens is a challenge that is of interest to the fields of human and veterinary medicine alike. We report that gamma delta T cells from virulent Mycobacterium bovis-infected cattle respond specifically and directly to complex, pro...

Generation of transgenic cattle expressing human β-defensin 3 as an approach to reducing susceptibility to Mycobacterium bovis infection.

PubMed

Su, Feng; Wang, Yongsheng; Liu, Guanghui; Ru, Kun; Liu, Xin; Yu, Yuan; Liu, Jun; Wu, Yongyan; Quan, Fusheng; Guo, Zekun; Zhang, Yong

2016-03-01

Bovine tuberculosis results from infection with Mycobacterium bovis, a member of the Mycobacterium tuberculosis family. Worldwide, M. bovis infections result in economic losses in the livestock industry; cattle production is especially hard-hit by this disease. Generating M. bovis-resistant cattle may potentially mitigate the impact of this disease by reducing M. bovis infections. In this study, we used transgenic somatic cell nuclear transfer to generate cattle expressing the gene encoding human β-defensin 3 (HBD3), which confers resistance to mycobacteria in vitro. We first generated alveolar epithelial cells expressing HBD3 under the control of the bovine MUC1 promoter, and confirmed that these cells secreted HBD3 and possessed anti-mycobacterial capacity. We then generated and identified transgenic cattle by somatic cell nuclear transfer. The cleavage and blastocyst formation rates of genetically modified embryos provided evidence that monoclonal transgenic bovine fetal fibroblast cells have an integral reprogramming ability that is similar to that of normal cells. Five genetically modified cows were generated, and their anti-mycobacterial capacities were evaluated. Alveolar epithelial cells and macrophages from these cattle expressed higher levels of HBD3 protein compared with non-transgenic cells and possessed effective anti-mycobacterial capacity. These results suggest that the overall risk of M. bovis infection in transgenic cattle is efficiently reduced, and support the development of genetically modified animals as an effective tool to reduce M. bovis infection. © 2016 Federation of European Biochemical Societies.

Inactivation of Mycobacterium bovis ssp. caprae in high-temperature, short-term pasteurized pilot-plant milk.

PubMed

Hammer, P; Richter, E; Rüsch-Gerdes, S; Walte, H-G C; Matzen, S; Kiesner, C

2015-03-01

Experiments to determine the efficacy of high temperature, short time (HTST) pasteurization of milk in terms of inactivation of pathogenic microorganisms were mainly performed between 1930 and 1960. Among the target organisms were Mycobacterium bovis and Mycobacterium tuberculosis. As a result, the Codex Alimentarius prescribes that HTST treatment of milk should lead to a significant reduction of pathogenic microorganisms during milk pasteurization. Due to the development of improved methods for the detection of survivors and of more advanced heating technology, verification of this requirement seemed to be necessary. To address recent outbreaks of tuberculosis in cattle caused by M. bovis ssp. caprae (M. caprae) in the southern regions of Germany, this organism was tested and compared with M. bovis ssp. bovis (M. bovis). Experiments were performed in a pilot plant for HTST pasteurization of milk with 3 strains of M. caprae and 1 strain of M. bovis. In preliminary trials at a fixed holding time of 25 s, the temperature at which significant inactivation occurred was 62.5°C for all strains. To determine D-values (decimal reduction times) for the inactivation kinetics, the strains were tested at 65, 62.5, and 60°C at holding times of 16.5, 25, and 35 s. At 65°C, the D-values of all strains ranged from 6.8 to 7.8 s, and at 62.5°C, D-values ranged from 14.5 to 18.1 s. Low inactivation was observed at 60°C. When the low slope of the inactivation curve allowed calculation of a D-value, these ranged from 40.8 to 129.9 s. In terms of log10 reductions, the highest values for all strains were 4.1 to 4.9 log at 65°C, with a holding time of 35 s. The tested strains of M. caprae and M. bovis showed similar low resistance to heat. Standard HTST treatment should result in a high reduction of these organisms and thus the requirements of the Codex Alimentarius for inactivation of pathogens by this process are far exceeded. Copyright © 2015 American Dairy Science Association

Utility of a fecal real-time PCR protocol for detection of Mycobacterium bovis infection in African buffalo (Syncerus caffer).

PubMed

Roug, Annette; Geoghegan, Claire; Wellington, Elizabeth; Miller, Woutrina A; Travis, Emma; Porter, David; Cooper, David; Clifford, Deana L; Mazet, Jonna A K; Parsons, Sven

2014-01-01

A real-time PCR protocol for detecting Mycobacterium bovis in feces was evaluated in bovine tuberculosis-infected African buffalo (Syncerus caffer). Fecal samples spiked with 1.42 × 10(3) cells of M. bovis culture/g and Bacille Calmette-Guérin standards with 1.58 × 10(1) genome copies/well were positive by real-time PCR but all field samples were negative.

Biochemical characteristics among Mycobacterium bovis BCG substrains.

PubMed

Hayashi, Daisuke; Takii, Takemasa; Mukai, Tetsu; Makino, Masahiko; Yasuda, Emi; Horita, Yasuhiro; Yamamoto, Ryuji; Fujiwara, Akiko; Kanai, Keita; Kondo, Maki; Kawarazaki, Aya; Yano, Ikuya; Yamamoto, Saburo; Onozaki, Kikuo

2010-05-01

In order to evaluate the biochemical characteristics of 14 substrains of Mycobacterium bovis bacillus Calmette Guérin (BCG) - Russia, Moreau, Japan, Sweden, Birkhaug, Danish, Glaxo, Mexico, Tice, Connaught, Montreal, Phipps, Australia and Pasteur - we performed eight different biochemical tests, including those for nitrate reduction, catalase, niacin accumulation, urease, Tween 80 hydrolysis, pyrazinamidase, p-amino salicylate degradation and resistance to thiophene 2-carboxylic acid hydrazide. Catalase activities of the substrains were all low. Data for nitrate reduction, niacin accumulation, Tween 80 hydrolysis, susceptibility to hydrogen peroxide and nitrate, and optimal pH for growth were all variable among these substrains. These findings suggest that the heterogeneities of biochemical characteristics are relevant to the differences in resistance of BCG substrains to environmental stress. The study also contributes to the re-evaluation of BCG substrains for use as vaccines.

Population structure of Mycobacterium bovis isolates from cattle in Mexico.

PubMed

Gutiérrez Reyes, José A; García Casanova, Leticia; Romero Torres, Cecilia; Sosa Gallegos, Susana L; Cantó Alarcón, Germinal J; Mercado Pezzat, Marcela; Pizano Martínez, Oscar; Estrada Chávez, Ciro; Milián Suazo, Feliciano

2012-09-01

The molecular fingerprints of 878 isolates of Mycobacterium bovis collected from cattle between 2009 and 2010 in different regions of Mexico were used in this study. One hundred and ninety-four spoligotypes were observed in total with a high degree of heterogeneity. Sixty-four percent of the isolates grouped into just nine spoligotypes, and 27% fell into only two spoligotypes: SB0673 and SB0669; 149 were orphan spoligotypes. The two predominant spoligotypes were found in almost all states in Mexico, especially in central Mexico, where there is a high concentration of dairy cattle; however, some spoligotypes were closely associated with restricted geographical areas. The hypothetical evolutionary relationship among spoligotypes was estimated using the spoligoforest program in the spolTools webpage. Four trees with connected components and nine unconnected nodes were found. The biggest tree had SB0140 strain as a root, suggesting this as the oldest strain in the tree. However, the relationship of this spoligotype with SB0673 and SB0669 was weak. The discriminatory power of spoligotyping for this M. bovis sample of isolates was 0.94, and the recent transmission index (RTI) 0.83, suggesting a high rate of recent transmission of some strains of M. bovis in the population. This parameter indicates that new measures are required to stop the dissemination of tuberculosis in cattle. Copyright © 2012 Elsevier B.V. All rights reserved.

Mycobacterium bovis in Burkina Faso: epidemiologic and genetic links between human and cattle isolates.

PubMed

Sanou, Adama; Tarnagda, Zekiba; Kanyala, Estelle; Zingué, Dezemon; Nouctara, Moumini; Ganamé, Zakaria; Combary, Adjima; Hien, Hervé; Dembele, Mathurin; Kabore, Antoinette; Meda, Nicolas; Van de Perre, Philippe; Neveu, Dorine; Bañuls, Anne Laure; Godreuil, Sylvain

2014-10-01

In sub-Saharan Africa, bovine tuberculosis (bTB) is a potential hazard for animals and humans health. The goal of this study was to improve our understanding of bTB epidemiology in Burkina Faso and especially Mycobacterium bovis transmission within and between the bovine and human populations. Twenty six M. bovis strains were isolated from 101 cattle carcasses with suspected bTB lesions during routine meat inspections at the Bobo Dioulasso and Ouagadougou slaughterhouses. In addition, 7 M. bovis strains were isolated from 576 patients with pulmonary tuberculosis. Spoligotyping, RDAf1 deletion and MIRU-VNTR typing were used for strains genotyping. The isolation of M. bovis strains was confirmed by spoligotyping and 12 spoligotype signatures were detected. Together, the spoligotyping and MIRU-VNTR data allowed grouping the 33 M. bovis isolates in seven clusters including isolates exclusively from cattle (5) or humans (1) or from both (1). Moreover, these data (genetic analyses and phenetic tree) showed that the M. bovis isolates belonged to the African 1 (Af1) clonal complex (81.8%) and the putative African 5 (Af5) clonal complex (18.2%), in agreement with the results of RDAf1 deletion typing. This is the first detailed molecular characterization of M. bovis strains from humans and cattle in Burkina Faso. The distribution of the two Af1 and putative Af5 clonal complexes is comparable to what has been reported in neighbouring countries. Furthermore, the strain genetic profiles suggest that M. bovis circulates across the borders and that the Burkina Faso strains originate from different countries, but have a country-specific evolution. The genetic characterization suggests that, currently, M. bovis transmission occurs mainly between cattle, occasionally between cattle and humans and potentially between humans. This study emphasizes the bTB risk in cattle but also in humans and the difficulty to set up proper disease control strategies in Burkina Faso.

Oral vaccination of guinea pigs with a Mycobacterium bovis bacillus Calmette-Guerin vaccine in a lipid matrix protects against aerosol infection with virulent M. bovis.

PubMed

Clark, Simon; Cross, Martin L; Nadian, Allan; Vipond, Julia; Court, Pinar; Williams, Ann; Hewinson, R Glyn; Aldwell, Frank E; Chambers, Mark A

2008-08-01

Increased incidence of bovine tuberculosis (TB) in the United Kingdom caused by infection with Mycobacterium bovis is a cause of considerable economic loss to farmers and the government. The Eurasian badger (Meles meles) represents a wildlife source of recurrent M. bovis infections of cattle in the United Kingdom, and its vaccination against TB with M. bovis bacillus Calmette-Guérin (BCG) is an attractive disease control option. Delivery of BCG in oral bait holds the best prospect for vaccinating badgers over a wide geographical area. Using a guinea pig pulmonary challenge model, we evaluated the protective efficacy of candidate badger oral vaccines, based on broth-grown or ball-milled BCG, delivered either as aqueous suspensions or formulated in two lipids with differing fatty acid profiles (one being animal derived and the other being vegetable derived). Protection was determined in terms of increasing body weight after aerosol challenge with virulent M. bovis, reduced dissemination of M. bovis to the spleen, and, in the case of one oral formulation, restricted growth of M. bovis in the lungs. Only oral BCG formulated in lipid gave significant protection. These data point to the potential of the BCG-lipid formulation for further development as a tool for controlling tuberculosis in badgers.

High Throughput Phenotypic Analysis of Mycobacterium tuberculosis and Mycobacterium bovis Strains' Metabolism Using Biolog Phenotype Microarrays

PubMed Central

Khatri, Bhagwati; Fielder, Mark; Jones, Gareth; Newell, William; Abu-Oun, Manal; Wheeler, Paul R.

2013-01-01

Tuberculosis is a major human and animal disease of major importance worldwide. Genetically, the closely related strains within the Mycobacterium tuberculosis complex which cause disease are well-characterized but there is an urgent need better to understand their phenotypes. To search rapidly for metabolic differences, a working method using Biolog Phenotype MicroArray analysis was developed. Of 380 substrates surveyed, 71 permitted tetrazolium dye reduction, the readout over 7 days in the method. By looking for ≥5-fold differences in dye reduction, 12 substrates differentiated M. tuberculosis H37Rv and Mycobacterium bovis AF2122/97. H37Rv and a Beijing strain of M. tuberculosis could also be distinguished in this way, as could field strains of M. bovis; even pairs of strains within one spoligotype could be distinguished by 2 to 3 substrates. Cluster analysis gave three clear groups: H37Rv, Beijing, and all the M. bovis strains. The substrates used agreed well with prior knowledge, though an unexpected finding that AF2122/97 gave greater dye reduction than H37Rv with hexoses was investigated further, in culture flasks, revealing that hexoses and Tween 80 were synergistic for growth and used simultaneously rather than in a diauxic fashion. Potential new substrates for growth media were revealed, too, most promisingly N-acetyl glucosamine. Osmotic and pH arrays divided the mycobacteria into two groups with different salt tolerance, though in contrast to the substrate arrays the groups did not entirely correlate with taxonomic differences. More interestingly, these arrays suggested differences between the amines used by the M. tuberculosis complex and enteric bacteria in acid tolerance, with some hydrophobic amino acids being highly effective. In contrast, γ-aminobutyrate, used in the enteric bacteria, had no effect in the mycobacteria. This study proved principle that Phenotype MicroArrays can be used with slow-growing pathogenic mycobacteria and already has

Tracing the origins of Mycobacterium bovis tuberculosis in humans in the USA to cattle in Mexico using spoligotyping☆

PubMed Central

Rodwell, Timothy C.; Kapasi, Anokhi J.; Moore, Marisa; Milian-Suazo, Feliciano; Harris, Beth; Guerrero, L.P.; Moser, Kathleen; Strathdee, Steffanie A.; Garfein, Richard S.

2010-01-01

Objectives To compare genotypes of Mycobacterium bovis strains from humans in Southern California with genotypes of M. bovis strains in cattle in Mexico and the USA to explore the possible origins of human infections. Methods We conducted a descriptive analysis of M. bovis genotypes from a binational population of humans and cattle using spacer oligonucleotide typing (spoligotyping). Results One hundred six human M. bovis spoligotypes were compared to spoligotypes from 496 Mexican cattle and 219 US cattle. Twelve spoligotype patterns were identified among human cases and 126 spoligotype patterns were detected in cattle. Over 91% (97/106) of the human M. bovis isolates had spoligotypes that were identical to those found in Mexican cattle. Four human cases had spoligotypes that matched both cattle born in Mexico and in the USA. Nine human cases had spoligotypes that did not match cattle born in Mexico or the USA. Conclusions Our data indicate that the population of M. bovis strains causing human TB disease in Southern California is closely related to the M. bovis strain population found in Mexican cattle and supports existing epidemiological evidence that human M. bovis disease in San Diego likely originated from Mexican cattle. PMID:20399697

Mycobacterium bovis: A Model Pathogen at the Interface of Livestock, Wildlife, and Humans

PubMed Central

Palmer, Mitchell V.; Thacker, Tyler C.; Waters, W. Ray; Gortázar, Christian; Corner, Leigh A. L.

2012-01-01

Complex and dynamic interactions involving domestic animals, wildlife, and humans create environments favorable to the emergence of new diseases, or reemergence of diseases in new host species. Today, reservoirs of Mycobacterium bovis, the causative agent of tuberculosis in animals, and sometimes humans, exist in a range of countries and wild animal populations. Free-ranging populations of white-tailed deer in the US, brushtail possum in New Zealand, badger in the Republic of Ireland and the United Kingdom, and wild boar in Spain exemplify established reservoirs of M. bovis. Establishment of these reservoirs is the result of factors such as spillover from livestock, translocation of wildlife, supplemental feeding of wildlife, and wildlife population densities beyond normal habitat carrying capacities. As many countries attempt to eradicate M. bovis from livestock, efforts are impeded by spillback from wildlife reservoirs. It will not be possible to eradicate this important zoonosis from livestock unless transmission between wildlife and domestic animals is halted. Such an endeavor will require a collaborative effort between agricultural, wildlife, environmental, and political interests. PMID:22737588

Low cross-reactivity of T-cell responses against lipids from Mycobacterium bovis and M. avium paratuberculosis during natural infection

PubMed Central

Van Rhijn, Ildiko; Nguyen, Thi Kim Anh; Michel, Anita; Cooper, Dave; Govaerts, Marc; Cheng, Tan-Yun; van Eden, Willem; Moody, D. Branch; Coetzer, Jacobus A. W.; Rutten, Victor; Koets, Ad P.

2011-01-01

Although CD1 proteins are known to present mycobacterial lipid antigens to T cells, there is little understanding of the in vivo behavior of T cells restricted by CD1a, CD1b and CD1c, and the relative immunogenicity and immunodominance of individual lipids within the total array of lipids that comprise a bacterium. Because bovines express multiple CD1 proteins and are natural hosts of Mycobacterium bovis and Mycobacterium avium paratuberculosis (MAP), we used them as a new animal model of CD1 function. Here, we report the surprisingly divergent responses against lipids produced by these two pathogens during infection. Despite considerable overlap in lipid content, only three out of 69 animals cross-react with M. bovis and MAP total lipid preparations. The unidentified immunodominant compound of M. bovis is a hydrophilic compound, whereas the immunodominant lipid of MAP is presented by CD1b and was identified as glucose monomycolate (GMM). The preferential recognition of GMM antigen by MAP-infected cattle may be explained by the higher expression of GMM by MAP than by M. bovis. The bacterial species-specific nature of the CD1-restricted, adaptive T-cell response affects the approach to development of lipid based immunodiagnostic tests. PMID:19688747

Testing a molasses-based bait for oral vaccination of white-tailed deer (Odocoileus virginianus) against Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

White-tailed deer (Odocoileus virginianus) in Michigan, USA are wildlife reservoirs of bovine tuberculosis (bTB) with documented spread to cattle. In vaccine efficacy trials, Mycobacterium bovis bacillus Calmette Guerin (BCG) administered orally reduces colonization and bTB-associated lesions in whi...

Mycobacterium bovis in a European bison (Bison bonasus) raises concerns about tuberculosis in Brazilian captive wildlife populations: a case report.

PubMed

Zimpel, Cristina Kraemer; Brum, Juliana Sperotto; de Souza Filho, Antônio Francisco; Biondo, Alexander Welker; Perotta, João Henrique; Dib, Cristina Corsi; Bonat, Marcelo; Neto, José Soares Ferreira; Brandão, Paulo Eduardo; Heinemann, Marcos Bryan; Guimaraes, Ana Marcia Sa

2017-02-10

Tuberculosis caused by Mycobacterium bovis is an important worldwide zoonosis and has been reported to cause clinical disease in several animal species, including captive wildlife. This report describes a case of M. bovis infection in a European bison from a Brazilian zoo and compiles a number of literature reports that raise concern regarding tuberculosis among captive wildlife in Brazil. A 13 year-old captive-born male bison (Bison bonasus) from a Brazilian zoo began presenting weight loss, diarrhea and respiratory symptoms, which inevitably led to his death. At the animal's necropsy, inspection of the thoracic and abdominal cavities revealed multiple enlarged lymph nodes, ranging from 4 to 10 cm, and pulmonary nodules containing caseous masses with firm white materials consistent with mineralization. Histopathology findings showed a significant amount of acid-alcohol resistant bacilli compatible with Mycobacterium spp. Specimens from lymph nodes and lungs were cultured on Petragnani and Stonebrink media, and specific PCR assays of the bacterial isolate identified it as M. bovis. The European bison reported herein died from a severe form of disseminated tuberculosis caused by M. bovis. A review of the available literature indicates possible widespread occurrence of clinical disease caused by M. bovis or M. tuberculosis affecting multiple animal species in Brazilian wildlife-related institutions. These likely underestimated numbers raise concern regarding the control of the disease in captive animal populations from Brazil.

Alternative activation modifies macrophage resistance to Mycobacterium bovis.

PubMed

Castillo-Velázquez, Uziel; Aranday-Cortés, Elihú; Gutiérrez-Pabello, José A

2011-07-05

The aim of this study was to evaluate the influence of macrophage alternative activation in the intracellular pathogen natural disease resistance phenotype of the host. Macrophage monolayers from resistant (R) (3) or susceptible (S) (3) cattle donors were treated with 10 ng/ml of bovine recombinant IL-4 (rbIL-4), and infected with virulent and avirulent Mycobacterium bovis (MOI 10:1). Bactericidal assays were performed to assess the bacterial phagocytic index and intracellular survival. Total RNA was reverse transcribed and used to analyze the relative changes in gene expression of IL-10, IL-12, IL-18 IL-1β, TNF-α, MCP-1, MCP-2, IL-6, MIP-1, MIP-3, iNOS, ARGII and SLAM by real time PCR. Cell supernatants were collected and nitric oxide and arginase production was assessed. Apoptosis induction was measured by TUNEL. IL-4 treatment increased the phagocytic index in both R and S macrophages; however intracellular survival was augmented mainly in S macrophages. Alternative activation decreased gene expression of pro-inflammatory cytokines, nitric oxide production and DNA fragmentation mainly in R macrophages. On the other hand, arginase production was not different between R and S macrophages. Alternative activation modifies the macrophage response against M. bovis. IL-4 treatment minimized the functional differences that exist between R and S macrophages. Copyright © 2011. Published by Elsevier B.V.

Optimization of modified Middlebrook 7H11 agar for isolation of Mycobacterium bovis from raw milk cheese.

PubMed

Forgrave, R; Donaghy, J A; Fisher, A; Rowe, M T

2014-10-01

Reports have highlighted the absence of contemporary peer reviewed publications pertaining to Mycobacterium bovis culture from raw milk and cheese. By replicating traditional methods, cheese-making methodology and equipment were devised to produce Cheddar (n = 6) and Caerphilly (n = 3) artificially contaminated with M. bovis (three genotypes) under stringent laboratory-containment guidelines for handling hazardous microbiological material. Middlebrook 7H11, modified for M. bovis isolation, was assessed for capacity to enumerate M. bovis despite changing cheese microflora and prolonged M. bovis exposure to the cheese matrix using maturing cheese test portions (n = 63; up to 16 weeks). Malachite green (MG) containing media isolated M. bovis at significantly (P < 0·05) lower levels than unmodified Middlebrook 7H11 agar despite MG being a common adjunct of Middlebrook 7H11 agar modified for M. bovis growth. Subsequently, a selective MG-free Middlebrook 7H11 agar modified using haemolysed red cells and calf serum was demonstrated as the best performing (P < 0·05) medium for recovery of M. bovis from typical UK cheese types, Cheddar and Caerphilly. Significance and impact of the study: Following increased M. bovis infection of UK cattle, the risk posed to consumers from consumption of unpasteurized milk and dairy products has changed. Furthermore, published methods for the culture and molecular detection of M. bovis in raw milk products are limited. Cheese-making protocols and M. bovis culture media reported here provide tools for further investigation of M. bovis survival during all stages of cheese manufacture and could inform future assessment of the risk to consumers from M. bovis contamination of unpasteurized dairy products. © 2014 The Society for Applied Microbiology.

Antemortem diagnosis of Mycobacterium bovis infection in free-ranging African lions (Panthera leo) and implications for transmission.

PubMed

Miller, Michele; Buss, Peter; Hofmeyr, Jennifer; Olea-Popelka, Francisco; Parsons, Sven; van Helden, Paul

2015-04-01

Diagnosis of tuberculosis in wildlife often relies on postmortem samples because of logistical challenges and lack of field-friendly techniques for live animal testing. Confirmation of infection through detection of infectious organisms is essential for studying the pathogenesis and epidemiology of disease. We describe the application of a technique to obtain respiratory samples from free-ranging living lions to facilitate detection of viable Mycobacterium bovis under field conditions. We identified M. bovis by mycobacterial culture and PCR in tracheobronchial lavage samples from 8/134 (6.0%) lions tested in Kruger National Park, South Africa. This confirms the respiratory shedding of viable M. bovis in living lions. The implications of these results are that infected lions have the potential to transmit this disease and serve as maintenance hosts.

Molecular epidemiology of human cases of tuberculosis by Mycobacterium bovis in Mexico.

PubMed

Milián-Suazo, Feliciano; Pérez-Guerrero, Laura; Arriaga-Díaz, Camila; Escartín-Chávez, Minerva

2010-10-01

The purpose of this study was to evaluate the role of Mycobacterium bovis in human cases of tuberculosis (TB) in an endemic area of the disease in cattle. Sputum, urine and other tissue samples were obtained from: (1) TB-symptomatic patients, (2) dairy farm workers and (3) abattoir workers. Samples of macroscopic lesions suspicious of TB were also obtained from cattle at slaughter in the same geographic area. A total of 562 human samples were collected: 255 from symptomatic patients, 218 from farm workers and 93 from abattoir workers. Samples were analysed by the bacillus acido-alcohol resitant (BAAR) and polymerase chain reaction (PCR) tests and cultured in Stonebrink and Löwenstein-Jensen. Spoligotyping was performed in all isolates obtained by culture and the DNA obtained by PCR. From the total number of human cases, 34 (6%) showed M. bovis spoligotype; eight spoligotypes from cattle showed an identical pattern to three spoligotypes from humans; a different set of spoligotypes from cattle (n = 8) had only one spacer difference to a set of spoligotypes from humans (n = 2). These results provide further evidence that infected cattle represent a risk to public health and support previous reports about the role of M. bovis in Mexican patients. There is no doubt that genotyping M. bovis isolates collected from cattle may have a substantial impact on our understanding of the epidemiology of TB. Copyright © 2010 Elsevier B.V. All rights reserved.

Tracing the origins of Mycobacterium bovis tuberculosis in humans in the USA to cattle in Mexico using spoligotyping.

PubMed

Rodwell, Timothy C; Kapasi, Anokhi J; Moore, Marisa; Milian-Suazo, Feliciano; Harris, Beth; Guerrero, L P; Moser, Kathleen; Strathdee, Steffanie A; Garfein, Richard S

2010-09-01

To compare genotypes of Mycobacterium bovis strains from humans in Southern California with genotypes of M. bovis strains in cattle in Mexico and the USA to explore the possible origins of human infections. We conducted a descriptive analysis of M. bovis genotypes from a binational population of humans and cattle using spacer oligonucleotide typing (spoligotyping). One hundred six human M. bovis spoligotypes were compared to spoligotypes from 496 Mexican cattle and 219 US cattle. Twelve spoligotype patterns were identified among human cases and 126 spoligotype patterns were detected in cattle. Over 91% (97/106) of the human M. bovis isolates had spoligotypes that were identical to those found in Mexican cattle. Four human cases had spoligotypes that matched both cattle born in Mexico and in the USA. Nine human cases had spoligotypes that did not match cattle born in Mexico or the USA. Our data indicate that the population of M. bovis strains causing human TB disease in Southern California is closely related to the M. bovis strain population found in Mexican cattle and supports existing epidemiological evidence that human M. bovis disease in San Diego likely originated from Mexican cattle. Copyright © 2010 International Society for Infectious Diseases. All rights reserved.

Longevity of Mycobacterium bovis in Raw and Traditional Souring Milk as a Function of Storage Temperature and Dose

PubMed Central

Hlokwe, Tiny; Raseleka, Keneilwe; Getz, Wayne M.; Marcotty, Tanguy

2015-01-01

Background Unpasteurised fresh and souring dairy products form an essential component of household diets throughout many rural communities in southern Africa. The presence of milk-borne zoonotic pathogens such as Mycobacterium bovis (M. bovis), the causative agent of bovine tuberculosis and zoonotic tuberculosis in humans, constitute a public health threat, especially in remote areas with poor disease surveillance in livestock and highly compromised human health due to HIV/AIDS. Methods In this study we used culture to determine the longevity of M. bovis in experimentally inoculated fresh and naturally souring milk obtained from communal cattle in the KwaZulu-Natal province of South Africa. The effect of bacterial load and storage temperature on the survival of M. bovis was evaluated by spiking mixtures of fresh milk and starter soured milk (aMasi) culture with three concentrations of bacteria (102, 104, 107 colony forming units/ml), followed by incubation under controlled laboratory conditions that mimicked ambient indoor (20°C) and outdoor (33°C) temperatures and periodic sampling and testing over time (0-56 days). Results M. bovis cultured from samples of the fresh and souring milk was identified by PCR analysis. At the highest spiking concentration (107cfu/ml), M. bovis survived for at least 2 weeks at 20°C; but, at all concentrations in the 33°C treatment, M. bovis was absent by three days after inoculation. Logistic regression analysis was used to assess the effects of bacterial concentration and time since inoculation, as well as determine the potential half-life of M. bovis in raw souring milk. Given the most favourable tested conditions for bacterial survival (20°C), approximately 25% of mycobacteria were alive after one day of storage (95% CI: 9-53%), giving an estimated half-life of M. bovis in raw souring milk of approximately 12 hours (95% CI: 7-27 hours). Conclusions This study demonstrates that M. bovis may survive in fresh and souring milk for

Antimicrobial Susceptibility Testing of Mycobacterium bovis Isolates from Michigan White-Tailed Deer during the 2009 Hunting Season.

PubMed

Fitzgerald, Scott D; Schooley, Angie M; Berry, Dale E; Kaneene, John B

2010-12-02

Michigan has had an ongoing outbreak of endemic Mycobacterium bovis which has been recognized within and sustained by its free-ranging white-tailed deer population since 1994. Worldwide, organisms within the Mycobacterium tuberculosis complex have exhibited the ability to develop resistance to antimicrobial agents, resulting in both the multidrug-resistant (MDR) and extensively drug-resistant (XDR) strains of human tuberculosis. Michigan's Bovine Tuberculosis Working Group has conducted active antimicrobial susceptibility testing on wildlife isolates of the endemic M. bovis organism at five-year intervals to detect any emerging drug resistance patterns. The results of 33 white-tailed deer origin isolates collected from the 2009 hunting season are reported here. There continues to be no evidence of any drug resistance except for pyrazinamide resistance. These results are likely due to the lack of antibacterial treatment applied to either wildlife or domestic animals which would provide selection pressure for the development of drug resistance.

Human tuberculosis caused by Mycobacterium bovis: a retrospective comparison with Mycobacterium tuberculosis in a Mexican tertiary care centre, 2000-2015.

PubMed

Torres-Gonzalez, Pedro; Cervera-Hernandez, Miguel E; Martinez-Gamboa, Areli; Garcia-Garcia, Lourdes; Cruz-Hervert, Luis P; Bobadilla-Del Valle, Miriam; Ponce-de Leon, Alfredo; Sifuentes-Osornio, Jose

2016-11-08

Human tuberculosis caused by Mycobacterium bovis is believed to be frequent in developing countries. Transmission is usually through ingestion of unpasteurized dairy products, although airborne contagion is possible. Disease caused by M. tuberculosis or M. bovis is clinically indistinguishable from each other. The aim of this study was to determine the factors associated with M. bovis disease. Retrospective analysis of all culture-positive cases of M. bovis and M. tuberculosis from 2000 to 2015, in a Mexican tertiary-care centre. Sociodemographic, clinical, and radiographic data from medical records were compared. Disease site was classified as pulmonary, extrapulmonary, or pulmonary and extrapulmonary, based on cultures. We evaluated 533 cases, 372 (69.7 %) of which were caused by M. tuberculosis and 161 (30.2 %) by M. bovis. Characteristics associated with M. bovis disease were: younger age (aOR 0.97, 95 % CI 0.95-0.98), glucocorticoid use (aOR 2.27, 95 % CI 1.42-3.63), and extrapulmonary disease (aOR 1.80, 95 % CI 1.21-2.69). M. tuberculosis was associated with lower socioeconomic status (aOR 0.52, 95 % CI 0.28-0.97). When we analysed only pulmonary cases, younger age (aOR 0.97, 95 % CI 0.96-0.99), glucocorticoid use (aOR 2.41, 95 % CI 1.30-4.46), and smoking (aOR 1.94, CI 95 % 1.15-3.27) were associated with M. bovis. Both groups showed similar proportions of direct microscopy smear results (respiratory samples) and chest X-ray cavitations. Younger age, glucocorticoid use, and extrapulmonary disease were associated with M. bovis as the causative agent of tuberculosis in a group of patients from a tertiary care centre in a country where bovine tuberculosis is endemic. Further studies must be conducted in the general population to determine pathogen-specific associated factors and outcomes.

Different Strategies for Molecular Differentiation of Mycobacterium bovis Strains Isolated in Sardinia, Italy

PubMed Central

Sechi, Leonardo A.; Leori, Guido; Lollai, Stefano A.; Duprè, Ilaria; Molicotti, Paola; Fadda, Giovanni; Zanetti, Stefania

1999-01-01

Different genetic markers were used to analyze 22 Mycobacterium bovis strains isolated from cattle in Sardinia and one human isolate. IS6110 DNA fingerprinting differentiated the strains into six patterns, whereas with enterobacterial repetitive consensus sequence primers produced seven clusters. PCR ribotyping followed by digestion with HaeIII and PvuII produced five and seven patterns, respectively. PCR with the (GTG)5 oligonucleotide primer showed the best discriminatory power, generating eight clusters among the strains analyzed. PMID:10103282

Mycobacterium bovis infections in domesticated non-bovine mammalian species. Part 2: A review of diagnostic methods.

PubMed

Broughan, J M; Crawshaw, T R; Downs, S H; Brewer, J; Clifton-Hadley, R S

2013-11-01

Despite the large host range of Mycobacterium bovis, ante-mortem diagnostic tests for the infection mostly lack sensitivity/specificity and/or remain unvalidated in non-bovine species. The epidemiology and importance of M. bovis infection in these species are discussed in the first part of this two-part review. This second part focuses on the diagnostic options available to identify infected species such as sheep, goats, dogs, cats, and camelids, and highlights the significant challenges posed, both in establishing estimates of disease prevalence and in controlling infections in these species, in the absence of fully validated tests. Crown Copyright © 2013. Published by Elsevier Ltd. All rights reserved.

Molecular and epidemiological population-based integrative analysis of human and animal Mycobacterium bovis infections in a low-prevalence setting.

PubMed

Palacios, Juan José; Navarro, Yurena; Romero, Beatriz; Penedo, Ana; Menéndez González, Ángela; Pérez Hernández, M Dolores; Fernández-Verdugo, Ana; Copano, Francisca; Torreblanca, Aurora; Bouza, Emilio; Domínguez, Lucas; de Juan, Lucía; García-de-Viedma, Darío

2016-11-15

Human Mycobacterium bovis infections are considered to be due to reactivations, when involve elderly people, or to recent transmissions, when exposure is occupational. We determined the cause of M. bovis infections by genotyping M. bovis isolates in a population-based study integrating human and animal databases. Among the 1,586 tuberculosis (TB) cases in Asturias, Northern Spain (1,080,000 inhabitants), 1,567 corresponded to M. tuberculosis and 19 to M. bovis. The number of human isolates sharing genotype with cattle isolates was higher than expected (47%) for a setting with low prevalence of bovine TB and efficient control programs in cattle. The risk of exposure to infected animals was probable/possible in most of these matched cases (77.7%). Recent transmission was the likely explanation of most M. bovis infections in elderly people. A potential human-to-human transmission was found. Our study illustrates a model of collaboration between human and animal health professionals to provide a precise snapshot of the transmission of M. bovis in the human-animal interface. Copyright © 2016 Elsevier B.V. All rights reserved.

Persistence of Mycobacterium bovis Bacillus Calmette-Guerin (BCG) in White-tailed Deer (Odocoileus virginianus) After Oral or Parenteral Vaccination

USDA-ARS?s Scientific Manuscript database

Mycobacterium bovis is the cause of tuberculosis in cattle and a serious zoonotic pathogen, most commonly contracted through consumption of unpasteurized dairy products. To control this zoonosis, many countries have developed bovine tuberculosis eradication programs. Although relatively successful, ...

Use of released pigs as sentinels for Mycobacterium bovis.

PubMed

Nugent, Graham; Whitford, Jackie; Young, Nigel

2002-10-01

Identifying the presence of bovine tuberculosis (TB; Mycobacterium bovis) in wildlife is crucial in guiding management aimed at eradicating the disease from New Zealand. Unfortunately, surveys of the principal wildlife host, the introduced brushtail possum (Trichosurus vulpecula), require large samples (> 95% of the population) before they can provide reasonable confidence that the disease is absent. In this study, we tested the feasibility of using a more wide-ranging species, feral pig (Sus scrofa), as an alternative sentinel capable of indicating TB presence. In January 2000, 17 pigs in four groups were released into a forested area with a low density of possums in which TB was known to be present. The pigs were radiotracked at 2 wk intervals from February to October 2000, and some of them were killed and necropsied at various intervals after release. Of the 15 pigs successfully recovered and necropsied, one killed 2 mo after release had no gross lesions typical of TB, and the only other pig killed at that time had greatly enlarged mandibular lymph nodes. The remainder were killed at longer intervals after release and all had gross lesions typical of TB. Mycobacterium bovis was isolated from all 15 pigs by mycobacterial culture. Home range sizes of pigs varied widely and increased with the length of time the pigs were in the forest, with minimum convex polygon range-size estimates averaging 10.7 km2 (range 4.7-20.3 km2) for the pigs killed after 6 mo. A 6 km radius around the kill site of each pig would have encompassed 95% of all of their previous locations at which they could have become infected. However, one pig shifted 35 km, highlighting the main limitation of using unmarked feral pigs as sentinels. This trial indicates use of resident and/or released free-ranging pigs is a feasible alternative to direct prevalence surveys of possums for detecting TB presence.

An Effort to Isolate Mycobacterium bovis from Environmental Substrates during Investigations of Bovine Tuberculosis Transmission Sites (Cattle Farms and Wildlife Areas) in Michigan, USA

PubMed Central

Fine, Amanda E.; O'Brien, Daniel J.; Winterstein, Scott R.; Kaneene, John B.

2011-01-01

Deer movements on cattle farms, wildlife feeding, and livestock management practices in Michigan are thought to create opportunities for indirect transmission of Mycobacterium bovis via environmental substrates. To confirm the presence of viable M. bovis in the environment, substrates were collected from 13 farms with culture-confirmed M. bovis in cattle and 5 sites with high prevalence of M. bovis in free-ranging deer. None of the samples processed for mycobacterial culture were positive for M. bovis. Agent, host, and landscape-level factors decrease the probability of detecting M. bovis in the environment using conventional mycobacterial culture. Molecular techniques that increase the probability of M. bovis detection in environmental substrates should be applied to known sites of M. bovis transmission in Michigan. In the interim, epidemiological investigations informed by experimental studies will be most effective in characterizing M. bovis persistence in the environment and its role in the indirect interspecies transmission of M. bovis. PMID:23738108

Identification of Mycobacterium bovis in bovine clinical samples by PCR species-specific primers.

PubMed Central

Romero, R E; Garzón, D L; Mejía, G A; Monroy, W; Patarroyo, M E; Murillo, L A

1999-01-01

Tuberculosis, caused by Mycobacterium bovis is emerging as the most important disease affecting cattle. Furthermore, it results in a major public health problem when transmitted to humans. Due to its difficult and non-specific diagnosis, M. bovis has been declared to be one of the etiologic agents causing significant economic loss in the cattle industry. Our group evaluated a more rapid and specific method, based on a new polymerase chain reaction species-specific primers, which amplifies a 470-base pair fragment of the M. bovis genome. A total of 275 milk-producing cows were studied by intradermal tuberculin test (ITT) which gave 184 positive and 91 negative cases. From them, 50 animals were taken from a cattle ranch free of tuberculosis. Three different samples were collected from each animal (blood, nasal mucus, and milk). Positive results were obtained from 26 animals by PCR (11.4%), 1 by bacteriological culturing (0.4%) and 1 by bacilloscopy (0.4%). This finding suggests, as in previous reports, that ITT, normally used for bovine tuberculosis detection, has the inconvenience of having a broad range of specificity and sensitivity, and the PCR technique is a more specific and sensitive test to detect infection associated with M. bovis. Therefore, we propose this PCR assay as a useful tool in the epidemiological characterization of infected animals in areas considered to be at high risk of transmission. Images Figure 1. PMID:10369566

Toxicogenomic response of Mycobacterium bovis BCG to peracetic acid and a comparative analysis of the M. bovis BCG response to three oxidative disinfectants.

PubMed

Nde, Chantal W; Toghrol, Freshteh; Jang, Hyeung-Jin; Bentley, William E

2011-04-01

Tuberculosis is a leading cause of death worldwide and infects thousands of Americans annually. Mycobacterium bovis causes tuberculosis in humans and several animal species. Peracetic acid is an approved tuberculocide in hospital and domestic environments. This study presents for the first time the transcriptomic changes in M. bovis BCG after treatment with 0.1 mM peracetic acid for 10 and 20 min. This study also presents for the first time a comparison among the transcriptomic responses of M. bovis BCG to three oxidative disinfectants: peracetic acid, sodium hypochlorite, and hydrogen peroxide after 10 min of treatment. Results indicate that arginine biosynthesis, virulence, and oxidative stress response genes were upregulated after both peracetic acid treatment times. Three DNA repair genes were downregulated after 10 and 20 min and cell wall component genes were upregulated after 20 min. The devR-devS signal transduction system was upregulated after 10 min, suggesting a role in the protection against peracetic acid treatment. Results also suggest that peracetic acid and sodium hypochlorite both induce the expression of the ctpF gene which is upregulated in hypoxic environments. Further, this study reveals that in M. bovis BCG, hydrogen peroxide and peracetic acid both induce the expression of katG involved in oxidative stress response and the mbtD and mbtI genes involved in iron regulation/virulence.

Prevalence and Risk Factors for Mycobacterium bovis Infection in African Lions ( Panthera leo ) in the Kruger National Park.

PubMed

Sylvester, Tashnica Taime; Martin, Laura Elizabeth Rosen; Buss, Peter; Loxton, Andre Gareth; Hausler, Guy Anton; Rossouw, Leana; van Helden, Paul; Parsons, Sven David Charles; Olea-Popelka, Francisco; Miller, Michele Ann

2017-04-01

Mycobacterium bovis, the causative agent of bovine tuberculosis (BTB), is endemic in the Kruger National Park (KNP), South Africa. African lions ( Panthera leo ) are susceptible to BTB, but the impact of the disease on lion populations is unknown. In this study, we used a novel gene expression assay for chemokine (C-X-C motif) ligand 9 (CXCL9) to measure the prevalence of M. bovis infection in 70 free-ranging lions that were opportunistically sampled in the southern and central regions of the KNP. In the southern region of the KNP, the apparent prevalence of M. bovis infection was 54% (95% confidence interval [CI]=36.9-70.5%), compared with 33% (95% CI=18.0-51.8%) in the central region, an important difference (P=0.08). Prevalence of M. bovis infection in lions showed similar patterns to estimated BTB prevalence in African buffaloes ( Syncerus caffer ) in the same areas. Investigation of other risk factors showed a trend for older lions, males, or lions with concurrent feline immunodeficiency virus infection to have a higher M. bovis prevalence. Our findings demonstrate that the CXCL9 gene expression assay is a useful tool for the determination of M. bovis status in free-ranging lions and identifies important epidemiologic trends for future studies.

Development of a Gene Expression Assay for the Diagnosis of Mycobacterium bovis Infection in African Lions (Panthera leo).

PubMed

Olivier, T T; Viljoen, I M; Hofmeyr, J; Hausler, G A; Goosen, W J; Tordiffe, A S W; Buss, P; Loxton, A G; Warren, R M; Miller, M A; van Helden, P D; Parsons, S D C

2017-06-01

Mycobacterium bovis infection, the cause of bovine tuberculosis (BTB), is endemic in wildlife in the Kruger National Park (KNP), South Africa. In lions, a high infection prevalence and BTB mortalities have been documented in the KNP; however, the ecological consequences of this disease are currently unknown. Sensitive assays for the detection of this infection in this species are therefore required. Blood from M. bovis-exposed, M. bovis-unexposed, M. tuberculosis-exposed and M. bovis-infected lions was incubated in QuantiFERON ® -TB Gold (QFT) tubes containing either saline or ESAT-6/CFP-10 peptides. Using qPCR, selected reference genes were evaluated for expression stability in these samples and selected target genes were evaluated as markers of antigen-dependent immune activation. The abundance of monokine induced by gamma interferon (MIG/CXCL9) mRNA, measured in relation to that of YWHAZ, was used as a marker of ESAT-6/CFP-10 sensitization. The gene expression assay results were compared between lion groups, and lenient and stringent diagnostic cut-off values were calculated. This CXCL9 gene expression assay combines a highly specific stimulation platform with a sensitive diagnostic marker that allows for discrimination between M. bovis-infected and M. bovis-uninfected lions. © 2015 Blackwell Verlag GmbH.

Evaluation of gamma interferon (IFN-gamma)-induced protein 10 (IP-10) responses for detection of cattle infected with Mycobacterium bovis: comparisons to IFN-gamma responses

USDA-ARS?s Scientific Manuscript database

Gamma interferon (IFN-gamma)-induced protein 10 (IP-10) has recently shown promise as a diagnostic biomarker of Mycobacterium tuberculosis infection of humans. The aim of the current study was to compare IP-10 and IFN-gamma responses upon Mycobacterium bovis infection in cattle using archived sample...

Short communication: Proteomic characterization of tuberculin purified protein derivative from Mycobacterium bovis.

PubMed

Cho, Yun Sang; Jang, Young-Boo; Lee, Sang-Eun; Cho, Je-Yoel; Ahn, Jung-Mo; Hwang, Inyeong; Heo, Eunjeong; Nam, Hyang-Mi; Cho, Donghee; Her, Moon; Jean, Young Hwa; Jung, Suk Chan; Kim, Jong Man; Lee, Hee Soo; Lee, Keechan; Belisle, John T

2015-08-01

Bovine tuberculin purified protein derivative (bPPD) is used as an intradermal test (IT) reagent to detect bovine tuberculosis (bTB) in most countries. Identification of bPPD proteins is critical to understanding the immunological reaction of IT at the molecular level. While bPPD from the United Kingdom (UK) and Brazil (BR) have been recently defined at the proteomic level, bPPD from the Republic of Korea (KR) has not yet been analyzed. Here, bPPD KR proteome was examined for the first time. In total, 271 proteins were identified, including Mycobacterium bovis-specific proteins Mb0854c and Mb2898, and 42 known T cell antigens. On comparing with proteomes of bPPD UK and BR, 33 proteins were found to be common among all three bPPDs, of which 15 proteins were T cell antigens. M. bovis-specific antigens with T cell activity in bPPD may be novel candidates for use as alternatives to currently available bPPD in diagnostics. Copyright © 2015 Elsevier Ltd. All rights reserved.

Characterization of bovine gamma delta T cells phenotype during post-natal development and following Mycobacterium bovis vaccination or virulent infection

USDA-ARS?s Scientific Manuscript database

Bovine tuberculosis caused by Mycobacterium bovis is a globally significant veterinary health problem. Gamma delta T cells are known to participate in the immune control of mycobacterial infections. Data in human and non-human primates suggest that mycobacterial infection regulates memory/effector p...

Direct Detection and Identification of Mycobacterium tuberculosis and Mycobacterium bovis in Bovine Samples by a Novel Nested PCR Assay: Correlation with Conventional Techniques

PubMed Central

Mishra, A.; Singhal, A.; Chauhan, D. S.; Katoch, V. M.; Srivastava, K.; Thakral, S. S.; Bharadwaj, S. S.; Sreenivas, V.; Prasad, H. K.

2005-01-01

Mycobacterium tuberculosis and M. bovis infect animals and humans. Their epidemiologies in developed and developing countries differ, owing to differences in the implementation of preventive measures (World Health Organization, 1999). Identification and differentiation of these closely related mycobacterial species would help to determine the source, reservoirs of infection, and disease burden due to diverse mycobacterial pathogens. The utility of the hupB gene (Rv2986c in M. tuberculosis, or Mb3010c in M. bovis) to differentiate M. tuberculosis and M. bovis was evaluated by a PCR-restriction fragment length polymorphism (RFLP) assay with 56 characterized bovine isolates (S. Prabhakar et al., J. Clin. Microbiol. 42:2724-2732, 2004). The degree of concordance between the PCR-RFLP assay and the microbiological characterization was 99.0% (P < 0.001). A nested PCR (N-PCR) assay was developed, replacing the PCR-RFLP assay for direct detection of M. tuberculosis and M. bovis in bovine samples. The N-PCR products of M. tuberculosis and M. bovis corresponded to 116 and 89 bp, respectively. The detection limit of mycobacterial DNA by N-PCR was 50 fg, equivalent to five tubercle bacilli. M. tuberculosis and/or M. bovis was detected in 55.5% (105/189) of the samples by N-PCR, compared to 9.4% (18/189) by culture. The sensitivities of N-PCR and culture were 97.3 and 29.7, respectively, and their specificities were 22.2 and 77.7%, respectively. The percentages of animals or samples identified as infected with M. tuberculosis or M. bovis by N-PCR and culture reflected the clinical categorizations of the cattle (P of <0.05 to <0.01). Mixed infection by N-PCR was detected in 22 animals, whereas by culture mixed infection was detected in 1 animal. PMID:16272503

Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination

USDA-ARS?s Scientific Manuscript database

Polyfunctional T cells simultaneously produce IFN-gamma, IL-2 and TNF-alpha and play relevant roles in several chronic infections, including TB. Mycobacterium bovis infection of cattle elicits ex vivo polyfunctional T cell responses. Vaccine-elicited IFN-gamma Tcm (CD4+ CD45RO+ CCR7+) responses corr...

Use of the Intradermal Tuberculin Test in a Herd of Captive Elk (Cervus elaphus nelsoni) Naturally Infected with Mycobacterium bovis

USDA-ARS?s Scientific Manuscript database

Tuberculosis of captive Cervidae, caused by Mycobacterium bovis, attracted attention in 1991 in the United States when investigations, prompted by the identification of a tuberculous elk (Cervus elaphus nelsoni) in Canada, revealed infected captive elk herds in 8 different states. Based on methods u...

An oral Mycobacterium bovis BCG vaccine for wildlife produced in the absence of animal-derived reagents.

PubMed

Cross, Martin L; Lambeth, Matthew R; Aldwell, Frank E

2009-09-01

Cultures of Mycobacterium bovis BCG, comprising predominantly single-cell bacilli, were prepared in broth without animal-derived reagents. When formulated into a vegetable-derived lipid matrix, the vaccine was stable in vitro and was immunogenic in vivo upon feeding it to mice. This formulation could be useful for oral vaccination of wildlife against tuberculosis, where concern over transmissible prions may preclude the field use of vaccines containing animal products.

Polyfunctional cytokine production by central memory T cells from cattle in response to Mycobacterium bovis infection and BCG vaccination

USDA-ARS?s Scientific Manuscript database

Polyfunctional T cells simultaneously produce IFN-gamma, IL-2 and TNF-alpha and play relevant roles in several chronic infections, including TB. Mycobacterium bovis infection of cattle elicits ex vivo polyfunctional T cell responses. Vaccine-elicited IFN-gamma Tcm (CD4 plus CD45RO plus CCR7 plus) re...

The variability and seasonality of the environmental reservoir of Mycobacterium bovis shed by wild European badgers.

PubMed

King, Hayley C; Murphy, Andrew; James, Phillip; Travis, Emma; Porter, David; Hung, Yu-Jiun; Sawyer, Jason; Cork, Jennifer; Delahay, Richard J; Gaze, William; Courtenay, Orin; Wellington, Elizabeth M

2015-08-06

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, has been increasing in UK cattle herds resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir of infection. One likely route of transmission to cattle is through exposure to infected badger urine and faeces. The relative importance of the environment in transmission remains unknown, in part due to the lack of information on the distribution and magnitude of environmental reservoirs. Here we identify potential infection hotspots in the badger population and quantify the heterogeneity in bacterial load; with infected badgers shedding between 1 × 10(3)- 4 × 10(5) M. bovis cells g(-1) of faeces, creating a substantial and seasonally variable environmental reservoir. Our findings highlight the potential importance of monitoring environmental reservoirs of M. bovis which may constitute a component of disease spread that is currently overlooked and yet may be responsible for a proportion of transmission amongst badgers and onwards to cattle.

The variability and seasonality of the environmental reservoir of Mycobacterium bovis shed by wild European badgers

PubMed Central

King, Hayley C.; Murphy, Andrew; James, Phillip; Travis, Emma; Porter, David; Hung, Yu-Jiun; Sawyer, Jason; Cork, Jennifer; Delahay, Richard J.; Gaze, William; Courtenay, Orin; Wellington, Elizabeth M.

2015-01-01

The incidence of Mycobacterium bovis, the causative agent of bovine tuberculosis, has been increasing in UK cattle herds resulting in substantial economic losses. The European badger (Meles meles) is implicated as a wildlife reservoir of infection. One likely route of transmission to cattle is through exposure to infected badger urine and faeces. The relative importance of the environment in transmission remains unknown, in part due to the lack of information on the distribution and magnitude of environmental reservoirs. Here we identify potential infection hotspots in the badger population and quantify the heterogeneity in bacterial load; with infected badgers shedding between 1 × 103 − 4 × 105 M. bovis cells g−1 of faeces, creating a substantial and seasonally variable environmental reservoir. Our findings highlight the potential importance of monitoring environmental reservoirs of M. bovis which may constitute a component of disease spread that is currently overlooked and yet may be responsible for a proportion of transmission amongst badgers and onwards to cattle. PMID:26247348

Cross reactive immune responses in cattle arising from exposure to Mycobacterium bovis and non-tuberculous mycobacteria.

PubMed

Jenkins, A O; Gormley, E; Gcebe, N; Fosgate, G T; Conan, A; Aagaard, C; Michel, A L; Rutten, V P M G

2018-04-01

Accurate diagnosis of tuberculosis in cattle may be compromised in areas where there are high rates of exposure to environmental/non-tuberculous mycobacteria (NTM). This cross reaction of immune responses to Mycobacterium bovis antigens shared with NTMs can result in reduced specificity of commonly used diagnostic tests including tuberculin skin tests and the interferon gamma assay (IFN-ɣ). In this study we assessed the cross-reactive immune responses of M. bovis (infected) and NTM exposed animals to M. bovis and M. avium tuberculin, the ESAT6/CFP10 cocktail antigen, tuberculin derived from cultures of selected NTMs, and a panel of recombinant mycobacterium tuberculosis complex (MTBC) antigens sharing homology with orthologues in NTM. Gamma interferon (IFN-ɣ) responses were measured in whole blood cultures using the IFN-ɣ assay and the IFN-ɣ elispot assay on purified peripheral blood mononuclear cells (PBMC). We observed the expected strong IFN-ɣ response to PPD-B in the M. bovis infected animals that distinguished this group from non-infected NTM exposed cattle. The IFN-ɣ responses to PPD-N (M. nonchromogenicum), were relatively high in both infected and non-infected NTM exposed cattle, but were not significantly different to classify the true infection status of each group. The results indicated that the cross-reactive responses to PPD-B and/or PPD-A with PPD-N, likely arose from prior exposure to environmental non-tuberculous mycobacteria. The IFN-ɣ immune responses to the 10 R-Mag measured by the IFN-ɣ elispot assay revealed that three of the selected antigens, Rv3615 (ESpC), Rv0287 (esxG) and the ESAT6/CFP10, were immunogenic in the infected cattle, and distinguished the infected cattle from the non-infected NTM exposed animals. The combined data of PPDs and R-Mags derived from NTM mycobacteria may prove useful in future development of novel bTB diagnostic tests. Copyright © 2018 Elsevier B.V. All rights reserved.

Evaluation of MIRU-VNTR for typing of Mycobacterium bovis isolated from Sika deer in Northeast China.

PubMed

Yang, Li; Wang, Chunyu; Wang, Haijun; Meng, Qingfeng; Wang, Quankai

2015-04-11

Bovine tuberculosis has led to serious economic losses for Sika Deer producers in China. Strategies for controlling the spread of Mycobacterium bovis are often hampered by a lack of epidemiological data. Specifically, tracing infections requires the ability to trace back infections, which, in turn, requires the ability to determine isolates with a common source. This study was planned to assess the discriminatory power of each mycobacterial interspersed repetitive unit (MIRU)-variable number tandem repeats (VNTR) locus and evaluate the most appropriate combination of MIRU-VNTR loci for molecular epidemiological studies on Sika Deer in China. The discriminatory power of MIRU-VNTR typing based on 22 known loci (12 MIRUs, 2 ETRs, 4 QUBs, and 4 Mtubs) were assessed in 96 Mycobacterium bovis strains collected sequentially from Sika Deer at a slaughterhouse in northeastern China. We defined four loci (MIRU4, ETRA, QUB11b, and Mtub4) as highly discriminative, eight loci (MIRU2, MIRU23, MIRU27, MIRU31, MIRU39, MIRU40, QUB26, and Mtub21) as moderately discriminative, and three loci (MIRU16, Mtub30, and Mtub34) as poorly discriminative. The final locus showed no polymorphism between strains. MIRU-VNTR typing as a whole was highly discriminative, with an overall allelic diversity of 0.897. Of the loci tested, the four highly discriminative loci and eight moderately discriminative loci proved to be most appropriate for first line typing of M. bovis from Sika Deer, with the same resolving ability as all 22 loci (H = 0.897). MIRU-VNTR typing is quick and effective for typing bovine tuberculosis isolates from Sika Deer in China.

Efficacy of parenteral vaccination against tuberculosis with heat-inactivated Mycobacterium bovis in experimentally challenged goats.

PubMed

Arrieta-Villegas, Claudia; Perálvarez, Tania; Vidal, Enric; Puighibet, Zoë; Moll, Xavier; Canturri, Albert; Sevilla, Iker A; Espada, Yvonne; Juste, Ramón A; Domingo, Mariano; Pérez de Val, Bernat

2018-01-01

Tuberculosis (TB) in animals is a re-emerging disease with a wide range of hosts that causes large economic losses in livestock. Goats are particularly susceptible to TB and, in endemic areas, vaccination may be a valuable measure to control the disease. The main aim of this study was to evaluate the efficacy of parenteral vaccination of goats with a heat-inactivated Mycobacterium bovis (HIMB) vaccine, and compare it to M. bovis Bacille Calmette-Guérin (BCG) vaccine. Twenty-four goat kids were divided in 3 groups as following: HIMB vaccinated group (n = 8), BCG vaccinated group (n = 8) and unvaccinated group (n = 8). Afterwards, goats were experimentally challenged with Mycobacterium caprae by the endobronchial route. Antigen specific interferon-γ release assays and serology were performed after vaccination and challenge. Pathological and bacteriological parameters were evaluated after necropsy at 9 weeks post-challenge (p.c.). HIMB vaccine showed similar levels of protection to BCG in terms of volume reduction of thoracic TB lesions, presence of extra-pulmonary lesions, as well as a slight reduction of bacterial load in pulmonary lymph nodes. Moreover, HIMB vaccine did not induce interferences on the interferon-γ release assay based on reagents previously developed to differentiate infected from BCG vaccinated individuals. The results indicate that HIMB is a suitable vaccine candidate for further larger-scale trials under field conditions in goats.

Assessment of Mycobacterium bovis deleted in p27-p55 virulence operon as candidate vaccine against tuberculosis in animal models.

PubMed

Bianco, María V; Clark, Simon; Blanco, Federico C; Garbaccio, Sergio; García, Elizabeth; Cataldi, Angel A; Williams, Ann; Bigi, Fabiana

2014-01-01

A Mycobacterium bovis knockout in p27-p55 operon was tested as an antituberculosis experimental vaccine in animal models. The mutant MbΔp27-p55 was significantly more attenuated in nude mice than its parental strain but more virulent than BCG Pasteur. Challenge experiments in mice and guinea pigs using M. bovis or M. tuberculosis strains showed similar protection conferred by MbΔp27-p55 mutant than BCG in terms of pathology and bacterial loads in spleen but lower protection than BCG in lungs. When tested in cattle, MbΔp27-p55 did not induce IL-2 expression and induced a very low production of IFNγ, suggesting that the lack of P27/P55 reduces the capacity of M. bovis of triggering an adequate Th1 response.

Mycobacterium bovis infection in the lion (Panthera leo): Current knowledge, conundrums and research challenges.

PubMed

Viljoen, Ignatius M; van Helden, Paul D; Millar, Robert P

2015-06-12

Mycobacterium bovis has global public-health and socio-economic significance and can infect a wide range of species including the lion (Panthera leo) resulting in tuberculosis. Lions are classified as vulnerable under the IUCN Red List of Threatened Species and have experienced a 30% population decline in the past two decades. However, no attempt has been made to collate and critically evaluate the available knowledge of M. bovis infections in lions and potential effects on population. In this review we set out to redress this. Arguments suggesting that ingestion of infected prey animals are the main route of infection for lions have not been scientifically proven and research is needed into other possible sources and routes of infection. The paucity of knowledge on host susceptibility, transmission directions and therefore host status, manifestation of pathology, and epidemiology of the disease in lions also needs to be addressed. Advances have been made in diagnosing the presence of M. bovis in lions. However, these diagnostic tests are unable to differentiate between exposure, presence of infection, or stage of disease. Furthermore, there are contradictory reports on the effects of M. bovis on lion populations with more data needed on disease dynamics versus the lion population's reproductive dynamics. Knowledge on disease effects on the lion reproduction and how additional stressors such as drought or co-morbidities may interact with tuberculosis is also lacking. Filling these knowledge gaps will contribute to the understanding of mycobacterial infections and disease in captive and wild lions and assist in lion conservation endeavours. Copyright © 2015 Elsevier B.V. All rights reserved.

DIAGNOSIS AND IMPLICATIONS OF MYCOBACTERIUM BOVIS INFECTION IN BANDED MONGOOSES (MUNGOS MUNGO) IN THE KRUGER NATIONAL PARK, SOUTH AFRICA.

PubMed

Brüns, Angela C; Tanner, Manfred; Williams, Mark C; Botha, Louise; O'Brien, Amanda; Fosgate, Geoffrey T; van Helden, Paul D; Clarke, John; Michel, Anita L

2017-01-01

Bovine tuberculosis (bTB) was first diagnosed in the Kruger National Park (KNP) in 1990. Research has since focused on the maintenance host, the African buffalo ( Syncerus caffer ) and clinically affected lion ( Panthera leo ). However, little is known about the role of small predators in tuberculosis epidemiology. During 2011-12, we screened banded mongooses ( Mungos mungo ) in the bTB high-prevalence zone of the KNP for Mycobacterium tuberculosis complex members. Fecal swabs, tracheal swabs, and tracheal lavages of 76 banded mongooses caught in cage traps within a 2-km radius of Skukuza Rest Camp were submitted for Mycobacterium culture, isolation, and species identification. Lesions and lymph node samples collected from 12 animals at postmortem examination were submitted for culture and histopathology. In lung and lymph nodes of two banded mongooses, well demarcated, irregularly margined, gray-yellow nodules of up to 5 mm diameter were identified with either central necrosis or calcification, characterized on histopathology as caseating necrosis with epithelioid macrophages or necrogranuloma with calcified centre. No acid fast bacteria were identified with Ziehl-Neelsen stain. We isolated Mycobacterium bovis from lung, lymph node, and liver samples, as well as from tracheal lavages and tracheal swab from the same two banded mongooses. Blood samples were positive by ElephantTB STAT-PAK® Assay for 12 and Enferplex™ TB Assay for five animals. Only the two banded mongooses positive on pathology and M. bovis culture were positive on both serologic assays. We provide evidence of bTB infection in banded mongooses in the KNP, demonstrate their ability to shed M. bovis , and propose a possible antemortem diagnostic algorithm. Our findings open the discussion around possible sources of infection and their significance at the human/wildlife interface in and around Skukuza.

Intradermal tuberculin testing of wild African lions (Panthera leo) naturally exposed to infection with Mycobacterium bovis.

PubMed

Keet, D F; Michel, A L; Bengis, R G; Becker, P; van Dyk, D S; van Vuuren, M; Rutten, V P M G; Penzhorn, B L

2010-08-26

African lions in the southern half of Kruger National Park (KNP) are infected with Mycobacterium bovis. Historically, reliable detection of mycobacteriosis in lions was limited to necropsy and microbiological analysis of lesion material collected from emaciated and ailing or repeat-offender lions. We report on a method of cervical intradermal tuberculin testing of lions and its interpretation capable of identifying natural exposure to M. bovis. Infected lions (n=52/95) were identified by detailed necropsy and mycobacterial culture. A large proportion of these confirmed infected lions (45/52) showed distinct responses to bovine tuberculin purified protein derivative (PPD) while responses to avian tuberculin PPD were variable and smaller. Confirmed uninfected lions from non-infected areas (n=11) responded variably to avian tuberculin PPD only. Various non-tuberculous mycobacteria (NTM) were cultured from 45/95 lions examined, of which 21/45 were co-infected with M. bovis. Co-infection with M. bovis and NTM did not influence skin reactions to bovine tuberculin PPD. Avian tuberculin PPD skin reactions were larger in M. bovis-infected lions compared to uninfected ones. Since NTM co-infections are likely to influence the outcome of skin testing, stricter test interpretation criteria were applied. When test data of bovine tuberculin PPD tests were considered on their own, as for a single skin test, sensitivity increased (80.8-86.5%) but false positive rate for true negatives (18.75%) remained unchanged. Finally, the adapted skin test procedure was shown not to be impeded by persistent Feline Immunodeficiency Virus(Ple) co-infection. Copyright 2010 Elsevier B.V. All rights reserved.

A Pilot Study Exploring the Use of Breath Analysis to Differentiate Healthy Cattle from Cattle Experimentally Infected with Mycobacterium bovis

PubMed Central

Ellis, Christine K.; Stahl, Randal S.; Nol, Pauline; Waters, W. Ray; Palmer, Mitchell V.; Rhyan, Jack C.; VerCauteren, Kurt C.; McCollum, Matthew; Salman, M. D.

2014-01-01

Bovine tuberculosis, caused by Mycobacterium bovis, is a zoonotic disease of international public health importance. Ante-mortem surveillance is essential for control; however, current surveillance tests are hampered by limitations affecting ease of use or quality of results. There is an emerging interest in human and veterinary medicine in diagnosing disease via identification of volatile organic compounds produced by pathogens and host-pathogen interactions. The objective of this pilot study was to explore application of existing human breath collection and analysis methodologies to cattle as a means to identify M. bovis infection through detection of unique volatile organic compounds or changes in the volatile organic compound profiles present in breath. Breath samples from 23 male Holstein calves (7 non-infected and 16 M. bovis-infected) were collected onto commercially available sorbent cartridges using a mask system at 90 days post-inoculation with M. bovis. Samples were analyzed using gas chromatography-mass spectrometry, and chromatographic data were analyzed using standard analytical chemical and metabolomic analyses, principle components analysis, and a linear discriminant algorithm. The findings provide proof of concept that breath-derived volatile organic compound analysis can be used to differentiate between healthy and M. bovis-infected cattle. PMID:24586655

Trends of Mycobacterium bovis Isolation and First-Line Anti-tuberculosis Drug Susceptibility Profile: A Fifteen-Year Laboratory-Based Surveillance.

