RESUMO
BACKGROUND: Oral vaccination with Mycobacterium bovis Bacille of Calmette and Guerin (BCG) has provided protection against M. bovis to badgers both experimentally and in the field. There is also evidence suggesting that the persistence of live BCG within the host is important for maintaining protection against TB. Here we investigated the capacity of badger inductive mucosal sites to absorb and maintain live BCG. The targeted mucosae were the oropharyngeal cavity (tonsils and sublingual area) and the small intestine (ileum). RESULTS: We showed that significant quantities of live BCG persisted within badger in tissues of vaccinated badgers for at least 8 weeks following oral vaccination with only very mild pathological features and induced the circulation of IFNγ-producing mononuclear cells. The uptake of live BCG by tonsils and drainage to retro-pharyngeal lymph nodes was repeatable in the animal group vaccinated by oropharyngeal instillation whereas those vaccinated directly in the ileum displayed a lower frequency of BCG detection in the enteric wall or draining mesenteric lymph nodes. No faecal excretion of live BCG was observed, including when BCG was delivered directly in the ileum. CONCLUSIONS: The apparent local loss of BCG viability suggests an unfavorable gastro-enteric environment for BCG in badgers, which should be taken in consideration when developing an oral vaccine for use in this species.
Assuntos
Administração Oral , Vacina BCG/administração & dosagem , Mustelidae/microbiologia , Mycobacterium bovis/isolamento & purificação , Animais , Vacina BCG/imunologia , Preparações de Ação Retardada , Fezes/microbiologia , Feminino , Íleo/microbiologia , Interferon gama/metabolismo , Linfonodos/microbiologia , Mycobacterium bovis/imunologia , Tuberculose/microbiologia , Tuberculose/prevenção & controle , Tuberculose/veterinária , Vacinação/veterináriaRESUMO
Mycobacterium bovis infection in wild red foxes was found in southern France, where livestock and other wildlife species are infected. Foxes frequently interact with cattle but have been underestimated as a reservoir of M. bovis. Our results suggest a possible role of the red fox in the epidemiology of bovine tuberculosis.
Assuntos
Doenças dos Animais/epidemiologia , Doenças dos Animais/microbiologia , Reservatórios de Doenças/microbiologia , Raposas/microbiologia , Mycobacterium bovis , Tuberculose/veterinária , Doenças dos Animais/diagnóstico , Animais , Técnicas de Tipagem Bacteriana , França/epidemiologia , Gado , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , ZoonosesRESUMO
We describe here 35 animal cases of tuberculosis due to Mycobacterium microti in France (2002-2014). Recently, molecular tools that overcome the difficulty of confirming infection by this potentially zoonotic agent have revealed an increasing number of cases, suggesting that its prevalence may have been underestimated.
Assuntos
Mycobacterium/isolamento & purificação , Tuberculose/veterinária , Animais , Animais Domésticos , Animais Selvagens , França/epidemiologia , Mycobacterium/classificação , Prevalência , Estudos Retrospectivos , Tuberculose/epidemiologia , Tuberculose/microbiologiaRESUMO
Mycobacterium bovis infects cattle and wildlife, and also causes a small proportion of tuberculosis cases in humans. In most European countries, M. bovis infections in cattle have been drastically reduced, but not eradicated. Here, to determine the M. bovis circulation within and between the human, cattle, and wildlife compartments, we characterized by spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) typing the genetic diversity of M. bovis isolates collected from humans, cattle, and wildlife in France from 2000 to 2010. We also assessed their genetic structure within and among the different host groups, and across time and space. The M. bovis genetic structure and its spatiotemporal variations showed different dynamics in the human and animal compartments. Most genotypes detected in human isolates were absent in cattle and wildlife isolates, possibly because in patients, M. bovis infection was contracted abroad or was the reactivation of an old lesion. Therefore, they did not match the genetic pool present in France during the study period. However, some human-cattle exchanges occurred because some genotypes were common to both compartments. This study provides new elements for understanding M. bovis epidemiology in France, and calls for increased efforts to control this pathogen worldwide.
RESUMO
Voles are maintenance hosts of Mycobacterium microti. In line with the goal to eradicate tuberculosis (TB) in livestock, the role of this mycobacteria needs to be assessed since it might interfere with current M. bovis/M. caprae surveillance strategies. To better understand the pathogenesis of TB in voles, an experimental infection model was set up to reproduce M. microti infection in laboratory Bank voles (Myodes glareolus). Two infection routes (intragastric and intraperitoneal) and doses (105 and 106 CFU/0.1 mL) were assessed. Voles were culled at different post-infection time points. Serology, histopathology, acid-fast bacilli staining, qPCR, and mycobacterial culture from tissues were performed. In addition, qPCR from feces and oral swabs were conducted to assess bacterial shedding. The model allowed us to faithfully reproduce the disease phenotype described in free-ranging voles and characterize the pathogenesis of the infection. Most animals showed multifocal and diffuse granulomatous lesions in the liver and spleen, respectively. Less frequently, granulomas were observed in lungs, lymph nodes, muscles, and salivary gland. Mycobacterial DNA was detected in feces from a few animals but not in oral swabs. However, one contact uninfected vole seroconverted and showed incipient TB compatible lesions, suggesting horizontal transmission between voles.