PubMed

Bobadilla-del Valle, Miriam; Torres-González, Pedro; Cervera-Hernández, Miguel Enrique; Martínez-Gamboa, Areli; Crabtree-Ramirez, Brenda; Chávez-Mazari, Bárbara; Ortiz-Conchi, Narciso; Rodríguez-Cruz, Luis; Cervantes-Sánchez, Axel; Gudiño-Enríquez, Tomasa; Cinta-Severo, Carmen; Sifuentes-Osornio, José; Ponce de León, Alfredo

2015-09-01

Mycobacterium tuberculosis causes the majority of tuberculosis (TB) cases in humans; however, in developing countries, human TB caused by M. bovis may be frequent but undetected. Human TB caused by M. bovis is considered a zoonosis; transmission is mainly through consumption of unpasteurized dairy products, and it is less frequently attributed to animal-to-human or human-to-human contact. We describe the trends of M. bovis isolation from human samples and first-line drug susceptibility during a 15-year period in a referral laboratory located in a tertiary care hospital in Mexico City. Data on mycobacterial isolates from human clinical samples were retrieved from the laboratory's database for the 2000-2014 period. Susceptibility to first-line drugs: rifampin, isoniazid, streptomycin (STR) and ethambutol was determined. We identified 1,165 isolates, 73.7% were M. tuberculosis and 26.2%, M. bovis. Among pulmonary samples, 16.6% were M. bovis. The proportion of M. bovis isolates significantly increased from 7.8% in 2000 to 28.4% in 2014 (X(2)trend, p<0.001). Primary STR resistance was higher among M. bovis compared with M. tuberculosis isolates (10.9% vs.3.4%, p<0.001). Secondary multidrug resistance (MDR) rates were 38.5% and 34.4% for M. bovis and M. tuberculosis, respectively (p = 0.637). A rising trend of primary STR monoresistance was observed for both species (3.4% in 2000-2004 vs. 7.6% in 2010-2014; p = 0.02). There is a high prevalence and a rising trend of M. bovis isolates in our region. The proportion of pulmonary M. bovis isolates is higher than in previous reports. Additionally, we report high rates of primary anti-tuberculosis resistance and secondary MDR in both M. tuberculosis and M. bovis. This is one of the largest reports on drug susceptibility of M. bovis from human samples and shows a significant proportion of first-line anti-tuberculosis drug resistance.

Mycobacterium bovis Infection of Cattle and White-Tailed Deer: Translational Research of Relevance to Human Tuberculosis.

PubMed

Waters, W Ray; Palmer, Mitchell V

2015-01-01

Tuberculosis (TB) is a premier example of a disease complex with pathogens primarily affecting humans (i.e., Mycobacterium tuberculosis) or livestock and wildlife (i.e., Mycobacterium bovis) and with a long history of inclusive collaborations between physicians and veterinarians. Advances in the study of bovine TB have been applied to human TB, and vice versa. For instance, landmark discoveries on the use of Koch's tuberculin and interferon-γ release assays for diagnostic purposes, as well as Calmette and Guérin's attenuated M. bovis strain as a vaccine, were first evaluated in cattle for control of bovine TB prior to wide-scale use in humans. Likewise, recent discoveries on the role of effector/memory T cell subsets and polyfunctional T cells in the immune response to human TB, particularly as related to vaccine efficacy, have paved the way for similar studies in cattle. Over the past 15 years, substantial funding for development of human TB vaccines has led to the emergence of multiple promising candidates now in human clinical trials. Several of these vaccines are being tested for immunogenicity and efficacy in cattle. Also, the development of population-based vaccination strategies for control of M. bovis infection in wildlife reservoirs will undoubtedly have an impact on our understanding of herd immunity with relevance to the control of both bovine and human TB in regions of the world with high prevalence of TB. Thus, the one-health approach to research on TB is mutually beneficial for our understanding and control of TB in humans, livestock, and wildlife. Published by Oxford University Press 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

Testing Eurasian wild boar piglets for serum antibodies against Mycobacterium bovis.

PubMed

Che' Amat, A; González-Barrio, D; Ortiz, J A; Díez-Delgado, I; Boadella, M; Barasona, J A; Bezos, J; Romero, B; Armenteros, J A; Lyashchenko, K P; Venteo, A; Rueda, P; Gortázar, C

2015-09-01

Animal tuberculosis (TB) caused by infection with Mycobacterium bovis and closely related members of the M. tuberculosis complex (MTC), is often reported in the Eurasian wild boar (Sus scrofa). Tests detecting antibodies against MTC antigens are valuable tools for TB monitoring and control in suids. However, only limited knowledge exists on serology test performance in 2-6 month-old piglets. In this age-class, recent infections might cause lower antibody levels and lower test sensitivity. We examined 126 wild boar piglets from a TB-endemic site using 6 antibody detection tests in order to assess test performance. Bacterial culture (n=53) yielded a M. bovis infection prevalence of 33.9%, while serum antibody prevalence estimated by different tests ranged from 19% to 38%, reaching sensitivities between 15.4% and 46.2% for plate ELISAs and between 61.5% and 69.2% for rapid immunochromatographic tests based on dual path platform (DPP) technology. The Cohen kappa coefficient of agreement between DPP WTB (Wildlife TB) assay and culture results was moderate (0.45) and all other serological tests used had poor to fair agreements. This survey revealed the ability of several tests for detecting serum antibodies against the MTC antigens in 2-6 month-old naturally infected wild boar piglets. The best performance was demonstrated for DPP tests. The results confirmed our initial hypothesis of a lower sensitivity of serology for detecting M. bovis-infected piglets, as compared to older wild boar. Certain tests, notably the rapid animal-side tests, can contribute to TB control strategies by enabling the setup of test and cull schemes or improving pre-movement testing. However, sub-optimal test performance in piglets as compared to that in older wild boar should be taken into account. Copyright © 2015 Elsevier B.V. All rights reserved.

Diagnostic value of animal-side antibody assays for rapid detection of Mycobacterium bovis or Mycobacterium microti infection in South American camelids.

PubMed

Lyashchenko, Konstantin P; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, H Martin; Zanolari, Patrik

2011-12-01

Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB.

Genotyping and rifampicin and isoniazid resistance in Mycobacterium bovis strains isolated from the lymph nodes of slaughtered cattle.

PubMed

Franco, Marília Masello Junqueira; Ribeiro, Márcio Garcia; Pavan, Fernando Rogério; Miyata, Marcelo; Heinemann, Marcos Bryan; de Souza Filho, Antonio Francisco; Cardoso, Rosilene Fressatti; de Almeida, Aryadne Larissa; Sakate, Ricardo Ichiro; Paes, Antonio Carlos

2017-05-01

In developing nations, 10-20% of the human cases of tuberculosis are caused by Mycobacterium bovis. However, this percentage may be underestimated because most laboratories in developing countries do not routinely perform mycobacterial cultures, and only a few have the systems in place to identify M. bovis. There are few studies investigating genotypic diversity and drug resistance in M. bovis from animal and/or human infections. The genotypic diversity of M. bovis strains obtained from bovine lymph nodes were investigated by spacer oligonucleotide typing (spoligotyping) and mycobacterial interspersed repetitive unit-variable-number tandem repeat typing (MIRU-VNTR). The phenotypic resistance to isoniazid and rifampicin and MIC values of the isolates were determined using the resazurin microtiter assay plate method (REMA). The evaluation of the possible genetic basis for such resistance was performed with GenoType MTBDRplus. Sixty-seven isolates were obtained, of which 11 (16%) were MDR-TB, 8 (12%) were isoniazid-resistant, and 2 (3%) were rifampicin-resistant. Mutations associated with drug resistance were not found. Genotyping techniques enabled the grouping of the strains into 12 clusters and 21 isolates with unique profiles. The high frequency of M. bovis reinforces the impact of the pathogen as a major causal agent of bovine tuberculosis in the study area. The resistance of the strains to drugs used for first-line treatment of human tuberculosis raises public health concerns. Further studies are required to elucidate the basis of drug resistance and genotypic diversity in M. bovis. Copyright © 2017 Elsevier Ltd. All rights reserved.

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins.

PubMed

Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C; Rutten, Victor

2016-01-01

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

Effect of milk fermentation by kefir grains and selected single strains of lactic acid bacteria on the survival of Mycobacterium bovis BCG.

PubMed

Macuamule, C L S; Wiid, I J; van Helden, P D; Tanner, M; Witthuhn, R C

2016-01-18

Mycobacterium bovis that causes Bovine tuberculosis (BTB) can be transmitted to humans thought consumption of raw and raw fermented milk products from diseased animals. Lactic acid bacteria (LAB) used in popular traditional milk products in Africa produce anti-microbial compounds that inhibit some pathogenic and spoilage bacteria. M. bovis BCG is an attenuated non-pathogenic vaccine strain of M. bovis and the aim of the study was to determine the effect of the fermentation process on the survival of M. bovis BCG in milk. M. bovis BCG at concentrations of 6 log CFU/ml was added to products of kefir fermentation. The survival of M. bovis BCG was monitored at 12-h intervals for 72 h by enumerating viable cells on Middlebrook 7H10 agar plates enriched with 2% BD BACTEC PANTA™. M. bovis BCG was increasingly reduced in sterile kefir that was fermented for a period of 24h and longer. In the milk fermented with kefir grains, Lactobacillus paracasei subsp. paracasei or Lactobacillus casei, the viability of M. bovis BCG was reduced by 0.4 logs after 24h and by 2 logs after 48 h of fermentation. No viable M. bovis BCG was detected after 60 h of fermentation. Results from this study show that long term fermentation under certain conditions may have the potential to inactivate M. bovis BCG present in the milk. However, to ensure safety of fermented milk in Africa, fermentation should be combined with other hurdle technologies such as boiling and milk pasteurisation. Copyright © 2015 Elsevier B.V. All rights reserved.

Whole genome sequencing of Mycobacterium bovis to obtain molecular fingerprints in human and cattle isolates from Baja California, Mexico.

PubMed

Sandoval-Azuara, Sarai Estrella; Muñiz-Salazar, Raquel; Perea-Jacobo, Ricardo; Robbe-Austerman, Suelee; Perera-Ortiz, Alejandro; López-Valencia, Gilberto; Bravo, Doris M; Sanchez-Flores, Alejandro; Miranda-Guzmán, Daniela; Flores-López, Carlos Alberto; Zenteno-Cuevas, Roberto; Laniado-Laborín, Rafael; de la Cruz, Fabiola Lafarga; Stuber, Tod P

2017-10-01

To determine genetic diversity by comparing the whole genome sequences of cattle and human Mycobacterium bovis isolates from Baja California. A whole genome sequencing strategy was used to obtain the molecular fingerprints of 172 isolates of M. bovis obtained from Baja California, Mexico; 155 isolates were from cattle and 17 isolates were from humans. Spoligotypes were characterized in silico and single nucleotide polymorphism (SNP) differences between the isolates were evaluated. A total of 12 M. bovis spoligotype patterns were identified in cattle and humans. Two predominant spoligotypes patterns were seen in both cattle and humans: SB0145 and SB1040. The SB0145 spoligotype represented 59% of cattle isolates (n=91) and 65% of human isolates (n=11), while the SB1040 spoligotype represented 30% of cattle isolates (n=47) and 30% of human isolates (n=5). When evaluating SNP differences, the human isolates were intimately intertwined with the cattle isolates. All isolates from humans had spoligotype patterns that matched those observed in the cattle isolates, and all human isolates shared common ancestors with cattle in Baja California based on SNP analysis. This suggests that most human tuberculosis caused by M. bovis in Baja California is derived from M. bovis circulating in Baja California cattle. These results reinforce the importance of bovine tuberculosis surveillance and control in this region. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

PubMed

Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

2016-12-01

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

Structural features of lipoarabinomannan from Mycobacterium bovis BCG. Determination of molecular mass by laser desorption mass spectrometry.

PubMed

Venisse, A; Berjeaud, J M; Chaurand, P; Gilleron, M; Puzo, G

1993-06-15

It was recently shown that mycobacterial lipoarabinomannan (LAM) can be classified into two types (Chatterjee, D., Lowell, K., Rivoire B., McNeil M. R., and Brennan, P. J. (1992) J. Biol. Chem. 267, 6234-6239) according to the presence or absence of mannosyl residues (Manp) located at the nonreducing end of the oligoarabinosyl side chains. These two types of LAM were found in a pathogenic Mycobacterium tuberculosis strain and in an avirulent M. tuberculosis strain, respectively, suggesting that LAM with Manp characterizes virulent and "disease-inducing strains." We now report the structure of the LAM from Mycobacterium bovis Bacille Calmette-Guérin (BCG) strain Pasteur, largely used throughout the world as vaccine against tuberculosis. Using an up-to-date analytical approach, we found that the LAM of M. bovis BCG belongs to the class of LAMs capped with Manp. By means of two-dimensional homonuclear and heteronuclear scalar coupling NMR analysis and methylation data, the sugar spin system assignments were partially established, revealing that the LAM contained two types of terminal Manp and 2-O-linked Manp. From the following four-step process: (i) partial hydrolysis of deacylated LAM (dLAM), (ii) oligosaccharide derivatization with aminobenzoic ethyl ester, (iii) HPLC purification, (iv) FAB/MS-MS analysis; it was shown that the dimannosyl unit alpha-D-Manp-(1-->2)-alpha-D-Manp is the major residue capping the termini of the arabinan of the LAM. In this report, LAM molecular mass determination was established using matrix-assisted UV-laser desorption/ionization mass spectrometry which reveals that the LAM molecular mass is around 17.4 kDa. The similarity of the LAM structures between M. bovis BCG and M. tuberculosis H37Rv is discussed in regard to their function in the immunopathology of mycobacterial infection.

Endogenous and Exogenous KdpF Peptide Increases Susceptibility of Mycobacterium bovis BCG to Nitrosative Stress and Reduces Intramacrophage Replication

PubMed Central

Rosas Olvera, Mariana; Vivès, Eric; Molle, Virginie; Blanc-Potard, Anne-Béatrice; Gannoun-Zaki, Laila

2017-01-01

Emerging antibiotic resistance in pathogenic bacteria like Mycobacterium sp., poses a threat to human health and therefore calls for the development of novel antibacterial strategies. We have recently discovered that bacterial membrane peptides, such as KdpF, possess anti-virulence properties when overproduced in pathogenic bacterial species. Overproduction of the KdpF peptide in Mycobacterium bovis BCG decreased bacterial replication within macrophages, without presenting antibacterial activity. We propose that KdpF functions as a regulatory molecule and interferes with bacterial virulence, potentially through interaction with the PDIM transporter MmpL7. We demonstrate here that KdpF overproduction in M. bovis BCG, increased bacterial susceptibility to nitrosative stress and thereby was responsible for lower replication rate within macrophages. Moreover, in a bacterial two-hybrid system, KdpF was able to interact not only with MmpL7 but also with two membrane proteins involved in nitrosative stress detoxification (NarI and NarK2), and a membrane protein of unknown function that is highly induced upon nitrosative stress (Rv2617c). Interestingly, we showed that the exogenous addition of KdpF synthetic peptide could affect the stability of proteins that interact with this peptide. Finally, the exogenous KdpF peptide presented similar biological effects as the endogenously expressed peptide including nitrosative stress susceptibility and reduced intramacrophage replication rate for M. bovis BCG. Taken together, our results establish a link between high levels of KdpF and nitrosative stress susceptibility to further highlight KdpF as a potent molecule with anti-virulence properties. PMID:28428950

Comparative Genomics and Proteomic Analysis of Four Non-tuberculous Mycobacterium Species and Mycobacterium tuberculosis Complex: Occurrence of Shared Immunogenic Proteins

PubMed Central

Gcebe, Nomakorinte; Michel, Anita; Gey van Pittius, Nicolaas C.; Rutten, Victor

2016-01-01

The Esx and PE/PPE families of proteins are among the most immunodominant mycobacterial antigens and have thus been the focus of research to develop vaccines and immunological tests for diagnosis of bovine and human tuberculosis, mainly caused by Mycobacterium bovis and Mycobacterium tuberculosis, respectively. In non-tuberculous mycobacteria (NTM), multiple copies of genes encoding homologous proteins have mainly been identified in pathogenic Mycobacterium species phylogenically related to Mycobacterium tuberculosis and Mycobacterium bovis. Only ancestral copies of these genes have been identified in nonpathogenic NTM species like Mycobacterium smegmatis, Mycobacterium sp. KMS, Mycobacterium sp. MCS, and Mycobacterium sp. JLS. In this study we elucidated the genomes of four nonpathogenic NTM species, viz Mycobacterium komanii sp. nov., Mycobacterium malmesburii sp. nov., Mycobacterium nonchromogenicum, and Mycobacterium fortuitum ATCC 6841. These genomes were investigated for genes encoding for the Esx and PE/PPE (situated in the esx cluster) family of proteins as well as adjacent genes situated in the ESX-1 to ESX-5 regions. To identify proteins actually expressed, comparative proteomic analyses of purified protein derivatives from three of the NTM as well as Mycobacterium kansasii ATCC 12478 and the commercially available purified protein derivatives from Mycobacterium bovis and Mycobacterium avium was performed. The genomic analysis revealed the occurrence in each of the four NTM, orthologs of the genes encoding for the Esx family, the PE and PPE family proteins in M. bovis and M. tuberculosis. The identification of genes of the ESX-1, ESX-3, and ESX-4 region including esxA, esxB, ppe68, pe5, and pe35 adds to earlier reports of these genes in nonpathogenic NTM like M. smegmatis, Mycobacterium sp. JLS and Mycobacterium KMS. This report is also the first to identify esxN gene situated within the ESX-5 locus in M. nonchromogenicum. Our proteomics analysis

Improved Specificity for Detection of Mycobacterium bovis in Fresh Tissues Using IS6110 Real-time PCR

USDA-ARS?s Scientific Manuscript database

Background: Culture of M. bovis from diagnostic specimens is the gold standard for bovine tuberculosis diagnostics in the US. Detection of M. bovis by PCR in tissue homogenates may provide a simple, rapid method to complement diagnostic culture. A significant impediment to PCR based assays on tissue...

Diagnostic Value of Animal-Side Antibody Assays for Rapid Detection of Mycobacterium bovis or Mycobacterium microti Infection in South American Camelids▿

PubMed Central

Lyashchenko, Konstantin P.; Greenwald, Rena; Esfandiari, Javan; Rhodes, Shelley; Dean, Gillian; de la Rua-Domenech, Ricardo; Meylan, Mireille; Vordermeier, HMartin; Zanolari, Patrik

2011-01-01

Tuberculosis (TB) in South American camelids (SAC) is caused by Mycobacterium bovis or Mycobacterium microti. Two serological methods, rapid testing (RT) and the dual-path platform (DPP) assay, were evaluated using naturally infected SAC. The study population included 156 alpacas and 175 llamas in Great Britain, Switzerland, and the United States. TB due to M. bovis (n = 44) or M. microti (n = 8) in 35 alpacas and 17 llamas was diagnosed by gross pathology examination and culture. Control animals were from herds with no TB history. The RT and the DPP assay showed sensitivities of 71% and 74%, respectively, for alpacas, while the sensitivity for llamas was 77% for both assays. The specificity of the DPP assay (98%) was higher than that of RT (94%) for llamas; the specificities of the two assays were identical (98%) for alpacas. When the two antibody tests were combined, the parallel-testing interpretation (applied when either assay produced a positive result) enhanced the sensitivities of antibody detection to 89% for alpacas and 88% for llamas but at the cost of lower specificities (97% and 93%, respectively), whereas the serial-testing interpretation (applied when both assays produced a positive result) maximized the specificity to 100% for both SAC species, although the sensitivities were 57% for alpacas and 65% for llamas. Over 95% of the animals with evidence of TB failed to produce skin test reactions, thus confirming concerns about the validity of this method for testing SAC. The findings suggest that serological assays may offer a more accurate and practical alternative for antemortem detection of camelid TB. PMID:22012976

Comparison of semi-automated commercial rep-PCR fingerprinting, spoligotyping, 12-locus MIRU-VNTR typing and single nucleotide polymorphism analysis of the embB gene as molecular typing tools for Mycobacterium bovis.

PubMed

Armas, Federica; Camperio, Cristina; Coltella, Luana; Selvaggini, Serena; Boniotti, Maria Beatrice; Pacciarini, Maria Lodovica; Di Marco Lo Presti, Vincenzo; Marianelli, Cinzia

2017-08-04

Highly discriminatory genotyping strategies are essential in molecular epidemiological studies of tuberculosis. In this study we evaluated, for the first time, the efficacy of the repetitive sequence-based PCR (rep-PCR) DiversiLab Mycobacterium typing kit over spoligotyping, 12-locus mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing and embB single nucleotide polymorphism (SNP) analysis for Mycobacterium bovis typing. A total of 49 M. bovis animal isolates were used. DNA was extracted and genomic DNA was amplified using the DiversiLab Mycobacterium typing kit. The amplified fragments were separated and detected using a microfluidics chip with Agilent 2100. The resulting rep-PCR-based DNA fingerprints were uploaded to and analysed using web-based DiversiLab software through Pearson's correlation coefficient. Rep-PCR DiversiLab grouped M. bovis isolates into ten different clusters. Most isolates sharing identical spoligotype, MIRU-VNTR profile or embB gene polymorphism were grouped into different rep-PCR clusters. Rep-PCR DiversiLab displayed greater discriminatory power than spoligotyping and embB SNP analysis but a lower resolution power than the 12-locus MIRU-VNTR analysis. MIRU-VNTR confirmed that it is superior to the other PCR-based methods tested here. In combination with spoligotyping and 12-locus MIRU-VNTR analysis, rep-PCR improved the discriminatory power for M. bovis typing.

Field evaluation of the efficacy of Mycobacterium bovis BCG vaccine against tuberculosis in goats.

PubMed

Vidal, Enric; Arrieta-Villegas, Claudia; Grasa, Miriam; Mercader, Irene; Domingo, Mariano; Pérez de Val, Bernat

2017-08-17

Control of animal tuberculosis (TB) through vaccination has emerged as a long-term strategy to complement test and slaughter control strategy. A pilot trial under field conditions was conducted in a goat herd with high TB prevalence to assess the efficacy of the Mycobacterium bovis BCG vaccine. Twenty-three goat kids vaccinated with BCG and other 22 unvaccinated control kids were euthanized at 18 months post-vaccination. Gross pathological and histopathological examination of target tissues was performed for detection of tuberculous lesions and assessment of vaccine efficacy. Mycobacterial culture and DNA detection were used to confirm Mycobacterium caprae infection. Vaccination significantly reduced the number of animals with TB lesions compared to unvaccinated controls (35% and 77%, respectively; P < 0.01). This reduction was even higher if only extra-pulmonary infection was considered (17% and 68%, respectively; P < 0.001). This trial demonstrates that BCG vaccination of goats can significantly reduce the TB lesion rates in high disease exposure conditions, indicating that vaccination could contribute to the control of TB in domestic goats.

Recombinant Mycobacterium bovis BCG for immunotherapy in nonmuscle invasive bladder cancer.

PubMed

Begnini, K R; Buss, J H; Collares, T; Seixas, F K

2015-05-01

In the past three decades, intravesical instillation of Mycobacterium bovis bacille Calmette-Guérin (BCG) has been used for treating bladder cancer and it still remains at the forefront of immunotherapy for cancer patients. Although BCG-based therapy is the most effective intravesical therapy for this kind of tumor and represents the only agent known to reduce progression into muscle invasive bladder cancer, BCG is ineffective in approximately 30-40 % of cases and disease recurs in up to 50 % of patients. Since that BCG is considered an effective vehicle for delivery of antigens due to its unique characteristics, the genetic manipulation of these mycobacteria has been appealing in the search for less toxic and more potent therapeutic agents for bladder cancer immunotherapy. Herein, we discuss current advances in recombinant BCG construction, research, concerns, and future directions to promote the development of this promising immunotherapeutic approach for bladder cancer.

Development of a model to simulate infection dynamics of Mycobacterium bovis in cattle herds in the United States

PubMed Central

Smith, Rebecca L.; Schukken, Ynte H.; Lu, Zhao; Mitchell, Rebecca M.; Grohn, Yrjo T.

2013-01-01

Objective To develop a mathematical model to simulate infection dynamics of Mycobacterium bovis in cattle herds in the United States and predict efficacy of the current national control strategy for tuberculosis in cattle. Design Stochastic simulation model. Sample Theoretical cattle herds in the United States. Procedures A model of within-herd M bovis transmission dynamics following introduction of 1 latently infected cow was developed. Frequency- and density-dependent transmission modes and 3 tuberculin-test based culling strategies (no test-based culling, constant (annual) testing with test-based culling, and the current strategy of slaughterhouse detection-based testing and culling) were investigated. Results were evaluated for 3 herd sizes over a 10-year period and validated via simulation of known outbreaks of M bovis infection. Results On the basis of 1,000 simulations (1000 herds each) at replacement rates typical for dairy cattle (0.33/y), median time to detection of M bovis infection in medium-sized herds (276 adult cattle) via slaughterhouse surveillance was 27 months after introduction, and 58% of these herds would spontaneously clear the infection prior to that time. Sixty-two percent of medium-sized herds without intervention and 99% of those managed with constant test-based culling were predicted to clear infection < 10 years after introduction. The model predicted observed outbreaks best for frequency-dependent transmission, and probability of clearance was most sensitive to replacement rate. Conclusions and Clinical Relevance Although modeling indicated the current national control strategy was sufficient for elimination of M bovis infection from dairy herds after detection, slaughterhouse surveillance was not sufficient to detect M bovis infection in all herds and resulted in subjectively delayed detection, compared with the constant testing method. Further research is required to economically optimize this strategy. PMID:23865885

Evaluation of Immunogenicity and Protective Efficacy Elicited by Mycobacterium bovis BCG Overexpressing Ag85A Protein against Mycobacterium tuberculosis Aerosol Infection.

PubMed

Xu, Zheng Zhong; Chen, Xiang; Hu, Ting; Meng, Chuang; Wang, Xiao Bo; Rao, Yan; Zhang, Xiao Ming; Yin, Yue Lan; Pan, Zhi Ming; Jiao, Xin An

2016-01-01

Mycobacterium bovis bacillus Calmette-Guérin (BCG) is currently the only vaccine available for preventing tuberculosis (TB), however, BCG has varying success in preventing pulmonary TB. In this study, a recombinant BCG (rBCG::Ag85A) strain overexpressing the immunodominant Ag85A antigen was constructed, and its immunogenicity and protective efficacy were evaluated. Our results indicated that the Ag85A protein was successfully overexpressed in rBCG::Ag85A, and the Ag85A peptide-MHC complexes on draining lymph node dendritic cells of C57BL/6 mice infected with rBCG::Ag85A were detectable 4 h post-infection. The C57BL/6 mice infected with this strain had stronger antigen-specific interferon-gamma (IFN-γ) responses and higher antibody titers than those immunized with BCG, and the protective experiments showed that rBCG::Ag85A can enhance protection against Mycobacterium tuberculosis (M. tuberculosis) H37Rv infection compared to the BCG vaccine alone. Our results demonstrate the potential of rBCG::Ag85A as a candidate vaccine against TB.

The toxicity of rifampicin polylactic acid nanoparticles against Mycobacterium bovis BCG and human macrophage THP-1 cell line

NASA Astrophysics Data System (ADS)

Erokhina, M.; Rybalkina, E.; Barsegyan, G.; Onishchenko, G.; Lepekha, L.

2015-11-01

Tuberculosis is rapidly becoming a major health problem. The rise in tuberculosis incidence stimulates efforts to develop more effective delivery systems for the existing antituberculous drugs while decreasing the side effects. The nanotechnology may provide novel drug delivery tools allowing controlled drug release. Rifampicin is one of the main antituberculous drugs, characterized by high toxicity, and Poly (L-lactic acid) (PLLA) is a biodegradable polymer used for the preparation of encapsulated drugs. The aim of our work was to evaluate the toxicity of rifampicin-PLLA nanoparticles against Mycobacterium bovis BCG using human macrophage THP-1 cell line. Our data demonstrate that rifampicin-PLLA is effective against M. bovis BCG in the infected macrophages. The drug is inducing the dysfunction of mitochondria and apoptosis in the macrophages and is acting as a potential substrate of Pgp thereby modulating cell chemosensitivity. The severity of the toxic effects of the rifampicin-PLLA nanoparticles is increasing in a dose-dependent manner. We suggest that free rifampicin induces death of M. bovis BCG after PLLA degradation and diffusion from phago-lysosomes to cytoplasm causing mitochondria dysfunction and affecting the Pgp activity.

Association of quantitative interferon-γ responses with the progression of naturally acquired Mycobacterium bovis infection in wild European badgers (Meles meles)

PubMed Central

Tomlinson, Alexandra J; Chambers, Mark A; McDonald, Robbie A; Delahay, Richard J

2015-01-01

Bovine tuberculosis is one of the biggest challenges facing cattle farming in Great Britain. European badgers (Meles meles) are a reservoir host for the causal agent, Mycobacterium bovis. There have been significant recent advances in diagnostic testing for tuberculosis in humans, cattle and badgers, with the development of species-specific assays for interferon-γ (IFN-γ), an important cytokine in tuberculous infections. Using data collected from longitudinal studies of naturally infected wild badgers, we report that the magnitude of the IFN-γ response to M. bovis antigens at the disclosing test event was positively correlated with subsequent progression of disease to a seropositive or excreting state. In addition, we show that the magnitude of the IFN-γ response, despite fluctuation, declined with time after the disclosing event for all badgers, but remained significantly higher in those animals with evidence of disease progression. We discuss how our findings may be related to the immunopathogenesis of natural M. bovis infection in badgers. PMID:25109384

Antibody response to a sterile filtered PPD tuberculin in M. bovis infected and M. bovis sensitized cattle.

PubMed

Rennie, Bryan; Filion, Lionel G; Smart, Nonie

2010-11-09

Bovine tuberculosis, caused by Mycobacterium bovis, afflicts approximately 50 million cattle worldwide and is detected by the tuberculin skin test (TST). While it has long been recognized that purified protein derivative (PPD) tuberculin is composed of a mixture of M. bovis derived protein components, little is known about the quality, relative quantity and identity of the proteins that make up PPD tuberculin. We manufactured a sterile filtered PPD tuberculin (SF-PPD) from a nine-week-old M. bovis culture supernatant in order to characterise the culture filtrate proteins (CFP) which make up M. bovis PPD tuberculin and to compare the antibody response of M. bovis infected versus M. bovis sensitized cattle. SF-PPD resolved into approximately 200 discrete spots using two-dimensional polyacrylamide gel electrophoresis (2-DE) while fewer than 65 spots could be discerned from 2-DE gels of tuberculin derived from autoclaved culture supernatant. Two dimensional Western blot analyses indicated that sera from M. bovis sensitized cattle recognized additional SF-PPD antigens as compared to M. bovis infected cattle at seven weeks post infection/sensitization. However, application of a comparative tuberculin skin test resulted in an antibody boosting response to the same set of M. bovis CFPs in both the M. bovis infected and M. bovis sensitized cattle. We concluded that it is the heat sterilization of the M. bovis CFPs that causes severe structural changes to the M. bovis proteins. This work suggests that M. bovis infected cattle and cattle artificially sensitized to M. bovis with an injection of heat killed cells exhibit similar antibody responses to M. bovis antigens.

Intracerebral Mycobacterium bovis bacilli Calmette-Guerin infection-induced immune responses in the CNS 1

PubMed Central

Lee, JangEun; Ling, Changying; Kosmalski, Michelle M.; Hulseberg, Paul; Schreiber, Heidi A.; Sandor, Matyas; Fabry, Zsuzsanna

2010-01-01

To study whether cerebral mycobacterial infection induces granuloma and protective immunity similar to systemic infection, we intracerebrally infected mice with Mycobacterium bovis bacilli Calmette-Guerin. Granuloma and IFN-γ+CD4+ T cell responses are induced in the central nervous system (CNS) similar to periphery, but the presence of IFN-γIL-17 double-positive CD4+ T cells is unique to the CNS. The major CNS source of TNF-α is microglia, with modest production by CD4+ T cells and macrophage. Protective immunity is accompanied by accumulation of Foxp3+CD4+ T cells and PD-L2+ dendritic cells, suggesting that both inflammatory and anti-inflammatory responses develop in the CNS following mycobacterial infection. PMID:19535154

Global gene expression and systems biology analysis of bovine monocyte-derived macrophages in response to in vitro challenge with Mycobacterium bovis.

PubMed

Magee, David A; Taraktsoglou, Maria; Killick, Kate E; Nalpas, Nicolas C; Browne, John A; Park, Stephen D E; Conlon, Kevin M; Lynn, David J; Hokamp, Karsten; Gordon, Stephen V; Gormley, Eamonn; MacHugh, David E

2012-01-01

Mycobacterium bovis, the causative agent of bovine tuberculosis, is a major cause of mortality in global cattle populations. Macrophages are among the first cell types to encounter M. bovis following exposure and the response elicited by these cells is pivotal in determining the outcome of infection. Here, a functional genomics approach was undertaken to investigate global gene expression profiles in bovine monocyte-derived macrophages (MDM) purified from seven age-matched non-related females, in response to in vitro challenge with M. bovis (multiplicity of infection 2:1). Total cellular RNA was extracted from non-challenged control and M. bovis-challenged MDM for all animals at intervals of 2 hours, 6 hours and 24 hours post-challenge and prepared for global gene expression analysis using the Affymetrix® GeneChip® Bovine Genome Array. Comparison of M. bovis-challenged MDM gene expression profiles with those from the non-challenged MDM controls at each time point identified 3,064 differentially expressed genes 2 hours post-challenge, with 4,451 and 5,267 differentially expressed genes detected at the 6 hour and 24 hour time points, respectively (adjusted P-value threshold ≤ 0.05). Notably, the number of downregulated genes exceeded the number of upregulated genes in the M. bovis-challenged MDM across all time points; however, the fold-change in expression for the upregulated genes was markedly higher than that for the downregulated genes. Systems analysis revealed enrichment for genes involved in: (1) the inflammatory response; (2) cell signalling pathways, including Toll-like receptors and intracellular pathogen recognition receptors; and (3) apoptosis. The increased number of downregulated genes is consistent with previous studies showing that M. bovis infection is associated with the repression of host gene expression. The results also support roles for MyD88-independent signalling and intracellular PRRs in mediating the host response to M. bovis.

Evaluation the virulence of Mycobacterium bovis isolated from milk samples through histopathological study in laboratory animals.

PubMed

Al-Saqur, I M; Al-Thwani, A N; Al-Attar, I M; Al-Mashhadani, M S

2016-12-01

Mycobacterium bovis has a broad host range, and it is the principal agent responsible for tuberculosis (TB) in bovine, domestic and wild mammals. M. bovis also infects human, causing zoonotic TB through ingestion, inhalation and, less frequently by contact with mucous membranes and broken skin. Zoonotic TB was formerly an endemic disease, usually transmitted to man by consumption of raw cow's milk. It is indistinguishable clinically or pathologically from TB caused by M. tuberculosis. The aims of this study were, to isolate and identified M. bovis from raw milk samples by different methods, and evaluate the virulence of M. bovis in laboratory animals (Rabbit). To conduct the study, ninety three cow's milk samples were collected from farms around Baghdad governorate. The decontamination of milk samples was firstly carried out, then samples were subjected to routine tests which include, direct smear for Ziehl Neelsen acid fast stain, culture, each sample was cultured on Lowenstein Jensen media with Sodium pyruvite (All cultures incubated on 37°C for 4-10weeks with continuous observation), and biochemical testes as Nitrate reduction test, Niacin paper strip test and pyrazinamidase test, were employed to diagnose and identified the bacteria. Beside molecular assay was used to confirm the identification of the isolates by Polymerase Chain Reaction (PCR) using specific primers for M. bovis. The virulence of these isolates were investigated through inoculate it in group of laboratory animals consist of 8 rabbit in addition to other group of 4 animals as control (inoculate with Phosphate Buffer Saline). The animals were scarified after 6weeks of inoculation, post- mortem examination was carried out, smears were taken from lesions, and tissue samples were collected from lymph nodes and different organs. The results revealed five isolates of M. bovis in direct smear by acid fast Ziehl-Neelsen stain, while eight isolates observed by culture, the colonies appeared with

The effects of serial skin testing with purified protein derivative on the level and quality of antibodies to complex and defined antigens in Mycobacterium bovis-infected cattle

USDA-ARS?s Scientific Manuscript database

Several serologic tests designed to detect antibodies to immunodominant Mycobacterium bovis antigens have recently emerged as ancillary tests for the detection of bovine tuberculosis in cattle, particularly when applied after injection of purified protein derivative (PPD) for skin test that signific...

Tuberculosis transmission by Mycobacterium bovis in a mixed cattle and goat herd.

PubMed

Zanardi, Giorgio; Boniotti, Maria Beatrice; Gaffuri, Alessandra; Casto, Barbara; Zanoni, Mariagrazia; Pacciarini, Maria Lodovica

2013-10-01

A tuberculosis (TB) outbreak caused by Mycobacterium bovis occurred in a mixed herd of three cattle and eighteen goats in Northern Italy in 2005. All the cattle were removed, as opposed to the co-existing goats, who remained in the farm and were not subsequently tested by the official intradermal tuberculin test. At the beginning of May 2006, a 7-day old calf was introduced into the herd from an officially TB-free (OTF) farm. On October 2006, tuberculous lesions were detected at the slaughterhouse in the same animal. The following epidemiological investigation on the herd highlighted a clinical suspicion of TB in one goat out of 35, and visible lesions were found at necropsy in the respiratory and intestinal tracts. Bacteriological culture and molecular tests confirmed the presence of M. bovis in both animals. Spoligotyping and Mycobacterial Interspersed Repetitive Units - Variable Number of Tandem Repeats (MIRU-VNTR) showed the same genomic profile of the previous breakdown occurred in 2005. Since this profile has never been described in Italy, these findings suggest the probable transmission of TB within the farm among cattle and goats. The remaining 34 goats were also tested by single intradermal cervical comparative tuberculin (SICCT) test, Interferon (IFN)-γ assay and ELISA for antibody to M. bovis. The SICCT test and the IFN-γ showed a good concordance with 20 and 19 positive reactors, respectively. By ELISA we found 12Ab-positive animals, seven of which had not been detected by the tests for cell-mediated immunity. Finally, 15 goats were found positive for gross lesions at necropsy. The in vivo tests revealed a total of 27 positive animals out of 35, which highlights the usefulness of the serology in parallel with SICCT and IFN-γ when an advanced stage of infection is suspected. Moreover, our results confirm the necessity for adopting the official tuberculin test on goats co-existing with cattle. Copyright © 2013. Published by Elsevier India Pvt Ltd.

Gene Deletions in Mycobacterium bovis BCG Stimulate Increased CD8+ T Cell Responses

PubMed Central

Panas, Michael W.; Sixsmith, Jaimie D.; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T.; Moy, Brian T.; Lee, Sunhee; Schmitz, Joern E.; Jacobs, William R.; Porcelli, Steven A.; Haynes, Barton F.; Letvin, Norman L.

2014-01-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8+ T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8+ T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8+ T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8+ T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. PMID:25287928

Gene deletions in Mycobacterium bovis BCG stimulate increased CD8+ T cell responses.

PubMed

Panas, Michael W; Sixsmith, Jaimie D; White, KeriAnn; Korioth-Schmitz, Birgit; Shields, Shana T; Moy, Brian T; Lee, Sunhee; Schmitz, Joern E; Jacobs, William R; Porcelli, Steven A; Haynes, Barton F; Letvin, Norman L; Gillard, Geoffrey O

2014-12-01

Mycobacteria, the etiological agents of tuberculosis and leprosy, have coevolved with mammals for millions of years and have numerous ways of suppressing their host's immune response. It has been suggested that mycobacteria may contain genes that reduce the host's ability to elicit CD8(+) T cell responses. We screened 3,290 mutant Mycobacterium bovis bacillus Calmette Guerin (BCG) strains to identify genes that decrease major histocompatibility complex (MHC) class I presentation of mycobacterium-encoded epitope peptides. Through our analysis, we identified 16 mutant BCG strains that generated increased transgene product-specific CD8(+) T cell responses. The genes disrupted in these mutant strains had disparate predicted functions. Reconstruction of strains via targeted deletion of genes identified in the screen recapitulated the enhanced immunogenicity phenotype of the original mutant strains. When we introduced the simian immunodeficiency virus (SIV) gag gene into several of these novel BCG strains, we observed enhanced SIV Gag-specific CD8(+) T cell responses in vivo. This study demonstrates that mycobacteria carry numerous genes that act to dampen CD8(+) T cell responses and suggests that genetic modification of these genes may generate a novel group of recombinant BCG strains capable of serving as more effective and immunogenic vaccine vectors. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Visual format for detection of Mycobacterium tuberculosis and M. bovis in clinical samples using molecular beacons.

PubMed

Kumar, Parameet; Nath, Kapili; Rath, Bimba; Sen, Manas K; Vishalakshi, Potharuju; Chauhan, Devender S; Katoch, Vishwa M; Singh, Sarman; Tyagi, Sanjay; Sreenivas, Vishnubhatla; Prasad, Hanumanthappa K

2009-09-01

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete.

Visual Format for Detection of Mycobacterium tuberculosis and M. bovis in Clinical Samples Using Molecular Beacons

PubMed Central

Kumar, Parameet; Nath, Kapili; Rath, Bimba; Sen, Manas K.; Vishalakshi, Potharuju; Chauhan, Devender S.; Katoch, Vishwa M.; Singh, Sarman; Tyagi, Sanjay; Sreenivas, Vishnubhatla; Prasad, Hanumanthappa K.

2009-01-01

A real-time polymerase chain reaction (PCR) assay for the direct identification of Mycobacterium tuberculosis and M. bovis using molecular beacons was developed. The assay was modified for use in regular thermal cyclers. Molecular beacons that were specific for M. tuberculosis (Tb-B) and M. bovis (Bo-B) were designed. The fluorescence of the target PCR product-molecular beacon probe complex was detected visually using a transilluminator. The results were then compared with those of conventional multiplex PCR (CM-PCR) assays and biochemical identification. The detection limit of Tb-B and Bo-B beacons was 500 fg and 50 fg by the visual format and real-time PCR assay, respectively, compared with 5 pg by CM-PCR assay. Pulmonary and extrapulmonary samples were examined. The agreement between culture and the two assays was very good in sputum samples and fair in extrapulmonary samples. The agreement between clinical diagnoses with the two assays was moderate in extrapulmonary samples. There was very good agreement between CM-PCR and visual format assays for all samples used in the study. Concordance in the identification of isolates by the visual, CM-PCR assay, and biochemical identification was seen. Hence, the use of molecular beacon detection of M. tuberculosis and M. bovis in clinical samples is feasible by setting up two asymmetric PCRs concurrently. The assay is sensitive, specific, simple to interpret, and takes less than 3 hours to complete. PMID:19661384

Detection of Mycobacterium bovis in Bovine and Bubaline Tissues Using Nested-PCR for TbD1

PubMed Central

Araújo, Cristina P.; Osório, Ana Luiza A. R.; Jorge, Kláudia S. G.; Ramos, Carlos Alberto N.; Filho, Antonio Francisco S.; Vidal, Carlos Eugênio S.; Roxo, Eliana; Nishibe, Christiane; Almeida, Nalvo F.; Júnior, Antônio A. F.; Silva, Marcio R.; Neto, José Diomedes B.; Cerqueira, Valíria D.; Zumárraga, Martín J.; Araújo, Flábio R.

2014-01-01

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis. PMID:24618787

Detection of Mycobacterium bovis in bovine and bubaline tissues using nested-PCR for TbD1.

PubMed

Araújo, Cristina P; Osório, Ana Luiza A R; Jorge, Kláudia S G; Ramos, Carlos Alberto N; Filho, Antonio Francisco S; Vidal, Carlos Eugênio S; Roxo, Eliana; Nishibe, Christiane; Almeida, Nalvo F; Júnior, Antônio A F; Silva, Marcio R; Neto, José Diomedes B; Cerqueira, Valíria D; Zumárraga, Martín J; Araújo, Flábio R

2014-01-01

In the present study, a nested-PCR system, targeting the TbD1 region, involving the performance of conventional PCR followed by real-time PCR, was developed to detect Mycobacterium bovis in bovine/bubaline tissue homogenates. The sensitivity and specificity of the reactions were assessed with DNA samples extracted from tuberculous and non-tuberculous mycobacteria, as well as other actinomycetales species and DNA samples extracted directly from bovine and bubaline tissue homogenates. In terms of analytical sensitivity, the DNA of M. bovis AN5 was detected up to 1.56 ng with conventional PCR, 97.6 pg with real-time PCR, and 1.53 pg with nested-PCR in the reaction mixture. The nested-PCR exhibited 100% analytical specificity for M. bovis when tested with the DNA of reference strains of environmental mycobacteria and closely-related Actinomycetales. A clinical sensitivity value of 76.0% was detected with tissue samples from animals that exhibited positive results in the comparative intradermal tuberculin test (CITT), as well as from those with lesions compatible with tuberculosis (LCT) that rendered positive cultures. A clinical specificity value of 100% was detected with tissue samples from animals with CITT- results, with no visible lesions (NVL) and negative cultures. No significant differences were found between the nested-PCR and culture in terms of detecting CITT+ animals with LCT or with NVL. No significant differences were recorded in the detection of CITT- animals with NVL. However, nested-PCR detected a significantly higher number of positive animals than the culture in the group of animals exhibiting LCT with no previous records of CITT. The use of the nested-PCR assay to detect M. bovis in tissue homogenates provided a rapid diagnosis of bovine and bubaline tuberculosis.

Rapid presumptive identification of the Mycobacterium tuberculosis-bovis complex by radiometric determination of heat stable urease

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

Gandy, J.H.; Pruden, E.L.; Cox, F.R.

1983-12-01

Simple and rapid Bactec methodologies for the determination of neat (unaltered) and heat stable urease activity of mycobacteria are presented. Clinical isolates (63) and stock cultures (32)--consisting of: M. tuberculosis (19), M. bovis (5), M. kansasii (15), M. marinum (4), M. simiae (3), M. scrofulaceum (16), M. gordonae (6), M. szulgai (6), M. flavescens (1), M. gastri (1), M. intracellulare (6), M. fortuitum-chelonei complex (12), and M. smegmatis (1)--were tested for neat urease activity by Bactec radiometry. Mycobacterial isolates (50-100 mg wet weight) were incubated at 35 degrees C for 30 minutes with microCi14C-urea. Urease-positive mycobacteria gave Bactec growth indexmore » (GI) values greater than 100 units, whereas urease-negative species gave values less than 10 GI units. Eighty-three isolates possessing neat urease activity were heated at 80 degrees C for 30 minutes followed by incubation at 35 degrees C for 30 minutes with 1 microCi14C-urea. Mycobacterium tuberculosis-bovis complex demonstrated heat-stable urease activity (GI more than 130 units) and could be distinguished from mycobacteria other than tuberculosis (MOTT), which gave GI values equal to or less than 40 units.« less

Variation in the Early Host-Pathogen Interaction of Bovine Macrophages with Divergent Mycobacterium bovis Strains in the United Kingdom.

PubMed

Jensen, Kirsty; Gallagher, Iain J; Johnston, Nicholas; Welsh, Michael; Skuce, Robin; Williams, John L; Glass, Elizabeth J

2018-03-01

Bovine tuberculosis has been an escalating animal health issue in the United Kingdom since the 1980s, even though control policies have been in place for over 60 years. The importance of the genetics of the etiological agent, Mycobacterium bovis , in the reemergence of the disease has been largely overlooked. We compared the interaction between bovine monocyte-derived macrophages (bMDM) and two M. bovis strains, AF2122/97 and G18, representing distinct genotypes currently circulating in the United Kingdom. These M. bovis strains exhibited differences in survival and growth in bMDM. Although uptake was similar, the number of viable intracellular AF2122/97 organisms increased rapidly, while G18 growth was constrained for the first 24 h. AF2122/97 infection induced a greater transcriptional response by bMDM than G18 infection with respect to the number of differentially expressed genes and the fold changes measured. AF2122/97 infection induced more bMDM cell death, with characteristics of necrosis and apoptosis, more inflammasome activation, and a greater type I interferon response than G18. In conclusion, the two investigated M. bovis strains interact in significantly different ways with the host macrophage. In contrast to the relatively silent infection by G18, AF2122/97 induces greater signaling to attract other immune cells and induces host cell death, which may promote secondary infections of naive macrophages. These differences may affect early events in the host-pathogen interaction, including granuloma development, which could in turn alter the progression of the disease. Therefore, the potential involvement of M. bovis genotypes in the reemergence of bovine tuberculosis in the United Kingdom warrants further investigation. Copyright © 2018 Jensen et al.

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification

PubMed Central

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64°C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 103 cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 103 cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

A Simple and Rapid Identification Method for Mycobacterium bovis BCG with Loop-Mediated Isothermal Amplification.

PubMed

Kouzaki, Yuji; Maeda, Takuya; Sasaki, Hiroaki; Tamura, Shinsuke; Hamamoto, Takaaki; Yuki, Atsushi; Sato, Akinori; Miyahira, Yasushi; Kawana, Akihiko

2015-01-01

Bacillus Calmette-Guérin (BCG) is widely used as a live attenuated vaccine against Mycobacterium tuberculosis and is an agent for standard prophylaxis against the recurrence of bladder cancer. Unfortunately, it can cause severe infectious diseases, especially in immunocompromised patients, and the ability to immediately distinguish BCG from other M. tuberculosis complexes is therefore important. In this study, we developed a simple and easy-to-perform identification procedure using loop-mediated amplification (LAMP) to detect deletions within the region of difference, which is deleted specifically in all M. bovis BCG strains. Reactions were performed at 64 °C for 30 min and successful targeted gene amplifications were detected by real-time turbidity using a turbidimeter and visual inspection of color change. The assay had an equivalent detection limit of 1.0 pg of genomic DNA using a turbidimeter whereas it was 10 pg with visual inspection, and it showed specificity against 49 strains of 44 pathogens, including M. tuberculosis complex. The expected LAMP products were confirmed through identical melting curves in real-time LAMP procedures. We employed the Procedure for Ultra Rapid Extraction (PURE) kit to isolate mycobacterial DNA and found that the highest sensitivity limit with a minimum total cell count of mycobacterium (including DNA purification with PURE) was up to 1 × 10(3) cells/reaction, based on color changes under natural light with FDA reagents. The detection limit of this procedure when applied to artificial serum, urine, cerebrospinal fluid, and bronchoalveolar lavage fluid samples was also about 1 × 10(3) cells/reaction. Therefore, this substitute method using conventional culture or clinical specimens followed by LAMP combined with PURE could be a powerful tool to enable the rapid identification of M. bovis BCG as point-of-care testing. It is suitable for practical use not only in resource-limited situations, but also in any clinical situation

New Recombinant Mycobacterium bovis BCG Expression Vectors: Improving Genetic Control over Mycobacterial Promoters.

PubMed

Kanno, Alex I; Goulart, Cibelly; Rofatto, Henrique K; Oliveira, Sergio C; Leite, Luciana C C; McFadden, Johnjoe

2016-04-01

The expression of many antigens, stimulatory molecules, or even metabolic pathways in mycobacteria such as Mycobacterium bovis BCG or M. smegmatis was made possible through the development of shuttle vectors, and several recombinant vaccines have been constructed. However, gene expression in any of these systems relied mostly on the selection of natural promoters expected to provide the required level of expression by trial and error. To establish a systematic selection of promoters with a range of strengths, we generated a library of mutagenized promoters through error-prone PCR of the strong PL5 promoter, originally from mycobacteriophage L5. These promoters were cloned upstream of the enhanced green fluorescent protein reporter gene, and recombinant M. smegmatis bacteria exhibiting a wide range of fluorescence levels were identified. A set of promoters was selected and identified as having high (pJK-F8), intermediate (pJK-B7, pJK-E6, pJK-D6), or low (pJK-C1) promoter strengths in both M. smegmatis and M. bovisBCG. The sequencing of the promoter region demonstrated that it was extensively modified (6 to 11%) in all of the plasmids selected. To test the functionality of the system, two different expression vectors were demonstrated to allow corresponding expression levels of the Schistosoma mansoni antigen Sm29 in BCG. The approach used here can be used to adjust expression levels for synthetic and/or systems biology studies or for vaccine development to maximize the immune response. Copyright © 2016 Kanno et al.

Mycobacterium bovis infections in domesticated non-bovine mammalian species. Part 1: Review of epidemiology and laboratory submissions in Great Britain 2004-2010.

PubMed

Broughan, J M; Downs, S H; Crawshaw, T R; Upton, P A; Brewer, J; Clifton-Hadley, R S

2013-11-01

Mycobacterium bovis, the causative agent of bovine tuberculosis (bTB), can infect a broad range of mammalian species in addition to domestic and feral cattle and badgers. Since legislation introduced in 2006 in Great Britain requires animal keepers, meat inspectors and veterinarians to notify the authorities of suspect bTB lesions or the isolation of M. bovis in any mammal excluding humans, the organism has been increasingly identified in domestic species other than cattle. Although in most cases 'spill-over' hosts, these remain a potential source of infection for cattle, wildlife, and possibly humans. In this first part of a two-part review of M. bovis infections in non-bovine domestic species, current knowledge of the epidemiology of such infections is presented along with novel data relating to diagnostic submissions for mycobacterial culture between 2004 and 2010. Over this period M. bovis infection was identified in 116 cats, 7 dogs, 34 llamas, 133 alpacas, 35 goats, 24 sheep and 85 pigs and wild boar. The risk that such infections pose to the control of bTB, and as zoonoses, is discussed. In part two, the options available to diagnose bTB in these species, as well as the challenges posed to disease detection and control will be discussed in depth. Copyright © 2013. Published by Elsevier Ltd.

Efficacy of oral BCG vaccination in protecting free-ranging cattle from natural infection by Mycobacterium bovis.

PubMed

Nugent, Graham; Yockney, Ivor J; Whitford, Jackie; Aldwell, Frank E; Buddle, Bryce M

2017-09-01

Vaccination of cattle against bovine tuberculosis could be a valuable control strategy, particularly in countries faced with intractable ongoing infection from a disease reservoir in wildlife. A field vaccination trial was undertaken in New Zealand. The trial included 1286 effectively free-ranging cattle stocked at low densities in a remote 7600ha area, with 55% of them vaccinated using Mycobacterium bovis BCG (Danish strain 1311). Vaccine was administered orally in all but 34 cases (where it was injected). After inclusion, cattle were exposed to natural sources of M. bovis infection in cattle and wildlife, most notably the brushtail possum (Trichosurus vulpecula). Cattle were slaughtered at 3-5 years of age and were inspected for tuberculous lesions, with mycobacteriological culture of key tissues from almost all animals. The prevalence of M. bovis infection was 4.8% among oral BCG vaccinates, significantly lower than the 11.9% in non-vaccinates. Vaccination appeared to both reduce the incidence of detectable infection, and to slow disease progression. Based on apparent annual incidence, the protective efficacy of oral BCG vaccine was 67.4% for preventing infection, and was higher in cattle slaughtered soon after vaccination. Skin-test reactivity to tuberculin was high in vaccinates re-tested 70days after vaccination but not in non-vaccinates, although reactor animals had minimal response in gamma-interferon blood tests. In re- tests conducted more than 12 months after vaccination, skin-test reactivity among vaccinates was much lower. These results indicate that oral BCG vaccination could be an effective tool for greatly reducing detectable infection in cattle. Copyright © 2017 Elsevier B.V. All rights reserved.

Detection of antibodies to tuberculosis antigens in free-ranging lions (Panthera leo) infected with Mycobacterium bovis in Kruger National Park, South Africa.

PubMed

Miller, Michele; Joubert, Jennifer; Mathebula, Nomkhosi; De Klerk-Lorist, Lin-Marie; Lyashchenko, Konstantin P; Bengis, Roy; van Helden, Paul; Hofmeyr, Markus; Olea-Popelka, Francisco; Greenwald, Rena; Esfandiari, Javan; Buss, Peter

2012-06-01

Bovine tuberculosis (TB), caused by Mycobacterium bovis, has become established in Kruger National Park, South Africa, in the cape buffalo (Syncerus caffer) population and in other species. TB in prey species has resulted in infection and morbidity in the resident lion (Panthera leo) prides. The only validated live animal test currently available for lions is the intradermal tuberculin test. Because this test requires capture twice, 72 hr apart, of free-ranging lions to read results, it is logistically difficult to administer in a large ecosystem. Therefore, development of a rapid animal-side screening assay would be ideal in providing information for wildlife managers, veterinarians, and researchers working with free-living lion prides. This study reports preliminary descriptive results from an ongoing project evaluating two serologic tests for M. bovis (ElephantTB Stat-Pak and dual path platform VetTB). Disease status was determined by postmortem culture and presence of pathologic lesions in 14 free-ranging lions. Seropositivity was found to be associated with M. bovis infection. Extended field studies are underway to validate these rapid animal-side immunoassays for antemortem screening tests for TB in lions.

Field evaluation of the protective efficacy of Mycobacterium bovis BCG vaccine against bovine tuberculosis.

PubMed

Lopez-Valencia, G; Renteria-Evangelista, T; Williams, J de Jesús; Licea-Navarro, A; Mora-Valle, A De la; Medina-Basulto, G

2010-02-01

The protective efficacy of Mycobacterium bovis BCG (1 x 10(6) single dose) was evaluated under field conditions. A total of 140 male Holstein Friesian calves, one to two week-old were selected. Two groups of 70 each were formed, one group was vaccinated and the other was injected with a placebo during their second week of age and followed until 12 months of age. The study considered a positive case of tuberculosis to be an animal that had a positive reaction to the three following tests in a row: tuberculin, IFNgamma PPD-B and IFNgamma ESAT6-CFP10 during the 12 months of exposure. The results showed a 59.4% efficacy (IC95%: 47.64-71.16). The non-vaccinated calves were 2.4 times more at risk of becoming infected (IC95%: 1.07-5.68) compared to vaccinated animals. As a complementary test a PCR test was performed using nasal exudates in some animals from both groups using a Mycobacterium complex detection kit. All the positive PCR reactions (5/44) were found in the non-vaccinated animals. These findings suggest that the use of the BCG vaccine, even though it is not capable of protecting 100%, does prevent TB vaccinated animals from excreting bacilli in their nasal secretions at their first year of age. Copyright 2009 Elsevier Ltd. All rights reserved.

Viral booster vaccines improve Mycobacterium bovis BCG-induced protection against bovine tuberculosis.

PubMed

Vordermeier, H Martin; Villarreal-Ramos, Bernardo; Cockle, Paul J; McAulay, Martin; Rhodes, Shelley G; Thacker, Tyler; Gilbert, Sarah C; McShane, Helen; Hill, Adrian V S; Xing, Zhou; Hewinson, R Glyn

2009-08-01

Previous work with small-animal laboratory models of tuberculosis has shown that vaccination strategies based on heterologous prime-boost protocols using Mycobacterium bovis bacillus Calmette-Guérin (BCG) to prime and modified vaccinia virus Ankara strain (MVA85A) or recombinant attenuated adenoviruses (Ad85A) expressing the mycobacterial antigen Ag85A to boost may increase the protective efficacy of BCG. Here we report the first efficacy data on using these vaccines in cattle, a natural target species of tuberculous infection. Protection was determined by measuring development of disease as an end point after M. bovis challenge. Either Ad85A or MVA85A boosting resulted in protection superior to that given by BCG alone: boosting BCG with MVA85A or Ad85A induced significant reduction in pathology in four/eight parameters assessed, while BCG vaccination alone did so in only one parameter studied. Protection was particularly evident in the lungs of vaccinated animals (median lung scores for naïve and BCG-, BCG/MVA85A-, and BCG/Ad85A-vaccinated animals were 10.5, 5, 2.5, and 0, respectively). The bacterial loads in lymph node tissues were also reduced after viral boosting of BCG-vaccinated calves compared to those in BCG-only-vaccinated animals. Analysis of vaccine-induced immunity identified memory responses measured by cultured enzyme-linked immunospot assay as well as in vitro interleukin-17 production as predictors of vaccination success, as both responses, measured before challenge, correlated positively with the degree of protection. Therefore, this study provides evidence of improved protection against tuberculosis by viral booster vaccination in a natural target species and has prioritized potential correlates of vaccine efficacy for further evaluation. These findings also have implications for human tuberculosis vaccine development.

Comparison of the recovery of Mycobacterium bovis isolates using the BACTEC MGIT 960 system, BACTEC 460 system, and Middlebrook 7H10 and 7H11 solid media.

PubMed

Hines, Nichole; Payeur, Janet B; Hoffman, Lorraine J

2006-05-01

The BACTEC Microbacteria Growth Indicator Tube (MGIT) 960 system was evaluated to determine how it compares with the BACTEC 460 radiometric system and solid media for recovery of Mycobacterium bovis from tissue samples. A total of 506 bovine lymph node samples were collected from abattoirs in the United States and Mexico between November 2003 and September 2004. Processed samples were inoculated into an MGIT 960 tube, BACTEC 460 vial, and Middlebrook 7H10 and Middlebrook 7H11 solid media. Ziehl-Neelsen slides were prepared to check for contaminants and confirm the presence of acid-fast positive bacilli. Samples containing acid-fast bacilli were confirmed as members of the Mycobacterium tuberculosis complex by a nucleic acid assay. Niacin and nitrate biochemical tests were used to distinguish M. bovis from M. tuberculosis isolates. Statistical analyses were performed to compare recovery rate, mean time to detection, contamination rates, as well as pair-wise comparisons in each category. The results showed that the MGIT 960 system had a higher recovery rate of M. bovis (122/129) than did the BACTEC 460 (102/129) and solid media system (96/129). The average time to detection was 15.8 days for the MGIT 960 system, 28.2 days for the BACTEC 460 system, and 43.4 days for solid media. Contamination rates were 6.9% for the MGIT 960 system, 3.4% for the BACTEC 460 system, and 21.7% for solid media. These results indicate the MGIT 960 system can be used as an alternative to the BACTEC 460 system for recovering M. bovis from tissue samples.

Evaluation of the Mycobacterium smegmatis and BCG models for the discovery of Mycobacterium tuberculosis inhibitors.

PubMed

Altaf, Mudassar; Miller, Christopher H; Bellows, David S; O'Toole, Ronan

2010-11-01

The objective of this study was to measure the efficacy of Mycobacterium smegmatis as a surrogate in vitro model for the detection of compounds which are inhibitory to the growth of Mycobacterium tuberculosis. A chemical screen of the LOPAC library for anti-mycobacterial compounds was performed using M. smegmatis. Parallel screens were conducted with another tuberculosis model, Mycobacterium bovis BCG, and with M. tuberculosis under identical growth conditions and the inhibitors detected across the three species were compared. 50% of compounds that were detected as active against M. tuberculosis were not detected using M. smegmatis compared to 21% of compounds using M. bovis BCG. To examine whether these findings were unique to LOPAC, screens were performed with the NIH Diversity Set and Spectrum Collection. An even higher proportion of M. tuberculosis inhibitors were not detected from the NIH Diversity Set and Spectrum Collection using M. smegmatis compared to M. bovis BCG. These data reveal that a significant proportion of M. tuberculosis inhibitors are missed in library screening with M. smegmatis. The basis of the variation in the inhibitory profiles of M. smegmatis and M. tuberculosis has yet to be fully determined, however, our genomic comparisons indicate that approximately 30% of M. tuberculosis proteins lack conserved orthologues in M. smegmatis compared to 3% being absent in M. bovis BCG. In conclusion, although M. smegmatis offers some technical benefits such as a shorter generation time and negligible risk to laboratory workers, it is significantly less effective in the detection of anti-M. tuberculosis compounds relative to M. bovis BCG. This limitation needs to be taken into consideration when selecting an in vitro screening model for tuberculosis drug discovery. Copyright © 2010 Elsevier Ltd. All rights reserved.

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence.

PubMed

de la Fuente, José; Díez-Delgado, Iratxe; Contreras, Marinela; Vicente, Joaquín; Cabezas-Cruz, Alejandro; Tobes, Raquel; Manrique, Marina; López, Vladimir; Romero, Beatriz; Bezos, Javier; Dominguez, Lucas; Sevilla, Iker A; Garrido, Joseba M; Juste, Ramón; Madico, Guillermo; Jones-López, Edward; Gortazar, Christian

2015-11-01

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to

Comparative Genomics of Field Isolates of Mycobacterium bovis and M. caprae Provides Evidence for Possible Correlates with Bacterial Viability and Virulence

PubMed Central

de la Fuente, José; Díez-Delgado, Iratxe; Contreras, Marinela; Vicente, Joaquín; Cabezas-Cruz, Alejandro; Tobes, Raquel; Manrique, Marina; López, Vladimir; Romero, Beatriz; Bezos, Javier; Dominguez, Lucas; Sevilla, Iker A.; Garrido, Joseba M.; Juste, Ramón; Madico, Guillermo; Jones-López, Edward; Gortazar, Christian

2015-01-01

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly affect humans and animals worldwide. The life cycle of mycobacteria is complex and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Recently, comparative genomics analyses have provided new insights into the evolution and adaptation of the MTBC to survive inside the host. However, most of this information has been obtained using M. tuberculosis but not other members of the MTBC such as M. bovis and M. caprae. In this study, the genome of three M. bovis (MB1, MB3, MB4) and one M. caprae (MB2) field isolates with different lesion score, prevalence and host distribution phenotypes were sequenced. Genome sequence information was used for whole-genome and protein-targeted comparative genomics analysis with the aim of finding correlates with phenotypic variation with potential implications for tuberculosis (TB) disease risk assessment and control. At the whole-genome level the results of the first comparative genomics study of field isolates of M. bovis including M. caprae showed that as previously reported for M. tuberculosis, sequential chromosomal nucleotide substitutions were the main driver of the M. bovis genome evolution. The phylogenetic analysis provided a strong support for the M. bovis/M. caprae clade, but supported M. caprae as a separate species. The comparison of the MB1 and MB4 isolates revealed differences in genome sequence, including gene families that are important for bacterial infection and transmission, thus highlighting differences with functional implications between isolates otherwise classified with the same spoligotype. Strategic protein-targeted analysis using the ESX or type VII secretion system, proteins linking stress response with lipid metabolism, host T cell epitopes of mycobacteria, antigens and peptidoglycan assembly protein identified new genetic markers and candidate vaccine antigens that warrant further study to

Detection of Mycobacterium bovis infection in African buffaloes (Syncerus caffer) using QuantiFERON®-TB Gold (QFT) tubes and the Qiagen cattletype® IFN-gamma ELISA.

PubMed

Bernitz, Netanya; Clarke, Charlene; Roos, Eduard O; Goosen, Wynand J; Cooper, David; van Helden, Paul D; Parsons, Sven D C; Miller, Michele A

2018-02-01

African buffaloes (Syncerus caffer) are wildlife maintenance hosts of Mycobacterium bovis, the cause of bovine tuberculosis. Consequently, M. bovis infected buffaloes pose a transmission risk for cattle and other wildlife species. Previously, a modification to the Qiagen QuantiFERON ® -TB Gold (QFT) system, using QFT tubes and an in-house bovine interferon-gamma (IFN-γ) ELISA, was evaluated for the detection of M. bovis infection in buffaloes. Subsequently, Qiagen has developed a commercially available cattletype ® IFN-gamma ELISA for the detection of antigen-specific IFN-γ release in ruminants. The aim of this study was to investigate the use of QFT tubes and the cattletype ® IFN-gamma ELISA, in a cattletype IFN-γ release assay (IGRA), to detect M. bovis infection in African buffaloes. The test agreements between the cattletype IGRA, single comparative intradermal skin test (SCITT) and Bovigam ® 1G IGRA in two M. bovis-exposed buffalo populations (n = 134 and n = 92) were calculated and κ coefficients ranged from 0.65 (95% CI 0.48-0.82) to 0.86 (95% CI 0.72-0.99). Increasing the QFT incubation time in one M. bovis-exposed buffalo cohort (n = 92), from 20 to 40 h, had no effect on the cattletype IGRA test results. Inter-assay and intra-assay reproducibility determination for the cattletype IGRA produced coefficient of variations (CV) <9.1% and <1.7%, respectively. A total of 21/21 known M. bovis-unexposed buffaloes tested negative in the cattletype IGRA. Moreover, the cattletype IGRA test result values were significantly greater for 13 M. bovis culture-positive buffaloes compared with 14 M. bovis-exposed culture-negative (P < .01) and 21 M. bovis-unexposed (P < .001) buffaloes, respectively. These findings suggest that the combination of QFT tubes and the cattletype ® IFN-gamma ELISA is a promising new diagnostic assay for the detection of M. bovis infection in African buffaloes. However, further research is needed to evaluate the

Whole Genome Sequencing Reveals Local Transmission Patterns of Mycobacterium bovis in Sympatric Cattle and Badger Populations

PubMed Central

Wright, David; Mallon, Tom; McCormick, Carl; Orton, Richard J.; McDowell, Stanley; Trewby, Hannah; Skuce, Robin A.; Kao, Rowland R.

2012-01-01

Whole genome sequencing (WGS) technology holds great promise as a tool for the forensic epidemiology of bacterial pathogens. It is likely to be particularly useful for studying the transmission dynamics of an observed epidemic involving a largely unsampled ‘reservoir’ host, as for bovine tuberculosis (bTB) in British and Irish cattle and badgers. BTB is caused by Mycobacterium bovis, a member of the M. tuberculosis complex that also includes the aetiological agent for human TB. In this study, we identified a spatio-temporally linked group of 26 cattle and 4 badgers infected with the same Variable Number Tandem Repeat (VNTR) type of M. bovis. Single-nucleotide polymorphisms (SNPs) between sequences identified differences that were consistent with bacterial lineages being persistent on or near farms for several years, despite multiple clear whole herd tests in the interim. Comparing WGS data to mathematical models showed good correlations between genetic divergence and spatial distance, but poor correspondence to the network of cattle movements or within-herd contacts. Badger isolates showed between zero and four SNP differences from the nearest cattle isolate, providing evidence for recent transmissions between the two hosts. This is the first direct genetic evidence of M. bovis persistence on farms over multiple outbreaks with a continued, ongoing interaction with local badgers. However, despite unprecedented resolution, directionality of transmission cannot be inferred at this stage. Despite the often notoriously long timescales between time of infection and time of sampling for TB, our results suggest that WGS data alone can provide insights into TB epidemiology even where detailed contact data are not available, and that more extensive sampling and analysis will allow for quantification of the extent and direction of transmission between cattle and badgers. PMID:23209404

Assessment of different formulations of oral Mycobacterium bovis Bacille Calmette-Guérin (BCG) vaccine in rodent models for immunogenicity and protection against aerosol challenge with M. bovis.

PubMed

Clark, Simon; Cross, Martin L; Smith, Alan; Court, Pinar; Vipond, Julia; Nadian, Allan; Hewinson, R Glyn; Batchelor, Hannah K; Perrie, Yvonne; Williams, Ann; Aldwell, Frank E; Chambers, Mark A

2008-10-29

Bovine tuberculosis (bTB) caused by infection with Mycobacterium bovis is causing considerable economic loss to farmers and Government in the United Kingdom as its incidence is increasing. Efforts to control bTB in the UK are hampered by the infection in Eurasian badgers (Meles meles) that represent a wildlife reservoir and source of recurrent M. bovis exposure to cattle. Vaccination of badgers with the human TB vaccine, M. bovis Bacille Calmette-Guérin (BCG), in oral bait represents a possible disease control tool and holds the best prospect for reaching badger populations over a wide geographical area. Using mouse and guinea pig models, we evaluated the immunogenicity and protective efficacy, respectively, of candidate badger oral vaccines based on formulation of BCG in lipid matrix, alginate beads, or a novel microcapsular hybrid of both lipid and alginate. Two different oral doses of BCG were evaluated in each formulation for their protective efficacy in guinea pigs, while a single dose was evaluated in mice. In mice, significant immune responses (based on lymphocyte proliferation and expression of IFN-gamma) were only seen with the lipid matrix and the lipid in alginate microcapsular formulation, corresponding to the isolation of viable BCG from alimentary tract lymph nodes. In guinea pigs, only BCG formulated in lipid matrix conferred protection to the spleen and lungs following aerosol route challenge with M. bovis. Protection was seen with delivery doses in the range 10(6)-10(7) CFU, although this was more consistent in the spleen at the higher dose. No protection in terms of organ CFU was seen with BCG administered in alginate beads or in lipid in alginate microcapsules, although 10(7) in the latter formulation conferred protection in terms of increasing body weight after challenge and a smaller lung to body weight ratio at necropsy. These results highlight the potential for lipid, rather than alginate, -based vaccine formulations as suitable delivery

Field-isolated genotypes of Mycobacterium bovis vary in virulence and influence case pathology but do not affect outbreak size.

PubMed

Wright, David M; Allen, Adrian R; Mallon, Thomas R; McDowell, Stanley W J; Bishop, Stephen C; Glass, Elizabeth J; Bermingham, Mairead L; Woolliams, John A; Skuce, Robin A

2013-01-01

Strains of many infectious agents differ in fundamental epidemiological parameters including transmissibility, virulence and pathology. We investigated whether genotypes of Mycobacterium bovis (the causative agent of bovine tuberculosis, bTB) differ significantly in transmissibility and virulence, combining data from a nine-year survey of the genetic structure of the M. bovis population in Northern Ireland with detailed records of the cattle population during the same period. We used the size of herd breakdowns as a proxy measure of transmissibility and the proportion of skin test positive animals (reactors) that were visibly lesioned as a measure of virulence. Average breakdown size increased with herd size and varied depending on the manner of detection (routine herd testing or tracing of infectious contacts) but we found no significant variation among M. bovis genotypes in breakdown size once these factors had been accounted for. However breakdowns due to some genotypes had a greater proportion of lesioned reactors than others, indicating that there may be variation in virulence among genotypes. These findings indicate that the current bTB control programme may be detecting infected herds sufficiently quickly so that differences in virulence are not manifested in terms of outbreak sizes. We also investigated whether pathology of infected cattle varied according to M. bovis genotype, analysing the distribution of lesions recorded at post mortem inspection. We concentrated on the proportion of cases lesioned in the lower respiratory tract, which can indicate the relative importance of the respiratory and alimentary routes of infection. The distribution of lesions varied among genotypes and with cattle age and there were also subtle differences among breeds. Age and breed differences may be related to differences in susceptibility and husbandry, but reasons for variation in lesion distribution among genotypes require further investigation.

Circulating Mycobacterium bovis peptides and host response proteins as biomarkers for unambiguous detection of subclinical infection

USDA-ARS?s Scientific Manuscript database

Background: Bovine tuberculosis remains one of the most damaging zoonotic diseases. A critical need exists for rapid and inexpensive diagnostics capable of detecting and differentiating M. bovis infection from other pathogenic and environmental mycobacteria at multiple surveillance levels. Method...

Field evaluation of three blood-based assays for elk (Cervus canadensis) naturally infected with Mycobacterium bovis.

PubMed

Shury, Todd K; Bergeson, Doug; Surujballi, Om; Lyashchenko, Konstantin P; Greenwald, Rena

2014-08-01

Diagnosis of Mycobacterium bovis in wild populations is very challenging due to complications imposed by the use of traditional skin tests, poor sensitivity of gold standard tests which rely on culture of M. bovis from tissues and wide variations in severity of disease. Various combinations of a lymphocyte stimulation test (LST), fluorescence polarization assay (FPA) and the Cervid TB Stat-Pak were evaluated using two different validation approaches: a latent class analysis and classical statistical approach using culture as a gold standard. A validation subsample consisting of animals culled for population control and mortalities from capture provided an unbiased estimate of test performance for comparison. The sensitivity of the LST (0.83, 95% CI: [0.70-0.97] as a single test was similar to existing tuberculin skin tests, but the sensitivity of the FPA (0.40, 95% CI: [0.22-0.58]) and Cervid TB Stat-Pak (0.62, 95% CI: [0.41-0.83]) were lower in this population. Test performance of the LST and Cervid TB Stat-Pak in parallel was similar to the use of all three tests in parallel and inclusion of the FPA did not greatly enhance test performance. Prevalence of M. bovis in elk varied substantially between the high risk area of southern Manitoba (9.1%, 95% CI: [6.09-12.1%]) and lower risk areas outside this zone (0.76%, 95% CI: [0-2.26%]). Bayesian latent class analysis indicated lack of covariance between the two antibody tests (FPA and Cervid TB Stat-Pak) while the classical two-stage analysis indicated there was conditional dependence between the tests. All three tests when used in parallel resulted in 100% NPV using all three validation methods, indicating few elk were misclassified as false negative by post mortem culture. Similar to previous studies, this study found that combinations of blood tests that utilize cell mediated responses along with humoral antibody responses maximize the sensitivity of tests for diagnosis of M. bovis in wild cervid populations

Towards harmonised procedures in wildlife epidemiological investigations: a serosurvey of infection with Mycobacterium bovis and closely related agents in wild boar (Sus scrofa) in Switzerland.

PubMed

Beerli, Olivia; Blatter, Sohvi; Boadella, Mariana; Schöning, Janne; Schmitt, Sarah; Ryser-Degiorgis, Marie-Pierre

2015-01-01

Bovine tuberculosis (bTB) is a (re-)emerging disease in European countries, including Switzerland. This study assesses the seroprevalence of infection with Mycobacterium bovis and closely related agents in wild boar (Sus scrofa) in Switzerland, because wild boar are potential maintenance hosts of these pathogens. The study employs harmonised laboratory methods to facilitate comparison with the situation in other countries. Eighteen out of 743 blood samples tested seropositive (2.4%, CI: 1.5-3.9%) by ELISA, and the results for 61 animals previously assessed using culture and PCR indicated that this serological test was not 100% specific for M. bovis, cross-reacting with M. microti. Nevertheless, serology appears to be an appropriate test methodology in the harmonisation of wild boar testing throughout Europe. In accordance with previous findings, the low seroprevalence found in wild boar suggests wildlife is an unlikely source of the M. bovis infections recently detected in cattle in Switzerland. This finding contrasts with the epidemiological situation pertaining in southern Spain. Copyright © 2014 Elsevier Ltd. All rights reserved.

Comparison of the Membrane Proteome of Virulent Mycobacterium tuberculosis and the Attenuated Mycobacterium bovis BCG Vaccine Strain by Label-free Quantitative Proteomics

PubMed Central

Gunawardena, Harsha P.; Feltcher, Meghan E.; Wrobel, John A.; Gu, Sheng; Braunstein, Miriam; Chen, Xian

2015-01-01

The Mycobacterium tuberculosis (MTB) membrane is rich in antigens that are potential targets for diagnostics and the development of new vaccines. To better understand the mechanisms underlying MTB virulence and identify new targets for therapeutic intervention we investigated the differential composition of membrane proteomes between virulent M. tuberculosis H37Rv (MTB) and the Mycobacterium bovis BCG vaccine strain. To compare the membrane proteomes, we used LC-MS/MS analysis in combination with label-free quantitative (LFQ) proteomics, utilizing the area-under-curve (AUC) of the extracted ion chromatograms (XIC) of peptides obtained from m/z and retention time alignment of MS1 features. With this approach, we obtained relative abundance ratios for 2,203 identified membrane-associated proteins in high confidence. Of these proteins, 294 showed statistically significant differences of at least 2 fold, in relative abundance between MTB and BCG membrane fractions. Our comparative analysis detected several proteins associated with known genomic regions of difference between MTB and BCG as being absent, which validated the accuracy of our approach. In further support of our label-free quantitative data, we verified select protein differences by immunoblotting. To our knowledge we have generated the first comprehensive and high coverage profile of comparative membrane proteome changes between virulent MTB and its attenuated relative BCG, which helps elucidate the proteomic basis of the intrinsic virulence of the MTB pathogen. PMID:24093440

A novel strategy for screening-out raw milk contaminated with Mycobacterium bovis on dairy farms by double-tagging PCR and electrochemical genosensing.

PubMed

Lermo, Anabel; Liébana, Susana; Campoy, Susana; Fabiano, Silvia; García, M Inés; Soutullo, Adriana; Zumárraga, Martín J; Alegret, Salvador; Pividori, M Isabel

2010-06-01

A highly sensitive assay for rapidly screening-out Mycobacterium bovis in contaminated samples was developed based on electrochemical genosensing. The assay consists of specific amplification and double-tagging of the IS6110 fragment, highly related to M. bovis, followed by electrochemical detection of the amplified product. PCR amplification was carried out using a labeled set of primers and resulted in a amplicon tagged at each terminus with both biotin and digoxigenin. Two different electrochemical platforms for the detection of the double-tagged amplicon were evaluated: (i) an avidin biocomposite (Av-GEB) and (ii) a magneto sensor (m-GEC) combined with streptavidin magnetic beads. In both cases, the double- tagged amplicon was immobilized through its biotinylated end and electrochemically detected, using an antiDig-HRP conjugate, through its digoxigenin end. The assay was determined to be highly sensitive, based on the detection of 620 and 10 fmol of PCR amplicon using the Av-GEB and m-GEC strategies, respectively. Moreover, the m-GEC assay showed promising features for the detection of M. bovis on dairy farms by screening for the presence of the bacterium's DNA in milk samples. The obtained results are discussed and compared with respect to those of inter-laboratory PCR assays and tuberculin skin testing.

Exposure to human alveolar lining fluid enhances Mycobacterium bovis BCG vaccine efficacy against Mycobacterium tuberculosis infection in a CD8+ T-cell-dependent manner.

PubMed

Moliva, J I; Hossfeld, A P; Canan, C H; Dwivedi, V; Wewers, M D; Beamer, G; Turner, J; Torrelles, J B

2018-05-01

Current tuberculosis (TB) treatments include chemotherapy and preventative vaccination with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). In humans, however, BCG vaccination fails to fully protect against pulmonary TB. Few studies have considered the impact of the human lung mucosa (alveolar lining fluid (ALF)), which modifies the Mycobacterium tuberculosis (M.tb) cell wall, revealing alternate antigenic epitopes on the bacterium surface that alter its pathogenicity. We hypothesized that ALF-induced modification of BCG would induce better protection against aerosol infection with M.tb. Here we vaccinated mice with ALF-exposed BCG, mimicking the mycobacterial cell surface properties that would be present in the lung during M.tb infection. ALF-exposed BCG-vaccinated mice were more effective at reducing M.tb bacterial burden in the lung and spleen, and had reduced lung inflammation at late stages of M.tb infection. Improved BCG efficacy was associated with increased numbers of memory CD8 + T cells, and CD8 + T cells with the potential to produce interferon-γ in the lung in response to M.tb challenge. Depletion studies confirmed an essential role for CD8 + T cells in controlling M.tb bacterial burden. We conclude that ALF modifications to the M.tb cell wall in vivo are relevant in the context of vaccine design.

Characterization of the transcriptional regulator Rv3124 of Mycobacterium tuberculosis identifies it as a positive regulator of molybdopterin biosynthesis and defines the functional consequences of a non-synonymous SNP in the Mycobacterium bovis BCG orthologue

PubMed Central

Mendoza Lopez, Pablo; Golby, Paul; Wooff, Esen; Garcia, Javier Nunez; Garcia Pelayo, M. Carmen; Conlon, Kevin; Gema Camacho, Ana; Hewinson, R. Glyn; Polaina, Julio; Suárez García, Antonio; Gordon, Stephen V.

2010-01-01

A number of single-nucleotide polymorphisms (SNPs) have been identified in the genome of Mycobacterium bovis BCG Pasteur compared with the sequenced strain M. bovis 2122/97. The functional consequences of many of these mutations remain to be described; however, mutations in genes encoding regulators may be particularly relevant to global phenotypic changes such as loss of virulence, since alteration of a regulator's function will affect the expression of a wide range of genes. One such SNP falls in bcg3145, encoding a member of the AfsR/DnrI/SARP class of global transcriptional regulators, that replaces a highly conserved glutamic acid residue at position 159 (E159G) with glycine in a tetratricopeptide repeat (TPR) located in the bacterial transcriptional activation (BTA) domain of BCG3145. TPR domains are associated with protein–protein interactions, and a conserved core (helices T1–T7) of the BTA domain seems to be required for proper function of SARP-family proteins. Structural modelling predicted that the E159G mutation perturbs the third α-helix of the BTA domain and could therefore have functional consequences. The E159G SNP was found to be present in all BCG strains, but absent from virulent M. bovis and Mycobacterium tuberculosis strains. By overexpressing BCG3145 and Rv3124 in BCG and H37Rv and monitoring transcriptome changes using microarrays, we determined that BCG3145/Rv3124 acts as a positive transcriptional regulator of the molybdopterin biosynthesis moa1 locus, and we suggest that rv3124 be renamed moaR1. The SNP in bcg3145 was found to have a subtle effect on the activity of MoaR1, suggesting that this mutation is not a key event in the attenuation of BCG. PMID:20378651

Comparative Proteomics Identifies Host Immune System Proteins Affected by Infection with Mycobacterium bovis

PubMed Central

López, Vladimir; Villar, Margarita; Queirós, João; Vicente, Joaquín; Mateos-Hernández, Lourdes; Díez-Delgado, Iratxe; Contreras, Marinela; Alves, Paulo C.; Alberdi, Pilar; Gortázar, Christian; de la Fuente, José

2016-01-01

Mycobacteria of the Mycobacterium tuberculosis complex (MTBC) greatly impact human and animal health worldwide. The mycobacterial life cycle is complex, and the mechanisms resulting in pathogen infection and survival in host cells are not fully understood. Eurasian wild boar (Sus scrofa) are natural reservoir hosts for MTBC and a model for mycobacterial infection and tuberculosis (TB). In the wild boar TB model, mycobacterial infection affects the expression of innate and adaptive immune response genes in mandibular lymph nodes and oropharyngeal tonsils, and biomarkers have been proposed as correlates with resistance to natural infection. However, the mechanisms used by mycobacteria to manipulate host immune response are not fully characterized. Our hypothesis is that the immune system proteins under-represented in infected animals, when compared to uninfected controls, are used by mycobacteria to guarantee pathogen infection and transmission. To address this hypothesis, a comparative proteomics approach was used to compare host response between uninfected (TB-) and M. bovis-infected young (TB+) and adult animals with different infection status [TB lesions localized in the head (TB+) or affecting multiple organs (TB++)]. The results identified host immune system proteins that play an important role in host response to mycobacteria. Calcium binding protein A9, Heme peroxidase, Lactotransferrin, Cathelicidin and Peptidoglycan-recognition protein were under-represented in TB+ animals when compared to uninfected TB- controls, but protein levels were higher as infection progressed in TB++ animals when compared to TB- and/or TB+ adult wild boar. MHCI was the only protein over-represented in TB+ adult wild boar when compared to uninfected TB- controls. The results reported here suggest that M. bovis manipulates host immune response by reducing the production of immune system proteins. However, as infection progresses, wild boar immune response recovers to limit pathogen

Detection of interferon-γ response to tuberculosis in blood collected at commencement of exsanguination at slaughter from cattle sensitized with Mycobacterium bovis.

PubMed

Okafor, Chika C; Grooms, Daniel L; Bolin, Steven R; Kaneene, John B

2012-06-01

To determine whether an interferon (IFN)-γ response sufficient to categorize cattle as positive for tuberculosis can be detected in blood collected at commencement of exsanguination at slaughter. 15 Holstein cows. 12 cows were experimentally sensitized by SC injection with inactivated Mycobacterium bovis in mineral oil, which induced an immune response that mimicked natural infection with M bovis. Three nonsensitized control cows were injected SC with mineral oil alone. By 5 weeks after injection, only the 12 sensitized cows had positive results for tuberculosis with whole blood IFN-γ assay. At that time, all 15 cows were sent to slaughter and samples of blood were collected from each cow immediately before stunning and at commencement of exsanguination (within 90 seconds after stunning). A whole blood IFN-γ assay was performed on the samples. Conditional probability and paired t tests were used to analyze changes in the categorical test interpretation and qualitative IFN-γ production, respectively. All 12 sensitized cows had positive results for tuberculosis in samples obtained immediately before stunning, and 9 retained positive results for samples obtained at commencement of exsanguination. There was a significant decrease in the mean background-corrected IFN-γ ELISA optical density values for samples obtained at commencement of exsanguination. IFN-γ response sufficient to classify cattle as positive for tuberculosis could be detected in blood collected at commencement of exsanguination. These findings support further development and use of the IFN-γ assay on blood samples collected at exsanguination as part of a bovine tuberculosis surveillance program.

Tuberculosis in Alpacas (Lama pacos) Caused by Mycobacterium bovis▿

PubMed Central

García-Bocanegra, I.; Barranco, I.; Rodríguez-Gómez, I. M.; Pérez, B.; Gómez-Laguna, J.; Rodríguez, S.; Ruiz-Villamayor, E.; Perea, A.

2010-01-01

We report three cases of tuberculosis in alpacas from Spain caused by Mycobacterium bovis. The animals revealed two different lesional patterns. Mycobacterial culture and PCR assay yielded positive results for M. bovis. Molecular typing of the isolates identified spoligotype SB0295 and identical variable-number tandem repeat (VNTR) allele sizes. PMID:20237097

Human Mycobacterium bovis infection in the United Kingdom: Incidence, risks, control measures and review of the zoonotic aspects of bovine tuberculosis.

PubMed

de la Rua-Domenech, Ricardo

2006-03-01

Amongst the members of the Mycobacterium tuberculosis complex (MTBC), M. tuberculosis is mainly a human pathogen, whereas M. bovis has a broad host range and is the principal agent responsible for tuberculosis (TB) in domestic and wild mammals. M. bovis also infects humans, causing zoonotic TB through ingestion, inhalation and, less frequently, by contact with mucous membranes and broken skin. Zoonotic TB is indistinguishable clinically or pathologically from TB caused by M. tuberculosis. Differentiation between the causative organisms may only be achieved by sophisticated laboratory methods involving bacteriological culture of clinical specimens, followed by typing of isolates according to growth characteristics, biochemical properties, routine resistance to pyrazinamide (PZA) and specific non-commercial nucleic acid techniques. All this makes it difficult to accurately estimate the proportion of human TB cases caused by M. bovis infection, particularly in developing countries. Distinguishing between the various members of the MTBC is essential for epidemiological investigation of human cases and, to a lesser degree, for adequate chemotherapy of the human TB patient. Zoonotic TB was formerly an endemic disease in the UK population, usually transmitted to man by consumption of raw cows' milk. Human infection with M. bovis in the UK has been largely controlled through pasteurization of cows' milk and systematic culling of cattle reacting to compulsory tuberculin tests. Nowadays the majority of the 7000 cases of human TB annually reported in the UK are due to M. tuberculosis acquired directly from an infectious person. In the period 1990-2003, between 17 and 50 new cases of human M. bovis infection were confirmed every year in the UK. This represented between 0.5% and 1.5% of all the culture-confirmed TB cases, a proportion similar to that of other industrialized countries. Most cases of zoonotic TB diagnosed in the UK are attributed to (i) reactivation of long

Use of fecal volatile organic compound analysis to discriminate between non-vaccinated and BCG—Vaccinated cattle prior to and after Mycobacterium bovis challenge

PubMed Central

Stahl, Randal; Waters, W. Ray; Palmer, Mitchell V.; Nol, Pauline; Rhyan, Jack C.; VerCauteren, Kurt C.; Koziel, Jacek A.

2017-01-01

Bovine tuberculosis is a zoonotic disease of global public health concern. Development of diagnostic tools to improve test accuracy and efficiency in domestic livestock and enable surveillance of wildlife reservoirs would improve disease management and eradication efforts. Use of volatile organic compound analysis in breath and fecal samples is being developed and optimized as a means to detect disease in humans and animals. In this study we demonstrate that VOCs present in fecal samples can be used to discriminate between non-vaccinated and BCG-vaccinated cattle prior to and after Mycobacterium bovis challenge. PMID:28686691

MRP8/14 induces autophagy to eliminate intracellular Mycobacterium bovis BCG.

PubMed

Wang, Jinli; Huang, Chunyu; Wu, Minhao; Zhong, Qiu; Yang, Kun; Li, Miao; Zhan, Xiaoxia; Wen, Jinsheng; Zhou, Lin; Huang, Xi

2015-04-01

To explore the role of myeloid-related protein 8/14 in mycobacterial infection. The mRNA and protein expression levels of MRP8 or MRP14 were measured by real-time PCR and flow cytometry, respectively. Role of MRP8/14 was tested by overexpression or RNA interference assays. Flow cytometry and colony forming unit were used to test the phagocytosis and the survival of intracellular Mycobacterium bovis BCG (BCG), respectively. Autophagy mediated by MRP8/14 was detected by Western blot and immunofluorescence. The colocalization of BCG phagosomes with autophagosomes or lysosomes was by detected by confocal microscopy. ROS production was detected by flow cytometry. MRP8/14 expressions were up-regulated in human monocytic THP1 cells and primary macrophages after mycobacterial challenge. Silencing of MRP8/14 suppressed bacterial killing, but had no influence on the phagocytosis of BCG. Importantly, silencing MRP8/14 decreased autophagy and BCG phagosome maturation in THP1-derived macrophages, thereby increasing the BCG survival. Additionally, we demonstrated that MRP8/14 promoted autophagy in a ROS-dependent manner. The present study revealed a novel role of MRP8/14 in the autophagy-mediated elimination of intracellular BCG by promoting ROS generation, which may provide a promising therapeutic target for tuberculosis and other intracellular bacterial infectious diseases. Copyright © 2014 The British Infection Association. Published by Elsevier Ltd. All rights reserved.

Identification of novel proteins in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin in the isoelectric point range 6-11.

PubMed

Florio, Walter; Batoni, Giovanna; Esin, Semih; Bottai, Daria; Maisetta, Giuseppantonio; Pardini, Manuela; Campa, Mario

2003-05-01

Two-dimensional gel electrophoresis and mass spectrometry were used to identify proteins in the isoelectric point range 6-11 in culture filtrates of Mycobacterium bovis bacillus Calmette-Guérin (BCG). Twelve proteins were identified, three of which had not been described previously. The expression of the identified proteins was comparatively analyzed in culture filtrates of BCG in different growth phases and culture conditions. For some of these proteins, the relative protein abundance in the different culture filtrate preparations was significantly different. The differential expression of the identified proteins is discussed in relation to their putative localization and/or biological function.

Photodynamic inactivation of the models Mycobacterium phlei and Mycobacterium smegmatis in vitro

NASA Astrophysics Data System (ADS)

Bruce-Micah, R.; Gamm, U.; Hüttenberger, D.; Cullum, J.; Foth, H.-J.

2009-07-01

Photodynamic inactivation (PDI) of bacterial strains presents an attractive potential alternative to antibiotic therapies. Success is dependent on the effective accumulation in bacterial cells of photochemical substances called photosensitizers, which are usually porphyrins or their derivatives. The kinetics of porphyrin synthesis after treatment with the precursor ALA and the accumulation of the Chlorin e6 and the following illumination were studied. The goal was to estimate effectivity of the destructive power of these PS in vitro in respect of the physiological states of Mycobacteria. So the present results examine the cell destruction by PDI using ALA-induced Porphyrins and Chlorin e6 accumulated in Mycobacterium phlei and Mycobacterium smegmatis, which serve as models for the important pathogens Mycobacterium tuberculosis, Mycobacterium leprae and Mycobacterium bovis. We could show that both Mycobacterium after ALA and Chlorin e6 application were killed by illumination with light of about 662 nm. A reduction of about 97% could be reached by using a lightdose of 70 mW/cm2.

Intranasal boosting with an adenovirus-vectored vaccine markedly enhances protection by parenteral Mycobacterium bovis BCG immunization against pulmonary tuberculosis.

PubMed

Santosuosso, Michael; McCormick, Sarah; Zhang, Xizhong; Zganiacz, Anna; Xing, Zhou

2006-08-01

Parenterally administered Mycobacterium bovis BCG vaccine confers only limited immune protection from pulmonary tuberculosis in humans. There is a need for developing effective boosting vaccination strategies. We examined a heterologous prime-boost regimen utilizing BCG as a prime vaccine and our recently described adenoviral vector expressing Ag85A (AdAg85A) as a boost vaccine. Since we recently demonstrated that a single intranasal but not intramuscular immunization with AdAg85A was able to induce potent protection from pulmonary Mycobacterium tuberculosis challenge in a mouse model, we compared the protective effects of parenteral and mucosal booster immunizations following subcutaneous BCG priming. Protection by BCG prime immunization was not effectively boosted by subcutaneous BCG or intramuscular AdAg85A. In contrast, protection by BCG priming was remarkably boosted by intranasal AdAg85A. Such enhanced protection by intranasal AdAg85A was correlated to the numbers of gamma interferon-positive CD4 and CD8 T cells residing in the airway lumen of the lung. Our study demonstrates that intranasal administration of AdAg85A represents an effective way to boost immune protection by parenteral BCG vaccination.

[Tuberculosis caused by Mycobacterium bovis in workers of bovine tuberculosis sanitation farms in Antioquia, Boyacá and Cundinamarca].

PubMed

Leal-Bohórquez, Andrés F; Castro-Osorio, Claudia M; Wintaco-Martínez, Luz M; Villalobos, Rafael; Puerto-Castro, Gloria M

2016-01-01

To perform classic and molecular epidemiological surveillance of human tuberculosis caused by Mycobacterium bovis in bovine supply chains at farms with PPD positive bovines in the departments of Antioquia, Boyacá and Cundinamarca during a one-year period. Livestock farms with PPD positive bovines or buffalos were visited in the study departments according to information obtained in the "Programa Nacional de Tuberculosis bovina" (National program on bovine Tuberculosis) released by ICA (Colombian Agriculture and Livestock Institute). Data on socio-demographic information and tuberculosis risk factors associated to the occupation were collected through a survey applied to all workers at the visited farms. Sputum samples were obtained after informed consent. The sputa underwent microbiological and molecular testing to identify members of the M. tuberculosis complex. Thirty-three livestock farms were visited and information of 164 workers from the bovine supply chain was collected. Staying in a PPD positive farm for more than a year, ignorance about the disease and the presence of possible vectors, like dogs and cats, were identified as possible risk factors for developing tuberculosis. No cases of tuberculosis caused by M. bovis or M. tuberculosis in workers of the visited farms were found. No cases of the disease caused by this zoonotic agent were documented in the departments of Antioquia, Boyacá and Cundinamarca.

An investigation of the effects of secondary processing on Mycobacterium spp. in naturally infected game meat and organs.

PubMed

Van der Merwe, M; Michel, A L

2010-09-01

The risk for humans to contract bovine tuberculosis through the consumption of undercooked game meat as well as biltong (traditionally dried game meat) is a concern. The survival potential of Mycobacterium bovis during the cooking and drying processes was researched in a preceding study on beef and the positive results compelled the authors to investigate the results with a similar preliminary study on game meat. Muscular, lymphatic and visceral tissues from skin test positive African buffalo (Syncerus caffer) and greater kudu (Tragelaphus strepsiceros) with tuberculous lesions were collected from the Hluhluwe iMfolozi Park during the park's culling programme. The different tissues were exposed to cooking and the muscular tissue to the drying process prior to culture. All acid-fast isolates were analysed by polymerase chain reaction for the presence of Mycobacterium bovis. All tissues were found negative for Mycobacterium bovis but non-tuberculous mycobacteria were isolated from kidney, liver, heart and lymph nodes. The results showed that these processes will kill Mycobacterium bovis but the unexpected recovery of non-tuberculous mycobacteria suggests possible survival and resistance characteristics of these strains which might be of veterinary public health interest.

Assessment of BCG and inactivated Mycobacterium bovis vaccines in an experimental tuberculosis infection model in sheep.

PubMed

Balseiro, Ana; Altuzarra, Raúl; Vidal, Enric; Moll, Xavier; Espada, Yvonne; Sevilla, Iker A; Domingo, Mariano; Garrido, Joseba M; Juste, Ramón A; Prieto, Miguel; Pérez de Val, Bernat

2017-01-01

Animal tuberculosis (TB) is a complex animal health problem that causes disruption to trade and significant economic losses. TB involves a multi-host system where sheep, traditionally considered a rare host of this infection, have been recently included. The aims of this study were to develop an experimental TB infection model in sheep with a Mycobacterium caprae field strain isolated from a tuberculous diseased ewe, and to use this to evaluate the safety and efficacy of two vaccines against TB in sheep, the live-attenuated M. bovis BCG vaccine (Danish strain) and a heat-inactivated M. bovis (HIMB) vaccine. Eighteen 2 month-old lambs were experimentally challenged with M. caprae by the endotracheal route (1.5 × 103 CFU). They were separated per treatment group into parenterally vaccinated with a live BCG Danish strain vaccine (n = 6), orally vaccinated with a suspension of HIMB (n = 6) and unvaccinated controls (n = 6). Clinical, immunological, pathological and bacteriological parameters of infection were measured. All lambs were successfully infected and developed gross TB lesions in the respiratory system. The BCG vaccine conferred considerable protection against experimental TB in lambs, as measured by a reduction of the gross lesion volumes and bacterial load. However, HIMB vaccinated animals did not show protection. This study proposes a reliable new experimental model for a better understanding of tuberculosis in sheep. BCG vaccination offers an effective prospect for controlling the disease. Moreover alternative doses and/or routes of administration should be considered to evaluate the efficacy of the HIMB vaccine candidate.

The prevalence, distribution and severity of detectable pathological lesions in badgers naturally infected with Mycobacterium bovis

PubMed Central

JENKINS, H. E.; MORRISON, W. I.; COX, D. R.; DONNELLY, C. A.; JOHNSTON, W. T.; BOURNE, F. J.; CLIFTON-HADLEY, R. S.; GETTINBY, G.; McINERNEY, J. P.; WATKINS, G. H.; WOODROFFE, R.

2008-01-01

SUMMARY The Randomized Badger Culling Trial (RBCT) began in 1998 to determine the impact of badger culling in controlling bovine tuberculosis in cattle. A total of 1166 badgers (14% of total) proactively culled during the RBCT were found to be tuberculous, offering a unique opportunity to study the pathology caused by Mycobacterium bovis in a large sample of badgers. Of these, 39% of adults (~6% of all adults culled) had visible lesions (detectable at necropsy) of bovine tuberculosis; cubs had a lower prevalence of infection (9%) but a higher percentage of tuberculous cubs (55·5%) had visible lesions. Only ~1% of adult badgers had extensive, severe pathology. Tuberculous badgers with recorded bite wounds (~5%) had a higher prevalence of visible lesions and a different distribution of lesions, suggesting transmission via bite wounds. However, the predominance of lesions in the respiratory tract indicates that most transmission occurs by the respiratory route. PMID:18047751

Sonic hedgehog-Dependent Induction of MicroRNA 31 and MicroRNA 150 Regulates Mycobacterium bovis BCG-Driven Toll-Like Receptor 2 Signaling

PubMed Central

Ghorpade, Devram Sampat; Holla, Sahana; Kaveri, Srini V.; Bayry, Jagadeesh; Patil, Shripad A.

2013-01-01

Hedgehog (HH) signaling is a significant regulator of cell fate decisions during embryogenesis, development, and perpetuation of various disease conditions. Testing whether pathogen-specific HH signaling promotes unique innate recognition of intracellular bacteria, we demonstrate that among diverse Gram-positive or Gram-negative microbes, Mycobacterium bovis BCG, a vaccine strain, elicits a robust activation of Sonic HH (SHH) signaling in macrophages. Interestingly, sustained tumor necrosis factor alpha (TNF-α) secretion by macrophages was essential for robust SHH activation, as TNF-α−/− macrophages exhibited compromised ability to activate SHH signaling. Neutralization of TNF-α or blockade of TNF-α receptor signaling significantly reduced the infection-induced SHH signaling activation both in vitro and in vivo. Intriguingly, activated SHH signaling downregulated M. bovis BCG-mediated Toll-like receptor 2 (TLR2) signaling events to regulate a battery of genes associated with divergent functions of M1/M2 macrophages. Genome-wide expression profiling as well as conventional gain-of-function or loss-of-function analysis showed that SHH signaling-responsive microRNA 31 (miR-31) and miR-150 target MyD88, an adaptor protein of TLR2 signaling, thus leading to suppression of TLR2 responses. SHH signaling signatures could be detected in vivo in tuberculosis patients and M. bovis BCG-challenged mice. Collectively, these investigations identify SHH signaling to be what we believe is one of the significant regulators of host-pathogen interactions. PMID:23166298

Mycobacterium bovis BCG mycobacteria--new application.

PubMed

Kowalewicz-Kulbat, Magdalena; Pestel, Joël; Biet, Franck; Locht, Camille; Tonnel, André-Bernard; Druszczyńska, Magdalena; Rudnicka, Wiesława

2006-01-01

The polarized response of T helper-2 (Th2) lymphocytes to an allergen is considered to be the main cause of the pathogenesis of asthma. In this study, we asked a question whether M. bovis BCG mycobacteria which are known for the preferential stimulation of T helper-1 (Th1) immunity, diminish the effector functions of Th2 cells from allergic patients upon stimulation with a common house dust mite Der p-1 allergen. Our results allow a positive answer to this question. We demonstrate that BCG modulates the dendritic cell-dependent allergen presentation process and switches naive T lymphocytes towards an anti-allergic Th1 profile.

Deletion of zmp1 improves Mycobacterium bovis BCG-mediated protection in a guinea pig model of tuberculosis.

PubMed

Sander, Peter; Clark, Simon; Petrera, Agnese; Vilaplana, Cristina; Meuli, Michael; Selchow, Petra; Zelmer, Andrea; Mohanan, Deepa; Andreu, Nuria; Rayner, Emma; Dal Molin, Michael; Bancroft, Gregory J; Johansen, Pål; Cardona, Pere-Joan; Williams, Ann; Böttger, Erik C

2015-03-10

Having demonstrated previously that deletion of zinc metalloprotease zmp1 in Mycobacterium bovis BCG increased immunogenicity of BCG vaccines, we here investigated the protective efficacy of BCG zmp1 deletion mutants in a guinea pig model of tuberculosis infection. zmp1 deletion mutants of BCG provided enhanced protection by reducing the bacterial load of tubercle bacilli in the lungs of infected guinea pigs. The increased efficacy of BCG due to zmp1 deletion was demonstrated in both BCG Pasteur and BCG Denmark indicating that the improved protection by zmp1 deletion is independent from the BCG sub-strain. In addition, unmarked BCG Δzmp1 mutant strains showed a better safety profile in a CB-17 SCID mouse survival model than the parental BCG strains. Together, these results support the further development of BCG Δzmp1 for use in clinical trials. Copyright © 2015 Elsevier Ltd. All rights reserved.

[Differentiation of species within the Mycobacterium tuberculosis complex by molecular techniques].

PubMed

Herrera-León, Laura; Pozuelo-Díaz, Rodolfo; Molina Moreno, Tamara; Valverde Cobacho, Azucena; Saiz Vega, Pilar; Jiménez Pajares, María Soledad

2009-11-01

The Mycobacterium tuberculosis complex includes the following species: Mycobacterium tuberculosis, Mycobacterium africanum, Mycobacterium bovis, Mycobacterium bovis-BCG, Mycobacterium microti, Mycobacterium caprae, Mycobacterium pinnipedii, and Mycobacterium canettii. These species cause tuberculosis in humans and animals. Identification of mycobacterial strains has classically been performed by phenotype study. Over the last years, laboratories have developed several molecular techniques to differentiate between these species. The aim of this study is to evaluate these methods and develop a simple, fast, identification scheme. We analyzed 251 strains randomly obtained from the strains studied in 2004, and 797 strains received by the Reference Laboratory between 2005 and 2007. Phenotype characterization of 4183 strains isolated during that period was done by studying the colony morphology, characteristics in culture, nitrate reduction, niacin accumulation, and growth in the presence of thiophen-2-carboxylic acid hydrazide 10 microg/mL and pyrazinamide 50 microg/mL. The molecular identification scheme designed was as follows: 1) gyrB PCR-RFLP with RsaI, TaqI or SacII and hsp65 RFLP/PCR with HhaI., and 2) multiplex-PCR to determine the presence/absence of the RD9 and RD1 regions. The results showed 100% agreement between phenotype study and the molecular scheme. This molecular identification scheme is a simple and fast method, with 100% sensitivity and specificity, that can be implemented in most clinical laboratories at a low cost.

Macrophage-Inducible C-Type Lectin Mincle-Expressing Dendritic Cells Contribute to Control of Splenic Mycobacterium bovis BCG Infection in Mice

PubMed Central

Behler, Friederike; Maus, Regina; Bohling, Jennifer; Knippenberg, Sarah; Kirchhof, Gabriele; Nagata, Masahiro; Jonigk, Danny; Izykowski, Nicole; Mägel, Lavinia; Welte, Tobias; Yamasaki, Sho

2014-01-01

The macrophage-inducible C-type lectin Mincle has recently been identified to be a pattern recognition receptor sensing mycobacterial infection via recognition of the mycobacterial cell wall component trehalose-6′,6-dimycolate (TDM). However, its role in systemic mycobacterial infections has not been examined so far. Mincle-knockout (KO) mice were infected intravenously with Mycobacterium bovis BCG to mimic the systemic spread of mycobacteria under defined experimental conditions. After intravenous infection with M. bovis BCG, Mincle-KO mice responded with significantly higher numbers of mycobacterial CFU in spleen and liver, while reduced granuloma formation was observed only in the spleen. At the same time, reduced Th1 cytokine production and decreased numbers of gamma interferon-producing T cells were observed in the spleens of Mincle-KO mice relative to the numbers in the spleens of wild-type (WT) mice. The effect of adoptive transfer of defined WT leukocyte subsets generated from bone marrow cells of zDC+/DTR mice (which bear the human diphtheria toxin receptor [DTR] under the control of the classical dendritic cell-specific zinc finger transcription factor zDC) to specifically deplete Mincle-expressing classical dendritic cells (cDCs) but not macrophages after diphtheria toxin application on the numbers of splenic and hepatic CFU and T cell subsets was then determined. Adoptive transfer experiments revealed that Mincle-expressing splenic cDCs rather than Mincle-expressing macrophages contributed to the reconstitution of attenuated splenic antimycobacterial immune responses in Mincle-KO mice after intravenous challenge with BCG. Collectively, we show that expression of Mincle, particularly by cDCs, contributes to the control of splenic M. bovis BCG infection in mice. PMID:25332121

Definition of purified enzyme-linked immunosorbent assay antigens from the culture filtrate protein of Mycobacterium bovis by proteomic analysis.

PubMed

Cho, Yun Sang; Lee, Sang-Eun; Ko, Young Joon; Cho, Donghee; Lee, Hyang Shim; Hwang, Inyeong; Nam, Hyangmi; Heo, Eunjung; Kim, Jong Man; Jung, Sukchan

2009-01-01

Enzyme-linked immunosorbent assay (ELISA) has been developed as the ancillary diagnosis of bovine tuberculosis at ante-mortem to overcome the disadvantages of intradermal skin test. In this study, the antigenic proteins were purified, applied to bTB ELISA, and identified through proteomic analysis. Culture filtrate protein of Mycobacterium bovis was fractionated by MonoQ column chromatography, and examined the antigenicity by immunoblotting. The antigenic 20 kDa protein was in-gel digested and identified the antigenome by LTQ mass spectrometer and peptide match fingerprinting, which were MPB64, MPB70, MPB83, Fas, Smc, Nrp, RpoC, Transposase, LeuA, and MtbE. The 20 kDa protein exhibited the highest antigenicity to bTB positive cattle in ELISA and would be useful for bTB serological diagnosis.

Interferometry as a tool for evaluating effects of antimicrobial doses on Mycobacterium bovis growth.

PubMed

Machado, Rachel R P; Dutra, Rafael C; Raposo, Nádia R B; Lesche, Bernhard; Gomes, Marlei S; Duarte, Rafael S; Soares, Geraldo Luiz G; Kaplan, Maria Auxiliadora C

2015-12-01

Interferometry was used together with the conventional microplate resazurin assay to evaluate the antimycobacterial properties of essential oil (EO) from fruits of Pterodon emarginatus and also of rifampicin against Mycobacterium bovis. The aim of this work is not only to investigate the potential antimycobacterial activity of this EO, but also to test the interferometric method in comparison with the conventional one. The Minimum Inhibitory Concentration (MIC) values of EO (625 μg/mL) and rifampicin (4 ng/mL) were firstly identified with the microplate method. These values were used as parameters in Drug Susceptibility Tests (DST) with interferometry. The interferometry confirmed the MIC value of EO identified with microplate and revealed a bacteriostatic behavior for this concentration. At 2500 μg/mL interferometry revealed bactericidal activity of the EO. Mycobacterial growth was detected with interferometry at 4 ng/mL of rifampicin and even at higher concentrations. One important difference is that the interferometric method preserves the sample, so that after weeks of quantitative observation, the sample can be used to evaluate the bactericidal activity of the tested drug. Copyright © 2015 Elsevier Ltd. All rights reserved.

Vaccination of cattle with Mycobacterium bovis BCG by a combination of systemic and oral routes.

PubMed

Buddle, Bryce M; Denis, Michel; Aldwell, Frank E; Martin Vordermeier, H; Glyn Hewinson, R; Neil Wedlock, D

2008-11-01

Mycobacterium bovis bacille Calmette-Guérin (BCG) vaccine delivered to calves by the subcutaneous (s.c.) or by the oral route in a formulated lipid matrix has been previously shown to induce similar levels of protection against bovine tuberculosis. The current study was aimed at determining whether a combination of delivering BCG by s.c. and oral routes would enhance levels of protection, compared to only one route of vaccination. Forty calves were randomly divided into four groups (10/group). Calves were vaccinated with 10(6)colony forming units (CFU) of BCG Pasteur by the s.c. route or orally with 10(9)CFU BCG incorporated into a lipid formulation. One group received a combination of BCG administered by both the s.c. and oral routes and a non-vaccinated group served as a control. The two groups of calves that received s.c. BCG produced strong IFN-gamma responses in whole blood cultures stimulated with bovine purified protein derivative (PPD) 3 weeks after vaccination. Cattle vaccinated just with oral BCG in a lipid matrix produced a strong IFN-gamma response 8 weeks after vaccination, and peaking at 11 weeks after vaccination. All calves were challenged by the intratracheal route with M. bovis 15 weeks after vaccination and were euthanized and necropsied to assess protection at 17 weeks following challenge. BCG given s.c. or orally induced significant and comparable levels of protection against the virulent challenge. Vaccination of cattle by a combination of s.c./oral routes did not enhance protection beyond that achieved by s.c. or oral vaccination alone. We conclude that vaccination of cattle with BCG by a combination of routes has no beneficial additive effects, compared to a single s.c. administration of BCG or BCG given orally in a lipid formulation.

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur

PubMed Central

2011-01-01

Background Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. Results The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3 - 8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Conclusions Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB. PMID:21507239

The predominance of Ethiopian specific Mycobacterium tuberculosis families and minimal contribution of Mycobacterium bovis in tuberculous lymphadenitis patients in Southwest Ethiopia.

PubMed

Tadesse, Mulualem; Abebe, Gemeda; Bekele, Alemayehu; Bezabih, Mesele; de Rijk, Pim; Meehan, Conor J; de Jong, Bouke C; Rigouts, Leen

2017-11-01

Ethiopia has an extremely high rate of extrapulmonary tuberculosis, dominated by tuberculous lymphadenitis (TBLN). However, little is known about Mycobacterium tuberculosis complex (MTBc) lineages responsible for TBLN in Southwest Ethiopia. A total of 304 MTBc isolates from TBLN patients in Southwest Ethiopia were genotyped primarily by spoligotyping. Isolates of selected spoligotypes were further analyzed by 15-loci mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) (n=167) and qPCR-based single nucleotide polymorphism (n=38). Isolates were classified into main phylogenetic lineages and families by using the reference strain collections and identification tools available at MIRU-VNTRplus data base. Resistance to rifampicin was determined by Xpert MTB/RIF. The majority of isolates (248; 81.6%) belonged to the Euro-American lineage (Lineage 4), with the ill-defined T and Haarlem as largest families comprising 116 (38.2%) and 43 (14.1%) isolates respectively. Of the T family, 108 isolates were classified as being part of the newly described Ethiopian families, namely Ethiopia_2 (n=44), Ethiopia_3 (n=34) and Ethiopia_H 37 Rv-like (n=30). Other sub-lineages included URAL (n=18), S (n=17), Uganda I (n=16), LAM (n=13), X (n=5), TUR (n=5), Uganda II (n=4) and unknown (n=19). Lineage 3 (Delhi/CAS) was the second most common lineage comprising 44 (14.5%) isolates. Interestingly, six isolates (2%) were belonged to Lineage 7, unique to Ethiopia. Lineage 1 (East-African Indian) and Lineage 2 (Beijing) were represented by 3 and 1 isolates respectively. M. bovis was identified in only two (0.7%) TBLN cases. The cluster rate was highest for Ethiopia_3 isolates showing clonal similarity with isolates from North Ethiopia. Lineage 3 was significantly associated with rifampicin resistance. In TBLN in Southwest Ethiopia, the recently described Ethiopia specific Lineage 4 families were predominant, followed by Lineage 3 and Lineage 4-Haarlem. The

Knowledge, Attitudes and Practices Regarding Risk to Human Infection due to Mycobacterium bovis among Cattle Farming Communities in Western Uganda.

PubMed

Kazoora, H B; Majalija, S; Kiwanuka, N; Kaneene, J B

2016-12-01

A cross-sectional study involving multistage cluster sampling was undertaken in Kashari county, Mbarara district, western Uganda, in which quantitative and qualitative approaches were utilized to determine the knowledge, attitudes and practices regarding risk of human infection with zoonotic tuberculosis due to Mycobacterium bovis among cattle farmers. Of 496 respondents, 50% were farm owners and 50% herdsmen. Males were 70.9% of all the study participants. Among these, 37.5% had good knowledge, 41.4% had positive attitudes and 75.2% good practices regarding zoonotic tuberculosis. In the multivariable model, good knowledge was associated with having ever received health education, spending more than 5 years keeping cattle, having heard of cattle condemned at the abattoir due to tuberculosis and marital status. Positive attitudes were associated with having ever received health education, having heard of cattle condemned at the abattoir due to tuberculosis and being a farm owner versus being a herdsman. Good practices were associated with health education and good knowledge of the disease. Overall, knowledge and attitudes towards zoonotic tuberculosis due to M. bovis in humans was found to be low. While the majority of the respondents reported good practices, there were some still consuming raw milk and its products, which may predispose them to infection and indicates the need for greater outreach for zoonotic tuberculosis education. © 2016 Blackwell Verlag GmbH.

The use of PCR technique in the identification of Mycobacterium species responsible for bovine tuberculosis in cattle and buffaloes in Pakistan.

PubMed

Akhtar, Farah; Javed, Muhammad Tariq; Aziz-ur-Rehman; Khan, Muhammad Nisar; Akhtar, Pervez; Hussain, Sayed Misdaq; Aslam, Muhammad Sohaib; Kausar, Razia; Qamar, Mehwish; Cagiola, Monica

2015-08-01

Bovine tuberculosis is one of the important diseases of dairy and wild animals. The disease is prevalent all over the world, though developed countries have tremendously reduced the prevalence through eradication campaigns. The prevalence of disease in Pakistan on the basis of tuberculin testing or culture isolation of the organism has been reported previously. It is, however, important to use the latest diagnostic tools, i.e. PCR to confirm the type of Mycobacterium infecting the animals in Pakistan. Therefore, the present study was carried out to assess the utility of direct PCR on milk samples and nasal swabs to confirm the type of Mycobacterium infecting the animals. This study was carried out on 215 cattle and buffaloes of more than 2 years of age present at two livestock farms. The tuberculin results showed 22.5% prevalence at one farm and 25.9% at the other with an overall prevalence of 24.7%. The 92.5% of milk samples and/or nasal swabs showed positive PCR for Mycobacterium genus, 86.8% for Mycobacterium tuberculosis complex and 77.4% for Mycobacterium bovis. The M. bovis by PCR was detected in 13.2% of milk samples, 24.5% of nasal swabs and 39.6% of both milk samples + nasal swabs. The results suggested that there are 60% higher chance for a nasal swab to yield a positive PCR for M. bovis than the milk sample. It can be concluded from the present study that tuberculin testing is a useful method in studying the prevalence of disease as the PCR for Mycobacterium genus was positive in 92.5%, M. tuberculosis complex in 86.8% and Mycobacterium bovis in 77.4% cases.

Parenteral Vaccination with Heat-Inactivated Mycobacterium Bovis Reduces the Prevalence of Tuberculosis-Compatible Lesions in Farmed Wild Boar.

PubMed

Díez-Delgado, I; Rodríguez, O; Boadella, M; Garrido, J M; Sevilla, I A; Bezos, J; Juste, R; Domínguez, L; Gortázar, C

2017-10-01

In 2012, a wild boar (Sus scrofa) tuberculosis (TB) control programme was set up in a wild boar farm by means of intramuscular (IM) vaccination with a heat-inactivated Mycobacterium bovis vaccine (IV). The goal was to assess safety and efficacy of the parenterally administered IV in a large farm setting with natural M. bovis circulation. Based on preceding results under laboratory conditions, we hypothesized that vaccinated piglets would show smaller scores of TB-compatible lesions (TBCL) than unvaccinated controls. After vaccination, no adverse reactions were detected by visual inspection or at post-mortem examination (n = 668 and 97, respectively). Post-mortem data on TBCL were available for 97 vaccinated wild boar and 182 controls. The observed TBCL prevalence was 4.1% (95% CI = 0.2-8%) and 12.1% (95% CI = 7.1-17.1%) for vaccinated and control wild boar, respectively (P < 0.05). Among those animals with TBCL, no difference in the mean lesion score was found (P > 0.05). The results show that IV administered intramuscularly to wild boar piglets is safe and protects vaccinated individuals (66% reduction in TBCL prevalence) against natural challenge in a low-prevalence setting. In a context of increasing TB prevalence in wild boar in Mediterranean habitats, vaccination achieved a progressive though slow decline in lesion prevalence since the onset of the vaccination scheme. Hence, vaccination might contribute, along with other tools, to TB control in wild boar and in pigs. © 2016 Blackwell Verlag GmbH.

Goats challenged with different members of the Mycobacterium tuberculosis complex display different clinical pictures.

PubMed

Bezos, J; Casal, C; Díez-Delgado, I; Romero, B; Liandris, E; Álvarez, J; Sevilla, I A; Juan, L de; Domínguez, L; Gortázar, C

2015-10-15

Tuberculosis (TB) in goats (Capra hircus) is due to infection with members of the Mycobacterium tuberculosis complex (MTC), mainly Mycobacterium bovis and Mycobacterium caprae. We report a comparative experimental infection of goats with M. bovis, M. caprae and M. tuberculosis strains. We hypothesized that goats experimentally infected with different members of the MTC would display different clinical pictures. Three groups of goats were challenged with either M. bovis SB0134 (group 1, n=5), M. caprae SB0157 (group 2, n=5) and M. tuberculosis SIT58 (group 3, n=4). The highest mean total lesion score was observed in M. bovis challenged goats (mean 15.2, range 9-19), followed by those challenged with M. caprae (10.8, 2-23). The lowest score was recorded in goats challenged with M. tuberculosis (3, 1-6). Culture results coincided with the lesion scores in yielding more positive pools (7/15) in M. bovis challenged goats. By contrast, only three pools were positive from goats challenged M. tuberculosis (3/12) and with M. caprae (3/15), respectively. Differences in the performance of the intradermal and gamma-interferon (IFN-γ) tests depending of the group were observed since all goats from group 1 were diagnosed using intradermal test and these goats reacted earlier to the IFN-γ assay in comparison to the other groups. This study confirmed that goats experimentally infected with different members of the MTC display different clinical pictures and this fact may have implications for MTC maintenance and bacterial shedding. Copyright © 2015 Elsevier B.V. All rights reserved.

Development of an in vitro model of the early-stage bovine tuberculous granuloma using Mycobacterium bovis-BCG.

PubMed

Garza-Cuartero, Laura; McCarthy, Elaine; Brady, Joseph; Cassidy, Joseph; Hamilton, Clare; Sekiya, Mary; NcNair, Jim; Mulcahy, Grace

2015-12-15

Mycobacterium bovis causes 3.1% of human tuberculosis cases, as described by the World Health Organisation. In cattle, this organism causes bovine tuberculosis (BTB) which can have a prevalence of up to 39.5% in some developing countries. In developed countries, although the prevalence of BTB has been reduced through eradication programmes, complete eradication has in some cases proved elusive, with prevalences in cattle of 0.5% in the Republic of Ireland and of 4.3% in the UK. As the tuberculous granuloma is the fundamental lesion that reflects the pathogenesis, immune control and progression of BTB, we aimed to develop an in vitro model of the early-stage bovine tuberculous granuloma, in order to model the early stages of BTB, while also reducing the use of experimentally infected animals. In vitro models of human and ovine mycobacterial granulomas have previously been developed; however, so far, there is no model for the BTB granuloma. As the disease in cattle differs in a number of ways from that in other species, we consider this to be a significant gap in the tools available to study the pathogenesis of BTB. By combining bovine monocyte-derived macrophages infected with M. bovis-BCG and autologous lymphocytes we have developed an early-stage tuberculous bovine granuloma model. In the model, 3D cell aggregations formed a spherical-shape that grew for up to 11 days post-infection. This bovine tuberculous granuloma model can aid in the study of such lesion development, and in comparative studies of pathogenesis, such as, for example, the question of mycobacterial latency in bovine tuberculosis. Copyright © 2015 Elsevier B.V. All rights reserved.

Evidence of presence of Mycobacterium tuberculosis in bovine tissue samples by multiplex PCR: possible relevance to reverse zoonosis.

PubMed

Mittal, M; Chakravarti, S; Sharma, V; Sanjeeth, B S; Churamani, C P; Kanwar, N S

2014-04-01

Bovine tuberculosis, caused by Mycobacterium bovis, remains one of the most important zoonotic health concerns worldwide. The transmission of Mycobacterium tuberculosis from humans to animals also occurs especially in countries where there is close interaction of humans with the animals. In the present study, thirty bovine lung tissue autopsy samples from an organized dairy farm located in North India were screened for the presence of Mycobacterium tuberculosis complex by smear microscopy, histopathological findings and PCR. Differential diagnosis of M. tuberculosis and M. bovis was made based on the deletion of mce-3 operon in M. bovis. The present study found eight of these samples positive for M. tuberculosis by multiplex PCR. Sequencing was performed on two PCR-positive representative samples and on annotation, and BLAST analysis confirmed the presence of gene fragment specific to Mycobacterium tuberculosis. The presence of M. tuberculosis in all the positive samples raises the possibility of human-to-cattle transmission and possible adaptation of this organism in bovine tissues. This study accentuates the importance of screening and differential diagnosis of Mycobacterium tuberculosis complex in humans and livestock for adopting effective TB control and eradication programmes. © 2014 Blackwell Verlag GmbH.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil.

PubMed

Sgarioni, Sônia Aparecida; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Leite, Clarice Queico Fujimura; de Prince, Karina Andrade; de Andrade Leite, Sergio Roberto; Filho, Dirceu Vedovello; Siqueira, Vera Lucia Dias; Caleffi-Ferracioli, Katiany Rizzieri; Cardoso, Rosilene Fressatti

2014-01-01

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5-10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil.

Occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk in the northwestern region of Paraná, Brazil

PubMed Central

Sgarioni, Sônia Aparecida; Hirata, Rosario Dominguez Crespo; Hirata, Mario Hiroyuki; Leite, Clarice Queico Fujimura; de Prince, Karina Andrade; de Andrade Leite, Sergio Roberto; Filho, Dirceu Vedovello; Siqueira, Vera Lucia Dias; Caleffi-Ferracioli, Katiany Rizzieri; Cardoso, Rosilene Fressatti

2014-01-01

Milk is widely consumed in Brazil and can be the vehicle of agent transmission. In this study, was evaluated the occurrence of Mycobacterium bovis and non-tuberculous mycobacteria (NTM) in raw and pasteurized milk consumed in the northwestern region of Paraná, Brazil. Fifty-two milk samples (20 pasteurized and 32 raw) from dairy farms near the municipality of Maringa, Parana State, Brazil were collected. Milk samples were decontaminated using 5% oxalic acid method and cultured on Lowenstein-Jensen and Stonebrink media at 35 °C and 30 °C, with and without 5–10% CO2. Mycobacteria isolates were identified by morphological features, PCR-Restriction Fragment Length Polymorphism Analysis (PCR-PRA) and Mycolic acids analysis. Thirteen (25%) raw and 2 (4%) pasteurized milk samples were positive for acid fast bacilli growth. Nine different species of NTM were isolated (M. nonchromogenicum, M. peregrinum, M. smegmatis, M. neoaurum, M. fortuitum, M. chelonae, M. flavescens, M. kansasii and M. scrofulaceum). M. bovis was not detected. Raw and pasteurized milk may be considered one source for NTM human infection. The paper reinforces the need for intensification of measures in order to avoid the milk contamination and consequently prevent diseases in the south of Brazil. PMID:25242962

Detection of a Putative TetR-Like Gene Related to Mycobacterium bovis BCG Growth in Cholesterol Using a gfp-Transposon Mutagenesis System

PubMed Central

Otal, Isabel; Pérez-Herrán, Esther; Garcia-Morales, Lazaro; Menéndez, María C.; Gonzalez-y-Merchand, Jorge A.; Martín, Carlos; García, María J.

2017-01-01

In vitro transposition is a powerful genetic tool for identifying mycobacterial virulence genes and studying virulence factors in relation to the host. Transposon shuttle mutagenesis is a method for constructing stable insertions in the genome of different microorganisms including mycobacteria. Using an IS1096 derivative, we have constructed the Tngfp, a transposon containing a promoterless green fluorescent protein (gfp) gene. This transposon was able to transpose randomly in Mycobacterium bovis BCG. Bacteria with a single copy of the gfp gene per chromosome from an M. bovis BCG::Tngfp library were analyzed and cells exhibiting high levels of fluorescence were detected by flow cytometry. Application of this approach allowed for the selection of a mutant, BCG_2177c::Tngfp (BCG-Tn), on the basis of high level of long-standing fluorescence at stationary phase. This BCG-Tn mutant showed some particular phenotypic features compared to the wild type strain, mainly during stationary phase, when cholesterol was used as a sole carbon source, thus supporting the relationships of the targeted gene with the regulation of cholesterol metabolism in this bacteria. This approach showed that Tngfp is a potentially useful tool for studying the involvement of the targeted loci in metabolic pathways of mycobacteria. PMID:28321208

Combining blue native polyacrylamide gel electrophoresis with liquid chromatography tandem mass spectrometry as an effective strategy for analyzing potential membrane protein complexes of Mycobacterium bovis bacillus Calmette-Guérin

PubMed Central

2011-01-01

Background Tuberculosis is an infectious bacterial disease in humans caused primarily by Mycobacterium tuberculosis, and infects one-third of the world's total population. Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine has been widely used to prevent tuberculosis worldwide since 1921. Membrane proteins play important roles in various cellular processes, and the protein-protein interactions involved in these processes may provide further information about molecular organization and cellular pathways. However, membrane proteins are notoriously under-represented by traditional two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) and little is known about mycobacterial membrane and membrane-associated protein complexes. Here we investigated M. bovis BCG by an alternative proteomic strategy coupling blue native PAGE to liquid chromatography tandem mass spectrometry (LC-MS/MS) to characterize potential protein-protein interactions in membrane fractions. Results Using this approach, we analyzed native molecular composition of protein complexes in BCG membrane fractions. As a result, 40 proteins (including 12 integral membrane proteins), which were organized in 9 different gel bands, were unambiguous identified. The proteins identified have been experimentally confirmed using 2-D SDS PAGE. We identified MmpL8 and four neighboring proteins that were involved in lipid transport complexes, and all subunits of ATP synthase complex in their monomeric states. Two phenolpthiocerol synthases and three arabinosyltransferases belonging to individual operons were obtained in different gel bands. Furthermore, two giant multifunctional enzymes, Pks7 and Pks8, and four mycobacterial Hsp family members were determined. Additionally, seven ribosomal proteins involved in polyribosome complex and two subunits of the succinate dehydrogenase complex were also found. Notablely, some proteins with high hydrophobicity or multiple transmembrane helixes were identified well

Description of Mycobacterium chelonae subsp. bovis subsp. nov., isolated from cattle (Bos taurus coreanae), emended description of Mycobacterium chelonae and creation of Mycobacterium chelonae subsp. chelonae subsp. nov.

PubMed

Kim, Byoung-Jun; Kim, Ga-Na; Kim, Bo-Ram; Jeon, Che Ok; Jeong, Joseph; Lee, Seon Ho; Lim, Ji-Hun; Lee, Seung-Heon; Kim, Chang Ki; Kook, Yoon-Hoh; Kim, Bum-Joon

2017-10-01

Three rapidly growing mycobacterial strains, QIA-37 T , QIA-40 and QIA-41, were isolated from the lymph nodes of three separate Korean native cattle, Hanwoo (Bos taurus coreanae). These strains were previously shown to be phylogenetically distinct but closely related to Mycobacterium chelonae ATCC 35752 T by taxonomic approaches targeting three genes (16S rRNA, hsp6 and rpoB) and were further characterized using a polyphasic approach in this study. The 16S rRNA gene sequences of all three strains showed 99.7 % sequence similarity with that of the M. chelonae type strain. A multilocus sequence typing analysis targeting 10 housekeeping genes, including hsp65 and rpoB, revealed a phylogenetic cluster of these strains with M. chelonae. DNA-DNA hybridization values of 78.2 % between QIA-37 T and M. chelonae indicated that it belongs to M. chelonae but is a novel subspecies distinct from M. chelonae. Phylogenetic analysis based on whole-genome sequences revealed a 95.44±0.06 % average nucleotide identity (ANI) value with M. chelonae, slightly higher than the 95.0 % ANI criterion for determining a novel species. In addition, distinct phenotypic characteristics such as positive growth at 37 °C, at which temperature M. chelonae does not grow, further support the taxonomic status of these strains as representatives of a novel subspecies of M. chelonae. Therefore, we propose an emended description of Mycobacterium chelonae, and descriptions of M. chelonae subsp. chelonae subsp. nov. and M. chelonae subsp. bovis subsp. nov. are presented; strains ATCC 35752 T (=CCUG 47445 T =CIP 104535 T =DSM 43804 T =JCM 6388 T =NCTC 946 T ) and QIA-37 T (=KCTC 39630 T =JCM 30986 T ) are the type strains of the two novel subspecies.

Effect of skin test on serum antibody responses to Mycobacterium bovis infection in cattle

USDA-ARS?s Scientific Manuscript database

Recently, several serologic tests designed to detect immunodominant antibodies to M. bovis antigens (e.g., MPB83, MPB70, ESAT-6, and CFP10) have emerged for potential use with samples from cattle. Of these, a commercial ELISA to MPB83/MPB70 (M. bovis antibody ELISA) has gained approval for use in ca...

Investigation of the high rates of extrapulmonary tuberculosis in Ethiopia reveals no single driving factor and minimal evidence for zoonotic transmission of Mycobacterium bovis infection.

PubMed

Berg, Stefan; Schelling, Esther; Hailu, Elena; Firdessa, Rebuma; Gumi, Balako; Erenso, Girume; Gadisa, Endalamaw; Mengistu, Araya; Habtamu, Meseret; Hussein, Jemal; Kiros, Teklu; Bekele, Shiferaw; Mekonnen, Wondale; Derese, Yohannes; Zinsstag, Jakob; Ameni, Gobena; Gagneux, Sebastien; Robertson, Brian D; Tschopp, Rea; Hewinson, Glyn; Yamuah, Lawrence; Gordon, Stephen V; Aseffa, Abraham

2015-03-03

Ethiopia, a high tuberculosis (TB) burden country, reports one of the highest incidence rates of extra-pulmonary TB dominated by cervical lymphadenitis (TBLN). Infection with Mycobacterium bovis has previously been excluded as the main reason for the high rate of extrapulmonary TB in Ethiopia. Here we examined demographic and clinical characteristics of 953 pulmonary (PTB) and 1198 TBLN patients visiting 11 health facilities in distinct geographic areas of Ethiopia. Clinical characteristics were also correlated with genotypes of the causative agent, Mycobacterium tuberculosis. No major patient or bacterial strain factor could be identified as being responsible for the high rate of TBLN, and there was no association with HIV infection. However, analysis of the demographic data of involved patients showed that having regular and direct contact with live animals was more associated with TBLN than with PTB, although no M. bovis was isolated from patients with TBLN. Among PTB patients, those infected with Lineage 4 reported "contact with other TB patient" more often than patients infected with Lineage 3 did (OR = 1.6, CI 95% 1.0-2.7; p = 0.064). High fever, in contrast to low and moderate fever, was significantly associated with Lineage 4 (OR = 2.3; p = 0.024). On the other hand, TBLN cases infected with Lineage 4 tended to get milder symptoms overall for the constitutional symptoms than those infected with Lineage 3. The study suggests a complex role for multiple interacting factors in the epidemiology of extrapulmonary TB in Ethiopia, including factors that can only be derived from population-based studies, which may prove to be significant for TB control in Ethiopia.

BAYESIAN ANALYSIS TO EVALUATE TESTS FOR THE DETECTION OF MYCOBACTERIUM BOVIS INFECTION IN FREE-RANGING WILD BISON (BISON BISON ATHABASCAE) IN THE ABSENCE OF A GOLD STANDARD.

PubMed

Chapinal, Núria; Schumaker, Brant A; Joly, Damien O; Elkin, Brett T; Stephen, Craig

2015-07-01

We estimated the sensitivity and specificity of the caudal-fold skin test (CFT), the fluorescent polarization assay (FPA), and the rapid lateral-flow test (RT) for the detection of Mycobacterium bovis in free-ranging wild wood bison (Bison bison athabascae), in the absence of a gold standard, by using Bayesian analysis, and then used those estimates to forecast the performance of a pairwise combination of tests in parallel. In 1998-99, 212 wood bison from Wood Buffalo National Park (Canada) were tested for M. bovis infection using CFT and two serologic tests (FPA and RT). The sensitivity and specificity of each test were estimated using a three-test, one-population, Bayesian model allowing for conditional dependence between FPA and RT. The sensitivity and specificity of the combination of CFT and each serologic test in parallel were calculated assuming conditional independence. The test performance estimates were influenced by the prior values chosen. However, the rank of tests and combinations of tests based on those estimates remained constant. The CFT was the most sensitive test and the FPA was the least sensitive, whereas RT was the most specific test and CFT was the least specific. In conclusion, given the fact that gold standards for the detection of M. bovis are imperfect and difficult to obtain in the field, Bayesian analysis holds promise as a tool to rank tests and combinations of tests based on their performance. Combining a skin test with an animal-side serologic test, such as RT, increases sensitivity in the detection of M. bovis and is a good approach to enhance disease eradication or control in wild bison.

Prospects in Mycobacterium bovis Bacille Calmette et Guérin (BCG) Vaccine Diversity and Delivery: Why does BCG fail to protect against Tuberculosis?

PubMed Central

Moliva, Juan I.; Turner, Joanne; Torrelles, Jordi B.

2015-01-01

Mycobacterium tuberculosis (M.tb) infection leads to active tuberculosis (TB), a disease that kills one human every 18 seconds. Current therapies available to combat TB include chemotherapy and the preventative vaccine Mycobacterium bovis Bacille Calmette et Guérin (BCG). Increased reporting of drug resistant M.tb strains worldwide indicates that drug development cannot be the primary mechanism for eradication. BCG vaccination has been used globally for protection against childhood and disseminated TB, however, its efficacy at protecting against pulmonary TB in adult and aging populations is highly variable. In this regard, the immune response generated by BCG vaccination is incapable of sterilizing the lung post M.tb infection as indicated by the large proportion of individuals with latent TB infection that have received BCG. Although many new TB vaccine candidates have entered the development pipeline, only a few have moved to human clinical trials; where they showed no efficacy and/or were withdrawn due to safety regulations. These trials highlight our limited understanding of protective immunity against the development of active TB. Here, we discuss current vaccination strategies and their impact on the generation and sustainability of protective immunity against TB. PMID:26319069

Vaccination of cattle with a high dose of BCG vaccine 3 weeks after experimental infection with Mycobacterium bovis increased the inflammatory response, but not tuberculous pathology.

PubMed

Buddle, Bryce M; Shu, Dairu; Parlane, Natalie A; Subharat, Supatsak; Heiser, Axel; Hewinson, R Glyn; Vordermeier, H Martin; Wedlock, D Neil

2016-07-01

A study was undertaken to determine whether BCG vaccination of cattle post-challenge could have an effect on a very early Mycobacterium bovis infection. Three groups of calves (n = 12/group) were challenged endobronchially with M. bovis and slaughtered 13 weeks later to examine for tuberculous lesions. One group had been vaccinated prophylactically with BCG Danish vaccine 21 weeks prior to challenge; a second group was vaccinated with a 4-fold higher dose of BCG Danish 3 weeks post-challenge and the third group, remained non-vaccinated. Vaccination prior to challenge induced only minimal protection with just a significant reduction in the lymph node lesion scores. Compared to the non-vaccinated group, BCG vaccination post-challenge produced no reduction in gross pathology and histopathology, but did result in significant increases in mRNA expression of pro-inflammatory mediators (IFN-γ, IL-12p40, IL-17A, IRF-5, CXCL9, CXCL10, iNOs, and TNF-α) in the pulmonary lymph nodes. Although there was no significant differences in the gross pathology and histopathology between the post-challenge BCG and non-vaccinated groups, the enhanced pro-inflammatory immune responses observed in the post-challenge BCG group suggest caution in the use of high doses of BCG where there is a possibility that cattle may be infected with M. bovis prior to vaccination. Copyright © 2016 Elsevier Ltd. All rights reserved.

Use of cattle farm resources by badgers (Meles meles) and risk of bovine tuberculosis (Mycobacterium bovis) transmission to cattle.

PubMed

Garnett, B T; Delahay, R J; Roper, T J

2002-07-22

Nocturnal observations, radio telemetry and time-lapse camera surveillance were used to investigate visits by badgers (Meles meles L.) to two cattle farms. During 59 half-nights (ca. 295 h) of observation and 17 nights (ca. 154 h) of camera surveillance, 139 separate visits to farm buildings, by at least 26 individually identifiable badgers from two social groups, were recorded. The badgers, which included three individuals infected with bovine tuberculosis (Mycobacterium bovis), used cowsheds, feedsheds, barns, haystacks, slurry pits, cattle troughs and farmyards to exploit a range of food resources, including cattle feed and silage. Cattle feed was contaminated with badger faeces and badgers also came into close contact with cattle. The minimum number of badgers visiting farm buildings per night was negatively correlated with local 24 h rainfall. We conclude that exploitation by badgers of resources provided by cattle farms constitutes a potentially important mechanism for tuberculosis transmission from badgers to cattle.

Mycobacterium tuberculosis Infection in a Domesticated Korean Wild Boar ( Sus scrofa coreanus).

PubMed

Seo, Min-Goo; Ouh, In-Ohk; Kim, Munki; Lee, Jienny; Kim, Young-Hoan; Do, Jae-Cheul; Kwak, Dongmi

2017-06-01

Tuberculosis, a chronic progressive disease, has been reported in bovine, swine, and primate species. Here, we report the first case of Mycobacterium tuberculosis infection in a Korean wild boar ( Sus scrofa coreanus). The owners this domesticated boar brought it to the Gyeongbuk Veterinary Service Laboratory in Korea after it was found dead and severely emaciated. Demarcated yellowish white nodules were found around the larynx and retropharyngeal lymph node during necropsy. The lungs had diffuse fibrinous pleuritis, severe congestion, and scattered nodules. More nodules were found in the spleen. Tuberculosis is characterized by massive macrophage infiltration and central caseous necrosis; both characteristics were found in the lungs. Histopathologic examination revealed that the alveolar lumen had marked fibrosis and exudates. Examination of the fluid revealed extensive macrophage permeation. To confirm a Mycobacterium infection, PCR was performed using two primer sets specific to the rpoB gene of Mycobacterium; Mycobacterium was detected in the lungs and spleen. To identify the species of Mycobacterium, immunohistochemical evaluation was performed using antibodies against Mycobacterium tuberculosis and Mycobacterium bovis . The results revealed immunoreactivity against M. tuberculosis but not against M. bovis . The consumption of undercooked or raw meat from game animals may expose humans and other animals to sylvatic infection. Consequently, Koreans who ingest wild boar may be at risk of a tuberculosis infection. To reduce the risk of foodborne infection and maintain public health, continuous monitoring and control strategies are required.

Assessment of safety and interferon gamma responses of Mycobacterium bovis BCG vaccine in goat kids and milking goats.

PubMed

Pérez de Val, Bernat; Vidal, Enric; López-Soria, Sergio; Marco, Alberto; Cervera, Zoraida; Martín, Maite; Mercader, Irene; Singh, Mahavir; Raeber, Alex; Domingo, Mariano

2016-02-10

Vaccination of domestic animals has emerged as an alternative long-term strategy for the control of tuberculosis (TB). A trial under field conditions was conducted in a TB-free goat herd to assess the safety of the Mycobacterium bovis BCG vaccine. Eleven kids and 10 milking goats were vaccinated with BCG. Bacterial shedding and interferon gamma (IFN-γ) responses were monitored throughout the study. Comprehensive pathological examination and mycobacterial culture of target tissues were performed. BCG vaccine strain was only isolated from the draining lymph node of the injection site of a kid euthanized at week 8 post-vaccination. The remaining animals were euthanized at week 24. Six out of 20 showed small granulomas at the injection site. BCG shedding was not detected in either faeces or in milk throughout the study. All vaccinated kids showed BCG-induced IFN-γ responses at week 8 post-vaccination. BCG vaccination of goats showed no lack of biological safety for the animals, environment and public health, and local adverse reactions were negligible. Copyright © 2016 Elsevier Ltd. All rights reserved.

Detection of IS6110 and HupB gene sequences of Mycobacterium tuberculosis and bovis in the aortic tissue of patients with Takayasu’s arteritis

PubMed Central

2012-01-01

Background Takayasu’s arteritis (TA) is a chronic inflammatory disease affecting the large arteries and their branches; its etiology is still unknown. In individuals suffering from TA, arterial inflammation progresses to stenosis and/or occlusion, leading to organ damage and affecting survival. Relation of TA with Mycobacterium tuberculosis has been known, but there have been only a few systematic studies focusing on this association. The IS6110 sequence identifies the Mycobacterium tuberculosis complex and the HupB establishes the differences between M. tuberculosis and M. bovis. Our objective was to search the presence of IS6110 and HupB genes in aorta of patients with TA. Methods We analyzed aorta tissues embedded in paraffin from 5760 autopsies obtained from our institution, we divided the selected samples as cases and controls; Cases: aortic tissues of individuals with Takayasu’s arteritis. Control positive: aortic tissues (with tuberculosis disease confirmed) and control negative with other disease aortic (atherosclerosis). Results Of 181 selected aorta tissues, 119 fulfilled the corresponding criteria for TA, TB or atherosclerosis. Thus 33 corresponded to TA, 33 to tuberculosis (TB) and 53 to atherosclerosis. The mean age was 22 ± 13, 41 ± 19, and 57 ± 10, respectively. IS6110 and HupB sequences were detected in 70% of TA tissues, 82% in tuberculosis, and in 32% with atherosclerosis. Important statistical differences between groups with TA, tuberculosis versus atherosclerosis (p = 0.004 and 0.0001, respectively) were found. Conclusion We identified a higher frequency of IS6110 and HupB genes in aortic tissues of TA patients. This data suggests that arterial damage could occur due to previous infection with M. tuberculosis. PMID:22905864

First-Time Detection of Mycobacterium bovis in Livestock Tissues and Milk in the West Bank, Palestinian Territories

PubMed Central

Ereqat, Suheir; Nasereddin, Abedelmajeed; Levine, Hagai; Azmi, Kifaya; Al-Jawabreh, Amer; Greenblatt, Charles L.; Abdeen, Ziad; Bar-Gal, Gila Kahila

2013-01-01

Background Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area. Methodology/principal findings A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample. Significance Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel. PMID:24069475

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus.

PubMed

Bastos, Reginaldo G; Dellagostin, Odir A; Barletta, Raúl G; Doster, Allan R; Nelson, Eric; Osorio, Fernando A

2002-11-22

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.

Gene expression profiling of peripheral blood mononuclear cells (PBMC) from Mycobacterium bovis infected cattle after in vitro antigenic stimulation with purified protein derivative of tuberculin (PPD).

PubMed

Meade, Kieran G; Gormley, Eamonn; Park, Stephen D E; Fitzsimons, Tara; Rosa, Guilherme J M; Costello, Eamon; Keane, Joseph; Coussens, Paul M; MacHugh, David E

2006-09-15

Microarray analysis of messenger RNA (mRNA) abundance was used to investigate the gene expression program of peripheral blood mononuclear cells (PBMC) from cattle infected with Mycobacterium bovis, the causative agent of bovine tuberculosis. An immunospecific bovine microarray platform (BOTL-4) with spot features representing 1336 genes was used for transcriptional profiling of PBMC from six M. bovis-infected cattle stimulated in vitro with bovine purified protein derivative of tuberculin (PPD-bovine). Cells were harvested at four time points (3 h, 6 h, 12 h and 24 h post-stimulation) and a split-plot design with pooled samples was used for the microarray experiment to compare gene expression between PPD-bovine stimulated PBMC and unstimulated controls for each time point. Statistical analyses of these data revealed 224 genes (approximately 17% of transcripts on the array) differentially expressed between stimulated and unstimulated PBMC across the 24 h time course (P<0.05). Of the 224 genes, 87 genes were significantly upregulated and 137 genes were significantly downregulated in M. bovis-infected PBMC stimulated with PPD-bovine across the 24 h time course. However, perturbation of the PBMC transcriptome was most apparent at time points 3 h and 12 h post-stimulation, with 81 and 84 genes differentially expressed, respectively. In addition, a more stringent statistical threshold (P<0.01) revealed 35 genes (approximately 3%) that were differentially expressed across the time course. Real-time quantitative reverse transcription PCR (qRT-PCR) of selected genes validated the microarray results and demonstrated a wide range of differentially expressed genes in PPD-bovine-, PPD-avian- and Concanavalin A (ConA) stimulated PBMC, including the interferon-gamma gene (IFNG), which was upregulated in PBMC stimulated with PPD-bovine (40-fold), PPD-avian (10-fold) and ConA (8-fold) after in vitro culture for 12 h. The pattern of expression of these genes in PPD-bovine stimulated

Evaluation of the Epidemiological Relevance of Variable-Number Tandem-Repeat Genotyping of Mycobacterium bovis and Comparison of the Method with IS6110 Restriction Fragment Length Polymorphism Analysis and Spoligotyping†

PubMed Central

Allix, Caroline; Walravens, Karl; Saegerman, Claude; Godfroid, Jacques; Supply, Philip; Fauville-Dufaux, Maryse

2006-01-01

Sources of Mycobacterium bovis contamination remain unclear for many cases of animal and human disease. A major limitation is the lack of sufficiently informative or epidemiologically well evaluated molecular methods for typing. Here, we report an evaluation of a high-throughput method based on 29 mycobacterial interspersed repetitive unit-variable-number tandem-repeat (MIRU-VNTR) loci to genotype 127 M. bovis isolates from cattle from 77 different Belgian farms, representative of a nationwide collection obtained from 1995 to 2003. MIRU-VNTR stability was demonstrated by analyzing a series of 74 isolates in total, obtained from different animals from a single farm or from different farms with an identified epidemiological link. The genotyping results and the genotypic diversity (h) were compared with those obtained by IS6110 restriction fragment length polymorphism (RFLP) analysis and spoligotyping. Among 68 isolates with no known epidemiological link, MIRU-VNTR typing discriminated better than either RFLP analysis or spoligotyping, with isolates taken individually (32 versus 16 and 17 genotypes; h = 0.91 versus 0.73 and 0.85, respectively) or in combination (32 versus 28 genotypes; h = 0.91 versus 0.92). Maximal resolution was already achieved with a subset of 9 loci. The observed congruence of the genetic relationships based on IS6110 RFLP analysis, spoligotyping, and MIRU-VNTR markers is consistent with a clonal population structure of M. bovis. These results support MIRU-VNTR typing as a convenient and discriminatory technique for analysis of the population structure of M. bovis in much greater detail and for addressing some still unresolved issues in the epidemiology of the pathogen. PMID:16757584

Use of whole-genome sequencing and evaluation of the apparent sensitivity and specificity of antemortem tuberculosis tests in the investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd.

PubMed

Bruning-Fann, Colleen S; Robbe-Austerman, Suelee; Kaneene, John B; Thomsen, Bruce V; Tilden, John D; Ray, Jean S; Smith, Richard W; Fitzgerald, Scott D; Bolin, Steven R; O'Brien, Daniel J; Mullaney, Thomas P; Stuber, Tod P; Averill, James J; Marks, David

2017-07-15

OBJECTIVE To describe use of whole-genome sequencing (WGS) and evaluate the apparent sensitivity and specificity of antemortem tuberculosis tests during investigation of an unusual outbreak of Mycobacterium bovis infection in a Michigan dairy herd. DESIGN Bovine tuberculosis (bTB) outbreak investigation. ANIMALS Cattle, cats, dog, and wildlife. PROCEDURES All cattle in the index dairy herd were screened for bTB with the caudal fold test (CFT), and cattle ≥ 6 months old were also screened with a γ-interferon (γIFN) assay. The index herd was depopulated along with all barn cats and a dog that were fed unpasteurized milk from the herd. Select isolates from M bovis-infected animals from the index herd and other bTB-affected herds underwent WGS. Wildlife around all affected premises was examined for bTB. RESULTS No evidence of bTB was found in any wildlife examined. Within the index herd, 53 of 451 (11.8%) cattle and 12 of 21 (57%) cats were confirmed to be infected with M bovis. Prevalence of M bovis-infected cattle was greatest among 4- to 7-month-old calves (16/49 [33%]) followed by adult cows (36/203 [18%]). The apparent sensitivity and specificity were 86.8% and 92.7% for the CFT and 80.4% and 96.5% for the γIFN assay when results for those tests were interpreted separately and 96.1% and 91.7% when results were interpreted in parallel. Results of WGS revealed that M bovis-infected barn cats and cattle from the index herd and 6 beef operations were infected with the same strain of M bovis. Of the 6 bTB-affected beef operations identified during the investigation, 3 were linked to the index herd only by WGS results; there was no record of movement of livestock or waste milk from the index herd to those operations. CONCLUSIONS AND CLINICAL RELEVANCE Whole-genome sequencing enhanced the epidemiological investigation and should be used in all disease investigations. Performing the CFT and γIFN assay in parallel improved the antemortem ability to detect M bovis

Pathology and diagnosis of Mycobacterium bovis in naturally infected dromedary camels (Camelus dromedarius) in India.

PubMed

Narnaware, Shirish Dadarao; Dahiya, Shyam Singh; Tuteja, Fateh Chand; Nagarajan, Govindasamy; Nath, Kashi; Patil, Nitin Vasantrao

2015-12-01

The present study investigated the pathological features of tuberculosis (TB) caused by Mycobacterium bovis and its diagnosis in naturally infected dromedary camels from an organized farm in India. During the period of the 5-year study, a total of 18 (19.56 %) camels out of 92 examined showed gross lesions compatible with TB at post-mortem. The clinical signs and pathological lesions in these camels were studied, and the efficacy of different diagnostic tests was also assessed. On the basis of occurrence and distribution of gross TB lesions, the infected camels revealed two different lesional patterns as pulmonary (n = 15) and disseminated (n = 3) form. The histopathology of affected organs revealed typical granulomatous lesions wherein the giant cells and acid-fast bacilli were occasionally observed in pulmonary form whereas they frequently observed in disseminated form. The single intradermal tuberculin test (SIDT) detected TB in 10 (55.55 %) whereas the Ziehl-Neelsen (ZN) stain and IS6110 PCR from tissue lesions detected 13 (72.22 %) and 18 (100 %) of the infected camels, respectively. The study suggests that pulmonary form of the TB is more common in camels indicating respiratory route as the major source of exposure in camel herds. Moreover, very low sensitivity of SIDT was observed which highlights the difficulty for confirmation of TB in live camels.

Detection of Mycobacterium bovis in formalin-fixed, paraffin-embedded tissues of cattle and elk by PCR amplification of an IS6110 sequence specific for Mycobacterium tuberculosis complex organisms.

PubMed

Miller, J; Jenny, A; Rhyan, J; Saari, D; Suarez, D

1997-07-01

A presumptive diagnosis of tuberculosis can be made if a tissue has characteristic histopathologic changes and acid-fast organisms. However, definitive diagnosis requires culture and species identification of the causative mycobacterium, a process that takes several weeks to complete. The purpose of work reported here was to determine if formalin-fixed, paraffin-embedded tissues could be tested by polymerase chain reaction (PCR) to provide a more rapid diagnosis of tuberculosis. Nondecalcified tissues from cases of tuberculosis in cattle and elk (Cervus elaphus) were examined. The primers used for PCR amplified a 123-bp fragment of IS6110, an insertion sequence that is specific for organisms in the Mycobacterium tuberculosis complex (M. tuberculosis, M. bovis, M. microti, M. africanum). The PCR test detected this sequence in tissues from 92 of 99 (93%) tuberculosis cases, including 3 of 4 elk. In 80 tissues, the positive results were obtained using material prepared by immersion of paraffin sections in water containing a detergent, followed by alternating boil/freeze cycles. The remaining positive results were obtained with DNA isolated from the crude tissue extracts by proteinase K digestion and phenol/chloroform purification. Accuracy of the IS6110 PCR test was demonstrated by negative test results on 31 tissues that had either nonmycobacterial granulomas or granulomatous lesions caused by other mycobacteria (M. paratuberculosis or M. avium). The findings of this study show that a PCR test usually can provide a rapid diagnosis of tuberculosis when it is applied to paraffin sections that have characteristic lesions and acid-fast organisms.

Nitric Oxide and KLF4 Protein Epigenetically Modify Class II Transactivator to Repress Major Histocompatibility Complex II Expression during Mycobacterium bovis Bacillus Calmette-Guérin Infection*

PubMed Central

Ghorpade, Devram Sampat; Holla, Sahana; Sinha, Akhauri Yash; Alagesan, Senthil Kumar; Balaji, Kithiganahalli Narayanaswamy

2013-01-01

Pathogenic mycobacteria employ several immune evasion strategies such as inhibition of class II transactivator (CIITA) and MHC-II expression, to survive and persist in host macrophages. However, precise roles for specific signaling components executing down-regulation of CIITA/MHC-II have not been adequately addressed. Here, we demonstrate that Mycobacterium bovis bacillus Calmette-Guérin (BCG)-mediated TLR2 signaling-induced iNOS/NO expression is obligatory for the suppression of IFN-γ-induced CIITA/MHC-II functions. Significantly, NOTCH/PKC/MAPK-triggered signaling cross-talk was found critical for iNOS/NO production. NO responsive recruitment of a bifunctional transcription factor, KLF4, to the promoter of CIITA during M. bovis BCG infection of macrophages was essential to orchestrate the epigenetic modifications mediated by histone methyltransferase EZH2 or miR-150 and thus calibrate CIITA/MHC-II expression. NO-dependent KLF4 regulated the processing and presentation of ovalbumin by infected macrophages to reactive T cells. Altogether, our study delineates a novel role for iNOS/NO/KLF4 in dictating the mycobacterial capacity to inhibit CIITA/MHC-II-mediated antigen presentation by infected macrophages and thereby elude immune surveillance. PMID:23733190

Oxidative Stress in Wild Boars Naturally and Experimentally Infected with Mycobacterium bovis

PubMed Central

Gassó, Diana; Vicente, Joaquín; Mentaberre, Gregorio; Soriguer, Ramón; Jiménez Rodríguez, Rocío; Navarro-González, Nora; Tvarijonaviciute, Asta; Lavín, Santiago; Fernández-Llario, Pedro; Segalés, Joaquim; Serrano, Emmanuel

2016-01-01

Reactive oxygen and nitrogen species (ROS-RNS) are important defence substances involved in the immune response against pathogens. An excessive increase in ROS-RNS, however, can damage the organism causing oxidative stress (OS). The organism is able to neutralise OS by the production of antioxidant enzymes (AE); hence, tissue damage is the result of an imbalance between oxidant and antioxidant status. Though some work has been carried out in humans, there is a lack of information about the oxidant/antioxidant status in the presence of tuberculosis (TB) in wild reservoirs. In the Mediterranean Basin, wild boar (Sus scrofa) is the main reservoir of TB. Wild boar showing severe TB have an increased risk to Mycobacterium spp. shedding, leading to pathogen spreading and persistence. If OS is greater in these individuals, oxidant/antioxidant balance in TB-affected boars could be used as a biomarker of disease severity. The present work had a two-fold objective: i) to study the effects of bovine TB on different OS biomarkers (namely superoxide dismutase (SOD), catalasa (CAT), glutathione peroxidase (GPX), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS)) in wild boar experimentally challenged with Mycobacterium bovis, and ii) to explore the role of body weight, sex, population and season in explaining the observed variability of OS indicators in two populations of free-ranging wild boar where TB is common. For the first objective, a partial least squares regression (PLSR) approach was used whereas, recursive partitioning with regression tree models (RTM) were applied for the second. A negative relationship between antioxidant enzymes and bovine TB (the more severe lesions, the lower the concentration of antioxidant biomarkers) was observed in experimentally infected animals. The final PLSR model retained the GPX, SOD and GR biomarkers and showed that 17.6% of the observed variability of antioxidant capacity was significantly correlated with

Immunohistochemical characterization of tuberculous lesions in sheep naturally infected with Mycobacterium bovis.

PubMed

Vallejo, Raquel; García Marín, Juan Francisco; Juste, Ramón Antonio; Muñoz-Mendoza, Marta; Salguero, Francisco Javier; Balseiro, Ana

2018-05-04

Sheep have been traditionally considered as less susceptible to Mycobacterium bovis (Mbovis) infection than other domestic ruminants such as cattle and goats. However, there is increasing evidence for the role of this species as a domestic Mbovis reservoir, mostly when sheep share grazing fields with infected cattle and goats. Nevertheless, there is a lack of information about the pathogenesis and the immune response of Mbovis infection in sheep. The goals of this study were to characterize the granuloma stages produced by the natural infection of Mbovis in sheep, to compare them with other species and to identify possible differences in the sheep immune response. Samples from bronchial lymph nodes from twelve Mbovis-naturally infected sheep were used. Four immunohistochemical protocols for the specific detection of T-lymphocytes, B-lymphocytes, plasma cells and macrophages were performed to study the local immune reaction within the granulomas. Differences were observed in the predominant cell type present in each type of granuloma, as well as differences and similarities with the development of tuberculous granulomas in other species. Very low numbers of T-lymphocytes were observed in all granuloma types indicating that specific cellular immune response mediated by T-cells might not be of much importance in sheep in the early stages of infection, when macrophages are the predominant cell type within lesions. Plasma cells and mainly B lymphocytes increased considerably as the granuloma developed being attracted to the lesions in a shift towards a Th2 response against the increasing amounts of mycobacteria. Therefore, we have proposed that the granulomas could be defined as initial, developed and terminal. Results showed that the study of the lymphoid tissue granulomata reinforces the view that the three different types of granuloma represent stages of lesion progression and suggest an explanation to the higher resistance of sheep based on a higher effective innate

Genomic and proteomic analyses of Mycobacterium bovis BCG Mexico 1931 reveal a diverse immunogenic repertoire against tuberculosis infection

PubMed Central

2011-01-01

Background Studies of Mycobacterium bovis BCG strains used in different countries and vaccination programs show clear variations in the genomes and immune protective properties of BCG strains. The aim of this study was to characterise the genomic and immune proteomic profile of the BCG 1931 strain used in Mexico. Results BCG Mexico 1931 has a circular chromosome of 4,350,386 bp with a G+C content and numbers of genes and pseudogenes similar to those of BCG Tokyo and BCG Pasteur. BCG Mexico 1931 lacks Region of Difference 1 (RD1), RD2 and N-RD18 and one copy of IS6110, indicating that BCG Mexico 1931 belongs to DU2 group IV within the BCG vaccine genealogy. In addition, this strain contains three new RDs, which are 53 (RDMex01), 655 (RDMex02) and 2,847 bp (REDMex03) long, and 55 single-nucleotide polymorphisms representing non-synonymous mutations compared to BCG Pasteur and BCG Tokyo. In a comparative proteomic analysis, the BCG Mexico 1931, Danish, Phipps and Tokyo strains showed 812, 794, 791 and 701 protein spots, respectively. The same analysis showed that BCG Mexico 1931 shares 62% of its protein spots with the BCG Danish strain, 61% with the BCG Phipps strain and only 48% with the BCG Tokyo strain. Thirty-nine reactive spots were detected in BCG Mexico 1931 using sera from subjects with active tuberculosis infections and positive tuberculin skin tests. Conclusions BCG Mexico 1931 has a smaller genome than the BCG Pasteur and BCG Tokyo strains. Two specific deletions in BCG Mexico 1931 are described (RDMex02 and RDMex03). The loss of RDMex02 (fadD23) is associated with enhanced macrophage binding and RDMex03 contains genes that may be involved in regulatory pathways. We also describe new antigenic proteins for the first time. PMID:21981907

Balancing Trained Immunity with Persistent Immune Activation and the Risk of Simian Immunodeficiency Virus Infection in Infant Macaques Vaccinated with Attenuated Mycobacterium tuberculosis or Mycobacterium bovis BCG Vaccine

PubMed Central

Jensen, Kara; dela Pena-Ponce, Myra Grace; Piatak, Michael; Shoemaker, Rebecca; Oswald, Kelli; Jacobs, William R.; Fennelly, Glenn; Lucero, Carissa; Mollan, Katie R.; Hudgens, Michael G.; Amedee, Angela; Kozlowski, Pamela A.; Estes, Jacob D.; Lifson, Jeffrey D.; Van Rompay, Koen K. A.; Larsen, Michelle

2016-01-01

ABSTRACT Our goal is to develop a pediatric combination vaccine to protect the vulnerable infant population against human immunodeficiency virus type 1 (HIV-1) and tuberculosis (TB) infections. The vaccine consists of an auxotroph Mycobacterium tuberculosis strain that coexpresses HIV antigens. Utilizing an infant rhesus macaque model, we have previously shown that this attenuated M. tuberculosis (AMtb)-simian immunodeficiency virus (SIV) vaccine is immunogenic, and although the vaccine did not prevent oral SIV infection, a subset of vaccinated animals was able to partially control virus replication. However, unexpectedly, vaccinated infants required fewer SIV exposures to become infected compared to naive controls. Considering that the current TB vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG), can induce potent innate immune responses and confer pathogen-unspecific trained immunity, we hypothesized that an imbalance between enhanced myeloid cell function and immune activation might have influenced the outcome of oral SIV challenge in AMtb-SIV-vaccinated infants. To address this question, we used archived samples from unchallenged animals from our previous AMtb-SIV vaccine studies and vaccinated additional infant macaques with BCG or AMtb only. Our results show that vaccinated infants, regardless of vaccine strain or regimen, had enhanced myeloid cell responses. However, CD4+ T cells were concurrently activated, and the persistence of these activated target cells in oral and/or gastrointestinal tissues may have facilitated oral SIV infection. Immune activation was more pronounced in BCG-vaccinated infant macaques than in AMtb-vaccinated infant macaques, indicating a role for vaccine attenuation. These findings underline the importance of understanding the interplay of vaccine-induced immunity and immune activation and its effect on HIV acquisition risk and outcome in infants. PMID:27655885

Mycobacterium leprae phenolglycolipid-1 expressed by engineered M. bovis BCG modulates early interaction with human phagocytes.

PubMed

Tabouret, Guillaume; Astarie-Dequeker, Catherine; Demangel, Caroline; Malaga, Wladimir; Constant, Patricia; Ray, Aurélie; Honoré, Nadine; Bello, Nana Fatimath; Perez, Esther; Daffé, Mamadou; Guilhot, Christophe

2010-10-21

The species-specific phenolic glycolipid 1 (PGL-1) is suspected to play a critical role in the pathogenesis of leprosy, a chronic disease of the skin and peripheral nerves caused by Mycobacterium leprae. Based on studies using the purified compound, PGL-1 was proposed to mediate the tropism of M. leprae for the nervous system and to modulate host immune responses. However, deciphering the biological function of this glycolipid has been hampered by the inability to grow M. leprae in vitro and to genetically engineer this bacterium. Here, we identified the M. leprae genes required for the biosynthesis of the species-specific saccharidic domain of PGL-1 and reprogrammed seven enzymatic steps in M. bovis BCG to make it synthesize and display PGL-1 in the context of an M. leprae-like cell envelope. This recombinant strain provides us with a unique tool to address the key questions of the contribution of PGL-1 in the infection process and to study the underlying molecular mechanisms. We found that PGL-1 production endowed recombinant BCG with an increased capacity to exploit complement receptor 3 (CR3) for efficient invasion of human macrophages and evasion of inflammatory responses. PGL-1 production also promoted bacterial uptake by human dendritic cells and dampened their infection-induced maturation. Our results therefore suggest that M. leprae produces PGL-1 for immune-silent invasion of host phagocytic cells.

Priming-boosting vaccination with recombinant Mycobacterium bovis bacillus Calmette-Guérin and a nonreplicating vaccinia virus recombinant leads to long-lasting and effective immunity.

PubMed

Ami, Yasushi; Izumi, Yasuyuki; Matsuo, Kazuhiro; Someya, Kenji; Kanekiyo, Masaru; Horibata, Shigeo; Yoshino, Naoto; Sakai, Koji; Shinohara, Katsuaki; Matsumoto, Sohkichi; Yamada, Takeshi; Yamazaki, Shudo; Yamamoto, Naoki; Honda, Mitsuo

2005-10-01

Virus-specific T-cell responses can limit immunodeficiency virus type 1 (HIV-1) transmission and prevent disease progression and so could serve as the basis for an affordable, safe, and effective vaccine in humans. To assess their potential for a vaccine, we used Mycobacterium bovis bacillus Calmette-Guérin (BCG)-Tokyo and a replication-deficient vaccinia virus strain (DIs) as vectors to express full-length gag from simian immunodeficiency viruses (SIVs) (rBCG-SIVgag and rDIsSIVgag). Cynomolgus macaques were vaccinated with either rBCG-SIVgag dermally as a single modality or in combination with rDIsSIVgag intravenously. When cynomologus macaques were primed with rBCG-SIVgag and then boosted with rDIsSIVgag, high levels of gamma interferon (IFN-gamma) spot-forming cells specific for SIV Gag were induced. This combination regimen elicited effective protective immunity against mucosal challenge with pathogenic simian-human immunodeficiency virus for the 1 year the macaques were under observation. Antigen-specific intracellular IFN-gamma activity was similarly induced in each of the macaques with the priming-boosting regimen. Other groups receiving the opposite combination or the single-modality vaccines were not effectively protected. These results suggest that a recombinant M. bovis BCG-based vector may have potential as an HIV/AIDS vaccine when administered in combination with a replication-deficient vaccinia virus DIs vector in a priming-boosting strategy.

Novel potential diagnostic test for Mycobacterium tuberculosis complex using combined immunomagnetic separation (IMS) and PCR-CTPP.

PubMed

Intorasoot, S; Tharinjaroen, C S; Phunpae, P; Butr-Indr, B; Anukool, U; Intachai, K; Orrapin, S; Apiratmateekul, N; Arunothong, S; Suthachai, V; Saengsawang, K; Khamnoi, P; Pata, S; Kasinrerk, W; Tragoolpua, K

2016-08-01

To exploit immunomagnetic separation combined with PCR with confronting two-pair primers (IMS-PCR-CTPP) as a rapid method for detection of Mycobacterium tuberculosis complex (MTC) and identification of Mycobacterium bovis from sputum specimens. Monoclonal antibody (mAb) against the mycobacterial antigen, 85B (Ag85B), was coupled with magnetic particles for specific immunomagnetic separation (IMS) of Mycobacterium spp. Immunofluorescence assay indicated the capability of mAb to bind to Ag85B in both the recombinant and the native form. The IMS combined with PCR-CTPP targeting the mycobacterial lep B gene was further implemented using 133 sputum samples with acid-fast bacilli grading from negative to 3+. The results showed the sensitivity and specificity of IMS-PCR-CTPP vs gold standard culture method were 89·9 and 88·6% respectively. The IMS-PCR-CTPP method shortens the time for tuberculosis (TB) diagnosis from months to a day. This method is also suitable for investigation of MTC and epidemiological study of Myco. bovis in sputum specimens. This study is the first report emphasizing the combination of IMS and PCR-CTPP for the detection of MTC and simultaneous identification of Myco. bovis from sputum. It could be used for TB diagnosis in resource-limited countries with high TB burden. © 2016 The Society for Applied Microbiology.

Whole-transcriptome, high-throughput RNA sequence analysis of the bovine macrophage response to Mycobacterium bovis infection in vitro.

PubMed

Nalpas, Nicolas C; Park, Stephen D E; Magee, David A; Taraktsoglou, Maria; Browne, John A; Conlon, Kevin M; Rue-Albrecht, Kévin; Killick, Kate E; Hokamp, Karsten; Lohan, Amanda J; Loftus, Brendan J; Gormley, Eamonn; Gordon, Stephen V; MacHugh, David E

2013-04-08

Mycobacterium bovis, the causative agent of bovine tuberculosis, is an intracellular pathogen that can persist inside host macrophages during infection via a diverse range of mechanisms that subvert the host immune response. In the current study, we have analysed and compared the transcriptomes of M. bovis-infected monocyte-derived macrophages (MDM) purified from six Holstein-Friesian females with the transcriptomes of non-infected control MDM from the same animals over a 24 h period using strand-specific RNA sequencing (RNA-seq). In addition, we compare gene expression profiles generated using RNA-seq with those previously generated by us using the high-density Affymetrix® GeneChip® Bovine Genome Array platform from the same MDM-extracted RNA. A mean of 7.2 million reads from each MDM sample mapped uniquely and unambiguously to single Bos taurus reference genome locations. Analysis of these mapped reads showed 2,584 genes (1,392 upregulated; 1,192 downregulated) and 757 putative natural antisense transcripts (558 upregulated; 119 downregulated) that were differentially expressed based on sense and antisense strand data, respectively (adjusted P-value ≤ 0.05). Of the differentially expressed genes, 694 were common to both the sense and antisense data sets, with the direction of expression (i.e. up- or downregulation) positively correlated for 693 genes and negatively correlated for the remaining gene. Gene ontology analysis of the differentially expressed genes revealed an enrichment of immune, apoptotic and cell signalling genes. Notably, the number of differentially expressed genes identified from RNA-seq sense strand analysis was greater than the number of differentially expressed genes detected from microarray analysis (2,584 genes versus 2,015 genes). Furthermore, our data reveal a greater dynamic range in the detection and quantification of gene transcripts for RNA-seq compared to microarray technology. This study highlights the value of RNA-seq in

A New Screen for Tuberculosis Drug Candidates Utilizing a Luciferase-Expressing Recombinant Mycobacterium bovis Bacillus Calmette-Guéren.

PubMed

Ozeki, Yuriko; Igarashi, Masayuki; Doe, Matsumi; Tamaru, Aki; Kinoshita, Naoko; Ogura, Yoshitoshi; Iwamoto, Tomotada; Sawa, Ryuichi; Umekita, Maya; Enany, Shymaa; Nishiuchi, Yukiko; Osada-Oka, Mayuko; Hayashi, Tetsuya; Niki, Mamiko; Tateishi, Yoshitaka; Hatano, Masaki; Matsumoto, Sohkichi

2015-01-01

Tuberculosis (TB) is a serious infectious disease caused by a bacterial pathogen. Mortality from tuberculosis was estimated at 1.5 million deaths worldwide in 2013. Development of new TB drugs is needed to not only to shorten the medication period but also to treat multi-drug resistant and extensively drug-resistant TB. Mycobacterium tuberculosis (Mtb) grows slowly and only multiplies once or twice per day. Therefore, conventional drug screening takes more than 3 weeks. Additionally, a biosafety level-3 (BSL-3) facility is required. Thus, we developed a new screening method to identify TB drug candidates by utilizing luciferase-expressing recombinant Mycobacterium bovis bacillus Calmette-Guéren (rBCG). Using this method, we identified several candidates in 4 days in a non-BSL-3 facility. We screened 10,080 individual crude extracts derived from Actinomyces and Streptomyces and identified 137 extracts which possessed suppressive activity to the luciferase of rBCG. Among them, 41 compounds inhibited the growth of both Mtb H37Rv and the extensively drug-resistant Mtb (XDR-Mtb) strains. We purified the active substance of the 1904-1 extract, which possessed strong activity toward rBCG, Mtb H37Rv, and XDR-Mtb but was harmless to the host eukaryotic cells. The MIC of this substance was 0.13 μg/ml, 0.5 μg/ml, and 2.0-7.5 μg/ml against rBCG, H37Rv, and 2 XDR-strains, respectively. Its efficacy was specific to acid-fast bacterium except for the Mycobacterium avium intracellular complex. Mass spectrometry and nuclear magnetic resonance analyses revealed that the active substance of 1904-1 was cyclomarin A. To confirm the mode of action of the 1904-1-derived compound, resistant BCG clones were used. Whole genome DNA sequence analysis showed that these clones contained a mutation in the clpc gene which encodes caseinolytic protein, an essential component of an ATP-dependent proteinase, and the likely target of the active substance of 1904-1. Our method provides a rapid and

Proteomic profile of culture filtrate from the Brazilian vaccine strain Mycobacterium bovis BCG Moreau compared to M. bovis BCG Pasteur.

PubMed

Berrêdo-Pinho, Marcia; Kalume, Dario E; Correa, Paloma R; Gomes, Leonardo H F; Pereira, Melissa P; da Silva, Renata F; Castello-Branco, Luiz R R; Degrave, Wim M; Mendonça-Lima, Leila

2011-04-20

Bacille Calmette-Guerin (BCG) is currently the only available vaccine against tuberculosis (TB) and comprises a heterogeneous family of sub-strains with genotypic and phenotypic differences. The World Health Organization (WHO) affirms that the characterization of BCG sub-strains, both on genomic and proteomic levels, is crucial for a better comprehension of the vaccine. In addition, these studies can contribute in the development of a more efficient vaccine against TB. Here, we combine two-dimensional electrophoresis (2DE) and mass spectrometry to analyse the proteomic profile of culture filtrate proteins (CFPs) from M. bovis BCG Moreau, the Brazilian vaccine strain, comparing it to that of BCG Pasteur. CFPs are considered of great importance given their dominant immunogenicity and role in pathogenesis, being available for interaction with host cells since early infection. The 2DE proteomic map of M. bovis BCG Moreau CFPs in the pH range 3-8 allowed the identification of 158 spots corresponding to 101 different proteins, identified by MS/MS. Comparison to BCG Pasteur highlights the great similarity between these BCG strains. However, quantitative analysis shows a higher expression of immunogenic proteins such as Rv1860 (BCG1896, Apa), Rv1926c (BCG1965c, Mpb63) and Rv1886c (BCG1923c, Ag85B) in BCG Moreau when compared to BCG Pasteur, while some heat shock proteins, such as Rv0440 (BCG0479, GroEL2) and Rv0350 (BCG0389, DnaK), show the opposite pattern. Here we report the detailed 2DE profile of CFPs from M. bovis BCG Moreau and its comparison to BCG Pasteur, identifying differences that may provide relevant information on vaccine efficacy. These findings contribute to the detailed characterization of the Brazilian vaccine strain against TB, revealing aspects that may lead to a better understanding of the factors leading to BCG's variable protective efficacy against TB.

Kinetics of the immune response profile in guinea pigs after vaccination with Mycobacterium bovis BCG and infection with Mycobacterium tuberculosis.

PubMed

Grover, Ajay; Taylor, Jennifer; Troudt, JoLynn; Keyser, Andrew; Arnett, Kimberly; Izzo, Linda; Rholl, Drew; Izzo, Angelo

2009-11-01

The guinea pig model of tuberculosis is used extensively in assessing novel vaccines, since Mycobacterium bovis BCG vaccination effectively prolongs survival after low-dose aerosol infection with virulent M. tuberculosis. To better understand how BCG extends time to death after pulmonary infection with M. tuberculosis, we examined cytokine responses postvaccination and recruitment of activated T cells and cytokine response postinfection. At 10 weeks postvaccination, splenic gamma interferon (IFN-gamma) mRNA was significantly elevated compared to the levels at 5 weeks in ex vivo stimulation assays. At 15, 40, 60, and 120 days postinfection, T-cell activation (CD4+ CD62Llow and CD8+ CD62Llow) and mRNA expression of IFN-gamma, tumor necrosis factor alpha (TNF-alpha), interleukin-1 (IL-1), IL-10, IL-12, and eomesodermin were assessed. Our data show that at day 40, BCG-vaccinated guinea pigs had significantly increased levels of IFN-gamma mRNA expression but decreased TNF-alpha mRNA expression in their lungs compared to the levels in nonvaccinated animals. At day 120, a time when nonvaccinated guinea pigs succumbed to infection, low levels of IFN-gamma mRNA were observed even though there were increasing levels of IL-1, IL-12, and IL-10, and the numbers of activated T cells did not differ from those in BCG-vaccinated animals. BCG vaccination conferred the advantage of recruiting greater numbers of CD4+ CD62Llow T cells at day 40, although the numbers of CD8+ CD62Llow T cells were not elevated compared to the numbers in nonvaccinated animals. Our data suggest that day 40 postinfection may be a pivotal time point in determining vaccine efficacy and prolonged survival and that BCG promotes the capacity of T cells in the lungs to respond to infection.

Evolutionary landscape of the Mycobacterium tuberculosis complex from the viewpoint of PhoPR: implications for virulence regulation and application to vaccine development.

PubMed

Broset, Esther; Martín, Carlos; Gonzalo-Asensio, Jesús

2015-10-20

Different members of the Mycobacterium genus have evolved to cause tuberculosis in diverse human populations and in a variety of animal species. Our cumulative knowledge of mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired not only during the natural evolution of mycobacterial species but also during in vitro subculture, which has given rise to the attenuated reference strain H37Ra or to different daughter strains of Mycobacterium bovis BCG. PhoPR is a well-known regulator of pathogenic phenotypes, including secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalose-based lipids, and modulation of antigen export, in members of the Mycobacterium tuberculosis complex (MTBC). Evolutionarily conserved polymorphisms in PhoPR from Mycobacterium africanum, M. bovis, or M. tuberculosis H37Ra result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and, probably, to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream from phoPR in a particular M. bovis strain that is able to transmit between humans or polymorphisms in M. africanum and M. bovis that affect the regulatory region of the espACD operon, allowing PhoPR-independent ESAT-6 secretion. This review highlights the increasing knowledge of the significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation. In this context, the live attenuated vaccine MTBVAC, based on a phoP fadD26 deletion mutant of M. tuberculosis, is the first vaccine of this kind to successfully enter into clinical development, representing a historic milestone in the field of human vaccinology. Copyright © 2015 Broset et al.

Evolutionary Landscape of the Mycobacterium tuberculosis Complex from the Viewpoint of PhoPR: Implications for Virulence Regulation and Application to Vaccine Development

PubMed Central

Broset, Esther

2015-01-01

ABSTRACT Different members of the Mycobacterium genus have evolved to cause tuberculosis in diverse human populations and in a variety of animal species. Our cumulative knowledge of mycobacterial genomes indicates that mutations in the PhoPR two-component virulence system were acquired not only during the natural evolution of mycobacterial species but also during in vitro subculture, which has given rise to the attenuated reference strain H37Ra or to different daughter strains of Mycobacterium bovis BCG. PhoPR is a well-known regulator of pathogenic phenotypes, including secretion of the virulence factor ESAT-6, biosynthesis of acyltrehalose-based lipids, and modulation of antigen export, in members of the Mycobacterium tuberculosis complex (MTBC). Evolutionarily conserved polymorphisms in PhoPR from Mycobacterium africanum, M. bovis, or M. tuberculosis H37Ra result in loss of functional phenotypes. Interestingly, some members of the MTBC have acquired compensatory mutations to counteract these polymorphisms and, probably, to maintain their pathogenic potential. Some of these compensatory mutations include the insertion of the IS6110 element upstream from phoPR in a particular M. bovis strain that is able to transmit between humans or polymorphisms in M. africanum and M. bovis that affect the regulatory region of the espACD operon, allowing PhoPR-independent ESAT-6 secretion. This review highlights the increasing knowledge of the significance of PhoPR in the evolution of the MTBC and its potential application in the construction of new attenuated vaccines based on phoPR inactivation. In this context, the live attenuated vaccine MTBVAC, based on a phoP fadD26 deletion mutant of M. tuberculosis, is the first vaccine of this kind to successfully enter into clinical development, representing a historic milestone in the field of human vaccinology. PMID:26489860

In vivo activity of released cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin is due principally to trehalose mycolates.

PubMed

Geisel, Rachel E; Sakamoto, Kaori; Russell, David G; Rhoades, Elizabeth R

2005-04-15

The hallmark of Mycobacterium-induced pathology is granulomatous inflammation at the site of infection. Mycobacterial lipids are potent immunomodulators that contribute to the granulomatous response and are released in appreciable quantities by intracellular bacilli. Previously we investigated the granulomagenic nature of the peripheral cell wall lipids of Mycobacterium bovis bacillus Calmette-Guérin (BCG) by coating the lipids onto 90-microm diameter microspheres that were mixed into Matrigel matrix with syngeneic bone marrow-derived macrophages and injected i.p. into mice. These studies demonstrated that BCG lipids elicit proinflammatory cytokines and recruit leukocytes. In the current study we determined the lipids responsible for this proinflammatory effect. BCG-derived cell wall lipids were fractionated and purified by liquid chromatography and preparative TLC. The isolated fractions including phosphatidylinositol dimannosides, cardiolipin, phosphatidylglycerol, phosphatidylethanolamine, trehalose monomycolate, trehalose dimycolate, and mycoside B. Trehalose dimycolate, when delivered to bone marrow-derived murine macrophages, induced the greatest secretion of IL-1beta, IL-6, and TNF-alpha in vitro. Trehalose dimycolate similarly induced the greatest secretion of these proinflammatory cytokines in ex vivo matrices over the course of 12 days. Trehalose monomycolate and dimycolate also induced profound neutrophil recruitment in vivo. Experiments with TLR2 or TLR4 gene-deficient mice revealed no defects in responses to trehalose mycolates, although MyD88-deficient mice manifested significantly reduced cell recruitment and cytokine production. These results demonstrate that the trehalose mycolates, particularly trehalose dimycolate, are the most bioactive lipids in the BCG extract, inducing a proinflammatory cascade that influences granuloma formation.

Conflicting Role of Mycobacterium Species in Multiple Sclerosis

PubMed Central

Cossu, Davide; Yokoyama, Kazumasa; Hattori, Nobutaka

2017-01-01

Mycobacterium is a genus of aerobic and acid-fast bacteria, which include several pathogenic organisms that cause serious diseases in mammals. Previous studies have associated the immune response against mycobacteria with multiple sclerosis (MS), a chronic demyelinating disease of the central nervous system with unknown etiology. The role of mycobacteria in the pathological process has been controversial and often conflicting. We provide a detailed review of the mycobacteria that have been linked to MS over the last three decades, with a focus on Mycobacterium bovis bacille Calmette–Guérin vaccine for human and oral exposure to Mycobacterium avium subsp. paratuberculosis. We will also discuss the exposure and genetic susceptibility to mycobacterial infection, the protective role of vaccination, as well as the possible mechanisms involved in initiating or worsening MS symptoms, with particular emphasis on the molecular mimicry between mycobacterial and human proteins. Finally, we will introduce topics such as heat shock proteins and recognition by innate immunity, and toll-like receptor signaling-mediated responses to Mycobacterium exposure. PMID:28579973

Non-contiguous genome sequence of Mycobacterium simiae strain DSM 44165(T.).

PubMed

Sassi, Mohamed; Robert, Catherine; Raoult, Didier; Drancourt, Michel

2013-01-01

Mycobacterium simiae is a non-tuberculosis mycobacterium causing pulmonary infections in both immunocompetent and imunocompromized patients. We announce the draft genome sequence of M. simiae DSM 44165(T). The 5,782,968-bp long genome with 65.15% GC content (one chromosome, no plasmid) contains 5,727 open reading frames (33% with unknown function and 11 ORFs sizing more than 5000 -bp), three rRNA operons, 52 tRNA, one 66-bp tmRNA matching with tmRNA tags from Mycobacterium avium, Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium microti, Mycobacterium marinum, and Mycobacterium africanum and 389 DNA repetitive sequences. Comparing ORFs and size distribution between M. simiae and five other Mycobacterium species M. simiae clustered with M. abscessus and M. smegmatis. A 40-kb prophage was predicted in addition to two prophage-like elements, 7-kb and 18-kb in size, but no mycobacteriophage was seen after the observation of 10(6) M. simiae cells. Fifteen putative CRISPRs were found. Three genes were predicted to encode resistance to aminoglycosides, betalactams and macrolide-lincosamide-streptogramin B. A total of 163 CAZYmes were annotated. M. simiae contains ESX-1 to ESX-5 genes encoding for a type-VII secretion system. Availability of the genome sequence may help depict the unique properties of this environmental, opportunistic pathogen.

Deletion of nuoG from the Vaccine Candidate Mycobacterium bovis BCG ΔureC::hly Improves Protection against Tuberculosis

PubMed Central

Gengenbacher, Martin; Nieuwenhuizen, Natalie; Vogelzang, Alexis; Liu, Haipeng; Kaiser, Peggy; Schuerer, Stefanie; Lazar, Doris; Wagner, Ina; Mollenkopf, Hans-Joachim

2016-01-01

ABSTRACT The current tuberculosis (TB) vaccine, Mycobacterium bovis Bacillus Calmette-Guérin (BCG), provides insufficient protection against pulmonary TB. Previously, we generated a listeriolysin-expressing recombinant BCG strain, which to date has successfully completed phase I and phase IIa clinical trials. In an attempt to further improve efficacy, we deleted the antiapoptotic virulence gene nuoG, encoding NADH dehydrogenase 1 subunit G, from BCG ΔureC::hly. In vitro, deletion of nuoG unexpectedly led to strongly increased recruitment of the autophagosome marker LC3 to the engulfed vaccine, suggesting that nuoG also affects xenophagic pathways. In mice, BCG ΔureC::hly ΔnuoG vaccination was safer than BCG and improved protection over that of parental BCG ΔureC::hly, significantly reducing TB load in murine lungs, ameliorating pulmonary pathology, and enhancing immune responses. Transcriptome analysis of draining lymph nodes after vaccination with either BCG ΔureC::hly or BCG ΔureC::hly ΔnuoG demonstrated earlier and stronger induction of immune responses than that with BCG SSI and suggested upregulation of inflammasome activation and interferon-induced GTPases. In summary, BCG ΔureC::hly ΔnuoG is a promising next-generation TB vaccine candidate with excellent efficacy and safety. PMID:27222470

Agreement between the caudal fold test and serological tests for the detection of Mycobacterium bovis infection in bison.

PubMed

Chapinal, Núria; Elkin, Brett T; Joly, Damien O; Schumaker, Brant A; Stephen, Craig

2012-08-01

The objective of this study was to estimate agreement between the caudal fold test (CFT) and different serological tests for the detection of Mycobacterium bovis infection in bison by using prevalence-adjusted bias-adjusted kappa (PABAK). A total of 212 of wild wood bison from Wood Buffalo National Park were tested with the CFT as well as several serological tests: fluorescent polarization assay (FPA), multiantigen print immunoassay (MAPIA), rapid lateral-flow test (RT) and dual path platform test (DPP). For RT, 3 variations were conducted using 30μl of serum (RT 30), 20μl of serum (RT 20) and 20μl of serum considering only a strong reaction as positive (RT 20 ST). The McNemar's χ(2) test was conducted to assess whether the proportion of positive test results to 2 different tests differed. Two measures of agreement between pair of tests were estimated: the Cohen's kappa statistic and PABAK. The apparent prevalence of tuberculosis in the sampled animals varied depending on the diagnostic test from 6.1% (FPA and DPP) to 47.2% (CFT). The prevalence estimated by CFT differed from the prevalence estimated by the other tests, whereas the prevalence estimated by FPA, MAPIA, RT 20 ST and DPP were not significantly different. The kappa and PABAK estimates calculated between CFT and the rest of the tests suggested poor to slight agreement between tests (k and PABAK<0.25 in all cases). The PABAK estimates for the pairwise combinations among serological tests were numerically greater than the kappa estimates (and significantly greater when FPA was compared to the rest of serological tests), and suggested substantial to almost perfect agreement (PABAK>0.75 in all cases). The disagreement between the skin and serological tests for the detection of M. bovis infection could be partly because the tests measure different immunological responses (cell-mediated vs. humoral) that are predominant at different stages of the infection, and partly due to inaccuracy of the tests

Mycobacterium komaniense sp. nov., a rapidly growing non-tuberculous Mycobacterium species detected in South Africa.

PubMed

Gcebe, Nomakorinte; Rutten, Victor P M G; van Pittius, Nicolaas Gey; Naicker, Brendon; Michel, Anita L

2018-05-01

Some species of non-tuberculous mycobacteria (NTM) have been reported to be opportunistic pathogens of animals and humans. Recently there has been an upsurge in the number of cases of NTM infections, such that some NTM species are now recognized as pathogens of humans and animals. From a veterinary point of view, the major significance of NTM is the cross-reactive immune response they elicit against Mycobacterium bovis antigens, leading to misdiagnosis of bovine tuberculosis. Four NTM isolates were detected from a bovine nasal swab, soil and water, during an NTM survey in South Africa. These were all found using 16S rRNA gene sequence analysis to be closely related to Mycobacterium moriokaense. The isolates were further characterised by sequence analysis of the partial fragments of hsp65, rpoB and sodA. The genome of the type strain was also elucidated. Gene (16S rRNA, hsp65, rpoB and sodA) and protein sequence data analysis of 6 kDa early secretory antigenic target (ESAT 6) and 10 kDa culture filtrate protein (CFP-10) revealed that these isolates belong to a unique Mycobacterium species. Differences in phenotypic and biochemical traits between the isolates and closely related species further supported that these isolates belong to novel Mycobacterium species. We proposed the name Mycobacterium komaniense sp. nov. for this new species. The type strain is GPK 1020 T (=CIP 110823T=ATCC BAA-2758).

Composition and immunoreactivity of the A60 complex and other cell fractions from Mycobacterium bovis BCG.

PubMed

Cocito, C; Vanlinden, F

1995-02-01

Surface static cultures of Mycobacterium bovis BCG contained cells embedded in an extracellular matrix, whose mechanical removal yielded free cells that were pressure disrupted and fractionated into cytoplasm and walls. Cell envelopes were either mechanically disrupted or extracted with detergents. Intracellular and extracellular fractions were analysed for proteins, polysaccharides, and antigen 6O (A60), a major complex immunodominant in tuberculosis. A60 was present in extracellular matrix, cytoplasm and walls: it represented a substantial portion of the proteins and polysaccharides of these fractions. While the protein/polysaccharide ratio varied according to the origin of A60 preparations, the electrophoretic patterns of A60 proteins (which accounted for the immunogenicity of the complex) remained unchanged. Western blots pointed to the proteins present within the 29-45 kDa range as the A60 components endowed with the highest immunogenicity level. Since the most heavily stained protein bands in SDS-PAGE patterns were located outside the region best recognized by antisera, a striking discordance was found between concentration and immunogenicity patterns of A60 proteins. The electrophoretic patterns of A60- and non-A60-proteins from cytoplasm were also different. A60 complexes in dot blots and some electrophoresed A60 proteins reacted with monoclonal antibodies directed against lipoarabinomannan (LAM), a highly immunogenic polymer of cell envelope. This contaminating compound was removed from A60 with organic solvents and detergents. SDS-PAGE and Western blot patterns of proteins from delipidated A60 were similar to those of native A60 proteins.

Detection and discrimination of Mycobacterium tuberculosis complex.

PubMed

Issa, Rahizan; Mohd Hassan, Nurul Akma; Abdul, Hatijah; Hashim, Siti Hasmah; Seradja, Valentinus H; Abdul Sani, Athirah

2012-01-01

A real-time quantitative polymerase chain reaction (qPCR) was developed for detection and discrimination of Mycobacterium tuberculosis (H37Rv and H37Ra) and M. bovis bacillus Calmette-Guérin (BCG) of the Mycobacterium tuberculosis complex (MTBC) from mycobacterial other than tuberculosis (MOTT). It was based on the melting curve (Tm) analysis of the gyrB gene using SYBR(®) Green I detection dye and the LightCycler 1.5 system. The optimal conditions for the assay were 0.25 μmol/L of primers with 3.1 mmol/L of MgCl(2) and 45 cycles of amplification. For M. tuberculosis (H37Rv and H37Ra) and M. bovis BCG of the MTBC, we detected the crossing points (Cp) at cycles of 16.96 ± 0.07, 18.02 ± 0.14, and 18.62 ± 0.09, respectively, while the Tm values were 90.19 ± 0.06 °C, 90.27 ± 0.09 °C, and 89.81 ± 0.04 °C, respectively. The assay was sensitive and rapid with a detection limit of 10 pg of the DNA template within 35 min. In this study, the Tm analysis of the qPCR assay was applied for the detection and discrimination of MTBC from MOTT. Copyright © 2012 Elsevier Inc. All rights reserved.

A simple whole cell based high throughput screening protocol using Mycobacterium bovis BCG for inhibitors against dormant and active tubercle bacilli.

PubMed

Khan, Arshad; Sarkar, Dhiman

2008-04-01

This study aimed at developing a whole cell based high throughput screening protocol to identify inhibitors against both active and dormant tubercle bacilli. A respiratory type of nitrate reductase (NarGHJI), which was induced during dormancy, could reflect the viability of dormant bacilli of Mycobacterium bovis BCG in microplate adopted model of in vitro dormancy. Correlation between reduction in viability and nitrate reductase activity was seen clearly when dormant stage inhibitor metronidazole and itaconic anhydride were applied in this in vitro microplate model. Active replicating stage could also be monitored in the same assay by measuring the A(620) of the culture. MIC values of 0.08, 0.075, 0.3 and 3.0 microg/ml, determined through monitoring A(620) in this assay for rifampin, isoniazid, streptomycin and ethambutol respectively, were well in agreement with previously reported by BACTEC and Bio-Siv assays. S/N ratio and Z' factor for the assay were 8.5 and 0.81 respectively which indicated the robustness of the protocol. Altogether the assay provides an easy, inexpensive, rapid, robust and high content screening tool to search novel antitubercular molecules against both active and dormant bacilli.

Protection against bovine tuberculosis induced by oral vaccination of cattle with Mycobacterium bovis BCG is not enhanced by co-administration of mycobacterial protein vaccines.

PubMed

Wedlock, D Neil; Aldwell, Frank E; Vordermeier, H Martin; Hewinson, R Glyn; Buddle, Bryce M

2011-12-15

Mycobacterium bovis bacille Calmette-Guérin (BCG) delivered to calves by the oral route in a formulated lipid matrix has been previously shown to induce protection against bovine tuberculosis. A study was conducted in cattle to determine if a combination of a low dose of oral BCG and a protein vaccine could induce protective immunity to tuberculosis while not sensitising animals to tuberculin. Groups of calves (10 per group) were vaccinated by administering 2 × 10(7)colony forming units (CFU) of BCG orally or a combination of 2 × 10(7)CFU oral BCG and a protein vaccine comprised of M. bovis culture filtrate proteins (CFP) formulated with the adjuvants Chitin and Gel 01 and delivered by the intranasal route, or CFP formulated with Emulsigen and the TLR2 agonist Pam(3)CSK(4) and administered by the subcutaneous (s.c.) route. Two further groups were vaccinated with the CFP/Chitin/Gel 01 or CFP/Emulsigen/Pam(3)CSK(4) vaccines alone. Positive control groups were given 10(8)CFU oral BCG or 10(6)CFU s.c. BCG while a negative control group was non-vaccinated. All animals were challenged with M. bovis 15 weeks after vaccination and euthanized and necropsied at 16 weeks following challenge. Groups of cattle vaccinated with s.c. BCG, 10(8)CFU or 2 × 10(7)CFU oral BCG showed significant reductions in seven, three and four pathological or microbiological disease parameters, respectively, compared to the results for the non-vaccinated group. There was no evidence of protection in calves vaccinated with the combination of oral BCG and CFP/Emulsigen/Pam(3)CSK(4) or oral BCG and CFP/Chitin/Gel 01 or vaccinated with the protein vaccines alone. Positive responses in the comparative cervical skin test at 12 weeks after vaccination were only observed in animals vaccinated with s.c. BCG, 10(8)CFU oral BCG or a combination of 2 × 10(7)CFU oral BCG and CFP/Chitin/Gel 01. In conclusion, co-administration of a protein vaccine, administered by either systemic or mucosal routes with oral

Apparent growth phase-dependent phosphorylation of malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a major fatty acid synthase II component in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Boon, Calvin; Dick, Thomas

2003-10-10

Probing protein extracts from exponentially growing and stationary phase cultures of Mycobacterium bovis BCG with anti-phospho amino acid antibodies revealed a 31-kDa anti-phospho threonine antibody-reactive protein specific to growing culture. The corresponding protein was purified via two-dimensional gel electrophoresis and identified via mass spectrometry to be malonyl coenzyme A:acyl carrier protein transacylase (MCAT), a component of the fatty acid biosynthetic pathway. MCAT tagged with histidine reacted with anti-phospho threonine antibody and was positive in an in-gel chemical assay for phospho proteins. Analysis of the growth phase dependence of MCAT-His phosphorylation and protein levels showed that phosphorylated MCAT-His can be detected only in growing culture. In contrast, MCAT-His protein level was growth phase-independent. These results suggest that MCAT may be a substrate of a protein kinase and phosphatase, and that aspects of fatty acid synthesis in tubercle bacilli are regulated by protein phosphorylation.

Mycobacterium tuberculosis causing tuberculous lymphadenitis in Maputo, Mozambique.

PubMed

Viegas, Sofia Omar; Ghebremichael, Solomon; Massawo, Leguesse; Alberto, Matos; Fernandes, Fabíola Couto; Monteiro, Eliane; Couvin, David; Matavele, José Maiane; Rastogi, Nalin; Correia-Neves, Margarida; Machado, Adelina; Carrilho, Carla; Groenheit, Ramona; Källenius, Gunilla; Koivula, Tuija

2015-11-21

The zoonosis bovine tuberculosis (TB) is known to be responsible for a considerable proportion of extrapulmonary TB. In Mozambique, bovine TB is a recognised problem in cattle, but little has been done to evaluate how Mycobacterium bovis has contributed to human TB. We here explore the public health risk for bovine TB in Maputo, by characterizing the isolates from tuberculous lymphadenitis (TBLN) cases, a common manifestation of bovine TB in humans, in the Pathology Service of Maputo Central Hospital, in Mozambique, during one year. Among 110 patients suspected of having TBLN, 49 had a positive culture result. Of those, 48 (98%) were positive for Mycobacterium tuberculosis complex and one for nontuberculous mycobacteria. Of the 45 isolates analysed by spoligotyping and Mycobacterial Interspersed Repetitive Unit-Variable Number Tandem Repeat (MIRU-VNTR), all were M. tuberculosis. No M. bovis was found. Cervical TBLN, corresponding to 39 (86.7%) cases, was the main cause of TBLN and 66.7% of those where from HIV positive patients. We found that TBLN in Maputo was caused by a variety of M. tuberculosis strains. The most prevalent lineage was the EAI (n = 19; 43.2%). Particular common spoligotypes were SIT 48 (EAI1_SOM sublineage), SIT 42 (LAM 9), SIT 1 (Beijing) and SIT53 (T1), similar to findings among pulmonary cases. M. tuberculosis was the main etiological agent of TBLN in Maputo. M. tuberculosis genotypes were similar to the ones causing pulmonary TB, suggesting that in Maputo, cases of TBLN arise from the same source as pulmonary TB, rather than from an external zoonotic source. Further research is needed on other forms of extrapulmonary TB and in rural areas where there is high prevalence of bovine TB in cattle, to evaluate the risk of transmission of M. bovis from cattle to humans.

Cell wall lipids from Mycobacterium bovis BCG are inflammatory when inoculated within a gel matrix: characterization of a new model of the granulomatous response to mycobacterial components.

PubMed

Rhoades, Elizabeth R; Geisel, Rachel E; Butcher, Barbara A; McDonough, Sean; Russell, David G

2005-05-01

The chronic inflammatory response to Mycobacterium generates complex granulomatous lesions that balance containment with destruction of infected tissues. To study the contributing factors from host and pathogen, we developed a model wherein defined mycobacterial components and leukocytes are delivered in a gel, eliciting a localized response that can be retrieved and analysed. We validated the model by comparing responses to the cell wall lipids from Mycobacterium bovis bacillus Calmette-Guerin (BCG) to reported activities in other models. BCG lipid-coated beads and bone marrow-derived macrophages (input macrophages) were injected intraperitoneally into BALB/c mice. Input macrophages and recruited peritoneal exudate cells took up fluorescently tagged BCG lipids, and matrix-associated macrophages and neutrophils produced tumor necrosis factor, interleukin-1alpha, and interleukin-6. Leukocyte numbers and cytokine levels were greater in BCG lipid-bearing matrices than matrices containing non-coated or phosphatidylglycerol-coated beads. Leukocytes arrived in successive waves of neutrophils, macrophages and eosinophils, followed by NK and T cells (CD4(+), CD8(+), or gammadelta) at 7 days and B cells within 12 days. BCG lipids also predisposed matrices for adherence and vascularization, enhancing cellular recruitment. We submit that the matrix model presents pertinent features of the murine granulomatous response that will prove to be an adaptable method for study of this complex response.

rBCG30-Induced Immunity and Cross-Protection against Mycobacterium leprae Challenge Are Enhanced by Boosting with the Mycobacterium tuberculosis 30-Kilodalton Antigen 85B

PubMed Central

Gillis, Thomas P.; Tullius, Michael V.

2014-01-01

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy. PMID:25001602

rBCG30-induced immunity and cross-protection against Mycobacterium leprae challenge are enhanced by boosting with the Mycobacterium tuberculosis 30-kilodalton antigen 85B.

PubMed

Gillis, Thomas P; Tullius, Michael V; Horwitz, Marcus A

2014-09-01

Leprosy remains a major global health problem and typically occurs in regions in which tuberculosis is endemic. Vaccines are needed that protect against both infections and do so better than the suboptimal Mycobacterium bovis BCG vaccine. Here, we evaluated rBCG30, a vaccine previously demonstrated to induce protection superior to that of BCG against Mycobacterium tuberculosis and Mycobacterium bovis challenge in animal models, for efficacy against Mycobacterium leprae challenge in a murine model of leprosy. rBCG30 overexpresses the M. tuberculosis 30-kDa major secretory protein antigen 85B, which is 85% homologous with the M. leprae homolog (r30ML). Mice were sham immunized or immunized intradermally with BCG or rBCG30 and challenged 2.5 months later by injection of viable M. leprae into each hind footpad. After 7 months, vaccine efficacy was assessed by enumerating the M. leprae bacteria per footpad. Both BCG and rBCG30 induced significant protection against M. leprae challenge. In the one experiment in which a comparison between BCG and rBCG30 was feasible, rBCG30 induced significantly greater protection than did BCG. Immunization of mice with purified M. tuberculosis or M. leprae antigen 85B also induced protection against M. leprae challenge but less so than BCG or rBCG30. Notably, boosting rBCG30 with M. tuberculosis antigen 85B significantly enhanced r30ML-specific immune responses, substantially more so than boosting BCG, and significantly augmented protection against M. leprae challenge. Thus, rBCG30, a vaccine that induces improved protection against M. tuberculosis, induces cross-protection against M. leprae that is comparable or potentially superior to that induced by BCG, and boosting rBCG30 with antigen 85B further enhances immune responses and protective efficacy. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

Prevalence of latent and active tuberculosis among dairy farm workers exposed to cattle infected by Mycobacterium bovis.

PubMed

Torres-Gonzalez, Pedro; Soberanis-Ramos, Orbelin; Martinez-Gamboa, Areli; Chavez-Mazari, Barbara; Barrios-Herrera, Ma Teresa; Torres-Rojas, Martha; Cruz-Hervert, Luis Pablo; Garcia-Garcia, Lourdes; Singh, Mahavir; Gonzalez-Aguirre, Adrian; Ponce de Leon-Garduño, Alfredo; Sifuentes-Osornio, José; Bobadilla-Del-Valle, Miriam

2013-01-01

Human tuberculosis caused by M. bovis is a zoonosis presently considered sporadic in developed countries, but remains a poorly studied problem in low and middle resource countries. The disease in humans is mainly attributed to unpasteurized dairy products consumption. However, transmission due to exposure of humans to infected animals has been also recognized. The prevalence of tuberculosis infection and associated risk factors have been insufficiently characterized among dairy farm workers (DFW) exposed in settings with poor control of bovine tuberculosis. Tuberculin skin test (TST) and Interferon-gamma release assay (IGRA) were administered to 311 dairy farm and abattoir workers and their household contacts linked to a dairy production and livestock facility in Mexico. Sputa of individuals with respiratory symptoms and samples from routine cattle necropsies were cultured for M. bovis and resulting spoligotypes were compared. The overall prevalence of latent tuberculosis infection (LTBI) was 76.2% (95% CI, 71.4-80.9%) by TST and 58.5% (95% CI, 53.0-64.0%) by IGRA. Occupational exposure was associated to TST (OR 2.72; 95% CI, 1.31-5.64) and IGRA (OR 2.38; 95% CI, 1.31-4.30) adjusting for relevant variables. Two subjects were diagnosed with pulmonary tuberculosis, both caused by M. bovis. In one case, the spoligotype was identical to a strain isolated from bovines. We documented a high prevalence of latent and pulmonary TB among workers exposed to cattle infected with M. bovis, and increased risk among those occupationally exposed in non-ventilated spaces. Interspecies transmission is frequent and represents an occupational hazard in this setting.

A single dose of a DNA vaccine encoding apa coencapsulated with 6,6'-trehalose dimycolate in microspheres confers long-term protection against tuberculosis in Mycobacterium bovis BCG-primed mice.

PubMed

Carlétti, Dyego; Morais da Fonseca, Denise; Gembre, Ana Flávia; Masson, Ana Paula; Weijenborg Campos, Lívia; Leite, Luciana C C; Rodrigues Pires, Andréa; Lannes-Vieira, Joseli; Lopes Silva, Célio; Bonato, Vânia Luiza Deperon; Horn, Cynthia

2013-08-01

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6'-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB.

(1)H-NMR spectroscopy revealed Mycobacterium tuberculosis caused abnormal serum metabolic profile of cattle.

PubMed

Chen, Yingyu; Wu, Junfang; Tu, Lingling; Xiong, Xuekai; Hu, Xidan; Huang, Jiong; Xu, Zhiguang; Zhang, Xiansong; Hu, Changmin; Hu, Xueying; Guo, Aizhen; Wang, Yulan; Chen, Huanchun

2013-01-01

To re-evaluate virulence of Mycobacterium tuberculosis (M. tb) in cattle, we experimentally infected calves with M. tb andMycobacterium bovisvia intratracheal injection at a dose of 2.0×10(7) CFU and observed the animals for 33 weeks. The intradermal tuberculin test and IFN-γin vitro release assay showed that both M. tb and M. bovis induced similar responses. Immunohistochemical staining of pulmonary lymph nodes indicated that the antigen MPB83 of both M. tb and M. bovis were similarly distributed in the tissue samples. Histological examinations showed all of the infected groups exhibited neutrophil infiltration to similar extents. Although the infected cattle did not develop granulomatous inflammation, the metabolic profiles changed significantly, which were characterized by a change in energy production pathways and increased concentrations of N-acetyl glycoproteins. Glycolysis was induced in the infected cattle by decreased glucose and increased lactate content, and enhanced fatty acid β-oxidation was induced by decreased TG content, and decreased gluconeogenesis indicated by the decreased concentration of glucogenic and ketogenic amino acids promoted utilization of substances other than glucose as energy sources. In addition, an increase in acute phase reactive serum glycoproteins, together with neutrophil infiltration and increased of IL-1β production indicated an early inflammatory response before granuloma formation. In conclusion, this study indicated that both M. tb and M.bovis were virulent to cattle. Therefore, it is likely that cattle with M. tb infections would be critical to tuberculosis transmission from cattle to humans. Nuclear magnetic resonance was demonstrated to be an efficient method to systematically evaluate M. tb and M. bovi sinfection in cattle.

Factors that Influence Mycobacterium bovis Infection in Red Deer and Wild Boar in an Epidemiological Risk Area for Tuberculosis of Game Species in Portugal.

PubMed

Madeira, S; Manteigas, A; Ribeiro, R; Otte, J; Fonseca, A Pina; Caetano, P; Abernethy, D; Boinas, F

2017-06-01

Bovine tuberculosis (bTB) is a worldwide zoonotic disease of domestic and wild animals. Eradication has proved elusive in those countries with intensive national programmes but with ongoing transmission between wildlife and cattle. In Portugal, a high-risk area for bTB was defined and specific measures implemented to assess and minimize the risk from wildlife. Data from the 2011 to 2014 hunting seasons for red deer (Cervus elaphus) and wild boar (Sus scrofa) were analysed with bovine demographic and bTB information to assess factors that determined the occurrence and distribution of bTB in both species. The likelihood of bTB-like lesions in wild boar was positively associated with density of red deer, wild boar and cattle, while for red deer, only their density and age were significant factors. The likelihood of Mycobacterium bovis isolation in wild boar was associated with density of cattle and red deer and also with the anatomical location of lesions, while for red deer, none of the variables tested were statistically significant. Our results suggest that, in the study area, the role of red deer and wild boar may be different from the one previously suggested by other authors for the Iberian Peninsula, as red deer may be the driving force behind M. bovis transmission to wild boar. These findings may assist the official services and game managing bodies for the management of hunting zones, what could also impact the success of the bTB eradication programme. © 2015 Blackwell Verlag GmbH.

Evidence of Mycobacterium tuberculosis complex bacteraemia in intradermal skin test positive cattle detected using phage-RPA.

PubMed

Swift, Benjamin M C; Convery, Thomas W; Rees, Catherine E D

2016-10-02

Bovine tuberculosis is a zoonotic infectious disease caused by Mycobacterium bovis that affects cattle and can cause tuberculosis in a range of wildlife animals. A bacteriophage-based method combined with PCR (phage-PCR) has been recently used to detect and identify viable pathogenic mycobacteria in the peripheral blood mononuclear cells (PBMCs) of animals suffering from paratuberculosis. To adapt this method for the detection of M. bovis in blood, a new isothermal DNA amplification protocol using Recombinase Polymerase Amplification (RPA) was developed and was found to be able to detect M. bovis BCG within 48 h, with a limit of detection of approximately 10 cells per ml of blood for artificially inoculated blood samples. When blood samples (2 ml) from a Single Comparative Cervical Intradermal Tuberculin (SCCIT)- negative beef herd were tested, Mycobacterium tuberculosis complex (MTC) cells were not detected from any (45) of the blood samples. However when blood samples from SCCIT-positive animals were tested, viable MTC bacteria were detected in 66 % (27/41) of samples. Of these 41 animals sampled, 32 % (13) had visible lesions. In the visible lesion (VL) group, 85 % (11/13) had detectable levels of MTC whereas only 57 % (16/28) of animals which had no visible lesions (NVL) were found to have detectable mycobacteraemia. These results indicated that this simple, rapid method can be applied for the study of M. bovis infections. The frequency with which viable mycobacteria were detected in the peripheral blood of SCCIT-positive animals changes the paradigm of this disease.

Evaluation of Mycobacterium tuberculosis Early Secreted Antigenic Target 6 Recombinant Protein as a Diagnostic Marker in Skin Test.

PubMed

Moradi, Jale; Mosavari, Nader; Ebrahimi, Mahmoud; Arefpajohi, Reza; Tebianian, Majid

2015-02-01

Tuberculosis (TB) is the leading infectious disease in the developing world. Delayed-type hypersensitivity skin test diagnoses TB using tuberculin purified protein derivative (PPD), but this test is incapable of distinguishing Mycobacterium tuberculosis (MTB) infection from bacillus Calmette-Guérin (BCG) vaccination or an infection caused by nontuberculous mycobacteria (NTM). This study was performed to evaluate the use of recombinant early secretory antigenic target 6 (rESAT-6), a secretory protein found only in MTB, Mycobacterium bovis, and few other mycobacterial species, as a skin marker for MTB in guinea pigs. We prepared recombinant MTB ESAT-6 and evaluated its use as a specific antigen for MTB in guinea pigs. Our results show that the purified MTB rESAT-6 antigen is capable of inducing a positive reaction only in guinea pigs sensitized to MTB. No such reaction was observed in the animals sensitized to M. bovis, BCG vaccination, or NTM (Mycobacterium avium). Our study results confirm that the ESAT-6 antigen is more specific to MTB infection than PPD and could be used in more specific skin tests for detection of MTB in large animals and in humans.

Inactivation of Mycobacterium paratuberculosis and Mycobacterium tuberculosis in fresh soft cheese by gamma radiation

NASA Astrophysics Data System (ADS)

Badr, Hesham M.

2011-11-01

The effectiveness of gamma irradiation on the inactivation of Mycobacterium paratuberculosis, Mycobacterium bovis and Mycobacterium tuberculosis in fresh soft cheese that prepared from artificially inoculated milk samples was studied. Irradiation at dose of 2 kGy was sufficient for the complete inactivation of these mycobacteria as they were not detected in the treated samples during storage at 4±1 °C for 15 days. Moreover, irradiation of cheese samples, that were prepared from un-inoculated milk, at this effective dose had no significant effects on their gross composition and contents from riboflavin, niacin and pantothenic acid, while significant decreases in vitamin A and thiamin were observed. In addition, irradiation of cheese samples had no significant effects on their pH and nitrogen fractions contents, except for the contents of ammonia, which showed a slight, but significant, increases due to irradiation. The analysis of cheese fats indicated that irradiation treatment induced significant increase in their oxidation parameters and contents from free fatty acids; however, the observed increases were relatively low. On the other hand, irradiation of cheese samples induced no significant alterations on their sensory properties. Thus, irradiation dose of 2 kGy can be effectively applied to ensure the safety of soft cheese with regards to these harmful mycobacteria.

Cloning of the Gene Encoding a 22-Kilodalton Cell Surface Antigen of Mycobacterium bovis BCG and Analysis of Its Potential for DNA Vaccination against Tuberculosis

PubMed Central

Lefèvre, Philippe; Denis, Olivier; De Wit, Lucas; Tanghe, Audrey; Vandenbussche, Paul; Content, Jean; Huygen, Kris

2000-01-01

Using spleen cells from mice vaccinated with live Mycobacterium bovis BCG, we previously generated three monoclonal antibodies reactive against a 22-kDa protein present in mycobacterial culture filtrate (CF) (K. Huygen et al., Infect. Immun. 61:2687–2693, 1993). These monoclonal antibodies were used to screen an M. bovis BCG genomic library made in phage λgt11. The gene encoding a 233-amino-acid (aa) protein, including a putative 26-aa signal sequence, was isolated, and sequence analysis indicated that the protein was 98% identical with the M. tuberculosis Lppx protein and that it contained a sequence 94% identical with the M. leprae 38-mer polypeptide 13B3 recognized by T cells from killed M. leprae-immunized subjects. Flow cytometry and cell fractionation demonstrated that the 22-kDa CF protein is also highly expressed in the bacterial cell wall and membrane compartment but not in the cytosol. C57BL/6, C3H, and BALB/c mice were vaccinated with plasmid DNA encoding the 22-kDa protein and analyzed for immune response and protection against intravenous M. tuberculosis challenge. Whereas DNA vaccination induced elevated antibody responses in C57BL/6 and particularly in C3H mice, Th1-type cytokine response, as measured by interleukin-2 and gamma interferon secretion, was only modest, and no protection against intravenous M. tuberculosis challenge was observed in any of the three mouse strains tested. Therefore, the 22-kDa antigen seems to have little potential for a DNA vaccine against tuberculosis, but it may be a good candidate for a mycobacterial antigen detection test. PMID:10678905

Spherical body protein 2 truncated copy 11 as a specific babesia bovis attenuation marker

USDA-ARS?s Scientific Manuscript database

Background: Spherical body protein 2 (SBP-2) truncated copies 7, 9 and 11, gene transcripts in Babesia bovis, were recently reported to be significantly up-regulated in two geographically distinct attenuated B. bovis strains. Results: Sequence comparisons between the sbp2t7, 9 and 11 genes among geo...

A Single Dose of a DNA Vaccine Encoding Apa Coencapsulated with 6,6′-Trehalose Dimycolate in Microspheres Confers Long-Term Protection against Tuberculosis in Mycobacterium bovis BCG-Primed Mice

PubMed Central

Carlétti, Dyego; Morais da Fonseca, Denise; Gembre, Ana Flávia; Masson, Ana Paula; Weijenborg Campos, Lívia; Leite, Luciana C. C.; Rodrigues Pires, Andréa; Lannes-Vieira, Joseli; Lopes Silva, Célio; Bonato, Vânia Luiza Deperon

2013-01-01

Mycobacterium bovis BCG prime DNA (Mycobacterium tuberculosis genes)-booster vaccinations have been shown to induce greater protection against tuberculosis (TB) than BCG alone. This heterologous prime-boost strategy is perhaps the most realistic vaccination for the future of TB infection control, especially in countries where TB is endemic. Moreover, a prime-boost regimen using biodegradable microspheres seems to be a promising immunization to stimulate a long-lasting immune response. The alanine proline antigen (Apa) is a highly immunogenic glycoprotein secreted by M. tuberculosis. This study investigated the immune protection of Apa DNA vaccine against intratracheal M. tuberculosis challenge in mice on the basis of a heterologous prime-boost regimen. BALB/c mice were subcutaneously primed with BCG and intramuscularly boosted with a single dose of plasmid carrying apa and 6,6′-trehalose dimycolate (TDM) adjuvant, coencapsulated in microspheres (BCG-APA), and were evaluated 30 and 70 days after challenge. This prime-boost strategy (BCG-APA) resulted in a significant reduction in the bacterial load in the lungs, thus leading to better preservation of the lung parenchyma, 70 days postinfection compared to BCG vaccinated mice. The profound effect of this heterologous prime-boost regimen in the experimental model supports its development as a feasible strategy for prevention of TB. PMID:23740922

Characterization of the Apa antigen from M. avium subsp. paratuberculosis: a conserved Mycobacterium antigen that elicits a strong humoral response in cattle.

PubMed

Gioffré, A; Echeverría-Valencia, G; Arese, A; Morsella, C; Garbaccio, S; Delgado, F; Zumárraga, M; Paolicchi, F; Cataldi, A; Romano, M I

2009-12-15

Johne's disease or paratuberculosis is widespread in almost all countries and remains difficult to eradicate. Nowadays, diagnosis of Mycobacterium avium subsp. paratuberculosis (MPTB) infection is one of the main concerns. In this work, we evaluated the expression, biochemical properties and antigenicity of the Apa antigen, encoded by the gene annotated as MAP1569, in the MPTB genome. We confirmed its expression in MPTB and its glycosylation by the ConA binding assay. Although the MPTB-Apa is not an immunodominant antigen, MPTB-infected cattle showed a strong humoral response to recombinant Apa by Western blot and ELISA. Milk was also a suitable sample to be tested by ELISA. We comparatively analysed the humoral cross-reactivity to the Apa from MPTB (MPTB-Apa) and the orthologue from Mycobacterium tuberculosis (MT-Apa, identical to that from Mycobacterium bovis) in both infected and control cows. Response of M. bovis- and MPTB-infected animals against MT-Apa was similar (P=0.6985) but the response of the M. bovis-infected ones to MPTB-Apa was differential, being significantly diminished (P<0.0001). Although 6 out 45 animals from MPTB-infected herds responded to MPTB-Apa stimulation in the IFNgamma release assay, we found no significant differences when compared infected herds with non-infected ones (P=0.34). This antigen, in contrast to bovine Purified Protein Derivative (PPDb), was strongly represented in avian PPD (PPDa), as shown by the recognition of BALB/c mice hyperimmune sera against MPTB-Apa by Dot-blot immunoassay. We therefore demonstrated the antigenicity of Apa in MPTB-infected animals and a differential response to the recombinant antigen when compared to M. bovis-infected animals. These traits herein described, added to the usefulness of milk samples to detect IgG anti-Apa, could be important for routine screening in dairy cattle, considering a multiantigenic approach to overcome the lack of immunodominance.

Subcutaneous administration of a 10-fold-lower dose of a commercial human tuberculosis vaccine, Mycobacterium bovis bacillus Calmette-Guerin Danish, induced levels of protection against bovine tuberculosis and responses in the tuberculin intradermal test similar to those induced by a standard cattle dose.

PubMed

Buddle, Bryce M; Hewinson, R Glyn; Vordermeier, H Martin; Wedlock, D Neil

2013-10-01

Vaccination of cattle with a commercial human tuberculosis (TB) vaccine, Mycobacterium bovis bacillus Calmette-Guérin (BCG) Danish, at a dose equivalent to 5 human doses of BCG has protected these animals against TB in field and experimental trials. There is interest in determining whether a 10-fold-lower dose could still protect cattle but not induce a tuberculin intradermal test response. Two groups of calves (n = 9/group) were vaccinated subcutaneously with a lyophilized BCG Danish vaccine containing either 0.5 (1 × 10(5) to 4 × 10(5) CFU) or 5 (1 × 10(6) to 4 × 10(6) CFU) human doses of BCG Danish, with an additional group of 10 calves serving as nonvaccinated controls. Fifteen weeks after vaccination, these animals were challenged intratracheally with 5 × 10(3) CFU of virulent M. bovis and another 15 weeks later were slaughtered and examined for the presence of tuberculous lesions. Vaccination of the calves with either 0.5 or 5 equivalent human doses of BCG Danish induced similar levels of protection against challenge with M. bovis, with both groups showing significant reductions in the pathological and microbiological parameters compared to those for the the control group (P < 0.05). Vaccination with either of the two BCG doses induced similar numbers of animals responding to the tuberculin intradermal test at 11 weeks postvaccination. Vaccination with a 0.5 equivalent human dose of a commercial lyophilized BCG vaccine can protect cattle against challenge with M. bovis.

Revisiting Host Preference in the Mycobacterium tuberculosis Complex: Experimental Infection Shows M. tuberculosis H37Rv to Be Avirulent in Cattle

PubMed Central

Whelan, Adam O.; Coad, Michael; Cockle, Paul J.; Hewinson, Glyn; Vordermeier, Martin; Gordon, Stephen V.

2010-01-01

Experiments in the late 19th century sought to define the host specificity of the causative agents of tuberculosis in mammals. Mycobacterium tuberculosis, the human tubercle bacillus, was independently shown by Smith, Koch, and von Behring to be avirulent in cattle. This finding was erroneously used by Koch to argue the converse, namely that Mycobacterium bovis, the agent of bovine tuberculosis, was avirulent for man, a view that was subsequently discredited. However, reports in the literature of M. tuberculosis isolation from cattle with tuberculoid lesions suggests that the virulence of M. tuberculosis for cattle needs to be readdressed. We used an experimental bovine infection model to test the virulence of well-characterized strains of M. tuberculosis and M. bovis in cattle, choosing the genome-sequenced strains M. tuberculosis H37Rv and M. bovis 2122/97. Cattle were infected with approximately 106 CFU of M. tuberculosis H37Rv or M. bovis 2122/97, and sacrificed 17 weeks post-infection. IFN-γ and tuberculin skin tests indicated that both M. bovis 2122 and M. tuberculosis H37Rv were equally infective and triggered strong cell-mediated immune responses, albeit with some indication of differential antigen-specific responses. Postmortem examination revealed that while M. bovis 2122/97–infected animals all showed clear pathology indicative of bovine tuberculosis, the M. tuberculosis–infected animals showed no pathology. Culturing of infected tissues revealed that M. tuberculosis was able to persist in the majority of animals, albeit at relatively low bacillary loads. In revisiting the early work on host preference across the M. tuberculosis complex, we have shown M. tuberculosis H37Rv is avirulent for cattle, and propose that the immune status of the animal, or genotype of the infecting bacillus, may have significant bearing on the virulence of a strain for cattle. This work will serve as a baseline for future studies into the genetic basis of host preference

First-time detection of Mycobacterium bovis in livestock tissues and milk in the West Bank, Palestinian Territories.

PubMed

Ereqat, Suheir; Nasereddin, Abedelmajeed; Levine, Hagai; Azmi, Kifaya; Al-Jawabreh, Amer; Greenblatt, Charles L; Abdeen, Ziad; Bar-Gal, Gila Kahila

2013-01-01

Bovine tuberculosis, bTB, is classified by the WHO as one of the seven neglected zoonontic diseases that cause animal health problems and has high potential to infect humans. In the West Bank, bTB was not studied among animals and the prevalence of human tuberculosis caused by M. bovis is unknown. Therefore, the aim of this study was to estimate the prevalence of bTB among cattle and goats and identify the molecular characteristics of bTB in our area. A total of 208 tissue samples, representing 104 animals, and 150 raw milk samples, obtained from cows and goats were examined for the presence of mycobacteria. The tissue samples were collected during routine meat inspection from the Jericho abattoir. DNA was extracted from all samples, milk and tissue biopsies (n = 358), and screened for presence of TB DNA by amplifying a 123-bp segment of the insertion sequence IS6110. Eight out of 254 animals (3.1%) were found to be TB positive based on the IS6110-PCR. Identification of M. bovis among the positive TB samples was carried out via real time PCR followed by high resolution melt curve analysis, targeting the A/G transition along the oxyR gene. Spoligotyping analysis revealed a new genotype of M. bovis that was revealed from one tissue sample. Detection of M. bovis in tissue and milk of livestock suggests that apparently healthy cattle and goats are a potential source of infection of bTB and may pose a risk to public health. Hence, appropriate measures including meat inspection at abattoirs in the region are required together with promotion of a health campaign emphasizing the importance of drinking pasteurized milk. In addition, further studies are essential at the farm level to determine the exact prevalence of bTB in goats and cattle herds in the West Bank and Israel.

A non-sense mutation in the putative anti-mutator gene ada/alkA of Mycobacterium tuberculosis and M. bovis isolates suggests convergent evolution

PubMed Central

Nouvel, Laurent X; Vultos, Tiago Dos; Kassa-Kelembho, Eric; Rauzier, Jean; Gicquel, Brigitte

2007-01-01

Background Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR). Results In this study, 55 MDR and 194 non-MDR strains were analyzed. Variations in DNA repair genes ada/alkA and ogt were identified. Among them, by comparison to M. tuberculosis published sequences, we found a non-sense variation in ada/alkA gene which was also observed in M. bovis AF2122 strain. SNPs that are present in the adjacent regions to the amber variation are different in M. bovis and in M. tuberculosis strain. Conclusion An Amber codon was found in the ada/alkA locus of clustered M. tuberculosis isolates and in M. bovis strain AF2122. This is likely due to convergent evolution because SNP differences between strains are incompatible with horizontal transfer of an entire gene. This suggests that such a variation may confer a selective advantage and be implicated in hypermutator phenotype expression, which in turn contributes to adaptation to environmental changes. PMID:17506895

Mycobacterium tuberculosis infection in grazing cattle in central Ethiopia.

PubMed

Ameni, Gobena; Vordermeier, Martin; Firdessa, Rebuma; Aseffa, Abraham; Hewinson, Glyn; Gordon, Stephen V; Berg, Stefan

2011-06-01

A preliminary study to characterise mycobacteria infecting tuberculous cattle from two different management systems in central Ethiopia was carried out. Approximately 27% of isolates from grazing cattle were Mycobacterium tuberculosis, while cattle in a more intensive-production system were exclusively infected with M. bovis. The practice of local farmers discharging chewed tobacco directly into the mouths of pastured cattle was identified as a potential route of human-to-cattle transmission of M. tuberculosis. Copyright © 2010 Elsevier Ltd. All rights reserved.

Formulation of a mmaA4 Gene Deletion Mutant of Mycobacterium bovis BCG in Cationic Liposomes Significantly Enhances Protection against Tuberculosis

PubMed Central

Derrick, Steven C.; Dao, Dee; Yang, Amy; Kolibab, Kris; Jacobs, William R.; Morris, Sheldon L.

2012-01-01

A new vaccination strategy is urgently needed for improved control of the global tuberculosis (TB) epidemic. Using a mouse aerosol Mycobacterium tuberculosis challenge model, we investigated the protective efficacy of a mmaA4 gene deletion mutant of Mycobacterium bovis BCG (ΔmmaA4BCG) formulated in dimethyl dioctadecyl ammonium bromide (DDA) – D(+) trehalose 6,6 dibenenate (TDB) (DDA/TDB) adjuvant. In previous studies, deletion of the mmaA4 gene was shown to reduce the suppression of IL-12 production often seen after mycobacterial infections. While the non-adjuvanted ΔmmaA4BCG strain did not protect mice substantially better than conventional BCG against a tuberculous challenge in four protection experiments, the protective responses induced by the ΔmmaA4BCG vaccine formulated in DDA/TDB adjuvant was consistently increased relative to nonadjuvanted BCG controls. Furthermore, the ΔmmaA4BCG-DDA/TDB vaccine induced significantly higher frequencies of multifunctional (MFT) CD4 T cells expressing both IFNγ and TNFα (double positive) or IFNγ, TNFα and IL-2 (triple positive) than CD4 T cells derived from mice vaccinated with BCG. These MFT cells were characterized by having higher IFNγ and TNFα median fluorescence intensity (MFI) values than monofunctional CD4 T cells. Interestingly, both BCG/adjuvant and ΔmmaA4BCG/adjuvant formulations induced significantly higher frequencies of CD4 T cells expressing TNFα and IL-2 than nonadjuvanted BCG or ΔmmaA4BCG vaccines indicating that BCG/adjuvant mixtures may be more effective at inducing central memory T cells. Importantly, when either conventional BCG or the mutant were formulated in adjuvant and administered to SCID mice or immunocompromised mice depleted of IFNγ, significantly lower vaccine-derived mycobacterial CFU were detected relative to immunodeficient mice injected with non-adjuvanted BCG. Overall, these data suggest that immunization with the ΔmmaA4BCG/adjuvant formulation may be an effective, safe

Oral vaccination of cattle with heat inactivated Mycobacterium bovis does not compromise bovine TB diagnostic tests.

PubMed

Jones, Gareth J; Steinbach, Sabine; Sevilla, Iker A; Garrido, Joseba M; Juste, Ramon; Vordermeier, H Martin

2016-12-01

In this study we investigated whether oral uptake of a heat inactivated M. bovis wildlife vaccine by domestic cattle induced systemic immune responses that compromised the use of tuberculin or defined antigens in diagnostic tests for bovine TB. Positive skin test and blood-based IFN-γ release assay (IGRA) results were observed in all calves vaccinated via the parenteral route (i.e. intramuscular). In contrast, no positive responses to tuberculin or defined antigens were observed in either the skin test or IGRA test when performed in calves vaccinated via the oral route. In conclusion, our results suggest that the heat inactivated M. bovis vaccine could be used to vaccinate wildlife in a baited form in conjunction with the following in cattle: (i) continuation of existing tuberculin skin testing or novel skin test formats based on defined antigens; and (ii) the use of IGRA tests utilizing tuberculin or defined antigens. Crown Copyright © 2016. Published by Elsevier B.V. All rights reserved.

Mycobacterium smegmatis strain for detection of Mycobacterium tuberculosis by PCR used as internal control for inhibition of amplification and for quantification of bacteria.

PubMed Central

Kolk, A H; Noordhoek, G T; de Leeuw, O; Kuijper, S; van Embden, J D

1994-01-01

For the detection of Mycobacterium tuberculosis by PCR, the IS6110 sequence was used. A modified target was constructed by insertion of 56 nucleotides in the IS6110 insertion element of Mycobacterium bovis BCG. This modified insertion sequence was integrated into the genome of Mycobacterium smegmatis, a mycobacterium species which does not contain the IS6110 element. When DNA from the modified M. smegmatis 1008 strain was amplified with IS6110-specific primers INS1 and INS2, a band of 301 bp was seen on agarose gel, whereas the PCR product of M. tuberculosis complex DNA was a 245-bp fragment with these primers. The addition of a small number of M. smegmatis 1008 cells to clinical samples before DNA purification enables the detection of problems which may be due to the loss of DNA in the isolation procedure or to the presence of inhibitors. The presence of inhibitors of the amplification reaction can be confirmed by the addition of M. smegmatis 1008 DNA after the DNA isolation procedure. Furthermore, competition between the different target DNAs of M. smegmatis 1008 DNA and M. tuberculosis complex DNA enables the estimation of the number of IS6110 elements in the clinical sample. Images PMID:8051267

Gliding Motility of Babesia bovis Merozoites Visualized by Time-Lapse Video Microscopy

PubMed Central

Asada, Masahito; Goto, Yasuyuki; Yahata, Kazuhide; Yokoyama, Naoaki; Kawai, Satoru; Inoue, Noboru; Kaneko, Osamu; Kawazu, Shin-ichiro

2012-01-01

Background Babesia bovis is an apicomplexan intraerythrocytic protozoan parasite that induces babesiosis in cattle after transmission by ticks. During specific stages of the apicomplexan parasite lifecycle, such as the sporozoites of Plasmodium falciparum and tachyzoites of Toxoplasma gondii, host cells are targeted for invasion using a unique, active process termed “gliding motility”. However, it is not thoroughly understood how the merozoites of B. bovis target and invade host red blood cells (RBCs), and gliding motility has so far not been observed in the parasite. Methodology/Principal Findings Gliding motility of B. bovis merozoites was revealed by time-lapse video microscopy. The recorded images revealed that the process included egress of the merozoites from the infected RBC, gliding motility, and subsequent invasion into new RBCs. The gliding motility of B. bovis merozoites was similar to the helical gliding of Toxoplasma tachyzoites. The trails left by the merozoites were detected by indirect immunofluorescence assay using antiserum against B. bovis merozoite surface antigen 1. Inhibition of gliding motility by actin filament polymerization or depolymerization indicated that the gliding motility was driven by actomyosin dependent process. In addition, we revealed the timing of breakdown of the parasitophorous vacuole. Time-lapse image analysis of membrane-stained bovine RBCs showed formation and breakdown of the parasitophorous vacuole within ten minutes of invasion. Conclusions/Significance This is the first report of the gliding motility of B. bovis. Since merozoites of Plasmodium parasites do not glide on a substrate, the gliding motility of B. bovis merozoites is a notable finding. PMID:22506073

Toward eradication: the effect of Mycobacterium bovis infection in wildlife on the evolution and future direction of bovine tuberculosis management in New Zealand.

PubMed

Livingstone, P G; Hancox, N; Nugent, G; de Lisle, G W

2015-06-01

New Zealand's bovine tuberculosis (TB) control programme has greatly reduced the burden of tuberculosis on the farming industry, from 11% of mature cattle found with TB at slaughter in 1905 to <0.003% in 2012/13. New Zealand implemented TB control measures in cattle from the mid-twentieth century, and later in farmed deer. Control was based on established methods of tuberculin testing of herds, slaughter of suspect cases, and livestock movement control. Unexplained regional control failures and serious disease outbreaks were eventually linked to wildlife-vectored infection from the introduced Australian brushtail possum (Trichosurus vulpecula), which also triggered a wildlife disease complex involving a range of introduced species. This paper reviews the progressive elucidation of the epidemiology of Mycobacterium bovis in New Zealand's wildlife and farmed livestock, and the parallel development of research-led, multi-faceted TB control strategies required to protect New Zealand's livestock industries from damaging infection levels. The adoption of coordinated national pest management strategies, with increasingly ambitious objectives agreed between government and industry funders, has driven a costly but very successful management regime targeted at controlling TB in the possum maintenance host. This success has led to initiation of a strategy designed to eradicate TB from New Zealand's livestock and wildlife, which is considered a realistic long-term prospect.

The epidemiology of Mycobacterium bovis in wild deer and feral pigs and their roles in the establishment and spread of bovine tuberculosis in New Zealand wildlife

PubMed Central

Nugent, G; Gortazar, C; Knowles, G

2015-01-01

Abstract In New Zealand, wild deer and feral pigs are assumed to be spillover hosts for Mycobacterium bovis, and so are not targeted in efforts aimed at locally eradicating bovine tuberculosis (TB) from possums (Trichosurus vulpecula), the main wildlife host. Here we review the epidemiology of TB in deer and pigs, and assess whether New Zealand's TB management programme could be undermined if these species sometimes achieve maintenance host status. In New Zealand, TB prevalences of up to 47% have been recorded in wild deer sympatric with tuberculous possums. Patterns of lesion distribution, age-specific prevalences and behavioural observations suggest that deer become infected mainly through exposure to dead or moribund possums. TB can progress rapidly in some deer (<10%), but generalised disease is uncommon in wild deer; conversely some infected animals can survive for many years. Deer-to-deer transmission of M. bovis is rare, but transmission from tuberculous deer carcasses to scavengers, including possums, is likely. That creates a small spillback risk that could persist for a decade after transmission of new infection to wild deer has been halted. Tuberculosis prevalence in New Zealand feral pigs can reach 100%. Infections in lymph nodes of the head and alimentary tract predominate, indicating that TB is mostly acquired through scavenging tuberculous carrion, particularly possums. Infection is usually well contained, and transmission between pigs is rare. Large reductions in local possum density result in gradual declines (over 10 years) in TB prevalence among sympatric wild deer, and faster declines in feral pigs. Elimination of TB from possums (and livestock) therefore results in eventual disappearance of TB from feral pigs and wild deer. However, the risk of spillback infection from deer to possums substantially extends the time needed to locally eradicate TB from all wildlife (compared to that which would be required to eradicate disease from possums alone

The epidemiology of Mycobacterium bovis in wild deer and feral pigs and their roles in the establishment and spread of bovine tuberculosis in New Zealand wildlife.

PubMed

Nugent, G; Gortazar, C; Knowles, G

2015-06-01

In New Zealand, wild deer and feral pigs are assumed to be spillover hosts for Mycobacterium bovis, and so are not targeted in efforts aimed at locally eradicating bovine tuberculosis (TB) from possums (Trichosurus vulpecula), the main wildlife host. Here we review the epidemiology of TB in deer and pigs, and assess whether New Zealand's TB management programme could be undermined if these species sometimes achieve maintenance host status. In New Zealand, TB prevalences of up to 47% have been recorded in wild deer sympatric with tuberculous possums. Patterns of lesion distribution, age-specific prevalences and behavioural observations suggest that deer become infected mainly through exposure to dead or moribund possums. TB can progress rapidly in some deer (<10%), but generalised disease is uncommon in wild deer; conversely some infected animals can survive for many years. Deer-to-deer transmission of M. bovis is rare, but transmission from tuberculous deer carcasses to scavengers, including possums, is likely. That creates a small spillback risk that could persist for a decade after transmission of new infection to wild deer has been halted. Tuberculosis prevalence in New Zealand feral pigs can reach 100%. Infections in lymph nodes of the head and alimentary tract predominate, indicating that TB is mostly acquired through scavenging tuberculous carrion, particularly possums. Infection is usually well contained, and transmission between pigs is rare. Large reductions in local possum density result in gradual declines (over 10 years) in TB prevalence among sympatric wild deer, and faster declines in feral pigs. Elimination of TB from possums (and livestock) therefore results in eventual disappearance of TB from feral pigs and wild deer. However, the risk of spillback infection from deer to possums substantially extends the time needed to locally eradicate TB from all wildlife (compared to that which would be required to eradicate disease from possums alone), while

Abortion associated with Mycoplasma bovis (M. bovis) in a bison (Bison bison) herd

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis (M. bovis) has recently emerged as a significant health threat in bison and is an increasing concern and source of economic loss for producers. Clinical manifestations of infection documented in bison include pneumonia, respiratory distress and polyarthritis. The current study des...

Comparative Protein Profiling of Intraphagosomal Expressed Proteins of Mycobacterium bovis BCG.

PubMed

Singhal, Neelja; Kumar, Manish; Sharma, Divakar; Bisht, Deepa

2016-01-01

BCG, the only available vaccine against tuberculosis affords a variable protection which wanes with time. In this study we have analyzed and compared the proteins which are expressed differentially during broth-culture and intraphagosomal growth of M.bovis BCG. Eight proteins which showed increased expression during the intraphagosomal growth were identified by MALDI-TOF/MS. These were - a precursor of alanine and proline-rich secreted protein apa, isoforms of malate dehydrogenase, large subunit alpha (Alpha-ETF) of electron transfer flavoprotein, immunogenic protein MPB64 precursor, UPF0036 protein, and two proteins with unknown function. Based on these findings we speculate that higher expression of these proteins has a probable role in intracellular survival, adaptation and/or immunoprotective effect of BCG. Further, these proteins might also be used as gene expression markers for endosome trafficking events of BCG.

Mycobacterium bovis Bacille Calmette-Guérin Infection in the CNS Suppresses Experimental Autoimmune Encephalomyelitis and Th17 Responses in an IFN-gamma-independent Manner1

PubMed Central

Lee, JangEun; Reinke, Emily K.; Zozulya, Alla L.; Sandor, Matyas; Fabry, Zsuzsanna

2009-01-01

Multiple sclerosis (MS) and an animal model resembling MS, experimental autoimmune encephalomyelitis (EAE), are inflammatory demyelinating diseases of the central nervous system (CNS) that are suppressed by systemic mycobacterial infection in mice and BCG vaccination in humans. Host defense responses against Mycobacterium in mice are influenced by T lymphocytes and their cytokine products, particularly IFN-γ, which plays a protective regulatory role in EAE. To analyze the counter-regulatory role of mycobacterial infection-induced IFN-γ in the CNS on the function of the pathological Th17 cells and the clinical outcome of EAE, we induced EAE in mice that were intracerebrally infected with Mycobacterium bovis bacille Calmette-Guerin (BCG). Here we demonstrate that intracerebral (i.c.) BCG infection prevented inflammatory cell recruitment to the spinal cord and suppressed the development of EAE. Concomitantly, there was a significant decrease in the frequency of MOG-specific IFN-γ-producing CD4+ T cells in the CNS. IL-17+CD4+ T cell responses were significantly suppressed in i.c. BCG-infected mice following EAE induction regardless of T cell specificity. The frequency of Foxp3+CD4+ T cells in these mice was equivalent to that of control mice. The i.c. BCG infection-induced protection of EAE and suppression of MOG-specific IL-17+CD4+ T cell responses were similar in both wild type (WT) and IFN-γ deficient mice. These data show that live BCG infection in the brain suppresses CNS autoimmunity. These findings also reveal that the regulation of Th17-mediated autoimmunity in the CNS can be independent of IFN-γ-mediated mechanisms. PMID:18941210

BoLA class I polymorphism and in vitro immune response to M. bovis antigens.

PubMed

Longeri, M; Polli, M; Ponti, W; Zanotti, M

1993-01-12

From a sample of 119 Friesian calves, serologically typed for BoLA class I, 47 subjects were chosen expressing 9 different MHC types (A6, A6.9, A10, A11, A14, A15, A30, W16, M103) with the same age and reared in the same farm conditions. The animals were s.c. injected with a water in oil suspension of killed M. bovis and the treatment was repeated two days later. Before the treatment and 21 days later, calves were bled and on PBM (peripheral blood mononuclear leucocytes) were performed the following tests: 1. Lymphocyte Stimulation with bovine and avian PPDs (Purified protein derivative of Mycobacterium bovis and Mycobacterium avium, respectively). 2. Phagocytic activity towards M. bovis. 3. Class II molecules expression on cell surface. 4. Percentage of leucocyte populations and subpopulations. In the in vitro Lymphocyte Stimulation test, all the animals and classes were responders. Animals bearing A10 BoLA class I presented c.p.m. (counts per minute) and index values higher than the other cattle; these values were significantly positively related both to bovine and avian PPDs (P < .01). By variance analysis A14 BoLA type showed a slight positive significant correlation with more efficient phagocytic activity. BoLA class I type did not seem to significantly affect percentage of class II positive cells and leucocyte percentages on PBM. ZUSAMMENFASSUNG: Der BoLA Klasse I Polymorphismus und in vitro immunologische Antwort gegen die Antigene von M. bovis Aus einer Stichprobe von 119 für BoLA Klasse I serologisch typisierten Friesian Kälber, wurden 47 Subjekte ausgewählt, die 9 verschiedene MHC Typen ausdrückten (A6, A6.9, A10, A11, A14, A15, A30, W16, M103). Alle waren gleich alt und in gleichen Haltungsbedingungen. Die Tiere wurden mit einer Wasser-in-Ol Suspension abgetöteter M. bovis subkutan injiziert und die Behandlung nach zwei Tagen wiederholt. Vor und 21 Tage nach Behandlung wurden die folgenden Tests ausgeführt: 1. Lymphozyten-Stimulationstest mit

Role for malonyl coenzyme A:acyl carrier protein transacylase (MCAT) in the growth-inhibitory effect of the calmodulin antagonist trifluoperazine in Mycobacterium bovis BCG.

PubMed

Sinha, Indrajit; Dick, Thomas

2004-06-01

To determine whether the fatty acid synthesis enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT) is involved in the growth-inhibitory effect of trifluoperazine in the tubercle bacillus Mycobacterium bovis BCG. BCG was grown in liquid culture with various concentrations of trifluoperazine and growth was monitored by OD measurement. To determine the effect of trifluoperazine on MCAT protein level, total protein was extracted from BCG cultures and was analysed by 2D gel electrophoresis and western blot. To confirm trifluoperazine-dependent reduction in the MCAT protein level, two BCG strains overexpressing MCAT at a low and high constitutive level were similarly tested. The synergic effect of trifluoperazine and isoniazid was tested at sub-MIC levels in liquid cultures. Trifluoperazine inhibition of growth correlates with reduction in the steady-state level of MCAT protein. Overexpression of MCAT confers resistance to trifluoperazine. Trifluoperazine acts synergically (albeit weakly) with isoniazid and no resistance towards isoniazid alone was observed due to overexpression of MCAT. This suggests MCAT to be a specific target of trifluoperazine. These results indicate MCAT as a target of trifluoperazine and provide an explanation for the inhibitory effect of trifluoperazine on mycobacterial lipid synthesis observed earlier. This makes MCAT a potential target for new antimycobacterials.

Molecular Characterization of Heterologous HIV-1gp120 Gene Expression Disruption in Mycobacterium bovis BCG Host Strain: A Critical Issue for Engineering Mycobacterial Based-Vaccine Vectors

PubMed Central

Joseph, Joan; Fernández-Lloris, Raquel; Pezzat, Elías; Saubi, Narcís; Cardona, Pere-Joan; Mothe, Beatriz; Gatell, Josep Maria

2010-01-01

Mycobacterium bovis Bacillus Calmette-Guérin (BCG) as a live vector of recombinant bacterial vaccine is a promising system to be used. In this study, we evaluate the disrupted expression of heterologous HIV-1gp120 gene in BCG Pasteur host strain using replicative vectors pMV261 and pJH222. pJH222 carries a lysine complementing gene in BCG lysine auxotrophs. The HIV-1 gp120 gene expression was regulated by BCG hsp60 promoter (in plasmid pMV261) and Mycobacteria spp. α-antigen promoter (in plasmid pJH222). Among 14 rBCG:HIV-1gp120 (pMV261) colonies screened, 12 showed a partial deletion and two showed a complete deletion. However, deletion was not observed in all 10 rBCG:HIV-1gp120 (pJH222) colonies screened. In this study, we demonstrated that E. coli/Mycobacterial expression vectors bearing a weak promoter and lysine complementing gene in a recombinant lysine auxotroph of BCG could prevent genetic rearrangements and disruption of HIV 1gp120 gene expression, a key issue for engineering Mycobacterial based vaccine vectors. PMID:20617151

Revisiting the structure of the anti-neoplastic glucans of Mycobacterium bovis Bacille Calmette-Guerin. Structural analysis of the extracellular and boiling water extract-derived glucans of the vaccine substrains.

PubMed

Dinadayala, Premkumar; Lemassu, Anne; Granovski, Pierre; Cérantola, Stéphane; Winter, Nathalie; Daffé, Mamadou

2004-03-26

The attenuated strain of Mycobacterium bovis Bacille Calmette-Guérin (BCG), used worldwide to prevent tuberculosis and leprosy, is also clinically used as an immunotherapeutic agent against superficial bladder cancer. An anti-tumor polysaccharide has been isolated from the boiling water extract of the Tice substrain of BCG and tentatively characterized as consisting primarily of repeating units of 6-linked-glucosyl residues. Mycobacterium tuberculosis and other mycobacterial species produce a glycogen-like alpha-glucan composed of repeating units of 4-linked glucosyl residues substituted at some 6 positions by short oligoglucosyl units that also exhibits an anti-tumor activity. Therefore, the impression prevails that mycobacteria synthesize different types of anti-neoplastic glucans or, alternatively, the BCG substrains are singular in producing a unique type of glucan that may confer to them their immunotherapeutic property. The present study addresses this question through the comparative analysis of alpha-glucans purified from the extracellular materials and boiling water extracts of three vaccine substrains. The polysaccharides were purified, and their structural features were established by mono- and two-dimensional NMR spectroscopy and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of the enzymatic and chemical degradation products of the purified compounds. The glucans isolated by the two methods from the three substrains of BCG were shown to exhibit identical structural features shared with the glycogen-like alpha-glucan of M. tuberculosis and other mycobacteria. Incidentally, we observed an occasional release of dextrans from Sephadex columns that may explain the reported occurrence of 6-substituted alpha-glucans in mycobacteria.

Toward eradication: the effect of Mycobacterium bovis infection in wildlife on the evolution and future direction of bovine tuberculosis management in New Zealand

PubMed Central

Livingstone, PG; Hancox, N; Nugent, G; de Lisle, GW

2015-01-01

Abstract New Zealand's bovine tuberculosis (TB) control programme has greatly reduced the burden of tuberculosis on the farming industry, from 11% of mature cattle found with TB at slaughter in 1905 to <0.003% in 2012/13. New Zealand implemented TB control measures in cattle from the mid-twentieth century, and later in farmed deer. Control was based on established methods of tuberculin testing of herds, slaughter of suspect cases, and livestock movement control. Unexplained regional control failures and serious disease outbreaks were eventually linked to wildlife-vectored infection from the introduced Australian brushtail possum (Trichosurus vulpecula), which also triggered a wildlife disease complex involving a range of introduced species. This paper reviews the progressive elucidation of the epidemiology of Mycobacterium bovis in New Zealand's wildlife and farmed livestock, and the parallel development of research-led, multi-faceted TB control strategies required to protect New Zealand's livestock industries from damaging infection levels. The adoption of coordinated national pest management strategies, with increasingly ambitious objectives agreed between government and industry funders, has driven a costly but very successful management regime targeted at controlling TB in the possum maintenance host. This success has led to initiation of a strategy designed to eradicate TB from New Zealand's livestock and wildlife, which is considered a realistic long-term prospect. PMID:25273888

Genetic diversity of the Mycobacterium tuberculosis Complex in San Luis Potosí, México

PubMed Central

2013-01-01

Background Although epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce. Methods Complete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined. Results Pulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities. Conclusions In San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage. PMID:23635381

Transmission of Mycobacterium tuberculosis between Farmers and Cattle in Central Ethiopia

PubMed Central

Ameni, Gobena; Tadesse, Konjit; Hailu, Elena; Deresse, Yohannes; Medhin, Girmay; Aseffa, Abraham; Hewinson, Glyn; Vordermeier, Martin; Berg, Stefan

2013-01-01

Background Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia. Methodology/Principal Findings A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by “Vordermeier et al. (2002)” was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively. Conclusions/Significance The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows

Decreased capacity of recombinant 45/47-kDa molecules (Apa) of Mycobacterium tuberculosis to stimulate T lymphocyte responses related to changes in their mannosylation pattern.

PubMed

Horn, C; Namane, A; Pescher, P; Rivière, M; Romain, F; Puzo, G; Bârzu, O; Marchal, G

1999-11-05

The Apa molecules secreted by Mycobacterium tuberculosis, Mycobacterium bovis, or BCG have been identified as major immunodominant antigens. Mass spectrometry analysis indicated similar mannosylation, a complete pattern from 1 up to 9 hexose residues/mole of protein, of the native species from the 3 reference strains. The recombinant antigen expressed in M. smegmatis revealed a different mannosylation pattern: species containing 7 to 9 sugar residues/mole of protein were in the highest proportion, whereas species bearing a low number of sugar residues were almost absent. The 45/47-kDa recombinant antigen expressed in E. coli was devoid of sugar residues. The proteins purified from M. tuberculosis, M. bovis, or BCG have a high capacity to elicit in vivo potent delayed-type hypersensitivity (DTH) reactions and to stimulate in vitro sensitized T lymphocytes of guinea pigs immunized with living BCG. The recombinant Apa expressed in Mycobacterium smegmatis was 4-fold less potent in vivo in the DTH assay and 10-fold less active in vitro to stimulate sensitized T lymphocytes than the native proteins. The recombinant protein expressed in Escherichia coli was nearly unable to elicit DTH reactions in vivo or to stimulate T lymphocytes in vitro. Thus the observed biological effects were related to the extent of glycosylation of the antigen.

Robust immunity to an auxotrophic Mycobacterium bovis BCG-VLP prime-boost HIV vaccine candidate in a nonhuman primate model.

PubMed

Chege, Gerald K; Burgers, Wendy A; Stutz, Helen; Meyers, Ann E; Chapman, Rosamund; Kiravu, Agano; Bunjun, Rubina; Shephard, Enid G; Jacobs, William R; Rybicki, Edward P; Williamson, Anna-Lise

2013-05-01

We previously reported that a recombinant pantothenate auxotroph of Mycobacterium bovis BCG expressing human immunodeficiency virus type 1 (HIV-1) subtype C Gag (rBCGpan-Gag) efficiently primes the mouse immune system for a boost with a recombinant modified vaccinia virus Ankara (rMVA) vaccine. In this study, we further evaluated the immunogenicity of rBCGpan-Gag in a nonhuman primate model. Two groups of chacma baboons were primed or mock primed twice with either rBCGpan-Gag or a control BCG. Both groups were boosted with HIV-1 Pr55(gag) virus-like particles (Gag VLPs). The magnitude and breadth of HIV-specific cellular responses were measured using a gamma interferon (IFN-γ) enzyme-linked immunosorbent spot (ELISPOT) assay, and the cytokine profiles and memory phenotypes of T cells were evaluated by polychromatic flow cytometry. Gag-specific responses were detected in all animals after the second inoculation with rBCGpan-Gag. Boosting with Gag VLPs significantly increased the magnitude and breadth of the responses in the baboons that were primed with rBCGpan-Gag. These responses targeted an average of 12 Gag peptides per animal, compared to an average of 3 peptides per animal for the mock-primed controls. Robust responses of Gag-specific polyfunctional T cells capable of simultaneously producing IFN-γ, tumor necrosis alpha (TNF-α), and interleukin-2 (IL-2) were detected in the rBCGpan-Gag-primed animals. Gag-specific memory T cells were skewed toward a central memory phenotype in both CD4(+) and CD8(+) T cell populations. These data show that the rBCGpan-Gag prime and Gag VLP boost vaccine regimen is highly immunogenic, inducing a broad and polyfunctional central memory T cell response. This report further indicates the feasibility of developing a BCG-based HIV vaccine that is safe for childhood HIV immunization.

First insights into the genetic diversity of Mycobacterium tuberculosis isolates from HIV-infected Mexican patients and mutations causing multidrug resistance

PubMed Central

2010-01-01

Background The prevalence of infections with Mycobacterium tuberculosis (MTb) and nontuberculous mycobacteria (NTM) species in HIV-infected patients in Mexico is unknown. The aims of this study were to determine the frequency of MTb and NTM species in HIV-infected patients from Mexico City, to evaluate the genotypic diversity of the Mycobacterium tuberculosis complex strains, to determine their drug resistance profiles by colorimetric microplate Alamar Blue assay (MABA), and finally, to detect mutations present in katG, rpoB and inhA genes, resulting in isoniazid (INH) and rifampin (RIF) resistance. Results Of the 67 mycobacterial strains isolated, 48 were identified as MTb, 9 as M. bovis, 9 as M. avium and 1 as M. intracellulare. IS6110-RFLP of 48 MTb strains showed 27 profiles. Spoligotyping of the 48 MTb strains yielded 21 patterns, and 9 M. bovis strains produced 7 patterns. Eleven new spoligotypes patterns were found. A total of 40 patterns were produced from the 48 MTb strains when MIRU-VNTR was performed. Nineteen (39.6%) MTb strains were resistant to one or more drugs. One (2.1%) multidrug-resistant (MDR) strain was identified. A novel mutation was identified in a RIF-resistant strain, GAG → TCG (Glu → Ser) at codon 469 of rpoB gene. Conclusions This is the first molecular analysis of mycobacteria isolated from HIV-infected patients in Mexico, which describe the prevalence of different mycobacterial species in this population. A high genetic diversity of MTb strains was identified. New spoligotypes and MIRU-VNTR patterns as well as a novel mutation associated to RIF-resistance were found. This information will facilitate the tracking of different mycobacterial species in HIV-infected individuals, and monitoring the spread of these microorganisms, leading to more appropriate measures for tuberculosis control. PMID:20236539

Effects of dietary glutamine supplementation on the body composition and protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guerin.

PubMed

Rogero, Marcelo Macedo; Borges, Maria Carolina; de Castro, Inar Alves; Pires, Ivanir S O; Borelli, Primavera; Tirapegui, Julio

2011-09-01

Glutamine, one of the most abundant amino acids found in maternal milk, favors protein anabolism. Early-weaned babies are deprived of this source of glutamine, in a period during which endogenous biosynthesis may be insufficient for tissue needs in states of metabolic stress, mainly during infections. The objective of this study was to verify the effects of dietary glutamine supplementation on the body composition and visceral protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Mice were weaned early on their 14th day of life and seperated into two groups, one of which was fed a glutamine-free diet (n = 16) and the other a glutamine-supplemented diet (40 g/kg diet) (n = 16). At 21 days of age, some mice were intraperitoneally injected with BCG. Euthanasia was performed at the 28th day of age. BCG inoculation significantly reduced body weight (P < 0.001), lean mass (P = 0.002), water (P = 0.006), protein (P = 0.007) and lipid content (P = 0.001) in the carcass. Dietary glutamine supplementation resulted in a significant increase in serum IGF-1 (P = 0.019) and albumin (P = 0.025) concentration, muscle protein concentration (P = 0.035) and lipid content (P = 0.002) in the carcass. In conclusion, dietary glutamine supplementation had a positive influence on visceral protein status but did not affect body composition in early-weaned mice inoculated with BCG.

Effects of Dietary Glutamine Supplementation on the Body Composition and Protein Status of Early-Weaned Mice Inoculated with Mycobacterium bovis Bacillus Calmette-Guerin

PubMed Central

Rogero, Marcelo Macedo; Borges, Maria Carolina; de Castro, Inar Alves; Pires, Ivanir S. O.; Borelli, Primavera; Tirapegui, Julio

2011-01-01

Glutamine, one of the most abundant amino acids found in maternal milk, favors protein anabolism. Early-weaned babies are deprived of this source of glutamine, in a period during which endogenous biosynthesis may be insufficient for tissue needs in states of metabolic stress, mainly during infections. The objective of this study was to verify the effects of dietary glutamine supplementation on the body composition and visceral protein status of early-weaned mice inoculated with Mycobacterium bovis Bacillus Calmette-Guérin (BCG). Mice were weaned early on their 14th day of life and seperated into two groups, one of which was fed a glutamine-free diet (n = 16) and the other a glutamine-supplemented diet (40 g/kg diet) (n = 16). At 21 days of age, some mice were intraperitoneally injected with BCG. Euthanasia was performed at the 28th day of age. BCG inoculation significantly reduced body weight (P < 0.001), lean mass (P = 0.002), water (P = 0.006), protein (P = 0.007) and lipid content (P = 0.001) in the carcass. Dietary glutamine supplementation resulted in a significant increase in serum IGF-1 (P = 0.019) and albumin (P = 0.025) concentration, muscle protein concentration (P = 0.035) and lipid content (P = 0.002) in the carcass. In conclusion, dietary glutamine supplementation had a positive influence on visceral protein status but did not affect body composition in early-weaned mice inoculated with BCG. PMID:22254124

Induction of Unconventional T Cells by a Mutant Mycobacterium bovis BCG Strain Formulated in Cationic Liposomes Correlates with Protection against Mycobacterium tuberculosis Infections of Immunocompromised Mice

PubMed Central

Yabe, Idalia; Morris, Sheldon; Cowley, Siobhan

2016-01-01

Earlier studies aimed at defining protective immunity induced by Mycobacterium bovis BCG immunization have largely focused on the induction of antituberculosis CD4+ and CD8+ T cell responses. Here we describe a vaccine consisting of a BCGΔmmaA4 deletion mutant formulated in dimethyl dioctadecyl-ammonium bromide (DDA) with d-(+)-trehalose 6,6′-dibehenate (TDB) (DDA/TDB) adjuvant (A4/Adj) that protected TCRδ−/− mice depleted of CD4+, CD8+, and NK1.1+ T cells against an aerosol challenge with M. tuberculosis. These mice were significantly protected relative to mice immunized with a nonadjuvanted BCGΔmmaA4 (BCG-A4) mutant and nonvaccinated controls at 2 months and 9 months postvaccination. In the absence of all T cells following treatment with anti-Thy1.2 antibody, the immunized mice lost the ability to control the infection. These results indicate that an unconventional T cell population was mediating protection in the absence of CD4+, CD8+, NK1.1+, and TCRγδ T cells and could exhibit memory. Focusing on CD4− CD8− double-negative (DN) T cells, we found that these cells accumulated in the lungs postchallenge significantly more in A4/Adj-immunized mice and induced significantly greater frequencies of pulmonary gamma interferon (IFN-γ)-producing cells than were seen in the nonvaccinated or nonadjuvanted BCG control groups. Moreover, pulmonary DN T cells from the A4/Adj group exhibited significantly higher IFN-γ integrated median fluorescence intensity (iMFI) values than were seen in the control groups. We also showed that enriched DN T cells from mice immunized with A4/Adj could control mycobacterial growth in vitro significantly better than naive whole-spleen cells. These results suggest that formulating BCG in DDA/TDB adjuvant confers superior protection in immunocompromised mice and likely involves the induction of long-lived memory DN T cells. PMID:27226281

Molecular characterisation of Mycobacterium caprae strains isolated in Poland.

PubMed

Krajewska-Wędzina, Monika; Kozińska, Monika; Orłowska, Blanka; Weiner, Marcin; Szulowski, Krzysztof; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Smith, Noel H

2018-03-10

Bovine tuberculosis (bovine TB, bTB) is caused by bovine bacilli: Mycobacterium bovis and M caprae The studies conducted in Poland, in the National Bovine Tuberculosis Reference Laboratory in the Department of Microbiology of the National Veterinary Research Institute in Pulawy, show that animal tuberculosis in Poland is also caused by M caprae We here describe the identification and genotypic assessment of 52 isolates of M caprae obtained from Polish cattle and wild animals over the last five years. We show that strains isolated from bison have significant genotypic diversity and are distinct compared with the genotypes of strains isolated from cattle. Similarly, isolates from cattle herds can be highly genotypically variable. Formal designation of the members of the Mycobacterium tuberculosis complex is controversial in Poland; there is a gap in veterinary legislation with regard to bTB and no explicit mention of M caprae causing tuberculosis in animal. © British Veterinary Association (unless otherwise stated in the text of the article) 2018. All rights reserved. No commercial use is permitted unless otherwise expressly granted.

Dielectrophoretic characterization of antibiotic-treated Mycobacterium tuberculosis complex cells.

PubMed

Inoue, Shinnosuke; Lee, Hyun-Boo; Becker, Annie L; Weigel, Kris M; Kim, Jong-Hoon; Lee, Kyong-Hoon; Cangelosi, Gerard A; Chung, Jae-Hyun

2015-10-01

Multi-drug resistant tuberculosis (MDR-TB) has become a serious concern for proper treatment of patients. As a phenotypic method, dielectrophoresis can be useful but is yet to be attempted to evaluate Mycobacterium tuberculosis complex cells. This paper investigates the dielectrophoretic behavior of Mycobacterium bovis (Bacillus Calmette-Guérin, BCG) cells that are treated with heat or antibiotics rifampin (RIF) or isoniazid (INH). The experimental parameters are designed on the basis of our sensitivity analysis. The medium conductivity (σ(m)) and the frequency (f) for a crossover frequency (f(xo1)) test are decided to detect the change of σ(m)-f(xo1) in conjunction with the drug mechanism. Statistical modeling is conducted to estimate the distributions of viable and nonviable cells from the discrete measurement of f (xo1). Finally, the parameters of the electrophysiology of BCG cells, C(envelope) and σ(cyto), are extracted through a sampling algorithm. This is the first evaluation of the dielectrophoresis (DEP) approach as a means to assess the effects of antimicrobial drugs on M. tuberculosis complex cells.

Effects of positive results for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA on results of caudal fold tuberculin test and interferon-gamma assay for tuberculosis in cattle.

PubMed

Dunn, John R; Kaneene, John B; Grooms, Daniel L; Bolin, Steven R; Bolin, Carole A; Bruning-Fann, Colleen S

2005-02-01

To determine whether cattle testing positive for Mycobacterium avium subsp paratuberculosis as determined by microbial culture of feces or antibody ELISA were more likely to have false-positive responses on the caudal fold tuberculin (CFT) test or interferon-gamma (IFN-gamma) assay for Mycobacterium bovis than cattle testing negative for M paratuberculosis. 1043 cattle from 10 herds in Michigan. Feces and blood samples for plasma were collected from cattle > or =24 months old on the day the CFT test was read. Fecal samples were submitted for microbial culture for M paratuberculosis. Plasma samples were tested for antibody against M paratuberculosis, and IFN-gamma after stimulation with purified protein derivative tuberculin from M bovis or M avium. Of 1043 cattle, 180 (17.3%) had positive CFT test results (suspects) and 8 (0.8%) had positive IFN-gamma assay results after stimulation with purified protein derivative tuberculin from M bovis. Forty-five (4.3%) and 115 (11.0%) cattle tested positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA, respectively. Cattle with positive responses for M paratuberculosis appeared to have an increased likelihood of false-positive results on the CFT test, although this association was not significant. No significant association was detected among cattle testing positive for M paratuberculosis as determined by microbial culture of feces and antibody ELISA and positive CFT test and IFN-gamma assay results for M bovis.

Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus

PubMed Central

Behar, Samuel M.; Carpenter, Stephen M.; Booty, Matthew G.; Barber, Daniel L.; Jayaraman, Pushpa

2014-01-01

Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease – the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage. PMID:25311810

Orchestration of pulmonary T cell immunity during Mycobacterium tuberculosis infection: immunity interruptus.

PubMed

Behar, Samuel M; Carpenter, Stephen M; Booty, Matthew G; Barber, Daniel L; Jayaraman, Pushpa

2014-12-01

Despite the introduction almost a century ago of Mycobacterium bovis BCG (BCG), an attenuated form of M. bovis that is used as a vaccine against Mycobacterium tuberculosis, tuberculosis remains a global health threat and kills more than 1.5 million people each year. This is mostly because BCG fails to prevent pulmonary disease--the contagious form of tuberculosis. Although there have been significant advances in understanding how the immune system responds to infection, the qualities that define protective immunity against M. tuberculosis remain poorly characterized. The ability to predict who will maintain control over the infection and who will succumb to clinical disease would revolutionize our approach to surveillance, control, and treatment. Here we review the current understanding of pulmonary T cell responses following M. tuberculosis infection. While infection elicits a strong immune response that contains infection, M. tuberculosis evades eradication. Traditionally, its intracellular lifestyle and alteration of macrophage function are viewed as the dominant mechanisms of evasion. Now we appreciate that chronic inflammation leads to T cell dysfunction. While this may arise as the host balances the goals of bacterial sterilization and avoidance of tissue damage, it is becoming clear that T cell dysfunction impairs host resistance. Defining the mechanisms that lead to T cell dysfunction is crucial as memory T cell responses are likely to be subject to the same subject to the same pressures. Thus, success of T cell based vaccines is predicated on memory T cells avoiding exhaustion while at the same time not promoting overt tissue damage. Copyright © 2014 Elsevier Ltd. All rights reserved.

Analysis of breath volatile organic compounds as a screening tool for detection of Tuberculosis in cattle

USDA-ARS?s Scientific Manuscript database

• Keywords: bovine tuberculosis; Mycobacterium bovis; breath analysis; volatile organic compound; gas chromatography; mass spectrometry; NaNose • Introduction: This presentation describes two studies exploring the use of breath VOCs to identify Mycobacterium bovis infection in cattle. • Methods: ...

Comparative proteomic analysis of virulent Korean Mycobacterium tuberculosis K-strain with other mycobacteria strain following infection of U-937 macrophage.

PubMed

Ryoo, Sung Weon; Park, Young Kil; Park, Sue-Nie; Shim, Young Soo; Liew, Hyunjeong; Kang, Seongman; Bai, Gill-Han

2007-06-01

In Korea, the Mycobacterium tuberculosis K-strain is the most prevalent clinical isolates and belongs to the Beijing family. In this study, we conducted comparative porteomics of expressed proteins of clinical isolates of the K-strain with H37Rv, H37Ra as well as the vaccine strain of Mycobacterium bovis BCG following phagocytosis by the human monocytic cell line U-937. Proteins were analyzed by 2-D PAGE and MALDITOF-MS. Two proteins, Mb1363 (probable glycogen phosphorylase GlgP) and MT2656 (Haloalkane dehalogenase LinB) were most abundant after phagocytosis of M. tuberculosis K-strain. This approach provides a method to determine specific proteins that may have critical roles in tuberculosis pathogenesis.

Some South African Rubiaceae Tree Leaf Extracts Have Antimycobacterial Activity Against Pathogenic and Non-pathogenic Mycobacterium Species.

PubMed

Aro, Abimbola O; Dzoyem, Jean P; Hlokwe, Tiny M; Madoroba, Evelyn; Eloff, Jacobus N; McGaw, Lyndy J

2015-07-01

Tuberculosis (TB) caused by Mycobacterium tuberculosis remains an ongoing threat to human health. Many plant species contain antimycobacterial compounds, which may serve as template molecules for new anti-TB drugs. The Rubiaceae family is the largest family of trees in southern Africa, and preliminary evidence revealed antimycobacterial activity in several species of the genus, motivating further studies. Leaf extracts of 15 tree species from the Rubiaceae family were screened for antimycobacterial activity against pathogenic M. tuberculosis and non-pathogenic Mycobacterium smegmatis, Mycobacterium aurum and Mycobacterium bovis BCG (Bacillus Calmette-Guérin) using a twofold serial microdilution assay. Cytotoxicity was determined using a tetrazolium-based colorimetric assay against C3A liver cells and Vero kidney cells. Minimum inhibitory concentration values as low as 0.04 mg/mL against M. smegmatis and M. tuberculosis were recorded. Activity against M. aurum was the best predictor of activity against pathogenic M. tuberculosis (correlation coefficient = 0.9). Bioautography indicated at least 40 different antimycobacterial compounds in the extracts. Cytotoxicity of the extracts varied, and Oxyanthus speciosus had the most promising selectivity index values. Copyright © 2015 John Wiley & Sons, Ltd.

A novel modified-indirect ELISA based on spherical body protein 4 for detecting antibody during acute and long-term infections with diverse babesia bovis strains

USDA-ARS?s Scientific Manuscript database

Background: Cattle persistently infected with Babesia bovis are reservoirs for intra- and inter-herd transmission. Since B. bovis is considered a persistent infection, developing a reliable, high-throughput assay that detects antibody during all stages of the infection could be pivotal for establish...

Genetic Evolution of Mycobacterium bovis Causing Tuberculosis in Livestock and Wildlife in France since 1978

PubMed Central

Hauer, Amandine; De Cruz, Krystel; Cochard, Thierry; Godreuil, Sylvain; Karoui, Claudine; Henault, Sylvie; Bulach, Tabatha; Bañuls, Anne-Laure; Biet, Franck; Boschiroli, María Laura

2015-01-01

To study the dynamics of bovine tuberculosis (bTB) in France, 4,654 M. bovis strains isolated mainly from livestock and wildlife since 1978 were characterized by spoligotyping and MLVA based on MIRU-VNTR. In our study spoligotyping allowed the discrimination of 176 types although 3 spoligotypes are predominant and account for more than half of the total strain population: SB0120 (26%), SB0134 (11%) and SB0121 (6%). In addition, 11% of the isolates, principally from Southern France, showing close spoligotypes and MIRU-VNTR types have been gathered in a family designated as the “F4-family”. MLVA typing allowed extensive discrimination, particularly for strains with predominant spoligotypes, with a total of 498 genotypes, several of which were highly regionalized. The similarity of the strains’ genetic relationships based on spoligotyping and MIRU-VNTR markers supports the co-existence of different clonal populations within the French M. bovis population. A genetic evolution of the strains was observed both geographically and in time. Indeed, as a result of the reduction of bTB due to the national control campaigns, a large reduction of the strains’ genetic variability took place in the last ten years. However, in the regions were bTB is highly prevalent at present, cases in both livestock and in wildlife are due to the spread of unique local genotype profiles. Our results show that the highly discriminating genotyping tools used in this study for molecular studies of bTB are useful for addressing pending questions, which would lead to a better insight into the epidemiology of the disease, and for finding proper solutions for its sustainable control in France. PMID:25658691

Relative virulence in bison and cattle of bison-associated genotypes of Mycoplasma bovis

USDA-ARS?s Scientific Manuscript database

Background. Mycoplasma bovis is a cause of respiratory disease in cattle and the bacterium most frequently isolated from bovine respiratory disease complex. It has recently emerged as a major health problem in bison, causing pharyngitis, pneumonia, arthritis, dystocia and abortion. In cattle, M. b...

Heparin-Binding Haemagglutinin, a New Tool for the Detection of Latent Mycobacterium tuberculosis Infection in Hemodialysis Patients

PubMed Central

Dessein, Rodrigue; Corbière, Véronique; Nortier, Joëlle; Dratwa, Max; Gastaldello, Karine; Pozdzik, Agnieszka; Lecher, Sophie; Grandbastien, Bruno; Locht, Camille; Mascart, Françoise

2013-01-01

Background Patients with end-stage renal disease (ESRD) and latently infected with Mycobacterium tuberculosis (LTBI) are at higher risk to develop tuberculosis (TB) than healthy subjects. Interferon-gamma release assays (IGRAs) were reported to be more sensitive than tuberculin skin tests for the detection of infected individuals in dialysis patients. Methods On 143 dialysis patients prospectively enrolled, we compared the results from the QuantiFERON®-TB Gold assay (QFT), to those of an IGRA in response to in vitro stimulation of circulating mononuclear cells with the mycobacterial latency antigen Heparin-Binding Haemagglutinin purified from Mycobacterium bovis BCG (native HBHA, nHBHA). Results Seven patients had a past history of active TB and 1 had an undetermined result with both IGRAs. Among the other 135 patients, 94 had concordant results with the QFT and nHBHA-IGRA, 40.0% being negative and therefore not latently infected, and 29.6% being positive and thus LTBI. Discrepant results between these tests were found for 36 patients positive only with the nHBHA-IGRA and 5 only with the QFT. Conclusions The nHBHA-IGRA is more sensitive than the QFT for the detection of LTBI dialysis patients, and follow-up of the patients will allow us to define the clinical significance of discrepant results between the nHBHA-IGRA and the QFT. PMID:23940693

Bovine Tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti). Mycobacterium bovis is the species most often isolated from tuberculous catt...

Bovine tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB) in animals and humans may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedii, M. microti, M. caprae, or M. canetti) . Mycobacterium bovis is the species most often isolated from tuberculous cat...

Bovine Tuberculosis Vaccine Efficacy Studies: Neonatal Calves and White-tailed Deer

USDA-ARS?s Scientific Manuscript database

Introduction Tuberculosis (TB) in humans and animals may result from exposure to bacilli within the Mycobacterium tuberculosis complex (i.e., M. tuberculosis, M. bovis, M. africanum, M. pinnipedi, M. microti, M. caprae, or M. canetti)(#1). Mycobacterium bovis is the species most often isolated from ...

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

PubMed

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

2010-03-01

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

Comprehensive analysis of mycolic acid subclass and molecular species composition of Mycobacterium bovis BCG Tokyo 172 cell wall skeleton (SMP-105).

PubMed

Uenishi, Yuko; Fujita, Yukiko; Kusunose, Naoto; Yano, Ikuya; Sunagawa, Makoto

2008-02-01

The mycobacterial cell envelope consists of a characteristic cell wall skeleton (CWS), a mycoloyl arabinogalactan peptidoglycan complex, and related hydrophobic components that contribute to the cell surface properties. Since mycolic acids have recently been reported to play crucial roles in host immune response, detailed molecular characterization of mycolic acid subclasses and sub-subclasses of CWS from Mycobacterium bovis BCG Tokyo 172 (SMP-105) was performed. Mycolic acids were liberated by alkali hydrolysis from SMP-105, and their methyl esters were separated by silica gel TLC into three subclasses: alpha-, methoxy-, and keto-mycolates. Each mycolate subclass was further separated by silver nitrate (AgNO(3))-coated silica gel TLC into sub-subclasses. Molecular weights of individual mycolic acid were determined by MALDI-TOF mass spectrometry. alpha-Mycolates were sub-grouped into cis, cis-dicyclopropanoic (alpha1), and cis-monocyclopropanoic-cis-monoenoic (alpha2) series; methoxy-mycolates were sub-grouped into cis-monocyclopropanoic (m1), trans-monocyclopropanoic (m2), trans-monoenoic (m3), cis-monocyclopropanoic-trans-monoenoic (m4), cis-monoenoic (m5), and cis-monocyclopropanoic-cis-monoenoic (m6) series; and keto-mycolates were sub-grouped into cis-monocyclopropanoic (k1), trans-monocyclopropanoic (k2), trans-monoenoic (k3), cis-monoenoic (k4), and cis-monocyclopropanoic-cis-monoenoic (k5) series. The position of each functional group, including cyclopropane rings and methoxy and keto groups, was determined by analysis of the meromycolates with fast atom bombardment (FAB) mass spectrometry and FAB mass-mass spectrometry, and the cis/trans ratio of cyclopropane rings and double bonds were determined by NMR analysis of methyl mycolates. Mycolic acid subclass and molecular species composition of SMP-105 showed characteristic features including newly-identified cis-monocyclopropanoic-trans-monoenoic mycolic acid (m4).

Responses to diagnostic tests for bovine tuberculosis in dairy and non-dairy cattle naturally exposed to Mycobacterium bovis in Great Britain.

PubMed

Downs, S H; Broughan, J M; Goodchild, A V; Upton, P A; Durr, P A

2016-10-01

Field surveillance of British cattle using the single intradermal comparative cervical tuberculin (SICCT) test shows a higher incidence rate of bovine tuberculosis (bTB) in dairy compared to beef herds, but a lower probability of post-mortem examination confirmed (PMC) Mycobacterium bovis infection in dairy herds. A cross-sectional study was conducted to compare animal level differences in bTB detection between dairy and non-dairy cattle in Great Britain. During the period from 2002 to 2005, 200 (41% dairy) reactors in the SICCT test (standard interpretation) were randomly selected, and 200 in-contact cattle (43% dairy) were purposively selected from bTB-infected herds. Interferon (IFN)-γ responses in blood to bovine and avian purified protein derivative (PPD), and early secretory antigen target 6 kDa and culture filtrate protein 10 (ESAT-6/CFP10), were measured. The post-mortem examination included gross pathological examination, mycobacterial culture and histopathology. The proportions of cattle positive to ESAT6/CFP10 were 26% (95% confidence interval, CI, 15-39%) in dairy reactors and 62% (95% CI 51-72%) in non-dairy reactors (P <0.001). PMC risk was 34% (95% CI 24-45%) in dairy reactors and 69% (95% CI 60-78%) in non-dairy reactors (P <0.001). The odds ratio for PMC risk in dairy reactors compared to non-dairy reactors, after controlling for bTB prevalence, herd size and SICCT test response, was 0.27 (95% CI 0.14-0.53; P <0.001). In surveillance data, adjusted animal level PMC risks were lower for dairy reactors than for beef reactors aged >2 years (P <0.001). Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.

Identification of new antibacterial targets in RNA polymerase of Mycobacterium tuberculosis by detecting positive selection sites.

PubMed

Wang, QingBiao; Xu, Yiqin; Gu, Zhuoya; Liu, Nian; Jin, Ke; Li, Yao; Crabbe, M James C; Zhong, Yang

2018-04-01

Bacterial RNA polymerase (RNAP) is an effective target for antibacterial treatment. In order to search new potential targets in RNAP of Mycobacterium, we detected adaptive selections of RNAP related genes in 13 strains of Mycobacterium by phylogenetic analysis. We first collected sequences of 17 genes including rpoA, rpoB, rpoC, rpoZ, and sigma factor A-M. Then maximum likelihood trees were constructed, followed by positive selection detection. We found that sigG shows positive selection along the clade (M. tuberculosis, M. bovis), suggesting its important evolutionary role and its potential to be a new antibacterial target. Moreover, the regions near 933Cys and 935His on the rpoB subunit of M. tuberculosis showed significant positive selection, which could also be a new attractive target for anti-tuberculosis drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus, and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques▿

PubMed Central

Rosario, Maximillian; Hopkins, Richard; Fulkerson, John; Borthwick, Nicola; Quigley, Máire F.; Joseph, Joan; Douek, Daniel C.; Greenaway, Hui Yee; Venturi, Vanessa; Gostick, Emma; Price, David A.; Both, Gerald W.; Sadoff, Jerald C.; Hanke, Tomáš

2010-01-01

Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration. PMID:20375158

Immunology of bovine tuberculosis: Perspectives on one health approaches and defining correlates of protection versus infection

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is an exemplary model of the One Health Concept. The human TB vaccine, M. bovis bacille Calmette-Guerin (BCG), was first proven effective in cattle prior to use in humans. Recent experimental ...

Vaccine approaches for bovine tuberculosis: Correlates of protection and relevance to human tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is a classic model of the One Health Concept. M. bovis Bacillus Calmette Guerin (BCG) was first proven effective in cattle prior to use in humans. Recent experimental trials with cattle have d...

Tuberculosis in domestic livestock: pathogenesis, transmission, and vaccination

USDA-ARS?s Scientific Manuscript database

The Mycobacterium tuberculosis complex includes agents such as M. tuberculosis and M. bovis, the cause of tuberculosis in most animals and a zoonotic pathogen. Mycobacterium bovis has one of the broadest host ranges of any pathogen, infecting most mammals, including humans. Models are used to study ...

Competitive and indirect enzyme-linked immunosorbent assays for Mycobacterium bovis infections based on MPB70 and lipoarabinomannan antigens.

PubMed Central

Sugden, E A; Stilwell, K; Rohonczy, E B; Martineau, P

1997-01-01

A competitive enzyme-linked immunosorbent assay (C-ELISA) using M. bovis BCG Tokyo culture filtrate as antigen and anti-MPB70 4C3/17 monoclonal antibody was developed for use in multiple animal species. An analysis of the C-ELISA data for cattle and bison serum panels revealed specificities of 68% to 85% and sensitivities of 85% to 89%. Receiver operator characteristics (ROC) of this data revealed areas of 81% to 92% for C-ELISA and demonstrated that C-ELISA as well as the indirect ELISA protocols, MPB70-ELISA and LAM-ELISA, discriminate M. bovis infected animals from non-infected animals for these particular panels. The kappa statistic values for agreement beyond chance between C-ELISA and MPB70-ELISA were determined after ELISA cutoffs were adjusted to minimize false positives. There were poor to excellent agreements between C-ELISA and MPB70-ELISA in all species tested (Bovidae, Cervidae, and Camelidae) that were consistently higher than the kappa statistic between C-ELISA and LAM-ELISA. The humoral response to one antigen and little or no response to the other in many animals argued for a parallel interpretation of C-ELISA and LAM-ELISA to increase sensitivity. PMID:9008794

Rapid identification of Mycobacterium avium ssp paratuberculosis laboratory strains by IS900-Nested polymerase chain reaction.

PubMed

Taheri, Mohammad Mohammad; Mosavari, Nader; Feizabadi, Mohammad Mehdi; Tadayon, Keyvan; Keshavarz, Rouholah; Pajoohi, Reza Aref; Soleimani, Kioomars; Pour, Shojaat Dashti

2016-12-01

Mycobacterium avium ssp paratuberculosis (MAP) causes paratuberculosis (Johne's disease) in ruminants. As a species, M. avium comprises M. avium subsp. hominissuis and a number of clones that are known to have evolved from this subspecies, namely M. avium subsp. avium (MAA), M. avium subsp. silvaticum, and MAP. Despite the very high genomic similarity of MAP and MAA, the insertion sequence IS900, which is 1,451-bp long, is now understood to be exclusively present in 10-20 copies in the genome of MAP. In the present study, a multidiscipline polymerase chain reaction (PCR)-based algorithm targeting16SrRNA, IS6110, IS901, IS1245, and IS900 markers has been employed to differentiate between six laboratory strains of M. avium complex (including MAP 316F, III&V, and 2e plus MAA D4), Mycobacterium tuberculosis DT, and Mycobacterium bovis AN5 strains used at the Razi Institute (Tehran, Iran) for the preparation of paratuberculin, avian, human, and bovine tuberculin, respectively. Three laboratory strains of III&V, 2e, and 316F were subcultured on Herrold's egg yolk medium, whereas the MAA strain of D4 along with M. bovis AN5 and M. tuberculosis DT were subcultured on Lowenstein-Jensen slopes. All the inoculated culture tubes were incubated for 8weeks at 37°C. Eventually, their genomic DNA was extracted according to the method of van Soolingen. Five individual PCRs were conducted on these templates to amplify 16SrRNA (genus-specific marker shared by all mycobacteria), IS900 (MAP-specific marker), IS901 (MAA-specific marker), IS1245 (M. avium complex (MAC)-specific marker), and IS6110 (M. tuberculosis complex (MTC)-specific marker) loci. Consequently, a 543-bp amplicon was amplified by all the six strains in PCR against 16SrRNA, an indication of their identity as members of Mycobacterium genus. A 245-bp fragment was detected in only IS6110-PCR with M. bovis AN5 as well as M. tuberculosis DT. In the IS1245 assessment, the MAA strain of D4 produced a 427-bp amplicon, whereas

First molecular survey of Anaplasma bovis in small ruminants from Tunisia.

PubMed

Ben Said, Mourad; Belkahia, Hanène; Karaoud, Maroua; Bousrih, Maha; Yahiaoui, Mouna; Daaloul-Jedidi, Monia; Messadi, Lilia

2015-09-30

To date, no information is available regarding the presence of Anaplasma bovis in the South Mediterranean area. In this study, prevalence, risk factors, and genetic diversity of A. bovis were assessed in small ruminants. A total of 563 healthy small ruminants (260 sheep and 303 goats), from 25 randomly selected flocks located in 5 localities from two bioclimatic areas in Tunisia, were investigated for the detection of A. bovis in blood by nested polymerase chain reaction (nPCR) assay. The overall infection rates of A. bovis were 42.7 and 23.8% in sheep and goats, respectively. Goats located in a sub-humid area were statistically more infected than those located in a humid area. A. bovis prevalence rate varied significantly according to sheep and goat flocks, and to the sheep breed. Infection with A. bovis was validated by sequencing. Sequence analysis based on the 16S rRNA gene showed that A. bovis from Tunisian goats and sheep clustered with other strain sequences detected from wild and domestic animals and published in GenBank. This study gives the first insight of presence of A. bovis DNA in small ruminants in Tunisia and suggests that these animal species may be playing an important role in the bovine anaplasmosis natural cycle caused by A. bovis in the South Mediterranean ecosystem. Copyright © 2015 Elsevier B.V. All rights reserved.

Efficient activation of human T cells of both CD4 and CD8 subsets by urease-deficient recombinant Mycobacterium bovis BCG that produced a heat shock protein 70-M. tuberculosis-derived major membrane protein II fusion protein.

PubMed

Mukai, Tetsu; Tsukamoto, Yumiko; Maeda, Yumi; Tamura, Toshiki; Makino, Masahiko

2014-01-01

For the purpose of obtaining Mycobacterium bovis bacillus Calmette-Guérin (BCG) capable of activating human naive T cells, urease-deficient BCG expressing a fusion protein composed of Mycobacterium tuberculosis-derived major membrane protein II (MMP-II) and heat shock protein 70 (HSP70) of BCG (BCG-DHTM) was produced. BCG-DHTM secreted the HSP70-MMP-II fusion protein and effectively activated human monocyte-derived dendritic cells (DCs) by inducing phenotypic changes and enhanced cytokine production. BCG-DHTM-infected DCs activated naive T cells of both CD4 and naive CD8 subsets, in an antigen (Ag)-dependent manner. The T cell activation induced by BCG-DHTM was inhibited by the pretreatment of DCs with chloroquine. The naive CD8(+) T cell activation was mediated by the transporter associated with antigen presentation (TAP) and the proteosome-dependent cytosolic cross-priming pathway. Memory CD8(+) T cells and perforin-producing effector CD8(+) T cells were efficiently produced from the naive T cell population by BCG-DHTM stimulation. Single primary infection with BCG-DHTM in C57BL/6 mice efficiently produced T cells responsive to in vitro secondary stimulation with HSP70, MMP-II, and M. tuberculosis-derived cytosolic protein and inhibited the multiplication of subsequently aerosol-challenged M. tuberculosis more efficiently than did vector control BCG. These results indicate that the introduction of MMP-II and HSP70 into urease-deficient BCG may be useful for improving BCG for control of tuberculosis.

Identification of a New DNA Region Specific for Members of Mycobacterium tuberculosis Complex

PubMed Central

Magdalena, Juana; Vachée, Anne; Supply, Philip; Locht, Camille

1998-01-01

The successful use of DNA amplification for the detection of tuberculous mycobacteria crucially depends on the choice of the target sequence, which ideally should be present in all tuberculous mycobacteria and absent from all other bacteria. In the present study we developed a PCR procedure based on the intergenic region (IR) separating two genes encoding a recently identified mycobacterial two-component system named SenX3-RegX3. The senX3-regX3 IR is composed of a novel type of repetitive sequence, called mycobacterial interspersed repetitive units (MIRUs). In a survey of 116 Mycobacterium tuberculosis strains characterized by different IS6110 restriction fragment length polymorphisms, 2 Mycobacterium africanum strains, 3 Mycobacterium bovis strains (including 2 BCG strains), and 1 Mycobacterium microti strain, a specific PCR fragment was amplified in all cases. This collection included M. tuberculosis strains that lack IS6110 or mtp40, two target sequences that have previously been used for the detection of M. tuberculosis. No PCR fragment was amplified when DNA from other organisms was used, giving a sensitivity of 100% and a specificity of 100% in the confidence limit of this study. The numbers of MIRUs were found to vary among strains, resulting in six different groups of strains on the basis of the size of the amplified PCR fragment. However, the vast majority of the strains (approximately 90%) fell within the same group, containing two 77-bp MIRUs followed by one 53-bp MIRU. PMID:9542912

A specific DNA probe which identifies Babesia bovis in whole blood.

PubMed

Petchpoo, W; Tan-ariya, P; Boonsaeng, V; Brockelman, C R; Wilairat, P; Panyim, S

1992-05-01

A genomic library of Babesia bovis DNA from the Mexican strain M was constructed in plasmid pUN121 and cloned in Escherichia coli. Several recombinants which hybridized strongly to radioactively labeled B. bovis genomic DNA in an in situ screening were selected and further analyzed for those which specifically hybridized to B. bovis DNA. It was found that pMU-B1 had the highest sensitivity, detecting 25 pg of purified B. bovis DNA, and 300 parasites in 10 microliters of whole infected blood, or 0.00025% parasitemia. pMU-B1 contained a 6.0 kb B. bovis DNA insert which did not cross-hybridize to Babesia bigemina, Trypanosoma evansi, Plasmodium falciparum, Anaplasma marginale, Boophilus microplus and cow DNA. In the Southern blot analysis of genomic DNA, pMU-B1 could differentiate between two B. bovis geographic isolates, Mexican strain M and Thai isolate TS4. Thus, the pMU-B1 probe will be useful in the diagnosis of Babesia infection in cattle and ticks, and in the differentiation of B. bovis strains.

Risk profiling of cattle farms as a potential tool in risk-based surveillance for Mycobacterium bovis infection among cattle in tuberculosis-free areas.

PubMed

Ribeiro-Lima, Joao; Schwabenlander, Stacey; Oakes, Michael; Thompson, Beth; Wells, Scott J

2016-06-15

OBJECTIVE To develop a cattle herd risk-profiling system that could potentially inform risk-based surveillance strategies for Mycobacterium bovis infection in cattle and provide information that could be used to help direct resource allocation by a state agency for this purpose. DESIGN Cross-sectional study. SAMPLE Records for any size movement (importation) of cattle into Minnesota from other US states during 2009 (n = 7,185) and 2011 (8,107). PROCEDURES Data from certificates of veterinary inspection were entered into a spreadsheet. Movement data were summarized at premises and county levels, and for each level, the distribution of cattle moved and number of movements were evaluated. Risk profiling (assessment and categorization of risk for disease introduction) for each import movement was performed on the basis of known risk factors. Latent class analysis was used to assign movements to risk classifications with adjustment on the basis of expert opinions from personnel knowledgeable about bovine tuberculosis; these data were used to classify premises as very high, high, medium, or low risk for disease introduction. RESULTS In each year, approximately 1,500 premises imported cattle, typically beef and feeder types, with the peak of import movements during the fall season. The risk model identified 4 risk classes for cattle movements. Approximately 500 of the estimated 27,406 (2%) cattle premises in Minnesota were in the very high or high risk groups for either year; greatest density of these premises was in the southeast and southwest regions of the state. CONCLUSIONS AND CLINICAL RELEVANCE A risk-profiling approach was developed that can be applied in targeted surveillance efforts for bovine tuberculosis, particularly in disease-free areas.

Laparoscopic pyloromyotomy: comparing the arthrotomy knife to the Bovie blade.

PubMed

Thomas, Priscilla G; Sharp, Nicole E; St Peter, Shawn D

2014-07-01

Laparoscopic pyloromyotomy was performed at our institution using an arthrotomy knife until it became unavailable in 2010. Thus, we adapted the use of the blunt Bovie tip, which can be used with or without electrocautery to perform the myotomy. This study compared the outcomes between using the arthrotomy knife versus the Bovie blade in laparoscopic pyloromyotomies. Retrospective review was performed on all laparoscopic pyloromyotomy patients from October 2007 to September 2012. Arthrotomy knife pyloromyotomy patients were compared with those performed with the Bovie blade. Patient demographics, diagnostic measurements, electrolyte levels, length of stay, operative time, and complications were compared. A total of 381 patients were included, with 191 in the arthrotomy group and 190 in the Bovie blade group. No significant differences existed between groups in age, weight, gender, pyloric dimensions, electrolyte levels, or length of stay. Mean operative times were 15.8±5.6 min with knife and 16.4±5.3 min for Bovie blade (P=0.24). In the arthrotomy knife group, there was one incomplete pyloromyotomy and one omental herniation. There was one wound infection in each group. Readmission rate was greater in the arthrotomy knife group (5.7%) versus the Bovie blade group (3.1%). The Bovie blade appears to offer no objective disadvantages compared with the arthrotomy knife when performing laparoscopic pyloromyotomy. Copyright © 2014 Elsevier Inc. All rights reserved.

Assessment of the probability of introducing Mycobacterium tuberculosis into Danish cattle herds.

PubMed

Foddai, Alessandro; Nielsen, Liza Rosenbaum; Krogh, Kaspar; Alban, Lis

2015-11-01

Tuberculosis is a zoonosis caused by Mycobacterium spp. International trade in cattle is regulated with respect to Mycobacterium bovis (M. bovis) but not Mycobacterium tuberculosis (M. tuberculosis), despite that cattle can become infected with both species. In this study we estimated the annual probability (PIntro) of introducing M. tuberculosis into the Danish cattle population, by the import of cattle and/or by immigrants working in Danish cattle herds. Data from 2013 with date, number, and origin of imported live cattle were obtained from the Danish cattle database. Information on immigrants working in Danish cattle herds was obtained through a questionnaire sent to Danish cattle farmers. The gained inputs were fed into three stochastic scenario trees to assess the PIntro for the current and alternative test-and-manage strategies, such as testing of imported animals and/or testing immigrant workers with the tuberculin skin test. We considered the population of Danish farmers and practitioners free of tuberculosis, because in Denmark, the incidence of the disease in humans is low and primarily related to immigrants and socially disadvantaged people. The median annual probability of introducing M. tuberculosis into the Danish cattle population due to imported live cattle was 0.008% (90% P.I.: 0.0007%; 0.03%), while the probability due to immigrant workers was 4.1% (90% P.I.: 0.8%; 12.1%). The median combined probability (PIntro) due to imported cattle plus workers was 4.1% (90% P.I.: 0.8%; 12.6%). Hence, on average at least one introduction each 24 (90% P.I.: 8; 125) years could be expected. Imported live cattle appeared to play a marginal role on the overall annual PIntro, because they represented only approximately 0.2% of the median annual probability. By testing immigrant workers the overall annual PIntro could be reduced to 0.2% (90% P.I.: 0.04%; 0.7%). Thus, testing of immigrant workers could be considered as a risk mitigation strategy to markedly reduce

Lipoproteins of slow-growing Mycobacteria carry three fatty acids and are N-acylated by Apolipoprotein N-Acyltransferase BCG_2070c

PubMed Central

2013-01-01

Background Lipoproteins are virulence factors of Mycobacterium tuberculosis. Bacterial lipoproteins are modified by the consecutive action of preprolipoprotein diacylglyceryl transferase (Lgt), prolipoprotein signal peptidase (LspA) and apolipoprotein N- acyltransferase (Lnt) leading to the formation of mature triacylated lipoproteins. Lnt homologues are found in Gram-negative and high GC-rich Gram-positive, but not in low GC-rich Gram-positive bacteria, although N-acylation is observed. In fast-growing Mycobacterium smegmatis, the molecular structure of the lipid modification of lipoproteins was resolved recently as a diacylglyceryl residue carrying ester-bound palmitic acid and ester-bound tuberculostearic acid and an additional amide-bound palmitic acid. Results We exploit the vaccine strain Mycobacterium bovis BCG as model organism to investigate lipoprotein modifications in slow-growing mycobacteria. Using Escherichia coli Lnt as a query in BLASTp search, we identified BCG_2070c and BCG_2279c as putative lnt genes in M. bovis BCG. Lipoproteins LprF, LpqH, LpqL and LppX were expressed in M. bovis BCG and BCG_2070c lnt knock-out mutant and lipid modifications were analyzed at molecular level by matrix-assisted laser desorption ionization time-of-flight/time-of-flight analysis. Lipoprotein N-acylation was observed in wildtype but not in BCG_2070c mutants. Lipoprotein N- acylation with palmitoyl and tuberculostearyl residues was observed. Conclusions Lipoproteins are triacylated in slow-growing mycobacteria. BCG_2070c encodes a functional Lnt in M. bovis BCG. We identified mycobacteria-specific tuberculostearic acid as further substrate for N-acylation in slow-growing mycobacteria. PMID:24093492

An overview of Mycoplasma bovis mastitis in Israel (2004-2014).

PubMed

Lysnyansky, Inna; Freed, Mor; Rosales, Ruben S; Mikula, Inna; Khateb, Nihaya; Gerchman, Irena; van Straten, Michael; Levisohn, Sharon

2016-01-01

The prevalence of Mycoplasma bovis in milk samples submitted to the Israeli National Service for Udder Health and Milk Quality was determined during the period 2004-2014 and the genetic pattern of the obtained isolates was assessed by multilocus sequence typing (MLST). Mycoplasma spp. were identified in 66 herds including M. bovis (n = 60), M. cottewii (n = 3), M. bovigenitalium (n = 2), M. alkalescens (n = 2) and M. yeatsii (n = 1). The proportion of M. bovis infected herds was relatively low (0-0.68%) in 2004-2007, increased to 3.77% during the 2008 outbreak, and ranged from 0.77 to 2.77% during the 2009-2014 period. Since 2008, about eight M. bovis positive dairy herds have been identified in Israel annually, with six of which on average being newly infected. MLST of 57 M. bovis isolates revealed that sequence type 10 was the dominant genotype identified in 60% of the herds. In conclusion, these data show that M. bovis is the main mycoplasmal mastitic pathogen in Israel. Copyright © 2015 Elsevier Ltd. All rights reserved.

Analysis of Babesia bovis-induced gene expression changes in the cattle tick, Rhipicephalus (Boophilus) microplus

USDA-ARS?s Scientific Manuscript database

Background: Cattle babesiosis is a tick-borne disease of cattle that has severe economic impact on cattle producers throughout the world's tropical and subtropical countries. The most severe form of the disease is caused by the apicomplexan, Babesia bovis, and transmitted to cattle through the bite ...

Evaluation of the efficiency of nested q-PCR in the detection of Mycobacterium tuberculosis complex directly from tuberculosis-suspected lesions in post-mortem macroscopic inspections of bovine carcasses slaughtered in the state of Mato Grosso, Brazil.

PubMed

Carvalho, Ricardo César Tavares; Furlanetto, Leone Vinícius; Maruyama, Fernanda Harumy; Araújo, Cristina Pires de; Barros, Sílvia Letícia Bomfim; Ramos, Carlos Alberto do Nascimento; Dutra, Valéria; Araújo, Flábio Ribeiro de; Paschoalin, Vânia Margaret Flosi; Nakazato, Luciano; Figueiredo, Eduardo Eustáquio de Souza

2015-08-01

Bovine tuberculosis (BTB) is a zoonotic disease caused by Mycobacterium bovis, a member of the Mycobacterium tuberculosis complex (MTC). The quick and specific detection of this species is of extreme importance, since BTB may cause economic impacts, in addition to presenting imminent risks to human health. In the present study a nested real-time PCR test (nested q-PCR) was used in post-mortem evaluations to assess cattle carcasses with BTB-suspected lesions. A total of 41,193 cattle slaughtered in slaughterhouses located in the state of Mato Grosso, were examined. Of the examined animals, 198 (0.48%) showed BTB-suspected lesions. M. bovis was isolated in 1.5% (3/198) of the samples. Multiplex-PCR detected MTC in 7% (14/198) of the samples. The nested q-PCR test detected MTC in 28% (56/198) of the BTB-suspected lesions, demonstrating higher efficiency when compared to the multiplex-PCR and conventional microbiology. Nested q-PCR can therefore be used as a complementary test in the national program for control and eradication of bovine tuberculosis. Copyright © 2015 Elsevier Ltd. All rights reserved.

Targeted surface expression of an exogenous antigen in stably transfected babesia bovis

USDA-ARS?s Scientific Manuscript database

Babesia bovis is a tick-borne intraerythocytic protozoan responsible for acute disease in cattle which can be controlled by vaccination with attenuated B. bovis strains. Emerging B. bovis transfection technologies may increase the usefulness of these live vaccines. Here we propose using transfected ...

The ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis

PubMed Central

2013-01-01

Background Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (Boophilus) microplus, which is distributed throughout the tropical and subtropical countries of the world. The transmission of B. bovis is transovarian and a previous study of the R. microplus ovarian proteome identified several R. microplus proteins that were differentially expressed in response to infection. Through various approaches, we studied the reaction of the R. microplus ovarian transcriptome in response to infection by B. bovis. Methods A group of ticks were allowed to feed on a B. bovis-infected splenectomized calf while a second group fed on an uninfected splenectomized control calf. RNA was purified from dissected adult female ovaries of both infected and uninfected ticks and a subtracted B. bovis-infected cDNA library was synthesized, subtracting with the uninfected ovarian RNA. Four thousand ESTs were sequenced from the ovary subtracted library and annotated. Results The subtracted library dataset assembled into 727 unique contigs and 2,161 singletons for a total of 2,888 unigenes, Microarray experiments designed to detect B. bovis-induced gene expression changes indicated at least 15 transcripts were expressed at a higher level in ovaries from ticks feeding upon the B. bovis-infected calf as compared with ovaries from ticks feeding on an uninfected calf. We did not detect any transcripts from these microarray experiments that were expressed at a lower level in the infected ovaries compared with the uninfected ovaries. Using the technique called serial analysis of gene expression, 41 ovarian transcripts from infected ticks were differentially expressed when compared with transcripts of controls. Conclusion Collectively, our experimental approaches provide

Molecular identification of Mycobacterium tuberculosis in cattle.

PubMed

Sweetline Anne, N; Ronald, B S M; Kumar, T M A Senthil; Kannan, P; Thangavelu, A

2017-01-01

Bovine tuberculosis continued to be a re-emerging problem in some countries especially in endemic areas due to the fact that human and animal health surveillance system is not adopted to diagnose the infection. This crisis can be attributed due to sharing of the same habitat especially in rural areas. In the present study, a total of 148 samples were collected from cattle for isolation over a period of 3 years from cattle with and without lesions, of which 67 isolates were obtained by culture. Fifty one isolates were identified as Mycobacterium tuberculosis complex (MTBC) by IS6110 PCR of which 43 (84.3%) were identified as M. tuberculosis and 08 (15.6%) were identified as M. bovis by using 12.7kb fragment multiplex PCR. Among this, 31 isolates which were positive for IS6110 PCR were subjected to spoligotyping and revealed 28 isolates belonging to MANU1 strain of M. tuberculosis. This study clearly indicates that high prevalence of M. tuberculosis than M. bovis in bovine was identified by means of culture and by molecular methods M. tuberculosis can affect cattle producing lesion in contradiction to the earlier thoughts. This study speculates that M. tuberculosis MANU1 strain infection in cattle could be due to spill over from human or other non specific hosts in tuberculosis endemic areas. Though bovine tuberculosis due to M. tuberculosis in cattle is not considered a serious threat worldwide, in countries where human TB is endemic, M. tuberculosis infection of cattle needs to be considered. Copyright © 2016 Elsevier B.V. All rights reserved.

Evaluation of a new rapid kit, BD MGIT TBc identification test for confirmation of Mycobacterium tuberculosis complex.

PubMed

Kandhakumari, Gandhi; Stephen, Selvaraj

2017-01-01

At present, three rapid kits are available globally for the confirmation of Mycobacterium tuberculosis complex (MTBC) in cultures by MPT64 antigen (MPT64 Ag) detection. These include Capilia TB, SD Bioline, and BD MGIT TBc Identification (TBcID). The third kit is yet to be validated in India. We have tested this kit and compared with SD Bioline using conventional tests as gold standard. Seventy-one MTBC (70 M. tuberculosis and one Mycobacterium bovis) and four nontuberculous mycobacteria (NTM) were isolated from 649 clinical specimens in MGIT 960 and/or Lowenstein-Jensen slants (LJ). MPT64 Ag was detected by both TBcID and SD Bioline kits in all the 71 clinical isolates and the reference strain M. tuberculosis H37Rv. All NTM species tested were negative by the two different kits. Thus, TBcID kit showed 100% concordance in terms of sensitivity and specificity. Rapid kits confirm MTBC cultures within 15 min in contrast to several weeks' time required by conventional techniques.

Evaluation of the Mycobacterium tuberculosis SO2 vaccine using a natural tuberculosis infection model in goats.

PubMed

Bezos, J; Casal, C; Álvarez, J; Roy, A; Romero, B; Rodríguez-Bertos, A; Bárcena, C; Díez, A; Juste, R; Gortázar, C; Puentes, E; Aguiló, N; Martín, C; de Juan, L; Domínguez, L

2017-05-01

The development of new vaccines against animal tuberculosis (TB) is a priority for improving the control and eradication of this disease, particularly in those species not subjected to compulsory eradication programmes. In this study, the protection conferred by the Mycobacterium tuberculosis SO 2 experimental vaccine was evaluated using a natural infection model in goats. Twenty-six goats were distributed in three groups: (1) 10 goats served as a control group; (2) six goats were subcutaneously vaccinated with BCG; and (3) 10 goats were subcutaneously vaccinated with SO 2 . Four months after vaccination, all groups were merged with goats infected with Mycobacterium bovis or Mycobacterium caprae, and tested over a 40 week period using a tuberculin intradermal test and an interferon-γ assay for mycobacterial reactivity. The severity of lesions was determined at post-mortem examination and the bacterial load in tissues were evaluated by culture. The two vaccinated groups had significantly lower lesion and bacterial culture scores than the control group (P<0.05); at the end of the study, the SO 2 vaccinated goats had the lowest lesion and culture scores. These results suggest that the SO 2 vaccine provides some protection against TB infection acquired from natural exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Effect of Oral Vaccination with Mycobacterium bovis BCG on the Development of Tuberculosis in Captive European Badgers (Meles meles).

PubMed

Chambers, Mark A; Aldwell, Frank; Williams, Gareth A; Palmer, Si; Gowtage, Sonya; Ashford, Roland; Dalley, Deanna J; Davé, Dipesh; Weyer, Ute; Salguero, Francisco J; Nunez, Alejandro; Nadian, Allan K; Crawshaw, Timothy; Corner, Leigh A L; Lesellier, Sandrine

2017-01-01

The European badger ( Meles meles ) is a reservoir host of Mycobacterium bovis and responsible for a proportion of the tuberculosis (TB) cases seen in cattle in the United Kingdom and Republic of Ireland. An injectable preparation of the bacillus Calmette-Guérin (BCG) vaccine is licensed for use in badgers in the UK and its use forms part of the bovine TB eradication plans of England and Wales. However, there are practical limitations to the widespread application of an injectable vaccine for badgers and a research priority is the development of an oral vaccine deliverable to badgers in bait. Previous studies reported the successful vaccination of badgers with oral preparations of 10 8 colony forming units (CFU) of both Pasteur and Danish strains of BCG contained within a lipid matrix composed of triglycerides of fatty acids. Protection against TB in these studies was expressed as a reduction in the number and apparent progression of visible lesions, and reductions in the bacterial load and dissemination of infection. To reduce the cost of an oral vaccine and reduce the potential for environmental contamination with BCG, it is necessary to define the minimal efficacious dose of oral BCG for badgers. The objectives of the two studies reported here were to compare the efficacy of BCG Danish strain in a lipid matrix with unformulated BCG given orally, and to evaluate the efficacy of BCG Danish in a lipid matrix at a 10-fold lower dose than previously evaluated in badgers. In the first study, both BCG unformulated and in a lipid matrix reduced the number and apparent progression of visible lesions and the dissemination of infection from the lung. In the second study, vaccination with BCG in the lipid matrix at a 10-fold lower dose produced a similar outcome, but with greater intra-group variability than seen with the higher dose in the first study. Further research is needed before we are able to recommend a final dose of BCG for oral vaccination of badgers against TB

The Effect of Oral Vaccination with Mycobacterium bovis BCG on the Development of Tuberculosis in Captive European Badgers (Meles meles)

PubMed Central

Chambers, Mark A.; Aldwell, Frank; Williams, Gareth A.; Palmer, Si; Gowtage, Sonya; Ashford, Roland; Dalley, Deanna J.; Davé, Dipesh; Weyer, Ute; Salguero, Francisco J.; Nunez, Alejandro; Nadian, Allan K.; Crawshaw, Timothy; Corner, Leigh A. L.; Lesellier, Sandrine

2017-01-01

The European badger (Meles meles) is a reservoir host of Mycobacterium bovis and responsible for a proportion of the tuberculosis (TB) cases seen in cattle in the United Kingdom and Republic of Ireland. An injectable preparation of the bacillus Calmette-Guérin (BCG) vaccine is licensed for use in badgers in the UK and its use forms part of the bovine TB eradication plans of England and Wales. However, there are practical limitations to the widespread application of an injectable vaccine for badgers and a research priority is the development of an oral vaccine deliverable to badgers in bait. Previous studies reported the successful vaccination of badgers with oral preparations of 108 colony forming units (CFU) of both Pasteur and Danish strains of BCG contained within a lipid matrix composed of triglycerides of fatty acids. Protection against TB in these studies was expressed as a reduction in the number and apparent progression of visible lesions, and reductions in the bacterial load and dissemination of infection. To reduce the cost of an oral vaccine and reduce the potential for environmental contamination with BCG, it is necessary to define the minimal efficacious dose of oral BCG for badgers. The objectives of the two studies reported here were to compare the efficacy of BCG Danish strain in a lipid matrix with unformulated BCG given orally, and to evaluate the efficacy of BCG Danish in a lipid matrix at a 10-fold lower dose than previously evaluated in badgers. In the first study, both BCG unformulated and in a lipid matrix reduced the number and apparent progression of visible lesions and the dissemination of infection from the lung. In the second study, vaccination with BCG in the lipid matrix at a 10-fold lower dose produced a similar outcome, but with greater intra-group variability than seen with the higher dose in the first study. Further research is needed before we are able to recommend a final dose of BCG for oral vaccination of badgers against TB or

Tuberculosis in Antelopes in a Zoo in Poland--Problem of Public Health.

PubMed

Krajewska, Monika; Załuski, Michał; Zabost, Anna; Orłowska, Blanka; Augustynowicz-Kopeć, Ewa; Anusz, Krzysztof; Lipiec, Marek; Weiner, Marcin; Szulowski, Krzysztof

2015-01-01

Bovine tuberculosis is an infectious disease that occurs in many species of both domestic and wild animals, as well as those held in captivity. The etiological factor is the acid resistant bacillus (Mycobacterium bovis or Mycobacterium caprae), which is characterized by the major pathogenicity among mycobacteria belonging to the Mycobacterium tuberculosis complex. The material from 8 antelopes from the zoo, suspected for tuberculosis were examined, and M. bovis strains were isolated from 6 of them. The spoligotyping method showing spoligo pattern 676763777777600. In Poland, this spoligotype has not been observed so far.

Antimicrobial activity of bovine NK-lysin-derived peptides on Mycoplasma bovis

PubMed Central

Falkenberg, Shollie M.; Register, Karen B.; Samorodnitsky, Daniel; Nicholson, Eric M.; Reinhardt, Timothy A.

2018-01-01

Antimicrobial peptides (AMPs) are a diverse group of molecules which play an important role in the innate immune response. Bovine NK-lysins, a type of AMP, have been predominantly found in the granules of cytotoxic T-lymphocytes and NK-cells. Bovine NK-lysin-derived peptides demonstrate antimicrobial activity against various bacterial pathogens, including several involved in bovine respiratory disease complex (BRDC) in cattle; however, such studies are yet to be performed with one important contributor to the BRDC, Mycoplasma bovis. Therefore, the goal of this study was to assess the antimicrobial activity of bovine NK-lysin-derived peptides on M. bovis. Thirty-mer synthetic peptides corresponding to the functional region helices 2 and 3 of bovine NK-lysins NK1, NK2A, NK2B, and NK2C were evaluated for killing activity on M. bovis isolates. Among four peptides, NK2A and NK2C showed the highest antimicrobial activity against the M. bovis isolates tested. All four NK-lysin peptides induced rapid plasma membrane depolarization in M. bovis at two concentrations tested. However, based on propidium iodide uptake, only NK2A and NK2C appeared capable of causing structural damage to M. bovis plasma membrane. Confocal microscopy, flow cytometry, and transmission electron microscopy further suggested NK-lysin-induced damage to the plasma membrane. Taken together, the findings in this study suggest that plasma membrane depolarization alone was insufficient to induce lethality, but disruption/permeabilization of the M. bovis plasma membrane was the cause of lethality. PMID:29771981

Role of the pks15/1 gene in the biosynthesis of phenolglycolipids in the Mycobacterium tuberculosis complex. Evidence that all strains synthesize glycosylated p-hydroxybenzoic methyl esters and that strains devoid of phenolglycolipids harbor a frameshift mutation in the pks15/1 gene.

PubMed

Constant, Patricia; Perez, Esther; Malaga, Wladimir; Lanéelle, Marie-Antoinette; Saurel, Olivier; Daffé, Mamadou; Guilhot, Christophe

2002-10-11

Diesters of phthiocerol and phenolphthiocerol are important virulence factors of Mycobacterium tuberculosis and Mycobacterium leprae, the two main mycobacterial pathogens in humans. They are both long-chain beta-diols, and their biosynthetic pathway is beginning to be elucidated. Although the two classes of molecules share a common lipid core, phthiocerol diesters have been found in all the strains of the M. tuberculosis complex examined although phenolphthiocerol diesters are produced by only a few groups of strains. To address the question of the origin of this diversity 8 reference strains and 10 clinical isolates of M. tuberculosis were analyzed. We report the presence of glycosylated p-hydroxybenzoic acid methyl esters, structurally related to the type-specific phenolphthiocerol glycolipids, in the culture media of all reference strains of M. tuberculosis, suggesting that the strains devoid of phenolphthiocerol derivatives are unable to elongate the putative p-hydroxybenzoic acid precursor. We also show that all the strains of M. tuberculosis examined and deficient in the production of phenolphthiocerol derivatives are natural mutants with a frameshift mutation in pks15/1 whereas a single open reading frame for pks15/1 is found in Mycobacterium bovis BCG, M. leprae, and strains of M. tuberculosis that produce phenolphthiocerol derivatives. Complementation of the H37Rv strain of M. tuberculosis, which is devoid of phenolphthiocerol derivatives, with the fused pks15/1 gene from M. bovis BCG restored phenolphthiocerol glycolipids production. Conversely, disruption of the pks15/1 gene in M. bovis BCG led to the abolition of the synthesis of type-specific phenolphthiocerol glycolipid. These data indicate that Pks15/1 is involved in the elongation of p-hydroxybenzoic acid to give p-hydroxyphenylalkanoates, which in turn are converted, presumably by the PpsA-E synthase, to phenolphthiocerol derivatives.

Molecular diagnosis and phylogenetic analysis of Babesia bigemina and Babesia bovis hemoparasites from cattle in South Africa

PubMed Central

2013-01-01

Background Babesia parasites, mainly Babesia bovis and B. bigemina, are tick-borne hemoparasites inducing bovine babesiosis in cattle globally. The clinical signs of the disease include, among others, anemia, fever and hemoglobinuria. Babesiosis is known to occur in tropical and subtropical regions of the world. In this study, we aim to provide information about the occurrence and phylogenetic relationship of B. bigemina and B. bovis species in cattle from different locations in nine provinces of South Africa. A total of 430 blood samples were randomly collected from apparently healthy cattle. These samples were genetically tested for Babesia parasitic infections using nested PCR assays with species-specific primers. Results Nested PCR assays with Group I primer sets revealed that the overall prevalence of B. bigemina and B. bovis in all bovine samples tested was 64.7% (95% CI = 60.0-69.0) and 35.1% (95% CI = 30.6-39.8), respectively. Only 117/430 (27.2%) animals had a mixed infection. The highest prevalence of 87.5% (95% CI = 77.2-93.5) for B. bigemina was recorded in the Free State province collection sites (Ficksburg, Philippolis and Botshabelo), while North West collection sites had the highest number of animals infected with B. bovis (65.5%; 95% CI = 52.7-76.4). Phylograms were inferred based on B. bigemina-specific gp45 and B. bovis-specific rap-1 nucleotide sequences obtained with Group II nested PCR primers. Phylogenetic analysis of gp45 sequences revealed significant differences in the genotypes of B. bigemina isolates investigated, including those of strains published in GenBank. On the other hand, a phylogeny based on B. bovis rap-1 sequences indicated a similar trend of clustering among the sequences of B. bovis isolates investigated in this study. Conclusion This study demonstrates the occurrence of Babesia parasites in cattle from different provinces of South Africa. It was also noted that the situation of Babesia parasitic infection

Transfected Babesia bovis Expressing a Tick GST as a Live Vector Vaccine

PubMed Central

Oldiges, Daiane P.; Laughery, Jacob M.; Tagliari, Nelson Junior; Leite Filho, Ronaldo Viana; Davis, William C.; da Silva Vaz, Itabajara; Termignoni, Carlos; Knowles, Donald P.; Suarez, Carlos E.

2016-01-01

The Rhipicephalus microplus tick is a notorious blood-feeding ectoparasite of livestock, especially cattle, responsible for massive losses in animal production. It is the main vector for transmission of pathogenic bacteria and parasites, including Babesia bovis, an intraerythrocytic apicomplexan protozoan parasite responsible for bovine Babesiosis. This study describes the development and testing of a live B. bovis vaccine expressing the protective tick antigen glutathione-S-transferase from Haemaphysalis longicornis (HlGST). The B. bovis S74-T3B parasites were electroporated with a plasmid containing the bidirectional Ef-1α (elongation factor 1 alpha) promoter of B. bovis controlling expression of two independent genes, the selectable marker GFP-BSD (green fluorescent protein–blasticidin deaminase), and HlGST fused to the MSA-1 (merozoite surface antigen 1) signal peptide from B. bovis. Electroporation followed by blasticidin selection resulted in the emergence of a mixed B. bovis transfected line (termed HlGST) in in vitro cultures, containing parasites with distinct patterns of insertion of both exogenous genes, either in or outside the Ef-1α locus. A B. bovis clonal line termed HlGST-Cln expressing intracellular GFP and HlGST in the surface of merozoites was then derived from the mixed parasite line HlGST using a fluorescent activated cell sorter. Two independent calf immunization trials were performed via intravenous inoculation of the HlGST-Cln and a previously described control consisting of an irrelevant transfected clonal line of B. bovis designated GFP-Cln. The control GFP-Cln line contains a copy of the GFP-BSD gene inserted into the Ef-1α locus of B. bovis in an identical fashion as the HIGST-Cln parasites. All animals inoculated with the HlGST-Cln and GFP-Cln transfected parasites developed mild babesiosis. Tick egg fertility and fully engorged female tick weight was reduced significantly in R. microplus feeding on HlGST-Cln-immunized calves

Ecology and pathogenicity of gastrointestinal Streptococcus bovis.

PubMed

Herrera, Paul; Kwon, Young Min; Ricke, Steven C

2009-01-01

Streptococcus bovis is an indigenous resident in the gastrointestinal tracts of both humans and animals. S. bovis is one of the major causes of bacterial endocarditis and has been implicated in the incidence of human colon cancer, possibly due to chronic inflammatory response at the site of intestinal colonization. Certain feeding regimens in ruminants can lead to overgrowth of S. bovis in the rumen, resulting in the over-production of lactate and capsular polysaccharide causing acute ruminal acidosis and bloat, respectively. There are multiple strategies in controlling acute lactic acidosis and bloat. The incidence of the two diseases may be controlled by strict dietary management. Gradual introduction of grain-based diets and the feeding of coarsely chopped roughage decrease the incidence of the two disease entities. Ionophores, which have been used to enhance feed conversion and growth rate in cattle, have been shown to inhibit the growth of lactic acid bacteria in the rumen. Other methods of controlling lactic acid bacteria in the ruminal environment (dietary supplementation of long-chain fatty acids, induction of passive and active immune responses to the bacteria, and the use of lytic bacteriophages) have also been investigated. It is anticipated that through continued in-depth ecological analysis of S. bovis the characteristics responsible for human and animal pathogenesis would be sufficiently identified to a point where more effective control strategies for the control of this bacteria can be developed.

Streptococcus bovis septicemia and meningitis associated with chronic radiation enterocolitis

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

Jadeja, L.; Kantarjian, H.; Bolivar, R.

1983-12-01

We describe the first patient with simultaneous S bovis septicemia and meningitis associated with chronic radiation enterocolitis. This case underlines the value of a thorough gastrointestinal evaluation of all patients with S bovis infection, and the need for a neurologic investigation even with minor neurologic manifestations.

[Epidemiology of resistance to antituberculosis drugs in Mycobacterium tuberculosis complex strains isolated from adenopathies in Djibouti. Prospective study carried out in 1999].

PubMed

Koeck, J L; Bernatas, J J; Gerome, P; Fabre, M; Houmed, A; Herve, V; Teyssou, R

2002-01-01

Tuberculosis is a major cause of death in the Republic of Djibouti. Tuberculous lymphadenitis represents about 25% of the clinical forms of tuberculosis in this country. Between January 1999 and April 1999, 196 lymph node specimens were consecutively collected from 153 patients living in Djibouti. Testing of susceptibility to the major anti-tuberculosis drugs was performed by the proportion method. Growth of Mycobacterium tuberculosis complex strains was obtained from specimens of 85 patients including 9 with prior treatment. Strains were identified as Mycobacterium tuberculosis in 78 cases, Mycobacterium canetti in 3, Mycobacterium africanum in 3, and Mycobacterium bovis in 1. Prevalence of HIV infection was 15%. Assessment of primary resistance demonstrated that the overall resistance rate, i.e., resistance to 1 or more drugs, was 18 (21.2%). Results showed resistance to isoniazid (H) in 6 cases (7.1%), rifampicin (R) in 3 (3.5%), ethambutol (E) in 1 (1.2%), streptomycin (S) in 13 (15.3%) and pyrazinamide (Z) in 1 (1.2%). Multidrug resistance (MDR) was found in 2 cases (2.4%). Assessment of acquired resistance demonstrated resistance to H in 4 cases (44%), R in 2 (22%), S in 2 (22%), E in 0, Z in 0 and MDR in 1 (11%). These findings were not significantly different from data obtained from sputum samples analysed between 1997 and 2000 or from those described in a study conducted in 1985.

Pathogenicity and Genetic Variation of 3 Strains of Corynebacterium bovis in Immunodeficient Mice

PubMed Central

Dole, Vandana S; Henderson, Kenneth S; Fister, Richard D; Pietrowski, Michael T; Maldonado, Geomaris; Clifford, Charles B

2013-01-01

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice. PMID:23849444

Pathogenicity and genetic variation of 3 strains of Corynebacterium bovis in immunodeficient mice.

PubMed

Dole, Vandana S; Henderson, Kenneth S; Fister, Richard D; Pietrowski, Michael T; Maldonado, Geomaris; Clifford, Charles B

2013-07-01

Corynebacterium bovis has been associated with hyperkeratotic dermatitis and acanthosis in mice. We studied 3 different strains of C. bovis: one previously described to cause hyperkeratotic dermatitis (HAC), one that infected athymic nude mice without leading to the classic clinical signs, and one of bovine origin (ATCC 7715). The 3 strains showed a few biochemical and genetic differences. Immunodeficient nude mice were housed in 3 independent isolators and inoculated with pure cultures of the 3 strains. We studied the transmission of these C. bovis studies to isolator-bedding and contact sentinels housed for 5 to 12 wk in filter-top or wire-top cages in the respective isolators. Using a 16S rRNA-based qPCR assay, we did not find consistent differences in growth and transmission among the 3 C. bovis strains, and neither the incidence nor severity of hyperkeratosis or acanthosis differed between strains. Housing in filter-top compared with wire-top cages did not alter the morbidity associated with any of the strains. Our findings confirmed the variability in the gross and histologic changes associated with C. bovis infection of mice. Although bacteriology was a sensitive method for the detection of Corynebacterium spp., standard algorithms occasionally misidentified C. bovis and several related species. Our study demonstrates that PCR of skin swabs or feces is a sensitive and specific method for the detection of C. bovis infection in mice. An rpoB-based screen of samples from North American vivaria revealed that HAC is the predominant C. bovis strain in laboratory mice.

Genetic diversity of the Mycobacterium tuberculosis complex in San Luis Potosí, México.

PubMed

López-Rocha, Estela; Juárez-Álvarez, Julio; Riego-Ruiz, Lina; Enciso-Moreno, Leonor; Ortega-Aguilar, Francisco; Hernández-Nieto, Julián; Enciso-Moreno, José A; López-Revilla, Rubén

2013-05-01

Although epidemiologic and socioeconomic criteria and biomedical risk factors indicate high-priority for tuberculosis (TB) control in Mexico, molecular epidemiology studies of the disease in the country are scarce. Complete sociodemographic and clinical data were obtained from 248 of the 432 pulmonary TB (PTB) cases confirmed from 2006 to 2010 on the population under epidemiological surveillance in the state of San Luis Potosí, México. From most PTB cases with complete data Mycobacterium tuberculosis complex (MTC) isolates were recovered and their spoligotypes, lineages and families, geographic distribution and drug resistance determined. Pulmonary tuberculosis incidence ranged from 2.4 to 33.4 (cases per 100,000 inhabitants) in the six state sanitary jurisdictions that were grouped in regions of low (jurisdictions I-II-III), intermediate (jurisdictions IV-V) and high incidence (jurisdiction VI) with 6.2, 17.3 and 33.4 rates, respectively. Most patients were poor, 50-years-median-age males and housewives. Among the 237 MTC spoligotyped isolates, 232 corresponded to M. tuberculosis (104 spoligotypes in 24 clusters) and five to M. bovis. The predominant Euro-American lineage was distributed all over the state, the East-Asian lineage (Beijing family) in the capital city, the Indo-Oceanic (Manila family) in eastern localities, and M. bovis in rural localities. In San Luis Potosí TB affects mainly poor male adults and is caused by M. tuberculosis and to a minor extent by M. bovis. There is great genotypic diversity among M. tuberculosis strains, the Euro-American lineage being much more prevalent than the Indo-Oceanic and East-Asian lineages. The frequency of resistant strains is relatively low and not associated to any particular lineage.

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis.

PubMed

Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi

2017-08-09

Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas' infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO ( MBOV_RS00785 ) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells.

TrmFO, a Fibronectin-Binding Adhesin of Mycoplasma bovis

PubMed Central

Guo, Yongpeng; Zhu, Hongmei; Wang, Jiayao; Huang, Jing; Khan, Farhan Anwar; Zhang, Jingjing; Guo, Aizhen; Chen, Xi

2017-01-01

Mycoplasma bovis is an important pathogenic mycoplasma, causing the cattle industry serious economic losses. Adhesion is a crucial step in the mycoplasmas’ infection and colonization process; fibronectin (Fn), an extracellular matrix glycoprotein, is a molecular bridge between the bacterial adhesins and host cell receptors. The present study was designed to characterize the Fn-binding ability of methylenetetrahydrofolate-tRNA-(uracil-5-)-methyltransferase (TrmFO) and its role in M. bovis cytoadherence. The trmFO (MBOV_RS00785) gene was cloned and expressed in E. coli BL21, and polyclonal antibodies against the recombinant TrmFO (rTrmFO) were raised in rabbits. Immunoblotting demonstrated that TrmFO was an immunogenic component, and the TrmFO expression was conserved in different M. bovis isolates. The mycoplasmacidal assay further showed that in the presence of complement, rabbit anti-recombinant TrmFO serum exhibited remarkable mycoplasmacidal efficacy. TrmFO was detected in both the M. bovis membrane and cytoplasm. By ligand dot blot and enzyme-linked immunosorbent assay (ELISA) binding assay, we found that rTrmFO bound Fn in a dose-dependent manner. Immunostaining visualized by confocal laser scanning microscopy showed that rTrmFO had capacity to adhere to the embryonic bovine lung (EBL) cells. In addition, the adhesion of M. bovis and rTrmFO to EBL cells could be inhibited by anti-rTrmFO antibodies. To the best of our knowledge, this is the first report to characterize the Fn-binding ability of TrmFO and its role in the bacterial adhesion to host cells. PMID:28792486

In vitro effects of citrus oils against Mycobacterium tuberculosis and non-tuberculous Mycobacteria of clinical importance.

PubMed

Crandall, Philip G; Ricke, Steven C; O'Bryan, Corliss A; Parrish, Nicole M

2012-01-01

We evaluated the in vitro activity of citrus oils against Mycobacterium tuberculosis and other non-tuberculous Mycobacterium species. Citrus essential oils were tested against a variety of Mycobacterium species and strains using the BACTEC radiometric growth system. Cold pressed terpeneless Valencia oil (CPT) was further tested using the Wayne model of in vitro latency. Exposure of M. tuberculosis and M. bovis BCG to 0.025 % cold pressed terpeneless Valencia orange oil (CPT) resulted in a 3-log decrease in viable counts versus corresponding controls. Inhibition of various clinical isolates of the M. avium complex and M. abscessus ranged from 2.5 to 5.2-logs. Some species/strains were completely inhibited in the presence of CPT including one isolate each of the following: the M. avium complex, M. chelonae and M. avium subsp. paratuberculosis. CPT also inhibited the growth of BCG more than 99 % in an in vitro model of latency which mimics anaerobic dormancy thought to occur in vivo. The activity of CPT against drug-resistant strains of the M. avium complex and M. abscessus suggest that the mechanism of action for CPT is different than that of currently available drugs. Inhibition of latently adapted bacilli offers promise for treatment of latent infections of MTB. These results suggest that the antimycobacterial properties of CPT warrant further study to elucidate the specific mechanism of action and clarify the spectrum of activity.

9 CFR 311.23 - Tapeworm cysts (cysticercus bovis) in cattle.

Code of Federal Regulations, 2014 CFR

2014-01-01

... 9 Animals and Animal Products 2 2014-01-01 2014-01-01 false Tapeworm cysts (cysticercus bovis) in cattle. 311.23 Section 311.23 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of...

9 CFR 311.23 - Tapeworm cysts (cysticercus bovis) in cattle.

Code of Federal Regulations, 2012 CFR

2012-01-01

... 9 Animals and Animal Products 2 2012-01-01 2012-01-01 false Tapeworm cysts (cysticercus bovis) in cattle. 311.23 Section 311.23 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of...

9 CFR 311.23 - Tapeworm cysts (cysticercus bovis) in cattle.

Code of Federal Regulations, 2011 CFR

2011-01-01

... 9 Animals and Animal Products 2 2011-01-01 2011-01-01 false Tapeworm cysts (cysticercus bovis) in cattle. 311.23 Section 311.23 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of...

9 CFR 311.23 - Tapeworm cysts (cysticercus bovis) in cattle.

Code of Federal Regulations, 2013 CFR

2013-01-01

... 9 Animals and Animal Products 2 2013-01-01 2013-01-01 false Tapeworm cysts (cysticercus bovis) in cattle. 311.23 Section 311.23 Animals and Animal Products FOOD SAFETY AND INSPECTION SERVICE, DEPARTMENT... PARTS § 311.23 Tapeworm cysts (cysticercus bovis) in cattle. (a) Except as provided in paragraph (b) of...

The Ovarian transcriptome of the cattle tick, Rhipicephalus (Boophilus) microplus, feeding upon a bovine host infected with Babesia bovis

USDA-ARS?s Scientific Manuscript database

Background: Cattle babesiosis is a tick-borne disease of cattle with the most severe form of the disease caused by the apicomplexan, Babesia bovis. Babesiosis is transmitted to cattle through the bite of infected cattle ticks of the genus Rhipicephalus. The most prevalent species is Rhipicephalus (B...

Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds.

PubMed

Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K

2016-01-01

Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks' air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection.

Surveillance of a Ventilated Rack System for Corynebacterium bovis by Sampling Exhaust-Air Manifolds

PubMed Central

Manuel, Christopher A; Pugazhenthi, Umarani; Leszczynski, Jori K

2016-01-01

Corynebacterium bovis causes an opportunistic infection of nude (Foxn1, nu/nu) mice, leading to nude mouse hyperkeratotic dermatitis (scaly skin disease). Enzootic in many nude mouse colonies, C. bovis spreads rapidly to naive nude mice, despite modern husbandry practices, and is very difficult to eradicate. To facilitate rapid detection in support of eradication efforts, we investigated a surveillance method based on quantitative real-time PCR (qPCR) evaluation of swabs collected from the horizontal exhaust manifold (HEM) of an IVC rack system. We first evaluated the efficacy of rack sanitation methods for removing C. bovis DNA from the HEM of racks housing endemic colonies of infected nude mice. Pressurized water used to flush the racks’ air exhaust system followed by a standard rack-washer cycle was ineffective in eliminating C. bovis DNA. Only after autoclaving did all sanitized racks test negative for C. bovis DNA. We then measured the effects of stage of infection (early or established), cage density, and cage location on the rack on time-to-detection at the HEM. Stage of infection significantly affected time-to-detection, independent of cage location. Early infections required 7.3 ± 1.2 d whereas established infections required 1 ± 0 d for detection of C. bovis at the HEM. Cage density influenced the quantity of C. bovis DNA detected but not time-to-detection. The location of the cage on the rack affected the time-to-detection only during early C. bovis infections. We suggest that qPCR swabs of HEM are useful during the routine surveillance of nude mouse colonies for C. bovis infection. PMID:26817981

A multilocus sequence typing method and curated database for Mycoplasma bovis

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant problem in bison, causing necrotic pha...

Study on bioactive compounds of in vitro cultured Calculus Suis and natural Calculus Bovis.

PubMed

Wan, Tien-Chun; Cheng, Fu-Yuan; Liu, Yu-Tse; Lin, Liang-Chuan; Sakata, Ryoichi

2009-12-01

The purpose of the study was to investigate bioactive compounds of in vitro cultured Calculus Suis and natural Calculus Bovis obtained as valuable by-products from animals used for meat production. The results showed that the components of natural Calculus Bovis were rich in bilirubin and biliverdin and had higher content of essential amino acids. The major amino acids of in vitro cultured Calculus Suis were identified as glycine, alanine, glutamic acid and aspartic acid, and those for natural Calculus Bovis were found to be glutamic acid, aspartic acid, proline, and arginine. The methionine and cysteine contents of precursors for glutathione in natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The mineral contents of zinc, iron and manganese of natural Calculus Bovis were significantly higher than those of in vitro cultured Calculus Suis. The major bile acids in both products were cholic acid and dehydrocholic acid, respectively. The chenodeoxycholic and ursodeoxycholic acid content of in vitro cultured Calculus Suis was significantly higher than that of natural Calculus Bovis.

Molecular epidemiology of Mycobacterium tuberculosis complex strains isolated from livestock and wild animals in Italy suggests the need for a different eradication strategy for bovine tuberculosis.

PubMed

Amato, B; Di Marco Lo Presti, V; Gerace, E; Capucchio, M T; Vitale, M; Zanghì, P; Pacciarini, M L; Marianelli, C; Boniotti, M B

2018-04-01

Bovine tuberculosis (bTB) is an important zoonosis, which has been re-emerging in different ecological scenarios. In Sicily, Italy, from 2004 to 2014, an anatomopathological survey for tuberculosis-like lesions both in farmed and wild animals was performed. The isolates were genotyped using spoligotyping and Mycobacterial Interspersed Repetitive Units-Variable Number of Tandem Repeats (MIRU-VNTR) techniques. High prevalence of lesions was observed for cattle (4%), pigs (4.9%) and wild boars (6.8%), and a total of 625 Mycobacterium bovis isolates were identified. Genotyping analysis showed the presence of 37 different spoligotypes including fifteen spoligotypes not present in other Italian regions and 266 MIRU-VNTR profiles. Spoligotype SB0120 exhibited the highest prevalence in cattle (50%) and pigs (56%) and the highest genetic variety with 126 different MIRU-VNTR profiles. The isolation of M. bovis in a farmer underlines the importance of M. bovis identification during the human TB diagnostic processes. This study supported the use of the genotyping analysis as a valuable tool for the evaluation of the epidemiological role of pigs and other domestic reservoirs such as goats and the role of wildlife in the maintenance of bTB infection. © 2017 Blackwell Verlag GmbH.

[Single and combining effects of Calculus Bovis and zolpidem on inhibitive neurotransmitter of rat striatum corpora].

PubMed

Liu, Ping; He, Xinrong; Guo, Mei

2010-04-01

To investigate the correlation effects between single or combined administration of Calculus Bovis or zolpidem and changes of inhibitive neurotransmitter in rat striatum corpora. Sampling from rat striatum corpora was carried out through microdialysis. The content of two inhibitive neurotransmitters in rat corpus striatum- glycine (Gly) and gama aminobutyric acid (GABA), was determined by HPLC, which involved pre-column derivation with orthophthaladehyde, reversed-phase gradient elution and fluorescence detection. GABA content of rat striatum corpora in Calculus Bovis group was significantly increased compared with saline group (P < 0.01). GABA content of zolpidem group and Calculus Boris plus zolpidem group were increased largely compared with saline group as well (P < 0.05). GABA content of Calculus Bovis group was higher than combination group (P < 0.05). GABA content of zolpidem group was not significantly different from combination group. Gly content of Calculus Bovis or zolpidem group was markedly increased compared with saline group or combination group (P < 0.05). Contents of two inhibitive neurotransmitters in rat striatum corpora were all significantly increased in Calculus Bovis group, zolpidem group and combination group. The magnitude of increase was lower in combination group than in Calculus Bovis group and Zolpidem group, suggesting that Calculus Bovis promoted encephalon inhibition is more powerful than zolpidem. The increase in two inhibitive neurotransmitters did not show reinforcing effect in combination group, suggesting that Calculus Bovis and zolpidem may compete the same receptors. Therefore, combination of Calculus Bovis containing drugs and zolpidem has no clinical significance. Calculus Bovis shouldn't as an aperture-opening drugs be used for resuscitation therapy.

Mistaken identity of a PCR target proposed for identification of Mycoplasma bovis and the effect of sequence variation on assay performance

USDA-ARS?s Scientific Manuscript database

Background. Mycoplasma bovis is an important cause of disease in cattle and has recently emerged as a primary disease agent in bison. Because the bacterium requires specialized growth conditions many diagnostic laboratories use PCR to replace or complement traditional isolation and identification ...

An impedance spectroscopy method for the detection and evaluation of Babesia bovis antibodies in cattle

USDA-ARS?s Scientific Manuscript database

An immunosensor method for diagnosis of Babesia bovis in cattle based on impedance measurement is presented in this study. The method probes the interaction between serum antibodies against B. bovis infected cattle and recombinant protein, RAP-1, with C-terminal obtained from a Portuguese B. bovis s...

Delayed presentation of a bowel Bovie injury after laparoscopic ventral hernia repair.

PubMed

Bhullar, Jasneet Singh; Gayagoy, Jennifer; Chaudhary, Sushant; Kolachalam, Ramachandra B

2013-01-01

Bowel injury during laparoscopic surgery is a rare but serious complication. A Bovie injury to the bowel can cause delayed perforation of the viscus, thus increasing the possibility of a preventable morbidity. Patients presenting with perforation peritonitis within 24 hours and up to 2 to 3 weeks after laparoscopic Bovie injury to the bowel have been reported in the literature. A 74-year-old woman underwent a laparoscopic ventral hernia mesh repair. Intraoperatively, a small area of superficial Bovie injury to the small bowel was repaired with Lembert sutures and tissue glue. Postoperatively, the patient recovered well, but she presented with perforation peritonitis 3 months after surgery. An exploratory laparotomy showed a jejunal perforation in the same area that was injured with cautery and repaired during the previous surgery. The patient was only using inhaled steroids for asthma on and off but had a remote history of chemotherapy and radiation for colorectal cancer. Bovie injury to the bowel has a hidden depth, causing a slow transmural tissue necrosis, and it might also impair local healing and eventually lead to perforation. Thus, the patient may present later than the usual period for wound healing and remodeling as previously reported. Given the disastrous consequence, it is imperative to perform a good surgical repair of even a minor Bovie injury to the bowel. This is the first report of a delayed presentation (>1 month) of a Bovie injury of the bowel.

Delayed Presentation of a Bowel Bovie Injury After Laparoscopic Ventral Hernia Repair

PubMed Central

Gayagoy, Jennifer; Chaudhary, Sushant; Kolachalam, Ramachandra B.

2013-01-01

Introduction: Bowel injury during laparoscopic surgery is a rare but serious complication. A Bovie injury to the bowel can cause delayed perforation of the viscus, thus increasing the possibility of a preventable morbidity. Patients presenting with perforation peritonitis within 24 hours and up to 2 to 3 weeks after laparoscopic Bovie injury to the bowel have been reported in the literature. Case Description: A 74-year-old woman underwent a laparoscopic ventral hernia mesh repair. Intraoperatively, a small area of superficial Bovie injury to the small bowel was repaired with Lembert sutures and tissue glue. Postoperatively, the patient recovered well, but she presented with perforation peritonitis 3 months after surgery. An exploratory laparotomy showed a jejunal perforation in the same area that was injured with cautery and repaired during the previous surgery. The patient was only using inhaled steroids for asthma on and off but had a remote history of chemotherapy and radiation for colorectal cancer. Conclusion: Bovie injury to the bowel has a hidden depth, causing a slow transmural tissue necrosis, and it might also impair local healing and eventually lead to perforation. Thus, the patient may present later than the usual period for wound healing and remodeling as previously reported. Given the disastrous consequence, it is imperative to perform a good surgical repair of even a minor Bovie injury to the bowel. This is the first report of a delayed presentation (>1 month) of a Bovie injury of the bowel. PMID:24018096

Clinical Mycoplasma bovis mastitis in prepubertal heifers on 2 dairy herds

PubMed Central

Fox, Lawrence K.; Muller, Fredrick J.; Wedam, Michael L.; Schneider, Christopher S.; Biddle, Mary Kate

2008-01-01

Findings of herd investigations of heifers with prepubertal mastitis are presented. Mycoplasma bovis was isolated from lacteal secretions and tissue samples of necropsied heifers; the same strain infected dams and herd mates. Vertical transmission is suggested in this first report of intramammary infections of M. bovis in peripubertal heifers. PMID:19183734

Transmission of Mycobacterium tuberculosis between farmers and cattle in central Ethiopia.

PubMed

Ameni, Gobena; Tadesse, Konjit; Hailu, Elena; Deresse, Yohannes; Medhin, Girmay; Aseffa, Abraham; Hewinson, Glyn; Vordermeier, Martin; Berg, Stefan

2013-01-01

Transmission of Mycobacterium tuberculosis (M. tuberculosis) complex could be possible between farmers and their cattle in Ethiopia. A study was conducted in mixed type multi-purposes cattle raising region of Ethiopia on 287 households (146 households with case of pulmonary tuberculosis (TB) and 141 free of TB) and 287 herds consisting of 2,033 cattle belonging to these households to evaluate transmission of TB between cattle and farmers. Interview, bacteriological examinations and molecular typing were used for human subjects while comparative intradermal tuberculin (CIDT) test, post mortem and bacteriological examinations, and molecular typing were used for animal studies. Herd prevalence of CIDT reactors was 9.4% and was higher (p<0.01) in herds owned by households with TB than in herds owned by TB free households. Animal prevalence was 1.8% and also higher (p<0.01) in cattle owned by households with TB case than in those owned by TB free households. All mycobacteria (141) isolated from farmers were M. tuberculosis, while only five of the 16 isolates from cattle were members of the M. tuberculosis complex (MTC) while the remaining 11 were members of non-tuberculosis mycobacteria (NTM). Further speciation of the five MTC isolates showed that three of the isolates were M. bovis (strain SB1176), while the remaining two were M. tuberculosis strains (SIT149 and SIT53). Pathology scoring method described by "Vordermeier et al. (2002)" was applied and the average severity of pathology in two cattle infected with M. bovis, in 11 infected with NTM and two infected with M. tuberculosis were 5.5, 2.1 and 0.5, respectively. The results showed that transmission of TB from farmers to cattle by the airborne route sensitizes the cows but rarely leads to TB. Similarly, low transmission of M. bovis between farmers and their cattle was found, suggesting requirement of ingestion of contaminated milk from cows with tuberculous mastitis.

The seal tuberculosis agent, Mycobacterium pinnipedii, infects domestic cattle in New Zealand: epidemiologic factors and DNA strain typing.

PubMed

Loeffler, Scott H; de Lisle, Geoffrey W; Neill, Mark A; Collins, Desmond M; Price-Carter, Marian; Paterson, Brent; Crews, Kevin B

2014-04-01

The fur seal (Arctocephalus forsteri), which is abundant in coastal areas of New Zealand, harbors several zoonotic pathogens, including Mycobacterium pinnipedii, a member of the Mycobacterium tuberculosis complex. We describe the microbiology and epidemiology of seven cases of M. pinnipedii infection in beef cattle (Bos primigenius) in coastal areas of New Zealand in 1991-2011. Epidemiologic factors were analyzed on six case farms and a telephone survey of 55 neighboring farms. A DNA-strain typing, using analysis of variable number tandem repeats and the direct repeats (VNTR/DR) of those isolates, was used to compare them to M. bovis isolates commonly found in New Zealand cattle and wildlife. In all cases of M. pinnipedii in cattle, only one animal in the herd was found to be infected. In six of seven cases, the lesions were in the thoracic lymph nodes, indicating a likely aerosol pathway. The lack of multiple cases within a herd suggests that cow-to-cow transmission is uncommon, if it occurs at all. There was no significant difference between case and control farms in distance to sea, herd size, herd type, or farming practice. The odds ratio for access to the beach for cattle on the Chatham Islands was significantly higher than it was for farms on the mainland coastal areas (odds ratio [OR] = 3.6, 95% CI = 1.1-11.4) Likewise, the odds ratio for acquiring tuberculosis was increased when farmers had seen seals on the property (OR =  9, 95% CI = 1.4-56.1 ). In all case farms, cattle had access to seals by beach grazing areas or waterways connecting directly with the ocean. The VNTR/DR typing of the isolates showed some variation in the M. pinnipedii isolates, with only two being identical; all isolates were easily distinguishable from M. bovis isolates.

A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages

PubMed Central

Van der Geize, Robert; Yam, Katherine; Heuser, Thomas; Wilbrink, Maarten H.; Hara, Hirofumi; Anderton, Matthew C.; Sim, Edith; Dijkhuizen, Lubbert; Davies, Julian E.; Mohn, William W.; Eltis, Lindsay D.

2007-01-01

Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC− mutant accumulated 3,4-DHSA, indicating that HsaCRHA1, formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette–Guérin. M. bovis bacillus Calmette–Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaCH37Rv and HsaDH37Rv transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities ≈40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics. PMID:17264217

A gene cluster encoding cholesterol catabolism in a soil actinomycete provides insight into Mycobacterium tuberculosis survival in macrophages.

PubMed

Van der Geize, Robert; Yam, Katherine; Heuser, Thomas; Wilbrink, Maarten H; Hara, Hirofumi; Anderton, Matthew C; Sim, Edith; Dijkhuizen, Lubbert; Davies, Julian E; Mohn, William W; Eltis, Lindsay D

2007-02-06

Rhodococcus sp. strain RHA1, a soil bacterium related to Mycobacterium tuberculosis, degrades an exceptionally broad range of organic compounds. Transcriptomic analysis of cholesterol-grown RHA1 revealed a catabolic pathway predicted to proceed via 4-androstene-3,17-dione and 3,4-dihydroxy-9,10-seconandrost-1,3,5(10)-triene-9,17-dione (3,4-DHSA). Inactivation of each of the hsaC, supAB, and mce4 genes in RHA1 substantiated their roles in cholesterol catabolism. Moreover, the hsaC(-) mutant accumulated 3,4-DHSA, indicating that HsaC(RHA1), formerly annotated as a biphenyl-degrading dioxygenase, catalyzes the oxygenolytic cleavage of steroid ring A. Bioinformatic analyses revealed that 51 rhodococcal genes specifically expressed during growth on cholesterol, including all predicted to specify the catabolism of rings A and B, are conserved within an 82-gene cluster in M. tuberculosis H37Rv and Mycobacterium bovis bacillus Calmette-Guérin. M. bovis bacillus Calmette-Guérin grew on cholesterol, and hsaC and kshA were up-regulated under these conditions. Heterologously produced HsaC(H37Rv) and HsaD(H37Rv) transformed 3,4-DHSA and its ring-cleaved product, respectively, with apparent specificities approximately 40-fold higher than for the corresponding biphenyl metabolites. Overall, we annotated 28 RHA1 genes and proposed physiological roles for a similar number of mycobacterial genes. During survival of M. tuberculosis in the macrophage, these genes are specifically expressed, and many appear to be essential. We have delineated a complete suite of genes necessary for microbial steroid degradation, and pathogenic mycobacteria have been shown to catabolize cholesterol. The results suggest that cholesterol metabolism is central to M. tuberculosis's unusual ability to survive in macrophages and provide insights into potential targets for novel therapeutics.

Mycobacterial RNA isolation optimized for non-coding RNA: high fidelity isolation of 5S rRNA from Mycobacterium bovis BCG reveals novel post-transcriptional processing and a complete spectrum of modified ribonucleosides.

PubMed

Hia, Fabian; Chionh, Yok Hian; Pang, Yan Ling Joy; DeMott, Michael S; McBee, Megan E; Dedon, Peter C

2015-03-11

A major challenge in the study of mycobacterial RNA biology is the lack of a comprehensive RNA isolation method that overcomes the unusual cell wall to faithfully yield the full spectrum of non-coding RNA (ncRNA) species. Here, we describe a simple and robust procedure optimized for the isolation of total ncRNA, including 5S, 16S and 23S ribosomal RNA (rRNA) and tRNA, from mycobacteria, using Mycobacterium bovis BCG to illustrate the method. Based on a combination of mechanical disruption and liquid and solid-phase technologies, the method produces all major species of ncRNA in high yield and with high integrity, enabling direct chemical and sequence analysis of the ncRNA species. The reproducibility of the method with BCG was evident in bioanalyzer electrophoretic analysis of isolated RNA, which revealed quantitatively significant differences in the ncRNA profiles of exponentially growing and non-replicating hypoxic bacilli. The method also overcame an historical inconsistency in 5S rRNA isolation, with direct sequencing revealing a novel post-transcriptional processing of 5S rRNA to its functional form and with chemical analysis revealing seven post-transcriptional ribonucleoside modifications in the 5S rRNA. This optimized RNA isolation procedure thus provides a means to more rigorously explore the biology of ncRNA species in mycobacteria. © The Author(s) 2014. Published by Oxford University Press on behalf of Nucleic Acids Research.

Mycobacterium bovis BCG Scar Status and HLA Class II Alleles Influence Purified Protein Derivative-Specific T-Cell Receptor Vβ Expression in Pulmonary Tuberculosis Patients from Southern India

PubMed Central

Shanmugalakshmi, S.; Dheenadhayalan, V.; Muthuveeralakshmi, P.; Arivarignan, G.; Pitchappan, R.M.

2003-01-01

Purified protein derivative (PPD) RT23-recalled T-cell receptor (TCR) Vβ expression was studied in the peripheral blood of 42 pulmonary tuberculosis patients and 44 healthy controls from southern India, a region where tuberculosis is endemic. Forty-eight-hour whole-blood cultures in the presence or absence of PPD-RT23 were set up, and at the end of the culture period total RNA was extracted and cDNA was synthesized. Expression of various TCR Vβ families was assessed by using family-specific primers. PPD-specific expression (usage) of TCR Vβ families 4, 6, 8 to 12, and 14 was found in more controls than patients. Among the responders (individuals who showed PPD-specific expression), endemic controls had significantly higher responses than the patients had for TCR Vβ families 2, 3, 7, 13, and 17. The majority of the patients did not show usage of most of the TCR Vβ families, and this was attributed to T-cell downregulation. A four-way nested classification analysis revealed that TCR Vβ family 1, 5, 9, 12, and 13 usage in the context of HLA class II high-risk alleles (DRB1*1501, DRB1*08, and DQB1*0601) and Mycobacterium bovis BCG scar status were the determining factors in susceptibility and resistance to tuberculosis. The healthier status of controls was attributed to the wider usage of many TCR Vβ families readily recalled by PPD, while the disease status of the patients was attributed to TCR Vβ downregulation and the resultant T-cell (memory cell?) unresponsiveness. Host genetics (HLA status) and BCG vaccination (scar status) seem to play important roles in skewing the immune response in adult susceptibility to pulmonary tuberculosis through TCR Vβ usage. PMID:12874334

Comparative study on major bioactive components in natural, artificial and in-vitro cultured Calculus Bovis.

PubMed

Yan, Shi-Kai; Wu, Yan-Wen; Liu, Run-Hui; Zhang, Wei-Dong

2007-01-01

Major bioactive components in various Calculus Bovis, including natural, artificial and in-vitro cultured Calculus Bovis, were comparatively studied. An approach of high-performance liquid chromatography coupled with ultraviolet and evaporative light scattering detections (HPLC/UV/ELSD) was established to simultaneously determinate six bioactive components thereof, including five bile acids (cholic acid, deoxycholic acid, ursodeoxycholic, chenodeoxycholic acid, hyodeoxycholic acid) and bilirubin. ELSD and UV detector were applied to detect bile acids and bilirubin respectively. The assay was performed on a C(18) column with water-acetonitrile gradient elution and the investigated constituents were authenticated by comparing retention times and mass spectra with those of reference compounds. The proposed method was applied to analyze twenty-one Calculus Bovis extraction samples, and produced data with acceptable linearity, precision, repeatability and accuracy. The result indicated the variations among Calculus Bovis samples under different developmental conditions. Artificial and in-vitro cultured Calculus Bovis, especially in-vitro cultured ones, which contain total bioactive constituents no less than natural products and have the best batch-to-batch uniformity, suffice to be used as substitutes of natural Calculus Bovis.

Diagnostic efficiency of abattoir meat inspection service in Ethiopia to detect carcasses infected with Mycobacterium bovis: Implications for public health

PubMed Central

2010-01-01

Background Bovine Tuberculosis (BTB) is a widespread and endemic disease of cattle in Ethiopia posing a significant threat to public health. Regular surveillance by skin test, bacteriology and molecular methods is not feasible due to lack of resource. Thus, routine abattoir (RA) inspection will continue to play a key role for national surveillance. We evaluated efficiency of RA inspection for diagnosis of Mycobacterium bovis infection and discussed its public health implications in light of a high risk of human exposure. Methods The study was conducted in five abattoirs: Addis Ababa, Adama, Hawassa, Yabello and Melge-Wondo abattoirs. The efficiency of routine abattoir (RA) inspection was validated in comparison to detailed abattoir (DA) inspection, followed by culture and microscopy (CM) and region of difference (RD) deletion analysis. Diagnostic accuracies (with corresponding measures of statistical uncertainty) were determined by computing test property statistics (sensitivity and specificity) and likelihood estimations using web-based SISA diagnostic statistics software. Post-test probability of detecting TB infected carcasses was estimated using nomograms. Agreement between RA and DA inspections was measured using kappa statistics. The study was conducted and reported in accordance with standards for reporting of diagnostic accuracy (STARD) requirements. Both routine and detailed meat inspection protocols were performed on a subpopulation of 3322 cattle selected randomly from among 78,269 cattle slaughtered during the study period. Three hundred thirty seven carcasses identified through detailed meat inspection protocols were subjected to culture and microscopy; of the 337, a subset of 105 specimens for culture and microscopy were subjected to further molecular testing. Results There was a substantial agreement between RA and DA inspections in Addis Ababa (Kappa = 0.7) and Melge-Wondo abattoirs (Kappa = 0.67). In Adama, Hawassa and Yabello abattoirs, the

Adherence of Moraxella bovis to cell cultures of bovine origin.

PubMed

Annuar, B O; Wilcox, G E

1985-09-01

The adherence of five strains of Moraxella bovis to cell cultures was investigated. M bovis adhered to cultures of bovine corneal epithelial and Madin-Darby bovine kidney cells but not to cell types of non-bovine origin. Both piliated and unpiliated strains adhered but piliated strains adhered to a greater extent than unpiliated strains. Antiserum against pili of one strain inhibited adherence of piliated strains but caused only slight inhibition of adherence to the unpiliated strains. Treatment of bacteria with magnesium chloride caused detachment of pili from the bacterial cell and markedly inhibited adherence of piliated strains but caused only slight inhibition of adherence by the unpiliated strains. The results suggested that adhesion of piliated strains to cell cultures was mediated via pili but that adhesins other than pili may be involved in the attachment of unpiliated strains of M bovis to cells.

A Reduced Antigen Load In Vivo, Rather Than Weak Inflammation, Causes a Substantial Delay in CD8+ T Cell Priming against Mycobacterium bovis (Bacillus Calmette-Guérin)1

PubMed Central

Russell, Marsha S.; Iskandar, Monica; Mykytczuk, Oksana L.; Nash, John H. E.; Krishnan, Lakshmi; Sad, Subash

2014-01-01

Regardless of the dose of Ag, Ag presentation occurs rapidly within the first few days which results in rapid expansion of the CD8+ T cell response that peaks at day 7. However, we have previously shown that this rapid priming of CD8+ T cells is absent during infection of mice with Mycobacterium bovis (bacillus Calmette-Guérin (BCG)). In this study, we have evaluated the mechanisms responsible for the delayed CD8+ T cell priming. Because BCG replicates poorly and survives within phagosomes we considered whether 1) generation of reduced amounts of Ag or 2) weaker activation by pathogen-associated molecular patterns (PAMPs) during BCG infection is responsible for the delay in CD8+ T cell priming. Using rOVA-expressing bacteria, our results indicate that infection of mice with BCG-OVA generates greatly reduced levels of OVA, which are 70-fold lower in comparison to the levels generated during infection of mice with Listeria monocytogenes-expressing OVA. Furthermore, increasing the dose of OVA, but not PAMP signaling during BCG-OVA infection resulted in rapid Ag presentation and consequent expansion of the CD8+ T cell response, indicating that the generation of reduced Ag levels, not lack of PAMP-associated inflammation, was responsible for delayed priming of CD8+ T cells. There was a strong correlation between the relative timing of Ag presentation and the increase in the level of OVA in vivo. Taken together, these results reveal that some slowly replicating pathogens, such as mycobacteria, may facilitate their chronicity by generating reduced Ag levels which causes a substantial delay in the development of acquired immune responses. PMID:17579040

Tick Passage Results in Enhanced Attenuation of Babesia bovis

PubMed Central

McElwain, Terry F.; Ueti, Massaro W.; Scoles, Glen A.; Reif, Kathryn E.; Lau, Audrey O. T.

2014-01-01

Serial blood passage of virulent Babesia bovis in splenectomized cattle results in attenuated derivatives that do not cause neurologic disease. Tick transmissibility can be lost with attenuation, but when retained, attenuated B. bovis can revert to virulence following tick passage. This study provides data showing that tick passage of the partially attenuated B. bovis T2Bo derivative strain further decreased virulence compared with intravenous inoculation of the same strain in infected animals. Ticks that acquired virulent or attenuated parasites by feeding on infected cattle were transmission fed on naive, splenectomized animals. While there was no significant difference between groups in the number of parasites in the midgut, hemolymph, or eggs of replete female ticks after acquisition feeding, animals infected with the attenuated parasites after tick transmission showed no clinical signs of babesiosis, unlike those receiving intravenous challenge with the same attenuated strain prior to tick passage. Additionally, there were significantly fewer parasites in blood and tissues of animals infected with tick-passaged attenuated parasites. Sequencing analysis of select B. bovis genes before and after tick passage showed significant differences in parasite genotypes in both peripheral blood and cerebral samples. These results provide evidence that not only is tick transmissibility retained by the attenuated T2Bo strain, but also it results in enhanced attenuation and is accompanied by expansion of parasite subpopulations during tick passage that may be associated with the change in disease phenotype. PMID:25114111

Role of P27 -P55 operon from Mycobacterium tuberculosis in the resistance to toxic compounds

PubMed Central

2011-01-01

Background The P27-P55 (lprG-Rv1410c) operon is crucial for the survival of Mycobacterium tuberculosis, the causative agent of human tuberculosis, during infection in mice. P55 encodes an efflux pump that has been shown to provide Mycobacterium smegmatis and Mycobacterium bovis BCG with resistance to several drugs, while P27 encodes a mannosylated glycoprotein previously described as an antigen that modulates the immune response against mycobacteria. The objective of this study was to determine the individual contribution of the proteins encoded in the P27-P55 operon to the resistance to toxic compounds and to the cell wall integrity of M. tuberculosis. Method In order to test the susceptibility of a mutant of M. tuberculosis H37Rv in the P27-P55 operon to malachite green, sodium dodecyl sulfate, ethidium bromide, and first-line antituberculosis drugs, this strain together with the wild type strain and a set of complemented strains were cultivated in the presence and in the absence of these drugs. In addition, the malachite green decolorization rate of each strain was obtained from decolorization curves of malachite green in PBS containing bacterial suspensions. Results The mutant strain decolorized malachite green faster than the wild type strain and was hypersensitive to both malachite green and ethidium bromide, and more susceptible to the first-line antituberculosis drugs: isoniazid and ethambutol. The pump inhibitor reserpine reversed M. tuberculosis resistance to ethidium bromide. These results suggest that P27-P55 functions through an efflux-pump like mechanism. In addition, deletion of the P27-P55 operon made M. tuberculosis susceptible to sodium dodecyl sulfate, suggesting that the lack of both proteins causes alterations in the cell wall permeability of the bacterium. Importantly, both P27 and P55 are required to restore the wild type phenotypes in the mutant. Conclusions The results clearly indicate that P27 and P55 are functionally connected in

Characterization of Mycobacterium tuberculosis Complex DNAs from Egyptian Mummies by Spoligotyping

PubMed Central

Zink, Albert R.; Sola, Christophe; Reischl, Udo; Grabner, Waltraud; Rastogi, Nalin; Wolf, Hans; Nerlich, Andreas G.

2003-01-01

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum. PMID:12517873

Bovine tuberculosis and the establishment of an eradication program in the United States: Role of veterinarians

USDA-ARS?s Scientific Manuscript database

The significance of the identification of Mycobacterium bovis as a zoonotic pathogen in 1882 was not initially recognized. After years of research by veterinarians, and other scientists, the importance of M. bovis as a pathogen and the public health ramifications, were appreciated. Veterinarians pla...

Transmission and Progression to Disease of Mycobacterium tuberculosis Phylogenetic Lineages in The Netherlands.

PubMed

Nebenzahl-Guimaraes, Hanna; Verhagen, Lilly M; Borgdorff, Martien W; van Soolingen, Dick

2015-10-01

The aim of this study was to determine if mycobacterial lineages affect infection risk, clustering, and disease progression among Mycobacterium tuberculosis cases in The Netherlands. Multivariate negative binomial regression models adjusted for patient-related factors and stratified by patient ethnicity were used to determine the association between phylogenetic lineages and infectivity (mean number of positive contacts around each patient) and clustering (as defined by number of secondary cases within 2 years after diagnosis of an index case sharing the same fingerprint) indices. An estimate of progression to disease by each risk factor was calculated as a bootstrapped risk ratio of the clustering index by the infectivity index. Compared to the Euro-American reference, Mycobacterium africanum showed significantly lower infectivity and clustering indices in the foreign-born population, while Mycobacterium bovis showed significantly lower infectivity and clustering indices in the native population. Significantly lower infectivity was also observed for the East African Indian lineage in the foreign-born population. Smear positivity was a significant risk factor for increased infectivity and increased clustering. Estimates of progression to disease were significantly associated with age, sputum-smear status, and behavioral risk factors, such as alcohol and intravenous drug abuse, but not with phylogenetic lineages. In conclusion, we found evidence of a bacteriological factor influencing indicators of a strain's transmissibility, namely, a decreased ability to infect and a lower clustering index in ancient phylogenetic lineages compared to their modern counterparts. Confirmation of these findings via follow-up studies using tuberculin skin test conversion data should have important implications on M. tuberculosis control efforts. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Streptococcus bovis endocarditis associated with colonic adenocarcinoma: report of a case.

PubMed

Ma, H M; Shyu, K G; Hwang, J J; Huang, S H; Hsieh, W C; Lien, W P

1992-08-01

Streptococcus bovis (S. bovis) endocarditis has been increasing over recent decades, especially among the senile population. A 74-year-old man presented with intermittent fever for two months. He had a past history of aortic dissection and underwent a Bentall operation one year before admission. A Janeway lesion was noted on his right hand and six blood cultures grew S. bovis. He was treated with penicillin-G, 3 microU intravenously, every six hours, and became afebrile three days later. A colonofiberoscopy was carried out despite the absence of any gastrointestinal symptoms, and a 2 x 2 cm mass was found at the cecum, with pathologic proof of adenocarcinoma. The patient died from a massive intracranial hemorrhage on the 23rd hospital day. Review of the literature revealed an intimate association between S. bovis bacteremia (or endocarditis) and underlying colonic neoplasia. Failure to be aware of the possible consequences of this combination may lead to detrimental patient prognosis. We strongly advise that every patient presenting with bacteremia or endocarditis due to this organism, even if they are free from gastrointestinal symptoms, should undergo a thorough lower gastrointestinal investigation to rule out colonic neoplasia.

Molecular and serologic evidence for Babesia bovis-like parasites in white-tailed deer (Odocoileus virginianus) in south Texas.

PubMed

Ramos, Christina M; Cooper, Susan M; Holman, Patricia J

2010-09-20

The current study was undertaken to determine if white-tailed deer in south Texas harbor Babesia bovis, a causative agent of bovine babesiosis. Blood samples from free-ranging white-tailed deer (Odocoileus virginianus) on two ranches in LaSalle and Webb Counties were screened for B. bovis and other hemoparasites by the polymerase chain reaction (PCR) to detect the piroplasm 18S rDNA. Serology was conducted on selected samples to detect antibody activity to B. bovis by the immunofluorescent antibody test (IFAT). PCR revealed that 16% of the LaSalle County samples and 4% of the Webb County samples were positive for B. bovis. Five of the LaSalle County and the two Webb County B. bovis 18S rDNA amplicons were cloned and sequenced. The resulting clones shared 99% identity to B. bovis 18S rRNA gene sequences derived from cattle isolates. Weak seroreactivity to B. bovis was shown by the IFAT. The samples were also screened for additional hemoparasites of deer including Theileria cervi, Babesia odocoilei and other Babesia spp. A genotypically unique Theileria sp. was found, along with T. cervi and B. odocoilei. The finding of putative B. bovis in white-tailed deer necessitates further study to determine if deer may act as a transient host or even a reservoir of infection for B. bovis pathogenic to cattle.

Mycobacterium tuberculosis infection in cattle from the Eastern Cape Province of South Africa.

PubMed

Hlokwe, Tiny Motlatso; Said, Halima; Gcebe, Nomakorinte

2017-10-10

Mycobacterium tuberculosis is the main causative agent of tuberculosis (TB) in human and Mycobacterium bovis commonly causes tuberculosis in animals. Transmission of tuberculosis caused by both pathogens can occur from human to animals and vice versa. In the current study, M. tuberculosis, as confirmed by polymerase chain reaction (PCR) using primers targeting 3 regions of difference (RD4, RD9 and RD12) on the genomes, was isolated from cattle originating from two epidemiologically unrelated farms in the Eastern Cape (E.C) Province of South Africa. Although the isolates were genotyped with variable number of tandem repeat (VNTR) typing, no detailed epidemiological investigation was carried out on the respective farms to unequivocally confirm or link humans as sources of TB transmission to cattle, a move that would have embraced the 'One Health' concept. In addition, strain comparison with human M. tuberculosis in the database from the E.C Province and other provinces in the country did not reveal any match. This is the first report of cases of M. tuberculosis infection in cattle in South Africa. The VNTR profiles of the M. tuberculosis strains identified in the current study will form the basis for creating M. tuberculosis VNTR database for animals including cattle for future epidemiological studies. Our findings however, call for urgent reinforcement of collaborative efforts between the veterinary and the public health services of the country.

Detection of Multidrug Resistance in Mycobacterium tuberculosis▿

PubMed Central

Sekiguchi, Jun-ichiro; Miyoshi-Akiyama, Tohru; Augustynowicz-Kopeć, Ewa; Zwolska, Zofia; Kirikae, Fumiko; Toyota, Emiko; Kobayashi, Intetsu; Morita, Koji; Kudo, Koichiro; Kato, Seiya; Kuratsuji, Tadatoshi; Mori, Toru; Kirikae, Teruo

2007-01-01

We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis. PMID:17108078

Molecular epidemiology of Mycobacterium africanum in Ghana.

PubMed

Asante-Poku, Adwoa; Otchere, Isaac Darko; Osei-Wusu, Stephen; Sarpong, Esther; Baddoo, Akosua; Forson, Audrey; Laryea, Clement; Borrell, Sonia; Bonsu, Frank; Hattendorf, Jan; Ahorlu, Collins; Koram, Kwadwo A; Gagneux, Sebastien; Yeboah-Manu, Dorothy

2016-08-09

Mycobacterium africanum comprises two phylogenetic lineages within the M. tuberculosis complex (MTBC) and is an important cause of human tuberculosis (TB) in West Africa. The reasons for this geographic restriction of M. africanum remain unclear. Here, we performed a prospective study to explore associations between the characteristics of TB patients and the MTBC lineages circulating in Ghana. We genotyped 1,211 MTBC isolates recovered from pulmonary TB patients recruited between 2012 and 2014 using single nucleotide polymorphism typing and spoligotyping. Associations between patient and pathogen variables were assessed using univariate and multivariate logistic regression. Of the 1,211 MTBC isolates analysed, 71.9 % (871) belonged to Lineage 4; 12.6 % (152) to Lineage 5 (also known as M. africanum West-Africa 1), 9.2 % (112) to Lineage 6 (also known as M. africanum West-Africa 2) and 0.6 % (7) to Mycobacterium bovis. Univariate analysis revealed that Lineage 6 strains were less likely to be isoniazid resistant compared to other strains (odds ratio = 0.25, 95 % confidence interval (CI): 0.05-0.77, P < 0.01). Multivariate analysis showed that Lineage 5 was significantly more common in patients from the Ewe ethnic group (adjusted odds ratio (adjOR): 2.79; 95 % CI: 1.47-5.29, P < 0.001) and Lineage 6 more likely to be found among HIV-co-infected TB patients (adjOR = 2.2; 95 % confidence interval (CI: 1.32-3.7, P < 0.001). Our findings confirm the importance of M. africanum in Ghana and highlight the need to differentiate between Lineage 5 and Lineage 6, as these lineages differ in associated patient variables.

Identification of the missing trans-acting enoyl reductase required for phthiocerol dimycocerosate and phenolglycolipid biosynthesis in Mycobacterium tuberculosis.

PubMed

Siméone, Roxane; Constant, Patricia; Guilhot, Christophe; Daffé, Mamadou; Chalut, Christian

2007-07-01

Phthiocerol dimycocerosates (DIM) and phenolglycolipids (PGL) are functionally important surface-exposed lipids of Mycobacterium tuberculosis. Their biosynthesis involves the products of several genes clustered in a 70-kb region of the M. tuberculosis chromosome. Among these products is PpsD, one of the modular type I polyketide synthases responsible for the synthesis of the lipid core common to DIM and PGL. Bioinformatic analyses have suggested that this protein lacks a functional enoyl reductase activity domain required for the synthesis of these lipids. We have identified a gene, Rv2953, that putatively encodes an enoyl reductase. Mutation in Rv2953 prevents conventional DIM formation and leads to the accumulation of a novel DIM-like product. This product is unsaturated between C-4 and C-5 of phthiocerol. Consistently, complementation of the mutant with a functional pks15/1 gene from Mycobacterium bovis BCG resulted in the accumulation of an unsaturated PGL-like substance. When an intact Rv2953 gene was reintroduced into the mutant strain, the phenotype reverted to the wild type. These findings indicate that Rv2953 encodes a trans-acting enoyl reductase that acts with PpsD in phthiocerol and phenolphthiocerol biosynthesis.

Incidence and transmission of Mycoplasma bovis mastitis in Holstein dairy cows in a hospital pen: A case study.

PubMed

Punyapornwithaya, V; Fox, L K; Hancock, D D; Gay, J M; Wenz, J R; Alldredge, J R

2011-01-01

The objective was to determine the incidence and transmission of mycoplasma mastitis in the hospital pen in a dairy herd of 650 lactating cows after a hospital pen was established following an outbreak of this disease. Mycoplasma mastitis status was monitored for 3 months through repeated collection of milk samples from cows with clinical mastitis (CM) and from bulk tank milk. During the outbreak 13 cows were diagnosed with Mycoplasma bovis CM, 1 cow with Mycoplasma sp. mastitis and 8 cows showed signs of arthritis, 3 of which were confirmed as having M. bovis arthritis. M. bovis isolates from cows with CM, arthritis and bulk tank milk had indistinguishable chromosomal digest pattern fingerprints. Incidence rates of M. bovis CM cases in the milking and hospital pens were 0.01 and 1.7 cases per 100 cow-days at risk. Approximately 70% of cows with M. bovis CM became infected within 12 days of entering the hospital pen. Transmission of M. bovis in the hospital pen occurred as 3 episodes. Each episode corresponded to the introduction of a cow with M. bovis CM from a milking pen. Evidence indicates that cows with M. bovis CM from milking pens were the source of transmission of the disease in the hospital pen and thus their presence in the hospital pen appeared to be a risk factor for transmission of M. bovis mastitis in this single case study herd. Copyright © 2010 Elsevier B.V. All rights reserved.

Insertion sequence typing of Mycobacterium tuberculosis: characterization of a widespread subtype with a single copy of IS6110.

PubMed

Fomukong, N G; Tang, T H; al-Maamary, S; Ibrahim, W A; Ramayah, S; Yates, M; Zainuddin, Z F; Dale, J W

1994-12-01

DNA fingerprinting with the insertion sequence IS6110 (also known as IS986) has become established as a major tool for investigating the spread of tuberculosis. Most strains of Mycobacterium tuberculosis have multiple copies of IS6110, but a small minority carry a single copy only. We have examined selected strains from Malaysia, Tanzania and Oman, in comparison with M. bovis isolates and BCG strains carrying one or two copies of IS6110. The insertion sequence appears to be present in the same position in all these strains, which suggests that in these organisms the element is defective in transposition and that the loss of transposability may have occurred at an early stage in the evolution of the M. tuberculosis complex.

The association of Streptococcus bovis/gallolyticus with colorectal tumors: The nature and the underlying mechanisms of its etiological role

PubMed Central

2011-01-01

Streptococcus bovis (S. bovis) bacteria are associated with colorectal cancer and adenoma. S. bovis is currently named S. gallolyticus. 25 to 80% of patients with S. bovis/gallolyticus bacteremia have concomitant colorectal tumors. Colonic neoplasia may arise years after the presentation of bacteremia or infectious endocarditis of S. bovis/gallolyticus. The presence of S. bovis/gallolyticus bacteremia and/or endocarditis is also related to the presence of villous or tubular-villous adenomas in the large intestine. In addition, serological relationship of S. gallolyticus with colorectal tumors and direct colonization of S. gallolyticus in tissues of colorectal tumors were found. However, this association is still under controversy and has long been underestimated. Moreover, the etiological versus non-etiological nature of this associationis not settled yet. Therefore, by covering the most of up to date studies, this review attempts to clarify the nature and the core of S. bovis/gallolyicus association with colorectal tumors and analyze the possible underlying mechanisms. PMID:21247505

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids.

PubMed

Côtes, Karen; Dhouib, Rabeb; Douchet, Isabelle; Chahinian, Henri; de Caro, Alain; Carrière, Frédéric; Canaan, Stéphane

2007-12-15

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units x mg(-1) at 37 degrees C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids.

Tuberculosis in wild and captive deer

USDA-ARS?s Scientific Manuscript database

Deer are found on every continent, save for Antarctica and Australia. Of the over 50 species of deer worldwide, tuberculosis due to Mycobacterium bovis has been documented in at least 14. The broad host range of M. bovis includes most mammals, including humans and livestock. Eradication programs hav...

Incidence and nature of human tuberculosis due to Mycobacterium africanum in South-East England: 1977-87.

PubMed

Grange, J M; Yates, M D

1989-08-01

A total of 210 new cases of tuberculosis due to Mycobacterium africanum were registered at the South-East Regional Centre for Tuberculosis Bacteriology, Dulwich, between 1977 and 1987 inclusive. This represented 1.25% of bacteriologically-confirmed cases of tuberculosis in South-East England, an incidence slightly higher than that of disease due to M. bovis. Two variants were identified: 150 strains were typed as African I (a type associated with East Africa) and 60 as African II (a type more prevalent in West Africa). Over half the patients infected with African I strains were of Indian subcontinent ethnic origin; patients of African ethnic origin predominated in the African II group while about a fifth o patients infected with either type were of European origin. The European patients with tuberculosis due to M. africanum were notably younger than those in the same region with disease due to other tubercle bacilli. The distribution of lesions due to M. africanum was similar to that due to other tubercle bacilli in the various ethnic groups, except that genito-urinary tuberculosis was uncommon. The importance of a clinical awareness that M. africanum is a highly pathogenic and transmissible tubercle bacillus rather than an opportunist or 'atypical' mycobacterium is stressed.

Understanding the Mechanisms of Immunopathogenesis of Human and Bovine Tuberculosis

USDA-ARS?s Scientific Manuscript database

Extensive investigations have revealed that zoonotic pathogens in the Mycobacterium tuberculosis complex (MTBC) evolved from a common ancestor. Although all the members can cause disease in one or more species of mammals, Mycobacterium tuberculosis (Mtb) and M. bovis (Mbv) are the major pathogens ...

Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro.

PubMed Central

ter Laak, E A; Noordergraaf, J H; Verschure, M H

1993-01-01

The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline. PMID:8452363

Susceptibilities of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum strains to antimicrobial agents in vitro.

PubMed

ter Laak, E A; Noordergraaf, J H; Verschure, M H

1993-02-01

The purpose of this study was to determine the susceptibility of various strains of Mycoplasma bovis, Mycoplasma dispar, and Ureaplasma diversum, which are prevalent causes of pneumonia in calves, to 16 antimicrobial agents in vitro. The MICs of the antimicrobial agents were determined by a serial broth dilution method for 16 field strains and the type strain of M. bovis, for 19 field strains and the type strain of M. dispar, and for 17 field strains of U. diversum. Final MICs for M. bovis and M. dispar were read after 7 days and final MICs for U. diversum after 1 to 2 days. All strains tested were susceptible to tylosin, kitasamycin, and tiamulin but were resistant to nifuroquine and streptomycin. Most strains of U. diversum were intermediately susceptible to oxytetracycline but fully susceptible to chlortetracycline; most strains of M. bovis and M. dispar, however, were resistant to both agents. Strains of M. dispar and U. diversum were susceptible to doxycycline and minocycline, but strains of M. bovis were only intermediately susceptible. Susceptibility or resistance to chloramphenicol, spiramycin, spectinomycin, lincomycin, or enrofloxacin depended on the species but was not equal for the three species. The type strains of M. bovis and M. dispar were more susceptible to various antimicrobial agents, including tetracyclines, than the field strains. This finding might indicate that M. bovis and M. dispar strains are becoming resistant to these agents. Antimicrobial agents that are effective in vitro against all three mycoplasma species can be considered for treating mycoplasma infections in pneumonic calves. Therefore, tylosin, kitasamycin, and tiamulin may be preferred over oxytetracycline and chlortetracycline.

Development of molecular methods for the rapid detection of antibiotic susceptibility of Mycoplasma bovis.

PubMed

Sulyok, Kinga M; Bekő, Katinka; Kreizinger, Zsuzsa; Wehmann, Enikő; Jerzsele, Ákos; Rónai, Zsuzsanna; Turcsányi, Ibolya; Makrai, László; Szeredi, Levente; Jánosi, Szilárd; Nagy, Sára Ágnes; Gyuranecz, Miklós

2018-01-01

Determining the antibiotic susceptibility profile of Mycoplasma bovis isolates in vitro provides the basis for the appropriate choice of antibiotics in the therapy. Traditionally, the antibiotic susceptibility examination of mycoplasmas is technically demanding, time-consuming and rarely performed in diagnostic laboratories. The aim of the present study was to develop rapid molecular assays to determine mutations responsible for elevated minimal inhibitory concentrations (MICs) to fluoroquinolones, tetracyclines, aminocyclitols, macrolides, lincosamides, phenicols and pleuromutilins in M. bovis. The nine mismatch amplification mutation assays (MAMA) and seven high resolution melt (HRM) tests designed in the present study enable the simultaneous detection of these genetic markers. The sensitivity of the assays varied between 10 2 -10 5 copy numbers/reaction. Cross-reactions with other mycoplasmas occurring in cattle were detected in assays targeting universal regions (e.g. 16S rRNA). Nevertheless, results of the novel method were in accordance with sequence and MICs data of the M. bovis pure cultures. Also, the tests of clinical samples containing high amount of M. bovis DNA were congruent even in the presence of other Mycoplasma spp. The presented method is highly cost-effective and can provide an antibiogram to 12 antibiotics in approximately 3-4 days when previous isolation of M. bovis is applied. In order to assure the proper identification of the genetic markers at issue, the regions examined by the MAMA and HRM tests are overlapping. In conclusion, the developed assays have potential to be used in routine diagnostics for the detection of antibiotic susceptibility in M. bovis. Copyright © 2017 Elsevier B.V. All rights reserved.

Identification of Moraxella bovis by qualitative genetic transformation and nutritional assays.

PubMed Central

Juni, E; Heym, G A; Newcomb, R D

1988-01-01

Strains of Moraxella bovis were identified definitively through the combined use of a qualitative genetic transformation assay and determination of the ability of the organism under examination to grow in a defined medium (medium MB). Except for weak transformation by DNA from strains of M. lacunata, M. nonliquefaciens, and M. (Branhamella) ovis, DNA samples from all other members of the genus Moraxella failed to transform either of the two M. bovis auxotrophs used in this study. Images PMID:3389822

Bovine tuberculosis in free-ranging carnivores from Michigan.

PubMed

Bruning-Fann, C S; Schmitt, S M; Fitzgerald, S D; Fierke, J S; Friedrich, P D; Kaneene, J B; Clarke, K A; Butler, K L; Payeur, J B; Whipple, D L; Cooley, T M; Miller, J M; Muzo, D P

2001-01-01

During a survey of carnivores and omnivores for bovine tuberculosis conducted in Michigan (USA) since 1996, Mycobacterium bovis was cultured from lymph nodes pooled from six coyotes (Canis latrans) (four adult female, two adult male), two adult male raccoons (Procyon lotor), one adult male red fox (Vulpes vulpes), and one 1.5-yr-old male black bear (Ursus americanus). One adult, male bobcat (Felis rufus) with histologic lesions suggestive of tuberculosis was negative on culture but positive for organisms belonging to the Mycobacterium tuberculosis complex when tested by polymerase chain reaction. All the tuberculous animals were taken from three adjoining counties where M. bovis is known to be endemic in the free-ranging white-tailed deer (Odocoileus virginianus) population. There were two coyotes, one raccoon, one red fox, and one bobcat infected in Alpena county. Montmorency County had two coyotes and one raccoon with M. bovis. Two coyotes and a bear were infected from Alcona County. These free-ranging carnivores/omnivores probably became infected with M. bovis through consumption of tuberculous deer. Other species included in the survey were opossum (Didelphis virginiana), gray fox (Urocyon cinereoargenteus), and badger (Taxidea taxus); these were negative for M. bovis.

Diversity of Babesia bovis merozoite surface antigen genes in the Philippines.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Ybanez, Adrian Patalinghug; Ybanez, Rochelle Haidee Daclan; Perez, Zandro Obligado; Guswanto, Azirwan; Igarashi, Ikuo; Yokoyama, Naoaki

2014-02-01

Babesia bovis is the causative agent of fatal babesiosis in cattle. In the present study, we investigated the genetic diversity of B. bovis among Philippine cattle, based on the genes that encode merozoite surface antigens (MSAs). Forty-one B. bovis-positive blood DNA samples from cattle were used to amplify the msa-1, msa-2b, and msa-2c genes. In phylogenetic analyses, the msa-1, msa-2b, and msa-2c gene sequences generated from Philippine B. bovis-positive DNA samples were found in six, three, and four different clades, respectively. All of the msa-1 and most of the msa-2b sequences were found in clades that were formed only by Philippine msa sequences in the respective phylograms. While all the msa-1 sequences from the Philippines showed similarity to those formed by Australian msa-1 sequences, the msa-2b sequences showed similarity to either Australian or Mexican msa-2b sequences. In contrast, msa-2c sequences from the Philippines were distributed across all the clades of the phylogram, although one clade was formed exclusively by Philippine msa-2c sequences. Similarities among the deduced amino acid sequences of MSA-1, MSA-2b, and MSA-2c from the Philippines were 62.2-100, 73.1-100, and 67.3-100%, respectively. The present findings demonstrate that B. bovis populations are genetically diverse in the Philippines. This information will provide a good foundation for the future design and implementation of improved immunological preventive methodologies against bovine babesiosis in the Philippines. The study has also generated a set of data that will be useful for futher understanding of the global genetic diversity of this important parasite. © 2013.

Multilocus sequence typing of Mycoplasma bovis reveals host-specific genotypes in cattle versus bison

USDA-ARS?s Scientific Manuscript database

Mycoplasma bovis is a primary agent of mastitis, pneumonia and arthritis in cattle and is the bacterium isolated most frequently from the polymicrobial syndrome known as bovine respiratory disease complex (BRDC). Recently, M. bovis has emerged as a significant health problem in bison, causing necro...

Immune responses to Mycoplasma bovis vaccination and experimental infection in the bovine mammary gland.

PubMed Central

Boothby, J T; Schore, C E; Jasper, D E; Osburn, B I; Thomas, C B

1988-01-01

This study characterized the immune responses in four vaccinated and four control cows in response to vaccination and experimental intramammary inoculation with Mycoplasma bovis. Specific antibody responses occurred in serum and milk in response to vaccination and experimental infection. Lymphocytes from peripheral blood, but not from the mammary gland of vaccinated cows had increased responsiveness to mitogens. No lymphocytes tested were responsive to M. bovis antigen. Both vaccination and experimental infection resulted in skin test reactivity. These results imply that vaccination results in immune responses which may alter the course of experimental M. bovis mastitis, but may contribute to cellular inflammation. PMID:3167718

Development and Host Compatibility of Plasmids for Two Important Ruminant Pathogens, Mycoplasma bovis and Mycoplasma agalactiae

PubMed Central

Sharma, Shukriti; Citti, Chistine; Sagné, Eveline; Marenda, Marc S.

2015-01-01

Mycoplasma bovis is a cause of pneumonia, mastitis, arthritis and otitis media in cattle throughout the world. However, despite its clinical significance, there is a paucity of tools to genetically manipulate it, impeding our capacity to further explore the molecular basis of its virulence. To address this limitation, we developed a series of homologous and heterologous replicable plasmids from M. bovis and M. agalactiae. The shortest replicable oriC plasmid based on the region downstream of dnaA in M. bovis was 247 bp and contained two DnaA boxes, while oriC plasmids based on the region downstream of dnaA in M. agalactiae strains 5632 and PG2 were 219 bp and 217 bp in length, respectively, and contained only a single DnaA box. The efficiency of transformation in M. bovis and M. agalactiae was inversely correlated with the size of the oriC region in the construct, and, in general, homologous oriC plasmids had a higher transformation efficiency than heterologous oriC plasmids. The larger pWholeoriC45 and pMM21-7 plasmids integrated into the genomic oriC region of M. bovis, while the smaller oriC plasmids remained extrachromosomal for up to 20 serial passages in selective media. Although specific gene disruptions were not be achieved in M. bovis in this study, the oriC plasmids developed here could still be useful as tools in complementation studies and for expression of exogenous genes in both M. bovis and M. agalactiae. PMID:25746296

Cloning, sequencing, and expression of the apa gene coding for the Mycobacterium tuberculosis 45/47-kilodalton secreted antigen complex.

PubMed

Laqueyrerie, A; Militzer, P; Romain, F; Eiglmeier, K; Cole, S; Marchal, G

1995-10-01

Effective protection against a virulent challenge with Mycobacterium tuberculosis is induced mainly by previous immunization with living attenuated mycobacteria, and it has been hypothesized that secreted proteins serve as major targets in the specific immune response. To identify and purify molecules present in culture medium filtrate which are dominant antigens during effective vaccination, a two-step selection procedure was used to select antigens able to interact with T lymphocytes and/or antibodies induced by immunization with living bacteria and to counterselect antigens interacting with the immune effectors induced by immunization with dead bacteria. A Mycobacterium bovis BCG 45/47-kDa antigen complex, present in BCG culture filtrate, has been previously identified and isolated (F. Romain, A. Laqueyrerie, P. Militzer, P. Pescher, P. Chavarot, M. Lagranderie, G. Auregan, M. Gheorghiu, and G. Marchal, Infect. Immun. 61:742-750, 1993). Since the cognate antibodies recognize the very same antigens present in M. tuberculosis culture medium filtrates, a project was undertaken to clone, express, and sequence the corresponding gene of M. tuberculosis. An M. tuberculosis shuttle cosmid library was transferred in Mycobacterium smegmatis and screened with a competitive enzyme-linked immunosorbent assay to detect the clones expressing the proteins. A clone containing a 40-kb DNA insert was selected, and by means of subcloning in Escherichia coli, a 2-kb fragment that coded for the molecules was identified. An open reading frame in the 2,061-nucleotide sequence codes for a secreted protein with a consensus signal peptide of 39 amino acids and a predicted molecular mass of 28,779 Da. The gene was referred to as apa because of the high percentages of proline (21.7%) and alanine (19%) in the purified protein. Southern hybridization analysis of digested total genomic DNA from M. tuberculosis (reference strains H37Rv and H37Ra) indicated that the apa gene was present as a

Characterization of Streptococcus bovis from the rumen of the dromedary camel and Rusa deer.

PubMed

Ghali, M B; Scott, P T; Al Jassim, R A M

2004-01-01

Isolation and characterization of Streptococcus bovis from the dromedary camel and Rusa deer. Bacteria were isolated from the rumen contents of four camels and two deer fed lucerne hay by culturing on the semi-selective medium MRS agar. Based on Gram morphology and RFLP analysis seven isolates, MPR1, MPR2, MPR3, MPR4, MPR5, RD09 and RD11 were selected and putatively identified as Streptococcus. The identity of these isolates was later confirmed by comparative DNA sequence analysis of the 16S rRNA gene with the homologous sequence from S. bovis strains, JB1, C14b1, NCFB2476, SbR1, SbR7 and Sb5, from cattle and sheep, and the Streptococcus equinus strain NCD01037T. The percentage similarity amongst all strains was >99%, confirming the identification of the camel isolates as S. bovis. The strains were further characterized by their ability to utilize a range of carbohydrates, the production of volatile fatty acids (VFA) and lactate and the determination of the doubling time in basal medium 10 supplemented with glucose. All the isolates produced l-lactate as a major fermentation end product, while four of five camel isolates produced VFA. The range of carbohydrates utilized by all the strains tested, including those from cattle and sheep were identical, except that all camel isolates and the deer isolate RD11 were additionally able to utilize arabinose. Streptococcus bovis was successfully isolated from the rumen of camels and deer, and shown by molecular and biochemical characterization to be almost identical to S. bovis isolates from cattle and sheep. Streptococcus bovis is considered a key lactic acid producing bacterium from the gastrointestinal tract of ruminants, and has been implicated as a causative agent of lactic acidosis. This study is the first report of the isolation and characterization of S. bovis from the dromedary camel and Rusa deer, and suggests a major contributive role of this bacterium to fermentative acidosis.

Target-Based Screen Against a Periplasmic Serine Protease That Regulates Intrabacterial pH Homeostasis in Mycobacterium tuberculosis

PubMed Central

2015-01-01

Mycobacterium tuberculosis (Mtb) maintains its intrabacterial pH (pHIB) near neutrality in the acidic environment of phagosomes within activated macrophages. A previously reported genetic screen revealed that Mtb loses this ability when the mycobacterial acid resistance protease (marP) gene is disrupted. In the present study, a high throughput screen (HTS) of compounds against the protease domain of MarP identified benzoxazinones as inhibitors of MarP. A potent benzoxazinone, BO43 (6-chloro-2-(2′-methylphenyl)-4H-1,3-benzoxazin-4-one), acylated MarP and lowered Mtb’s pHIB and survival during incubation at pH 4.5. BO43 had similar effects on MarP-deficient Mtb, suggesting the existence of additional target(s). Reaction of an alkynyl-benzoxazinone, BO43T, with Mycobacterium bovis variant bacille Calmette-Guérin (BCG) followed by click chemistry with azido-biotin identified both the MarP homologue and the high temperature requirement A1 (HtrA1) homologue, an essential protein. Thus, the chemical probe identified through a target-based screen not only reacted with its intended target in the intact cells but also implicated an additional enzyme that had eluded a genetic screen biased against essential genes. PMID:25457457

Spatio-Temporal Distribution of Mycobacterium tuberculosis Complex Strains in Ghana.

PubMed

Yeboah-Manu, Dorothy; Asare, P; Asante-Poku, A; Otchere, I D; Osei-Wusu, S; Danso, E; Forson, A; Koram, K A; Gagneux, Sebastien

2016-01-01

There is a perception that genomic differences in the species/lineages of the nine species making the Mycobacterium tuberculosis complex (MTBC) may affect the efficacy of distinct control tools in certain geographical areas. We therefore analyzed the prevalence and spatial distribution of MTBC species and lineages among isolates from pulmonary TB cases over an 8-year period, 2007-2014. Mycobacterial species isolated by culture from consecutively recruited pulmonary tuberculosis patients presenting at selected district/sub-district health facilities were confirmed as MTBC by IS6110 and rpoß PCR and further assigned lineages and sub lineages by spoligotyping and large sequence polymorphism PCR (RDs 4, 9, 12, 702, 711) assays. Patient characteristics, residency, and risks were obtained with a structured questionnaire. We used SaTScan and ArcMap analyses to identify significantly clustered MTBC lineages within selected districts and spatial display, respectively. Among 2,551 isolates, 2,019 (79.1%), 516 (20.2%) and 16 (0.6%) were identified as M. tuberculosis sensu stricto (MTBss), M. africanum (Maf), 15 M. bovis and 1 M. caprae, respectively. The proportions of MTBss and Maf were fairly constant within the study period. Maf spoligotypes were dominated by Spoligotype International Type (SIT) 331 (25.42%), SIT 326 (15.25%) and SIT 181 (14.12%). We found M. bovis to be significantly higher in Northern Ghana (1.9% of 212) than Southern Ghana (0.5% of 2339) (p = 0.020). Using the purely spatial and space-time analysis, seven significant MTBC lineage clusters (p< 0.05) were identified. Notable among the clusters were Ghana and Cameroon sub-lineages found to be associated with north and south, respectively. This study demonstrated that overall, 79.1% of TB in Ghana is caused by MTBss and 20% by M. africanum. Unlike some West African Countries, we did not observe a decline of Maf prevalence in Ghana.

Mycobacterium tuberculosis Rv1987 induces Th2 immune responses and enhances Mycobacterium smegmatis survival in mice.

PubMed

Sha, Shanshan; Shi, Xiaoxia; Deng, Guoying; Chen, Lina; Xin, Yi; Ma, Yufang

2017-04-01

Mycobacterium tuberculosis can interfere with host immune response and escape clearance through its specific antigens. M. tuberculosis Rv1987 encoded by region of difference (RD)-2 gene is a secretory protein with immunogenic potency. Here, we investigated the impact of Rv1987 on host cytokine responses and T cell polarization in mouse aerosol model. A recombinant M. smegmatis mc 2 155 strain that overexpressed Rv1987 protein (named MS1987) was constructed and used to infect C57BL/6 mice. The mc 2 155 harbored the empty vector (named MSVec) was as a control. The results showed that MS1987 challenged mice promoted Th2-biased cytokine responses with lower secretion of IFN-γ but higher production of IL-4 and Rv1987-specific IgG antibody compared to MSVec infected mice. Neutrophilic inflammation and high bacterial burden were observed in the lung tissues of MS1987 infected mice probably own to the failed Th1 cell immunity. Besides, subcutaneous injection of Rv1987 protein could mediate the Th1 cytokine responses caused by M. bovis BCG in mice. These results indicated that M. tuberculosis Rv1987 protein could modulate host immune response towards Th2 profile, which probably contributed to the immune evasion of bacteria from host elimination. Copyright © 2017 Elsevier GmbH. All rights reserved.

Tuberculosis-resistant transgenic cattle

USDA-ARS?s Scientific Manuscript database

Tuberculosis is a devastating disease that affects humans and many animal species. In humans, tuberculosis (TB) is mainly caused by Mycobacterium tuberculosis, while most cases in cattle are caused by Mycobacterium bovis. However, Mb can also cause, albeit rarely, human TB. In this issue, Wu et al. ...

Antimicrobial susceptibility and molecular characterization of macrolide resistance of Mycoplasma bovis isolates from multiple provinces in China

PubMed Central

KONG, Ling-Cong; GAO, Duo; JIA, Bo-Yan; WANG, Zi; GAO, Yun-Hang; PEI, Zhi-Hua; LIU, Shu-Ming; XIN, Jiu-Qing; MA, Hong-Xia

2015-01-01

Mycoplasma bovis has spread widely throughout the world via animal movement and has become an important pathogen of bovine respiratory disease. However, the minimum inhibitory concentrations of antimicrobials for Mycoplasma bovis have not been studied in China. The objective of this study was to determine the prevalence and antibiotic resistance of Mycoplasma bovis isolated from young cattle with respiratory infection in China. Mycoplasma bovis was detected in 32/45 bovine respiratory infection outbreaks at beef farms in 8 provinces in China. The isolates were susceptible or had medium sensitivity to ciprofloxacin, enrofloxacin and doxycycline, but were frequently resistant to macrolides (13/32, 41%). An A2058G (Escherichia coli Numbering) mutation located in the rrnA operon in domain V of 23S rRNA was observed in strains that were resistant to macrolides. This single mutations at the rrnA operon in domain V of 23S rRNA may play an important role in the resistance of Mycoplasma bovis strains to macrolides. PMID:26346744

Characterization of an exported monoglyceride lipase from Mycobacterium tuberculosis possibly involved in the metabolism of host cell membrane lipids

PubMed Central

Côtes, Karen; Dhouib, Rabeb; Douchet, Isabelle; Chahinian, Henri; deCaro, Alain; Carrière, Frédéric; Canaan, Stéphane

2007-01-01

The Rv0183 gene of the Mycobacterium tuberculosis H37Rv strain, which has been implicated as a lysophospholipase, was cloned and expressed in Escherichia coli. The purified Rv0183 protein did not show any activity when lysophospholipid substrates were used, but preferentially hydrolysed monoacylglycerol substrates with a specific activity of 290 units·mg−1 at 37 °C. Rv0183 hydrolyses both long chain di- and triacylglycerols, as determined using the monomolecular film technique, although the turnover was lower than with MAG (monoacyl-glycerol). The enzyme shows an optimum activity at pH values ranging from 7.5 to 9.0 using mono-olein as substrate and is inactivated by serine esterase inhibitors such as E600, PMSF and tetrahydrolipstatin. The catalytic triad is composed of Ser110, Asp226 and His256 residues, as confirmed by the results of site-directed mutagenesis. Rv0183 shows 35% sequence identity with the human and mouse monoglyceride lipases and well below 15% with the other bacterial lipases characterized so far. Homologues of Rv0183 can be identified in other mycobacterial genomes such as Mycobacterium bovis, Mycobacterium smegmatis, and even Mycobacterium leprae, which is known to contain a low number of genes involved in the replication process within the host cells. The results of immunolocalization studies performed with polyclonal antibodies raised against the purified recombinant Rv0183 suggested that the enzyme was present only in the cell wall and culture medium of M. tuberculosis. Our results identify Rv0183 as the first exported lipolytic enzyme to be characterized in M. tuberculosis and suggest that Rv0183 may be involved in the degradation of the host cell lipids. PMID:17784850

STING-Dependent 2'-5' Oligoadenylate Synthetase-Like Production Is Required for Intracellular Mycobacterium leprae Survival.

PubMed

de Toledo-Pinto, Thiago Gomes; Ferreira, Anna Beatriz Robottom; Ribeiro-Alves, Marcelo; Rodrigues, Luciana Silva; Batista-Silva, Leonardo Ribeiro; Silva, Bruno Jorge de Andrade; Lemes, Robertha Mariana Rodrigues; Martinez, Alejandra Nóbrega; Sandoval, Felipe Galvan; Alvarado-Arnez, Lucia Elena; Rosa, Patrícia Sammarco; Shannon, Edward Joseph; Pessolani, Maria Cristina Vidal; Pinheiro, Roberta Olmo; Antunes, Sérgio Luís Gomes; Sarno, Euzenir Nunes; Lara, Flávio Alves; Williams, Diana Lynn; Ozório Moraes, Milton

2016-07-15

Cytosolic detection of nucleic acids elicits a type I interferon (IFN) response and plays a critical role in host defense against intracellular pathogens. Herein, a global gene expression profile of Mycobacterium leprae-infected primary human Schwann cells identified the genes differentially expressed in the type I IFN pathway. Among them, the gene encoding 2'-5' oligoadenylate synthetase-like (OASL) underwent the greatest upregulation and was also shown to be upregulated in M. leprae-infected human macrophage cell lineages, primary monocytes, and skin lesion specimens from patients with a disseminated form of leprosy. OASL knock down was associated with decreased viability of M. leprae that was concomitant with upregulation of either antimicrobial peptide expression or autophagy levels. Downregulation of MCP-1/CCL2 release was also observed during OASL knock down. M. leprae-mediated OASL expression was dependent on cytosolic DNA sensing mediated by stimulator of IFN genes signaling. The addition of M. leprae DNA enhanced nonpathogenic Mycobacterium bovis bacillus Calmette-Guerin intracellular survival, downregulated antimicrobial peptide expression, and increased MCP-1/CCL2 secretion. Thus, our data uncover a promycobacterial role for OASL during M. leprae infection that directs the host immune response toward a niche that permits survival of the pathogen. © The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America. All rights reserved. For permissions, e-mail journals.permissions@oup.com.

Correlates of Vaccine-Induced Protection against Mycobacterium tuberculosis Revealed in Comparative Analyses of Lymphocyte Populations

PubMed Central

Kurtz, Sherry L.

2015-01-01

A critical hindrance to the development of a novel vaccine against Mycobacterium tuberculosis is a lack of understanding of protective correlates of immunity and of host factors involved in a successful adaptive immune response. Studies from our group and others have used a mouse-based in vitro model system to assess correlates of protection. Here, using this coculture system and a panel of whole-cell vaccines with varied efficacy, we developed a comprehensive approach to understand correlates of protection. We compared the gene and protein expression profiles of vaccine-generated immune peripheral blood lymphocytes (PBLs) to the profiles found in immune splenocytes. PBLs not only represent a clinically relevant cell population, but comparing the expression in these populations gave insight into compartmentally specific mechanisms of protection. Additionally, we performed a direct comparison of host responses induced when immune cells were cocultured with either the vaccine strain Mycobacterium bovis BCG or virulent M. tuberculosis. These comparisons revealed host-specific and bacterium-specific factors involved in protection against virulent M. tuberculosis. Most significantly, we identified a set of 13 core molecules induced in the most protective vaccines under all of the conditions tested. Further validation of this panel of mediators as a predictor of vaccine efficacy will facilitate vaccine development, and determining how each promotes adaptive immunity will advance our understanding of antimycobacterial immune responses. PMID:26269537

A Novel Enzyme-Linked Immunosorbent Assay for Diagnosis of Mycobacterium avium subsp. paratuberculosis Infections (Johne's Disease) in Cattle

PubMed Central

Speer, C. A.; Scott, M. Cathy; Bannantine, John P.; Waters, W. Ray; Mori, Yasuyuki; Whitlock, Robert H.; Eda, Shigetoshi

2006-01-01

Enzyme-linked immunosorbent assays (ELISAs) for the diagnosis of Johne's disease (JD), caused by Mycobacterium avium subsp. paratuberculosis, were developed using whole bacilli treated with formaldehyde (called WELISA) or surface antigens obtained by treatment of M. avium subsp. paratuberculosis bacilli with formaldehyde and then brief sonication (called SELISA). ELISA plates were coated with either whole bacilli or sonicated antigens and tested for reactivity against serum obtained from JD-positive and JD-negative cattle or from calves experimentally inoculated with M. avium subsp. paratuberculosis, Mycobacterium avium subsp. avium, or Mycobacterium bovis. Because the initial results obtained from the WELISA and SELISA were similar, most of the subsequent experiments reported herein were performed using the SELISA method. To optimize the SELISA test, various concentrations (3.7 to 37%) of formaldehyde and intervals of sonication (2 to 300 s) were tested. With an increase in formaldehyde concentration and a decreased interval of sonication, there was a concomitant decrease in nonspecific binding by the SELISA. SELISAs prepared by treating M. avium subsp. paratuberculosis with 37% formaldehyde and then a 2-s burst of sonication produced the greatest difference (7×) between M. avium subsp. paratuberculosis-negative and M. avium subsp. paratuberculosis-positive serum samples. The diagnostic sensitivity and specificity for JD by the SELISA were greater than 95%. The SELISA showed subspecies-specific detection of M. avium subsp. paratuberculosis infections in calves experimentally inoculated with M. avium subsp. paratuberculosis or other mycobacteria. Based on diagnostic sensitivity and specificity, the SELISA appears superior to the commercial ELISAs routinely used for the diagnosis of JD. PMID:16682472

Widespread Environmental Contamination with Mycobacterium tuberculosis Complex Revealed by a Molecular Detection Protocol

PubMed Central

Santos, Nuno; Santos, Catarina; Valente, Teresa; Gortázar, Christian; Almeida, Virgílio; Correia-Neves, Margarida

2015-01-01

Environmental contamination with Mycobacterium tuberculosis complex (MTC) has been considered crucial for bovine tuberculosis persistence in multi-host-pathogen systems. However, MTC contamination has been difficult to detect due to methodological issues. In an attempt to overcome this limitation we developed an improved protocol for the detection of MTC DNA. MTC DNA concentration was estimated by the Most Probable Number (MPN) method. Making use of this protocol we showed that MTC contamination is widespread in different types of environmental samples from the Iberian Peninsula, which supports indirect transmission as a contributing mechanism for the maintenance of bovine tuberculosis in this multi-host-pathogen system. The proportion of MTC DNA positive samples was higher in the bovine tuberculosis-infected than in presumed negative area (0.32 and 0.18, respectively). Detection varied with the type of environmental sample and was more frequent in sediment from dams and less frequent in water also from dams (0.22 and 0.05, respectively). The proportion of MTC-positive samples was significantly higher in spring (p<0.001), but MTC DNA concentration per sample was higher in autumn and lower in summer. The average MTC DNA concentration in positive samples was 0.82 MPN/g (CI95 0.70–0.98 MPN/g). We were further able to amplify a DNA sequence specific of Mycobacterium bovis/caprae in 4 environmental samples from the bTB-infected area. PMID:26561038

A new method for identification of natural, artificial and in vitro cultured Calculus bovis using high-performance liquid chromatography-mass spectrometry

PubMed Central

Liu, Yonggang; Tan, Peng; Liu, Shanshan; Shi, Hang; Feng, Xin; Ma, Qun

2015-01-01

Objective: Calculus bovis have been widely used in Chinese herbology for the treatment of hyperpyrexia, convulsions, and epilepsy. Nowadays, due to the limited source and high market price, the substitutes, artificial and in vitro cultured Calculus bovis, are getting more and more commonly used. The adulteration phenomenon is serious. Therefore, it is crucial to establish a fast and simple method in discriminating the natural, artificial and in vitro cultured Calculus bovis. Bile acids, one of the main active constituents, are taken as an important indicator for evaluating the quality of Calculus bovis and the substitutes. Several techniques have been built to analyze bile acids in Calculus bovis. Whereas, as bile acids are with poor ultraviolet absorbance and high structural similarity, effective technology for identification and quality control is still lacking. Methods: In this study, high-performance liquid chromatography (HPLC) coupled with tandem mass spectrometry (LC/MS/MS) was applied in the analysis of bile acids, which effectively identified natural, artificial and in vitro cultured Calculus bovis and provide a new method for their quality control. Results: Natural, artificial and in vitro cultured Calculus bovis were differentiated by bile acids analysis. A new compound with protonated molecule at m/z 405 was found, which we called 3α, 12α-dihydroxy-7-oxo-5α-cholanic acid. This compound was discovered in in vitro cultured Calculus bovis, but almost not detected in natural and artificial Calculus bovis. A total of 13 constituents was identified. Among them, three bio-markers, including glycocholic acid, glycodeoxycholic acid and taurocholic acid (TCA) were detected in both natural and artificial Calculus bovis, but the density of TCA was different in two kinds of Calculus bovis. In addition, the characteristics of bile acids were illustrated. Conclusions: The HPLC coupled with tandem MS (LC/MS/MS) method was feasible, easy, rapid and accurate in

Development of a real-time PCR for detection of Mycoplasma bovis in bovine milk and lung samples.

PubMed

Cai, Hugh Y; Bell-Rogers, Patricia; Parker, Lois; Prescott, John F

2005-11-01

A real-time polymerase chain reaction (PCR) assay using hybridization probes on a LightCycler platform was developed for detection of Mycoplasma bovis from individual bovine mastitis milk and pneumonic lung tissues. The detection limit was 550 colony forming units (cfu)/ml of milk and 650 cfu/25 mg of lung tissue. A panel of bovine Mycoplasma and of other bovine-origin bacteria were tested; only M. bovis strains were positive, with a melting peak of 66.6 degrees C. Mycoplasma agalactiae PG2 was also positive and could be distinguished because it had a melting peak of 63.1 degrees C. In validation testing of clinical samples, the relative sensitivity and specificity were 100% and 99.3% for individual milks and 96.6% and 100% for the lung tissue. Using M. bovis real-time PCR, the M. bovis culture-positive milk samples were estimated to contain between 5 x 10(4) and 7.7 x 10(8) cfu/ml and the M. bovis culture-positive lungs between 1 x 10(3) and 1 x 10(9) cfu/25 mg. Isolation, confirmed with the real-time PCR and colony fluorescent antibody test, showed that at the herd level, the proportion of samples positive for M. bovis isolation in mastitis milk samples submitted to the Mastitis Laboratory, Animal Health Laboratory, University of Guelph, Ontario, Canada, was 2.4% (5/201). We conclude that this probe-based real-time PCR assay is a sensitive, specific, and rapid method to identify M. bovis infection in bovine milk and pneumonic lungs.

Nilgai antelope in northern Mexico as a possible carrier for cattle fever ticks and Babesia bovis and Babesia bigemina.

PubMed

Cárdenas-Canales, Elsa M; Ortega-Santos, J Alfonso; Campbell, Tyler A; García-Vázquez, Zeferino; Cantú-Covarrubias, Antonio; Figueroa-Millán, Julio V; DeYoung, Randall W; Hewitt, David G; Bryant, Fred C

2011-07-01

Of 20 blood samples from nilgais from México, five were polymerase chain reaction-positive for Babesia bigemina and one for Babesia bovis. Positive samples had the expected 170 (B. bigemina) and 291 (B. bovis) base pairs and were identical to Gen-Bank B. bigemina accession S45366 and B. bovis M38218.

The immune response to bovine tuberculosis: Correlates of protection and relevance to human tuberculosis

USDA-ARS?s Scientific Manuscript database

Tuberculosis (TB), primarily due to Mycobacterium tuberculosis in humans and Mycobacterium bovis in cattle, is a classic model for demonstration of the One Health Concept. Early studies with cattle were instrumental in the development of the use of Koch’s tuberculin as an in vivo measure of cell-med...

EVIDENCE FOR THE MACROPHAGE INDUCING GENE IN MYCOBACTERIUM INTRACELLULARE

EPA Science Inventory

Background: The Mycobacterium avium Complex (MAC) includes the species M. avium (MA), M. intracellulare (MI), and possibly others. Organisms belonging to the MAC are phylogenetically closely related, opportunistic pathogens. The macrophage inducing gene (mig) is the only well-des...

The Burden of Mycobacterial Disease in Ethiopian Cattle: Implications for Public Health

PubMed Central

Berg, Stefan; Firdessa, Rebuma; Habtamu, Meseret; Gadisa, Endalamaw; Mengistu, Araya; Yamuah, Lawrence; Ameni, Gobena; Vordermeier, Martin; Robertson, Brian D.; Smith, Noel H.; Engers, Howard; Young, Douglas; Hewinson, R. Glyn; Aseffa, Abraham; Gordon, Stephen V.

2009-01-01

Background Bovine tuberculosis (bTB), caused by Mycobacterium bovis, is a debilitating disease of cattle. Ethiopia has one of the largest cattle populations in the world, with an economy highly dependent on its livestock. Furthermore, Ethiopia has one of the highest incidence rates of human extrapulmonary TB in the world, a clinical presentation that is often associated with transmission of M. bovis from cattle to humans. Methodology/Principal Findings Here we present a comprehensive investigation of the prevalence of bTB in Ethiopia based on cases identified at slaughterhouses. Out of approximately 32,800 inspected cattle, ∼4.7% showed suspect tuberculous lesions. Culture of suspect lesions yielded acid-fast bacilli in ∼11% of cases, with M. bovis accounting for 58 of 171 acid-fast cultures, while 53 isolates were non-tuberculous mycobacteria. Strikingly, M. tuberculosis was isolated from eight cattle, an unusual finding that suggests human to animal transmission. Conclusions/Significance Our analysis has revealed that bTB is widely spread throughout Ethiopia, albeit at a low prevalence, and provides underpinning evidence for public health policy formulation. PMID:19352493

Expression of 6-Cys gene superfamily defines babesia bovis sexual stage development within rhipicephalus microplus

USDA-ARS?s Scientific Manuscript database

Babesia bovis, an intra-erythrocytic tick-borne apicomplexan protozoan, is one of the agents of bovine babesiosis. Its life cycle includes sexual reproduction within cattle fever ticks, Rhipicephalus spp. Six B. bovis 6-Cys gene superfamily members were previously identified (A, B, C, D, E, F) and t...

In vivo induction of neutrophil extracellular traps by Mycobacterium tuberculosis in a guinea pig model.

PubMed

Filio-Rodríguez, Georgina; Estrada-García, Iris; Arce-Paredes, Patricia; Moreno-Altamirano, María M; Islas-Trujillo, Sergio; Ponce-Regalado, M Dolores; Rojas-Espinosa, Oscar

2017-10-01

In 2004, a novel mechanism of cellular death, called 'NETosis', was described in neutrophils. This mechanism, different from necrosis and apoptosis, is characterized by the release of chromatin webs admixed with microbicidal granular proteins and peptides (NETs). NETs trap and kill a variety of microorganisms. Diverse microorganisms, including Mycobacterium tuberculosis, are NET inducers in vitro. The aim of this study was to examine whether M. tuberculosis can also induce NETs in vivo and if the NETs are bactericidal to the microorganism. Guinea pigs were intradermally inoculated with M. tuberculosis H37Rv, and the production of NETs was investigated at several time points thereafter. NETs were detected as early as 30 min post-inoculation and were clearly evident by 4 h post-inoculation. NETs produced in vivo contained DNA, myeloperoxidase, elastase, histones, ROS and acid-fast bacilli. Viable and heat-killed M. tuberculosis, as well as Mycobacterium bovis BCG were efficient NET inducers, as were unilamellar liposomes prepared with lipids from M. tuberculosis. In vitro, guinea pig neutrophils also produced NETs in response to M. tuberculosis. However, neither the in vivo nor the in vitro-produced NETs were able to kill M. tuberculosis. Nevertheless, in vivo, neutrophils might propitiate recruitment and activation of more efficient microbicidal cells.

Molecular detection and identification of Babesia bovis and Babesia bigemina in cattle in northern Thailand.

PubMed

Cao, Shinuo; Aboge, Gabriel Oluga; Terkawi, Mohamad Alaa; Yu, Longzheng; Kamyingkird, Ketsarin; Luo, Yuzi; Li, Yan; Goo, Youn-Kyoung; Yamagishi, Junya; Nishikawa, Yoshifumi; Yokoyama, Naoaki; Suzuki, Hiroshi; Igarashi, Ikuo; Maeda, Ryuichiro; Inpankaew, Tawin; Jittapalapong, Sathaporn; Xuan, Xuenan

2012-09-01

Although Babesia bovis and Babesia bigemina infections cause economic losses in the cattle industry in northern Thailand, there is inadequate information on Babesia isolates present in the area. Therefore, to determine the prevalence and genetic relationship between Babesia isolates, we screened 200 blood samples of cattle from Chiang Rai, Chiang Mai, and Lumpang provinces of northern Thailand. A nested polymerase chain reaction using primers targeting B. bovis spherical body protein 2 (BboSBP2) and B. bigemina rhoptry-associated protein 1a (BbiRAP-1a) genes revealed a prevalence of 12 and 21 % for B. bovis and B. bigemina, respectively, while that of mixed infections was 6.5 % samples. The prevalences of B. bovis in Chiang Rai, Chiang Mai, and Lumpang were 9.5, 3.7, and 25.5 %, respectively. For B. bigemina, the prevalences were 15.8, 12.9, and 39.2 % in Chiang Rai, Chiang Mai, and Lumpang, respectively. Mixed infections with B. bovis and B. bigemina were 6.3 % in Chiang Rai, 1.9 % in Chiang Mai, and 13.7 % in Lumpang. The identical sequences of either BboSBP2 gene or BbiRAP-1a gene were shared among the Babesia isolates in the three provinces of northern Thailand. Further analysis using the internal transcribed spacer gene revealed at least four genotypes for B. bovis and five genotypes for B. bigemina in northern Thailand, while the sequences present great genetic diversities in the different isolates. Overall, we have demonstrated a high prevalence and polymorphism of Babesia parasites in northern Thailand calling for the need to design effective control programs for bovine babesiosis.

Mycobacterium saopaulense sp. nov., a rapidly growing mycobacterium closely related to members of the Mycobacterium chelonae–Mycobacterium abscessus group

PubMed Central

Nogueira, Christiane Lourenço; Whipps, Christopher M.; Matsumoto, Cristianne Kayoko; Chimara, Erica; Droz, Sara; Tortoli, Enrico; de Freitas, Denise; Cnockaert, Margo; Palomino, Juan Carlos; Martin, Anandi; Vandamme, Peter

2015-01-01

Five isolates of non-pigmented, rapidly growing mycobacteria were isolated from three patients and, in an earlier study, from zebrafish. Phenotypic and molecular tests confirmed that these isolates belong to the Mycobacterium chelonae–Mycobacterium abscessus group, but they could not be confidently assigned to any known species of this group. Phenotypic analysis and biochemical tests were not helpful for distinguishing these isolates from other members of the M. chelonae–M. abscessus group. The isolates presented higher drug resistance in comparison with other members of the group, showing susceptibility only to clarithromycin. The five isolates showed a unique PCR restriction analysis pattern of the hsp65 gene, 100 % similarity in 16S rRNA gene and hsp65 sequences and 1–2 nt differences in rpoB and internal transcribed spacer (ITS) sequences. Phylogenetic analysis of a concatenated dataset including 16S rRNA gene, hsp65, and rpoB sequences from type strains of more closely related species placed the five isolates together, as a distinct lineage from previously described species, suggesting a sister relationship to a group consisting of M. chelonae, Mycobacterium salmoniphilum, Mycobacterium franklinii and Mycobacterium immunogenum. DNA–DNA hybridization values >70 % confirmed that the five isolates belong to the same species, while values < 70 % between one of the isolates and the type strains of M. chelonae and M. abscessus confirmed that the isolates belong to a distinct species. The polyphasic characterization of these isolates, supported by DNA–DNA hybridization results, demonstrated that they share characteristics with M. chelonae–M. abscessus members, but constitute a different species, for which the name Mycobacterium saopaulense sp. nov. is proposed. The type strain is EPM 10906T ( = CCUG 66554T = LMG 28586T = INCQS 0733T). PMID:26358475

Modulation of Roquin function in myeloid cells reduces Mycobacterium tuberculosis induced inflammation

PubMed Central

Nagalingam, Gayathri; Vinuesa, Carola G.; Britton, Warwick J; Saunders, Bernadette M.

2017-01-01

Damaging inflammation is a hallmark of Mycobacterium tuberculosis infection, and understanding how this is regulated is important for the development of new therapies to limit excessive inflammation. The E3 ubiquitin ligase, Roquin, is involved in immune regulation, however its role in immunity to M. tuberculosis is unknown. To address this we infected mice with a point mutation in Roquin1/Rc3h1 (sanroque). Aerosol-infected sanroque mice showed enhanced control of M. tuberculosis infection associated with delayed bacterial dissemination and upregulated TNF production in the lung after 2 weeks. However, this early control of infection was not maintained, and by 8 weeks post-infection sanroque mice demonstrated increased bacterial burden and dysregulated inflammation in the lung. As the inflammation in the lung of the sanroque mice could have been influenced by emerging autoimmune conditions that are characteristic of aging sanroque mice, the function of Roquin was examined in immune cell subsets in the absence of autoimmune complications. Mycobacterium bovis BCG-primed sanroque T cells transferred into Rag1-/- mice provided equivalent protection in the spleen and liver. Interestingly, the transfer of mycobacteria-specific (P25 CD4+ TCR transgenic) wild-type spleen cells into sanroque.Rag1-/- mice actually led to enhanced protection with reduced bacterial load, decreased chemokine expression and reduced inflammation in the lung compared with transfers into Rag1-/- mice expressing intact Roquin. These studies suggest that modulation of Roquin in myeloid cells may reduce both inflammation and bacterial growth during the chronic phase of M. tuberculosis infection. PMID:28747346

In vitro activities of levofloxacin used alone and in combination with first- and second-line antituberculous drugs against Mycobacterium tuberculosis.

PubMed Central

Rastogi, N; Goh, K S; Bryskier, A; Devallois, A

1996-01-01

By using the radiometric BACTEC 460-TB methodology, the inhibitory and bactericidal activity of the optically active L-isomer of ofloxacin (levofloxacin) was compared with those of the D-isomer and the commercially available mixture containing equal amounts of DL-isomers (ofloxacin) against the Mycobacterium tuberculosis complex (type strain H37Rv, a panel of drug-susceptible and -resistant clinical isolates including multidrug-resistant isolates of M. tuberculosis, as well as M. africanum, M. bovis, and M. bovis BCG). Levofloxacin MICs (range 0.50 to 0.75 microgram/ml) were about 1 dilution lower than those of ofloxacin (MIC range, 0.75 to 1.00 microgram/ml) and 5 to 6 dilutions lower than those of the D-isomer (MIC range, 32 to 60 micrograms/ml). The MICs of levofloxacin, ofloxacin, and D-ofloxacin at which 90% of the strains are inhibited were 0.50, 1.00, and 64 micrograms/ml, respectively. The multidrug-resistant M. tuberculosis strains resistant to first-line drugs were as susceptible to quinolones as the wild-type drug-susceptible isolates. Levofloxacin at 0.5 microgram/ml showed bactericidal activity comparable to the activities of 1.0 microgram of ofloxacin per ml and 64 micrograms of D-ofloxacin per ml, with MBCs within the range of 0.5 to 2.0 micrograms/ml, compared with MBCs of 0.75 to 4.0 micrograms of ofloxacin per ml for M. tuberculosis, M. africanum, M. bovis BCG. Combination testing of sub-MICs of levolofoxacin with other first-line (isoniazid, rifampin, and ethambutol) and second-line (amikacin and clofazimine) antituberculous drugs was evaluated with various two-, three-, and four-drug combinations; enhanced drug activity was observed in 8 of 25, 12 of 20, and 8 of 15 tests, respectively, indicating that levofloxacin acts in synergy with other antituberculous drugs. PMID:8807049

Descriptive epidemiology of Moraxella bovis, Moraxella bovoculi and Moraxella ovis in beef calves with naturally occurring infectious bovine keratoconjunctivitis (Pinkeye).

PubMed

O'Connor, A M; Shen, H G; Wang, C; Opriessnig, T

2012-03-23

Infectious bovine keratoconjunctivitis (IBK) is an ocular disease that causes substantial weight loss in beef calves. The objective of this study was to evaluate the association between Moraxella bovoculi, Moraxella bovis and Moraxella ovis and IBK incidence. A cohort design was used. From 239 calves and 478 eyes, 77 randomly chosen eyes were monitored for M. bovoculi, M. bovis, M. ovis and IBK incidence over 4 months. One hypothesis tested was that IBK hazard in eyes was not associated with detection of M. bovoculi, M. bovis and M. ovis. A secondary hypothesis tested that IBK cases were not associated with increased prevalence of M. bovoculi, M. bovis and M. ovis. 23% of 77 eyes developed IBK. M. ovis was identified in one IBK-negative eye. The adjusted hazard ratio (HR) for IBK incidence from eyes where M. bovoculi or M. bovis were recovered prior to disease occurrence were not statistically significant (M. bovoculi HR=1.38, 95% CI: 0.54-3.53, p=0.49, M. bovis HR=1.60, 95% CI: 0.48-5.53, p=0.44). The adjusted hazard ratio for M. bovoculi in IBK lesions was 6.45 (95% CI: 3.35-12.44, p<0.001). The adjusted hazard ratio for M. bovis in IBK lesions was 2.33 (95% CI: 1.22-4.45, p=0.01). A temporal association between prior exposure to M. bovoculi or M. bovis and subsequent IBK incidence was not demonstrated. However, M. bovoculi and M. bovis are more frequently recovered from eyes with IBK lesions than unaffected eyes and this provides weak evidence for a causal role. Copyright © 2011 Elsevier B.V. All rights reserved.

The susceptibility of bovine udder quarters colonized with Corynebacterium bovis to experimental infection with Staphylococcus aureus or Streptococcus agalactiae.

PubMed Central

Brooks, B W; Barnum, D A

1984-01-01

Twenty bovine udder quarters colonized with Corynebacterium bovis SR6 and 20 uncolonized quarters were challenged by inoculation of Staphylococcus aureus Newbould 305 (ATCC 29740) into the teat cistern. The percentage of infection in quarters colonized with C. bovis (50%) was significantly lower than that in controls (100%). By similar challenge no significant difference was observed between the percentage of infection with Streptococcus agalactiae ATCC 27956 in 33 quarters colonized with C. bovis (70%) compared to 33 controls (87.9%). A total of 37 quarters colonized with C. bovis and 37 control quarters were challenged with Staph. aureus Newbould 305 (ATCC 29740) and Maxi (ATCC 27543) and Strep. agalactiae (ATCC 27956) by exposure of the teat orifice. The percentage of teat ducts colonized with C. bovis which became infected with either pathogen was not different from that for controls. PMID:6372969

Mycobacterium tuberculosis Maltosyltransferase GlgE, a Genetically Validated Antituberculosis Target, Is Negatively Regulated by Ser/Thr Phosphorylation*

PubMed Central

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-01-01

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette–Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically. PMID:23609448

Mycobacterium tuberculosis maltosyltransferase GlgE, a genetically validated antituberculosis target, is negatively regulated by Ser/Thr phosphorylation.

PubMed

Leiba, Jade; Syson, Karl; Baronian, Grégory; Zanella-Cléon, Isabelle; Kalscheuer, Rainer; Kremer, Laurent; Bornemann, Stephen; Molle, Virginie

2013-06-07

GlgE is a maltosyltransferase involved in the biosynthesis of α-glucans that has been genetically validated as a potential therapeutic target against Mycobacterium tuberculosis. Despite also making α-glucan, the GlgC/GlgA glycogen pathway is distinct and allosterically regulated. We have used a combination of genetics and biochemistry to establish how the GlgE pathway is regulated. M. tuberculosis GlgE was phosphorylated specifically by the Ser/Thr protein kinase PknB in vitro on one serine and six threonine residues. Furthermore, GlgE was phosphorylated in vivo when expressed in Mycobacterium bovis bacillus Calmette-Guérin (BCG) but not when all seven phosphorylation sites were replaced by Ala residues. The GlgE orthologues from Mycobacterium smegmatis and Streptomyces coelicolor were phosphorylated by the corresponding PknB orthologues in vitro, implying that the phosphorylation of GlgE is widespread among actinomycetes. PknB-dependent phosphorylation of GlgE led to a 2 orders of magnitude reduction in catalytic efficiency in vitro. The activities of phosphoablative and phosphomimetic GlgE derivatives, where each phosphorylation site was substituted with either Ala or Asp residues, respectively, correlated with negative phosphoregulation. Complementation studies of a M. smegmatis glgE mutant strain with these GlgE derivatives, together with both classical and chemical forward genetics, were consistent with flux through the GlgE pathway being correlated with GlgE activity. We conclude that the GlgE pathway appears to be negatively regulated in actinomycetes through the phosphorylation of GlgE by PknB, a mechanism distinct from that known in the classical glycogen pathway. Thus, these findings open new opportunities to target the GlgE pathway therapeutically.

Risk of Mycoplasma bovis transmission from contaminated sand bedding to naive dairy calves.

PubMed

Wilson, D J; Justice-Allen, A; Goodell, G; Baldwin, T J; Skirpstunas, R T; Cavender, K B

2011-03-01

The objective of this study was to evaluate the possible transmission of Mycoplasma bovis from positive sand bedding to naïve dairy calves. Twelve preweaned Holstein bull calves were blocked in pairs and randomly assigned as unexposed controls (n=6) bedded with control sand, or exposed calves (n=6) bedded with sand previously positive for M. bovis at a dairy farm. Bedding sand was cultured weekly. Nasal and ear swabs and sera were collected weekly, tracheal swabs were collected monthly, and by the end of the 105-d study, all calves were euthanized (n=10) or died (n=2). Sera were tested for M. bovis-specific antibody. Mycoplasma spp. culture was performed on nasal and ear swabs; culture and a PCR differentiating multiple Mycoplasma spp. were performed on postmortem samples of lung, retropharyngeal lymph node, and trachea from each calf. A complete necropsy also was performed. During 6 wk, mycoplasma concentration in exposed group sand was between 200 and 32,000 cfu/g. All 166 tracheal swabs, nasal and ear swabs, and postmortem tests from all calves were negative for mycoplasma. All 94 sera were negative for M. bovis-specific antibody. No gross pathology suggestive of mycoplasma disease was detected. The probability of mycoplasma detection, if an exposed calf had become infected 4 wk after exposure, ranged between 97 and 99% depending on time of exposure for individual calves. There was no evidence that sand bedding contaminated with M. bovis might serve as a source of transmission to naïve dairy calves. Copyright © 2011 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.

Mycoplasma bovis NADH oxidase functions as both a NADH oxidizing and O2 reducing enzyme and an adhesin.

PubMed

Zhao, Gang; Zhang, Hui; Chen, Xi; Zhu, Xifang; Guo, Yusi; He, Chenfei; Anwar Khan, Farhan; Chen, Yingyu; Hu, Changmin; Chen, Huanchun; Guo, Aizhen

2017-03-03

Mycoplasma bovis causes considerable economic losses in the cattle industry worldwide. In mycoplasmal infections, adhesion to the host cell is of the utmost importance. In this study, the amino acid sequence of NOX was predicted to have enzymatic domains. The nox gene was then cloned and expressed in Escherichia coli. The enzymatic activity of recombinant NOX (rNOX) was confirmed based on its capacity to oxidize NADH to NAD + and reduce O 2 to H 2 O 2 . The adherence of rNOX to embryonic bovine lung (EBL) cells was confirmed with confocal laser scanning microscopy, enzyme-linked immunosorbent assay, and flow cytometry. Both preblocking EBL cells with purified rNOX and preneutralizing M. bovis with polyclonal antiserum to rNOX significantly reduced the adherence of M. bovis to EBL cells. Mycoplasma bovis NOX- expressed a truncated NOX protein at a level 10-fold less than that of the wild type. The capacities of M. bovis NOX- for cell adhesion and H 2 O 2 production were also significantly reduced. The rNOX was further used to pan phage displaying lung cDNA library and fibronectin was determined to be potential ligand. In conclusion, M. bovis NOX functions as both an active NADH oxidase and adhesin, and is therefore a potential virulence factor.

MYCOBACTERIUM AVIUM AND DRINKING WATER WHAT ARE THE CONNECTIONS?

EPA Science Inventory

Background: Human Mycobacterium avium infections are only known to be acquired from environmental sources such as water and soil. We compared M. avium isolates from clinical and drinking water sources using molecular tools. Methods: M. avium was isolated from water samples colle...

Interaction of Mycobacterium tuberculosis with human respiratory mucosa.

PubMed

Middleton, A M; Chadwick, M V; Nicholson, A G; Dewar, A; Groger, R K; Brown, E J; Ratliff, T L; Wilson, R

2002-01-01

Endobronchial infection is associated with pulmonary tuberculosis in the majority of cases. We have investigated the adherence of Mycobacterium tuberculosis to the human respiratory mucosa. Organ cultures constructed with human tissue were infected with M. tuberculosis in the presence or absence of mycobacterial fibronectin attachment cell surface proteins and examined by scanning electron microscopy. M. tuberculosis adhered mainly to extracellular matrix (ECM) in areas of mucosal damage, but not to ciliated mucosa, intact extruded cells, basement membrane or collagen fibres. Bacteria also adhered to fibrous but not globular mucus and occasionally to healthy unciliated mucosa, open tight junctions and to extruded cells that had degenerated, exposing their contents. There was a significant reduction (p<0.05) in the number of bacteria adhering to ECM after pre-incubation of bacteria with fibronectin and after pre-incubation of the tissue with M. avium fibronectin attachment protein (FAP) and M. bovis antigen 85B protein, in a concentration dependent manner. The combined effect of FAP and antigen 85B protein was significantly greater than either protein alone. Bacterial adherence to fibrous mucus was not influenced by fibronectin. We conclude that M. tuberculosis adheres to ECM in areas of mucosal damage at least in part via FAP and antigen 85B protein.

Detection of Corynebacterium bovis infection in athymic nude mice from a research animal facility in Korea.

PubMed

Kim, Tae-Hyoun; Kim, Dong-Su; Han, Ju-Hee; Chang, Seo-Na; Kim, Kyung-Sul; Seok, Seung-Hyeok; Kim, Dong-Jae; Park, Jong-Hwan; Park, Jae-Hak

2014-12-01

Corynebacterium (C.) bovis infection in nude mice causes hyperkeratosis and weight loss and has been reported worldwide but not in Korea. In 2011, nude mice from an animal facility in Korea were found to have white flakes on their dorsal skin. Histopathological testing revealed that the mice had hyperkeratosis and Gram-positive bacteria were found in the skin. We identified isolated bacteria from the skin lesions as C. bovis using PCR and 16S rRNA sequencing. To the best of our knowledge, this is the first report of C. bovis infection in nude mice from Korea.

Whole-genome sequence analysis of the Mycobacterium avium complex and proposal of the transfer of Mycobacterium yongonense to Mycobacterium intracellulare subsp. yongonense subsp. nov.

PubMed

Castejon, Maria; Menéndez, Maria Carmen; Comas, Iñaki; Vicente, Ana; Garcia, Maria J

2018-06-01

Bacterial whole-genome sequences contain informative features of their evolutionary pathways. Comparison of whole-genome sequences have become the method of choice for classification of prokaryotes, thus allowing the identification of bacteria from an evolutionary perspective, and providing data to resolve some current controversies. Currently, controversy exists about the assignment of members of the Mycobacterium avium complex, as is for the cases of Mycobacterium yongonense and 'Mycobacterium indicus pranii'. These two mycobacteria, closely related to Mycobacterium intracellulare on the basis of standard phenotypic and single gene-sequences comparisons, were not considered a member of such species on the basis on some particular differences displayed by a single strain. Whole-genome sequence comparison procedures, namely the average nucleotide identity and the genome distance, showed that those two mycobacteria should be considered members of the species M. intracellulare. The results were confirmed with other whole-genome comparison supplementary methods. According to the data provided, Mycobacterium yongonense and 'Mycobacterium indicus pranii' should be considered and renamed and included as members of M. intracellulare. This study highlights the problems caused when a novel species is accepted on the basis of a single strain, as was the case for M. yongonense. Based mainly on whole-genome sequence analysis, we conclude that M. yongonense should be reclassified as a subspecies of Mycobacterium intracellulareas Mycobacterium intracellularesubsp. yongonense and 'Mycobacterium indicus pranii' classified in the same subspecies as the type strain of Mycobacterium intracellulare and classified as Mycobacterium intracellularesubsp. intracellulare.

Detection of Babesia bovis carrier cattle by using polymerase chain reaction amplification of parasite DNA.

PubMed Central

Fahrimal, Y; Goff, W L; Jasmer, D P

1992-01-01

Carrier cattle infected with Babesia bovis are difficult to detect because of the low numbers of parasites that occur in peripheral blood. However, diagnosis of low-level infections with the parasite is important for evaluating the efficacies of vaccines and in transmission and epidemiological studies. We used the polymerase chain reaction (PCR) to amplify a portion of the apocytochrome b gene from the parasite and tested the ability of this method to detect carrier cattle. The target sequence is associated with a 7.4-kb DNA element in undigested B. bovis genomic DNA (as shown previously), and the amplified product was detected by Southern and dot blot hybridization. The assay was specific for B. bovis, since no amplification was detected with Babesia bigemina, Trypanosoma brucei, Anaplasma marginale, or leukocyte DNA. The target sequence was amplified in DNA from B. bovis Mexico, Texas, and Australia S and L strains, demonstrating the applicability of the method to strains from different geographic regions. The sensitivity of the method ranged from 1 to 10 infected erythrocytes extracted from 0.5 ml of blood. This sensitivity was about 1,000 times greater than that from the use of unamplified parasite DNA. By the PCR method, six B. bovis carrier cattle were detected 86% of the time (range, 66 to 100%) when they were tested 11 times, while with microscopic examination of thick blood smears, the same carrier cattle were detected only 36% of the time (range, 17 to 66%). The method provides a useful diagnostic tool for detecting B. bovis carrier cattle, and the sensitivity is significantly improved over that of current methods. The results also suggest that characteristics of the apocytchrome b gene may make this a valuable target DNA for PCR-based detection of other hemoparasites. Images PMID:1624551

Gut transcriptome of replete adult female cattle ticks, Rhipicephalus (Boophilus) microplus, feeding upon a Babesia bovis-infected bovine host.

PubMed

Heekin, Andrew M; Guerrero, Felix D; Bendele, Kylie G; Saldivar, Leo; Scoles, Glen A; Dowd, Scot E; Gondro, Cedric; Nene, Vishvanath; Djikeng, Appolinaire; Brayton, Kelly A

2013-09-01

As it feeds upon cattle, Rhipicephalus (Boophilus) microplus is capable of transmitting a number of pathogenic organisms, including the apicomplexan hemoparasite Babesia bovis, a causative agent of bovine babesiosis. The R. microplus female gut transcriptome was studied for two cohorts: adult females feeding on a bovine host infected with B. bovis and adult females feeding on an uninfected bovine. RNA was purified and used to generate a subtracted cDNA library from B. bovis-infected female gut, and 4,077 expressed sequence tags (ESTs) were sequenced. Gene expression was also measured by a microarray designed from the publicly available R. microplus gene index: BmiGI Version 2. We compared gene expression in the tick gut from females feeding upon an uninfected bovine to gene expression in tick gut from females feeding upon a splenectomized bovine infected with B. bovis. Thirty-three ESTs represented on the microarray were expressed at a higher level in female gut samples from the ticks feeding upon a B. bovis-infected calf compared to expression levels in female gut samples from ticks feeding on an uninfected calf. Forty-three transcripts were expressed at a lower level in the ticks feeding upon B. bovis-infected female guts compared with expression in female gut samples from ticks feeding on the uninfected calf. These array data were used as initial characterization of gene expression associated with the infection of R. microplus by B. bovis.

Identification of specific metabolites in culture supernatant of Mycobacterium tuberculosis using metabolomics: exploration of potential biomarkers

PubMed Central

Lau, Susanna KP; Lam, Ching-Wan; Curreem, Shirly OT; Lee, Kim-Chung; Lau, Candy CY; Chow, Wang-Ngai; Ngan, Antonio HY; To, Kelvin KW; Chan, Jasper FW; Hung, Ivan FN; Yam, Wing-Cheong; Yuen, Kwok-Yung; Woo, Patrick CY

2015-01-01

Although previous studies have reported the use of metabolomics for Mycobacterium species differentiation, little is known about the potential of extracellular metabolites of Mycobacterium tuberculosis (MTB) as specific biomarkers. Using an optimized ultrahigh performance liquid chromatography–electrospray ionization–quadruple time of flight–mass spectrometry (UHPLC–ESI–Q–TOF–MS) platform, we characterized the extracellular metabolomes of culture supernatant of nine MTB strains and nine non-tuberculous Mycobacterium (NTM) strains (four M. avium complex, one M. bovis Bacillus Calmette–Guérin (BCG), one M. chelonae, one M. fortuitum and two M. kansasii). Principal component analysis readily distinguished the metabolomes between MTB and NTM. Using multivariate and univariate analysis, 24 metabolites with significantly higher levels in MTB were identified. While seven metabolites were identified by tandem mass spectrometry (MS/MS), the other 17 metabolites were unidentified by MS/MS against database matching, suggesting that they may be potentially novel compounds. One metabolite was identified as dexpanthenol, the alcohol analog of pantothenic acid (vitamin B5), which was not known to be produced by bacteria previously. Four metabolites were identified as 1-tuberculosinyladenosine (1-TbAd), a product of the virulence-associated enzyme Rv3378c, and three previously undescribed derivatives of 1-TbAd. Two derivatives differ from 1-TbAd by the ribose group of the nucleoside while the other likely differs by the base. The remaining two metabolites were identified as a tetrapeptide, Val-His-Glu-His, and a monoacylglycerophosphoglycerol, phosphatidylglycerol (PG) (16∶0/0∶0), respectively. Further studies on the chemical structure and biosynthetic pathway of these MTB-specific metabolites would help understand their biological functions. Studies on clinical samples from tuberculosis patients are required to explore for their potential role as diagnostic

Acquired resistance to the 16-membered macrolides tylosin and tilmicosin by Mycoplasma bovis.

PubMed

Lerner, Uri; Amram, Eytan; Ayling, Roger D; Mikula, Inna; Gerchman, Irena; Harrus, Shimon; Teff, Dina; Yogev, David; Lysnyansky, Inna

2014-01-31

The molecular mechanism of acquired resistance to the 16-membered macrolides tylosin (Ty) and tilmicosin (Tm) was investigated in Mycoplasma bovis field isolates. Sequence analysis of domains II and V of the two 23S rRNA alleles and ribosomal proteins L4 and L22 was performed on 54 M. bovis isolates showing different minimal inhibitory concentrations (MIC). The presence of any one of the point mutations G748A, C752T, A2058G, A2059G or A2059C (Escherichia coli numbering) in one or both alleles of the 23S rRNAs was correlated with decreased susceptibility to Ty (8-1024 μg/ml) and to Tm (32 to >256 μg/ml) in 27/27 and 27/31 M. bovis isolates, respectively. Although a single mutation in domain II or V could be sufficient to cause decreased susceptibility to Ty, our data imply that a combination of mutations in two domains is necessary to achieve higher MICs (≥ 128 μg/ml). The influence of a combination of mutations in two domains II and V on enhancement of resistance to Tm was less clear. In addition, the amino acid (aa) substitution L22-Q90H was found in 24/32 representative M. bovis isolates with different MICs, but no correlation with decreased susceptibility to Ty or Tm was identified. Multiple aa substitutions were also identified in the L4 protein, including at positions 185-186 (positions 64 and 65 in E. coli) which are adjacent to the macrolide-binding site. This is the first description of the molecular mechanism of acquired resistance to the 16-membered macrolides in M. bovis. Copyright © 2013 Elsevier B.V. All rights reserved.

A novel screening method based on menadione mediated rapid reduction of tetrazolium salt for testing of anti-mycobacterial agents.

PubMed

Singh, Upasana; Akhtar, Shamim; Mishra, Abhishek; Sarkar, Dhiman

2011-02-01

A microplate-based rapid, inexpensive and robust technique is developed by using tetrazolium salt 2,3-bis[2-methyloxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide (XTT) and menadione to determine the viability of Mycobacterium tuberculosis, Mycobacterium bovis BCG and Mycobacterium smegmatis bacilli in microplate format. In general, XTT reduction is an extremely slow process which takes almost 24 h to produce a detectable signal. Menadione could drastically induce this reduction to an almost equal extent within a few minutes in a dose dependent manner. The reduction of XTT is directly proportional to the cell concentration in the presence of menadione. The standardized protocol used 200 μM of XTT and 60 μM of menadione in 250 μl of cell suspension grown either in aerobic or anaerobic conditions. The cell suspension of M. bovis BCG and M. tuberculosis were incubated for 40 min before reading the optical density at 470 nm whereas M. smegmatis was incubated for 20 min. Calculated Signal/Noise (S/N) ratios obtained by applying this protocol were 5.4, 6.4 and 9.4 using M. bovis BCG, M. tuberculosis and M. smegmatis respectively. The calculated Z' factors were >0.8 for all mycobacterium bacilli indicating the robustness of the XTT Reduction Menadione Assay (XRMA) for rapid screening of inhibitors. The assay protocol was validated by applying 10 standard anti-tubercular agents on M. tuberculosis, M. bovis BCG and M. smegmatis. The Minimum Inhibitory Concentration (MIC) values were found to be similar to reported values from Colony Forming Unit (CFU) and REMA (resazurin microplate assay) assays. Altogether, XRMA is providing a novel anti-tubercular screening protocol which could be useful in high throughput screening programs against different physiological stages of the bacilli. Copyright © 2010 Elsevier B.V. All rights reserved.

Bovine tuberculosis at the human-livestock-wildlife interface: is it a public health problem in Tanzania? A review.

PubMed

Katale, Bugwesa Z; Mbugi, Erasto V; Kendal, Sharon; Fyumagwa, Robert D; Kibiki, Gibson S; Godfrey-Faussett, Peter; Keyyu, Julius D; Van Helden, Paul; Matee, Mecky I

2012-06-20

Despite the apparent public health concern about Bovine tuberculosis (BTB) in Tanzania, little has been done regarding the zoonotic importance of the disease and raising awareness of the community to prevent the disease. Bovine tuberculosis is a potential zoonotic disease that can infect a variety of hosts, including humans. The presence of multiple hosts including wild animals, inefficient diagnostic techniques, absence of defined national controls and eradication programs could impede the control of bovine TB. In Tanzania, the diagnosis of Mycobacterium bovis in animals is mostly carried out by tuberculin skin testing, meat inspection in abattoirs and only rarely using bacteriological techniques. The estimated prevalence of BTB in animals in Tanzania varies and ranges across regions from 0.2% to 13.3%, which is likely to be an underestimate if not confirmed by bacteriology or molecular techniques. Mycobacterium bovis has been detected and isolated from different animal species and has been recovered in 10% of apparently healthy wildebeest that did not show lesions at post-mortem. The transmission of the disease from animals to humans can occur directly through the aerosol route and indirectly by consumption of raw milk. This poses an emerging disease threat in the current era of HIV confection in Tanzania and elsewhere. Mycobacterium bovis is one of the causative agents of human extra pulmonary tuberculosis. In Tanzania there was a significant increase (116.6%) of extrapulmonary cases reported between 1995 and 2009, suggesting the possibility of widespread M. bovis and Mycobacterium tuberculosis infection due to general rise of Human Immunodeficiency virus (HIV). This paper aims to review the potential health and economic impact of bovine tuberculosis and challenges to its control in order to safeguard human and animal population in Tanzania.

Mycobacterium bovis BCG promotes tumor cell survival from tumor necrosis factor-α-induced apoptosis.

PubMed

Holla, Sahana; Ghorpade, Devram Sampat; Singh, Vikas; Bansal, Kushagra; Balaji, Kithiganahalli Narayanaswamy

2014-09-11

Increased incidence of lung cancer among pulmonary tuberculosis patients suggests mycobacteria-induced tumorigenic response in the host. The alveolar epithelial cells, candidate cells that form lung adenocarcinoma, constitute a niche for mycobacterial replication and infection. We thus explored the possible mechanism of M. bovis Bacillus Calmette-Guérin (BCG)-assisted tumorigenicity in type II epithelial cells, human lung adenocarcinoma A549 and other cancer cells. Cancer cell lines originating from lung, colon, bladder, liver, breast, skin and cervix were treated with tumor necrosis factor (TNF)-α in presence or absence of BCG infection. p53, COP1 and sonic hedgehog (SHH) signaling markers were determined by immunoblotting and luciferase assays, and quantitative real time PCR was done for p53-responsive pro-apoptotic genes and SHH signaling markers. MTT assays and Annexin V staining were utilized to study apoptosis. Gain- and loss-of-function approaches were used to investigate the role for SHH and COP1 signaling during apoptosis. A549 xenografted mice were used to validate the contribution of BCG during TNF-α treatment. Here, we show that BCG inhibits TNF-α-mediated apoptosis in A549 cells via downregulation of p53 expression. Substantiating this observation, BCG rescued A549 xenografts from TNF-α-mediated tumor clearance in nude mice. Furthermore, activation of SHH signaling by BCG induced the expression of an E3 ubiquitin ligase, COP1. SHH-driven COP1 targeted p53, thereby facilitating downregulation of p53-responsive pro-apoptotic genes and inhibition of apoptosis. Similar effects of BCG could be shown for HCT116, T24, MNT-1, HepG2 and HELA cells but not for HCT116 p53(-/-) and MDA-MB-231 cells. Our results not only highlight possible explanations for the coexistence of pulmonary tuberculosis and lung cancer but also address probable reasons for failure of BCG immunotherapy of cancers.

Detection of Mycobacterium bovis in Organs of Slaughtered Cattle by DNA-Based Polymerase Chain Reaction and Ziehl-Neelsen Techniques in Bauchi State, Nigeria

PubMed Central

Sa'idu, A. S.; Okolocha, E. C.; Dzikwi, A. A.; Kwaga, J. K. P.; Gamawa, A. A.; Usman, A.; Maigari, S. A.; Ibrahim, S.

2015-01-01

Bovine tuberculosis is a chronic, infectious, and contagious zoonotic disease of domestic animals, wild animals, and humans. It also poses a public health threat and economic losses. This study was aimed at determining the prevalence of bovine tuberculosis in slaughtered cattle, based on PM meat inspection, Ziehl-Neelsen staining, and PCR techniques in Bauchi State, Nigeria. A Prospective study was conducted on 800 cattle slaughtered in the three Zonal abattoirs of Bauchi State, Nigeria. One hundred and twenty (15%) tissues from different organs had suspected bTB lesions at PM. Out of the samples examined 35 (29.2%) were AFB positive by ZN and 10 (8.3%) were confirmed positive for M. bovis by PCR, with an overall prevalence of 29.16% and 8.33%, respectively. Female had a higher prevalence rate than male cattle at 16.66% and 12.5 % by ZN and 5.00% and 3.33% by PCR, respectively (P>0.05, χ 2 = 0.218). However, there was a statistically significant association (P<0.05, χ 2 = 7.002) between detection of bTB and the age of cattle. ZN revealed that cattle aged 6 years and above had the highest number of positive bTB cases 67.9%, while cattle aged 3–5 years had the lowest 14.81%. PCR technique revealed that the cattle aged 6 and above years also had the highest percentage positive M. bovis cases of 22.84%, whereas cattle aged 3–5 years had the lowest and the overall prevalence rate of 8.33%. The study found a high infection rate of bTB among cattle and majority of the lesions 54.2% were from lungs. The prevalence of bTB was higher in Bauchi metropolitan abattoir which supplies larger population of the state with beef. PMID:26464955

Disinfective process of strongly acidic electrolyzed product of sodium chloride solution against Mycobacteria.

PubMed

Yamamoto, Tomoyo Matsushita; Nakano, Takashi; Yamaguchi, Masaki; Shimizu, Mitsuhide; Wu, Hong; Aoki, Hiroaki; Ota, Rie; Kobayashi, Toyohide; Sano, Kouichi

2012-12-01

Electrolyzed acid water (EAW) has been studied for its disinfective potential against pathogenic microbes; however, the bactericidal process against Mycobacteria has not been clearly presented. In this study, to clarify the disinfective process against Mycobacteria, EAW-treated bacteria were examined against laboratory strains of Mycobacterium bovis (M. bovis), Mycobacterium smegmatis (M. smegmatis), and Mycobacterium terrae (M. terrae) by recovery culture and observation of morphology, enzymatic assay, and the detection of DNA. All experiments were performed with the use of EAW containing 30 ppm free chlorine that kills Mycobacteria, including three pathogenic clinical isolates of Mycobacterium tuberculosis (M. tuberculosis) and six isolates of other Mycobacteria, within 5 min. In morphology, the bacterial surface became rough, and a longitudinal concavity-like structure appeared. The intrabacterial enzyme of EAW-contacted bacteria was inactivated, but chromosomal DNA was not totally denatured. These results suggest that the bactericidal effect of EAW against Mycobacteria occurs by degradation of the cell wall, followed by denaturation of cytoplasmic proteins, but degeneration of the nucleic acid is not always necessary.

Differential Expression of Three Members of the Multidomain Adhesion CCp Family in Babesia bigemina, Babesia bovis and Theileria equi

PubMed Central

Bastos, Reginaldo G.; Suarez, Carlos E.; Laughery, Jacob M.; Johnson, Wendell C.; Ueti, Massaro W.; Knowles, Donald P.

2013-01-01

Members of the CCp protein family have been previously described to be expressed on gametocytes of apicomplexan Plasmodium parasites. Knocking out Plasmodium CCp genes blocks the development of the parasite in the mosquito vector, making the CCp proteins potential targets for the development of a transmission-blocking vaccine. Apicomplexans Babesia bovis and Babesia bigemina are the causative agents of bovine babesiosis, and apicomplexan Theileria equi causes equine piroplasmosis. Bovine babesiosis and equine piroplasmosis are the most economically important parasite diseases that affect worldwide cattle and equine industries, respectively. The recent sequencing of the B. bovis and T. equi genomes has provided the opportunity to identify novel genes involved in parasite biology. Here we characterize three members of the CCp family, named CCp1, CCp2 and CCp3, in B. bigemina, B. bovis and T. equi. Using B. bigemina as an in vitro model, expression of all three CCp genes and proteins was demonstrated in temperature-induced sexual stages. Transcripts for all three CCp genes were found in vivo in blood stages of T. equi, and transcripts for CCp3 were detected in vivo in blood stages of B. bovis. However, no protein expression was detected in T. equi blood stages or B. bovis blood stages or B. bovis tick stages. Collectively, the data demonstrated a differential pattern of expression of three orthologous genes of the multidomain adhesion CCp family by B. bigemina, B. bovis and T. equi. The novel CCp members represent potential targets for innovative approaches to control bovine babesiosis and equine piroplasmosis. PMID:23844089

Immune Responses to Bacillus Calmette–Guérin Vaccination: Why Do They Fail to Protect against Mycobacterium tuberculosis?

PubMed Central

Moliva, Juan I.; Turner, Joanne; Torrelles, Jordi B.

2017-01-01

Mycobacterium tuberculosis (M.tb), the causative agent of tuberculosis (TB), is the current leading cause of death due to a single infectious organism. Although curable, the broad emergence of multi-, extensive-, extreme-, and total-drug resistant strains of M.tb has hindered eradication efforts of this pathogen. Furthermore, computational models predict a quarter of the world’s population is infected with M.tb in a latent state, effectively serving as the largest reservoir for any human pathogen with the ability to cause significant morbidity and mortality. The World Health Organization has prioritized new strategies for improved vaccination programs; however, the lack of understanding of mycobacterial immunity has made it difficult to develop new successful vaccines. Currently, Mycobacterium bovis bacillus Calmette–Guérin (BCG) is the only vaccine approved for use to prevent TB. BCG is highly efficacious at preventing meningeal and miliary TB, but is at best 60% effective against the development of pulmonary TB in adults and wanes as we age. In this review, we provide a detailed summary on the innate immune response of macrophages, dendritic cells, and neutrophils in response to BCG vaccination. Additionally, we discuss adaptive immune responses generated by BCG vaccination, emphasizing their specific contributions to mycobacterial immunity. The success of future vaccines against TB will directly depend on our understanding of mycobacterial immunity. PMID:28424703

Limitations of Using IL-17A and IFN-γ-Induced Protein 10 to Detect Bovine Tuberculosis

PubMed Central

Xin, Ting; Gao, Xintao; Yang, Hongjun; Li, Pingjun; Liang, Qianqian; Hou, Shaohua; Sui, Xiukun; Guo, Xiaoyu; Yuan, Weifeng; Zhu, Hongfei; Ding, Jiabo; Jia, Hong

2018-01-01

Bovine tuberculosis (bTB) is primarily caused by infection with Mycobacterium bovis, which belongs to the Mycobacterium tuberculosis complex. The airborne route is considered the most common for transmission of M. bovis, and more than 15% of cattle with bTB shed the Mycobacterium, which can be detect by nested PCR to amplify mycobacterial mpb70 from a nasal swab from a cow. To screen for cytokines fostering early and accurate detection of bTB, peripheral blood mononuclear cells were isolated from naturally M. bovis-infected, experimentally M. bovis 68002-infected, and uninfected cattle, then these cells were stimulated by PPD-B, CFP-10-ESAT-6 (CE), or phosphate-buffered saline (PBS) for 6 h. The levels of interferon gamma (IFN-γ), IFN-γ-induced protein 10 (IP-10), IL-6, IL-12, IL-17A, and tumor necrosis factor alpha mRNA were measured using real-time PCR. To explore the cytokines associated with different periods of M. bovis infection, cattle were divided into three groups: PCR-positive, PCR-negative, and uninfected using the tuberculin skin test, CFP-10/ESAT-6/TB10.4 protein cocktail-based skin test, IFN-γ release assay (IGRA), CFP-10/ESAT-6 (CE)-based IGRA, and nested PCR. The expression of IP-10, IL-17A, and IFN-γ proteins induced by PPD-B, CE, or PBS was detected by ELISA. The results showed that levels of PPD-B-stimulated IL-17A and IP-10 (mRNA and protein), and CE-induced IP-10 (mRNA and protein) were significantly higher in cattle naturally or experimentally infected with M. bovis than in those that were uninfected. The levels of PPD-B- or CE-induced IL-17A and IP-10 (protein) could be used to differentiate M. bovis-infected calves from uninfected ones for 6 to 30 weeks post-infection, whereas PPD-B- and CE-induced IP-10 and IL-17A mRNA expression could be used to differentiate M. bovis-infected calves from uninfected ones between 6 and 58 weeks post-infection. However, CE-induced IL-17A (protein) was not a reliable indicator of M. bovis infection

A dominant lineage of Mycoplasma bovis is associated with an increased number of severe mastitis cases in cattle.

PubMed

Bürki, Sibylle; Spergser, Joachim; Bodmer, Michèle; Pilo, Paola

2016-11-30

Mycoplasma bovis is the most frequent etiologic agent of bovine mycoplasmosis. It causes various diseases in bovines and considerable economic loss due to the lack of effective treatment or preventive measures such as vaccination. In contrast to the US, where M. bovis-mastitis has been reported for a long time, M. bovis infections in Switzerland and Austria were predominantly associated with pneumonia and subclinical mastitis. However, since 2007 the situation has changed with the emergence of severe M. bovis-associated mastitis cases in both countries. In order to evaluate the molecular epidemiology of the bacteria isolated from these infections, recent and old Swiss, along with recent Austrian M. bovis isolates were analyzed by a typing method displaying intermediate resolution of evolutionary relationships among isolates called Multi-Locus Sequence Typing (MLST). The analysis of Swiss and Austrian M. bovis isolates revealed two major lineages. Isolates collected since 2007 in both countries cluster in the lineage I including ST5, ST33, ST34, 36, and ST38-40 (clonal complex 1), while all Swiss isolates recovered before 2007 cluster in the lineage II comprising ST17 and ST35 (clonal complex 5). Further investigations are necessary to understand if lineage I has a higher predilection or virulence toward mammary gland cells than the old lineage or if other factors are involved in the increased number of severe mastitis cases. Copyright © 2016 Elsevier B.V. All rights reserved.

Genetic diversity and antigenicity variation of Babesia bovis merozoite surface antigen-1 (MSA-1) in Thailand.

PubMed

Tattiyapong, Muncharee; Sivakumar, Thillaiampalam; Takemae, Hitoshi; Simking, Pacharathon; Jittapalapong, Sathaporn; Igarashi, Ikuo; Yokoyama, Naoaki

2016-07-01

Babesia bovis, an intraerythrocytic protozoan parasite, causes severe clinical disease in cattle worldwide. The genetic diversity of parasite antigens often results in different immune profiles in infected animals, hindering efforts to develop immune control methodologies against the B. bovis infection. In this study, we analyzed the genetic diversity of the merozoite surface antigen-1 (msa-1) gene using 162 B. bovis-positive blood DNA samples sourced from cattle populations reared in different geographical regions of Thailand. The identity scores shared among 93 msa-1 gene sequences isolated by PCR amplification were 43.5-100%, and the similarity values among the translated amino acid sequences were 42.8-100%. Of 23 total clades detected in our phylogenetic analysis, Thai msa-1 gene sequences occurred in 18 clades; seven among them were composed of sequences exclusively from Thailand. To investigate differential antigenicity of isolated MSA-1 proteins, we expressed and purified eight recombinant MSA-1 (rMSA-1) proteins, including an rMSA-1 from B. bovis Texas (T2Bo) strain and seven rMSA-1 proteins based on the Thai msa-1 sequences. When these antigens were analyzed in a western blot assay, anti-T2Bo cattle serum strongly reacted with the rMSA-1 from T2Bo, as well as with three other rMSA-1 proteins that shared 54.9-68.4% sequence similarity with T2Bo MSA-1. In contrast, no or weak reactivity was observed for the remaining rMSA-1 proteins, which shared low sequence similarity (35.0-39.7%) with T2Bo MSA-1. While demonstrating the high genetic diversity of the B. bovis msa-1 gene in Thailand, the present findings suggest that the genetic diversity results in antigenicity variations among the MSA-1 antigens of B. bovis in Thailand. Copyright © 2016 Elsevier B.V. All rights reserved.

Unique Mechanism of Action of the Thiourea Drug Isoxyl on Mycobacterium tuberculosis*

PubMed Central

Phetsuksiri, Benjawan; Jackson, Mary; Scherman, Hataichanok; McNeil, Michael; Besra, Gurdyal S.; Baulard, Alain R.; Slayden, Richard A.; DeBarber, Andrea E.; Barry, Clifton E.; Baird, Mark S.; Crick, Dean C.; Brennan, Patrick J.

2016-01-01

The thiourea isoxyl (thiocarlide; 4,4′-diisoamyloxydiphenylthiourea) is known to be an effective anti-tuberculosis drug, active against a range of multidrug-resistant strains of Mycobacterium tuberculosis and has been used clinically. Little was known of its mode of action. We now demonstrate that isoxyl results in a dose-dependent decrease in the synthesis of oleic and, consequently, tuberculostearic acid in M. tuberculosis with complete inhibition at 3 μg/ml. Synthesis of mycolic acid was also affected. The anti-bacterial effect of isoxyl was partially reversed by supplementing growth medium with oleic acid. The specificity of this inhibition pointed to a Δ9-stearoyl desaturase as the drug target. Development of a cell-free assay for Δ9-desaturase activity allowed direct demonstration of the inhibition of oleic acid synthesis by isoxyl. Interestingly, sterculic acid, a known inhibitor of Δ9-desaturases, emulated the effect of isoxyl on oleic acid synthesis but did not affect mycolic acid synthesis, demonstrating the lack of a relationship between the two effects of the drug. The three putative fatty acid desaturases in the M. tuberculosis genome, desA1, desA2, and desA3, were cloned and expressed in Mycobacterium bovis BCG. Cell-free assays and whole cell labeling demonstrated increased Δ9-desaturase activity and oleic acid synthesis only in the desA3-overexpressing strain and an increase in the minimal inhibitory concentration for isoxyl, indicating that DesA3 is the target of the drug. These results validate membrane-bound Δ9-desaturase, DesA3, as a new therapeutic target, and the thioureas as anti-tuberculosis drugs worthy of further development. PMID:14559907

9 CFR 113.409 - Tuberculin-PPD Bovis, Intradermic.

Code of Federal Regulations, 2010 CFR

2010-01-01

... Tuberculin supplied by Animal and Plant Health Inspection Service. (1) Test animals. White female guinea pigs... used in a previous test, shall be used in the specificity test. Twenty-three guinea pigs (10 sensitized... being tested, and 20 guinea pigs (10 sensitized with M. bovis and 10 sensitized with M. avium) shall be...

Molecular diagnosis and phylogenetic analysis of Babesia bigemina and Babesia bovis hemoparasites from cattle in South Africa.

PubMed

Mtshali, Moses Sibusiso; Mtshali, Phillip Senzo

2013-08-08

Babesia parasites, mainly Babesia bovis and B. bigemina, are tick-borne hemoparasites inducing bovine babesiosis in cattle globally. The clinical signs of the disease include, among others, anemia, fever and hemoglobinuria. Babesiosis is known to occur in tropical and subtropical regions of the world. In this study, we aim to provide information about the occurrence and phylogenetic relationship of B. bigemina and B. bovis species in cattle from different locations in nine provinces of South Africa. A total of 430 blood samples were randomly collected from apparently healthy cattle. These samples were genetically tested for Babesia parasitic infections using nested PCR assays with species-specific primers. Nested PCR assays with Group I primer sets revealed that the overall prevalence of B. bigemina and B. bovis in all bovine samples tested was 64.7% (95% CI = 60.0-69.0) and 35.1% (95% CI = 30.6-39.8), respectively. Only 117/430 (27.2%) animals had a mixed infection. The highest prevalence of 87.5% (95% CI = 77.2-93.5) for B. bigemina was recorded in the Free State province collection sites (Ficksburg, Philippolis and Botshabelo), while North West collection sites had the highest number of animals infected with B. bovis (65.5%; 95% CI = 52.7-76.4). Phylograms were inferred based on B. bigemina-specific gp45 and B. bovis-specific rap-1 nucleotide sequences obtained with Group II nested PCR primers. Phylogenetic analysis of gp45 sequences revealed significant differences in the genotypes of B. bigemina isolates investigated, including those of strains published in GenBank. On the other hand, a phylogeny based on B. bovis rap-1 sequences indicated a similar trend of clustering among the sequences of B. bovis isolates investigated in this study. This study demonstrates the occurrence of Babesia parasites in cattle from different provinces of South Africa. It was also noted that the situation of Babesia parasitic infection in cattle from certain areas