RESUMO
In Europe, animal tuberculosis (TB) due to Mycobacterium bovis involves multi-host communities that include cattle and wildlife species, such as wild boar (Sus scrofa), badgers (Meles meles) and red deer (Cervus elaphus). Red fox (Vulpes vulpes) infections have also been recently reported in some TB endemic regions in the Iberian Peninsula and France, with some of the infected animals shedding M. bovis in urine and feces. In order to understand the pathogenesis of M. bovis infection in foxes and the associated risk of transmission, 12 captive foxes (6 females and 6 males) were inoculated orally with 2 × 107 colony-forming units of a French field isolate of M. bovis. Clinical samples (urine, feces and oropharyngeal swabs) were collected every four weeks and tested for molecular diagnosis and bacteriology. Serological responses were measured by IDEXX M. bovis Ab Test and Multi Antigen Print Immunoassay (MAPIA). At a post-mortem examination performed 12 weeks post infection (wpi), tissues were tested for the presence of M. bovis and associated gross and microscopic TB-like lesions. M. bovis was detected by PCR in bladder swabs of 3 animals at 12 wpi. It was also detected pre-mortem at different time points of the experiment in the oropharyngeal mucus of three individuals and in the feces of nine foxes, with two of them confirmed by bacteriology. All 12 foxes had at least 4 PCR positive samples (out of the 23 tested), and all but 1 fox had at least 1 culture positive sample. The culture negative fox was PCR positive in both retropharyngeal and mesenteric lymph nodes, in line with the results of the other animals. Seroconversion was observed in all foxes except one during the experiment, and in nine at the final time point. No gross visible lesions were found in any animal at the post-mortem examination. The histology showed small granulomas within the lymph nodes, tonsils, liver and lungs from eight animals, with the presence of few acid-fast bacilli. These results confirmed that all orally-infected foxes developed mild TB lesions but they were able to shed mycobacteria in about 75% of cases, 1 month post-infection (9 out 12 foxes). These results show that it is possible to induce typical TB infection experimentally in captive foxes, with measurable M. bovis excretion; such an experimental system could be useful for future evaluations of diagnostics and vaccines in this species.
RESUMO
Mycobacterium microti, member of the Mycobacterium tuberculosis, complex is known to interfere in the screening and diagnosis of bovine tuberculosis. This pathogen is increasingly detected in the frame of surveillance programs for tuberculosis in livestock and wildlife. Recently, red foxes (Vulpes vulpes) were found infected by Mycobacterium bovis in four French endemic areas. M. microti infection was concomitantly found during this investigation. Rates of infection by M. microti and M. bovis are not different except in one of the four areas (lower prevalence for M. microti in Charente). As for M. bovis infection, none of the infected foxes presented gross TB-like lesions. Infection of red foxes by M. microti seems to occur by ingestion of contaminated food, as mesenteric lymph nodes are mostly infected albeit no fecal excretion could be detected. Red foxes appear to be susceptible to Mycobacterium microti infection but seem to play a role of dead-end host for the transmission of this bacillus.
RESUMO
Members of the Mycobacterium avium complex (MAC) are ubiquitous bacteria that can be found in water, food, and other environmental samples and are considered opportunistic pathogens for numerous animal species, mainly birds and pigs, as well as for humans. We have recently demonstrated the usefulness of a PCR-based mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing for the molecular characterization of M. avium subsp. paratuberculosis and M. avium strains exclusively isolated from AIDS patients. In the present study we extended our analysis, based on eight MIRU-VNTR markers, to a strain collection comprehensively comprising the other M. avium subspecies, including M. avium subsp. avium, M. avium subsp. hominissuis, and M. avium subsp. silvaticum, isolated from numerous animal species, HIV-positive and HIV-negative humans, and environmental sources. All strains were fully typeable, with the discriminatory index being 0.885, which is almost equal to that obtained by IS1311 restriction fragment length polymorphism (RFLP) typing as a reference. In contrast to IS1311 RFLP typing, MIRU-VNTR typing was able to further discriminate M. avium subsp. avium strains. MIRU-VNTR alleles strongly associated with or specific for M. avium subspecies were detected in several markers. Moreover, the MIRU-VNTR typing-based results were consistent with a scenario of the independent evolution of M. avium subsp. avium/M. avium subsp. silvaticum and M. avium subsp. paratuberculosis from M. avium subsp. hominissuis, previously proposed on the basis of multilocus sequence analysis. MIRU-VNTR typing therefore appears to be a convenient typing method capable of distinguishing the three main subspecies and strains of the complex and providing new epidemiological knowledge on MAC.
Assuntos
Técnicas de Tipagem Bacteriana , Impressões Digitais de DNA , Complexo Mycobacterium avium/classificação , Complexo Mycobacterium avium/isolamento & purificação , Infecção por Mycobacterium avium-intracellulare/microbiologia , Infecção por Mycobacterium avium-intracellulare/veterinária , Polimorfismo Genético , Animais , Elementos de DNA Transponíveis , DNA Bacteriano/genética , Genótipo , Humanos , Repetições Minissatélites , Epidemiologia Molecular/métodos , Complexo Mycobacterium avium/genética , Polimorfismo de Fragmento de Restrição , Sensibilidade e EspecificidadeRESUMO
Mycobacterium microti, a member of the Mycobacterium tuberculosis complex, was originally described as the cause of tuberculosis in wild rodents. However, in the last few years, an increasing number of cases have been reported in wildlife (wild boars and badgers) and livestock (goat and cattle) in the frame of bovine tuberculosis (bTB) surveillance program, demonstrating the risk of interference with bTB diagnosis in France. In 2019, we detected four cattle infected with M.microti, from three different herds in three different distant regions. For all these cases, ante-mortem diagnosis by the skin test (single intradermal comparative cervical tuberculin (SICCT)) was positive. Confirmation of M.microti infection was based on molecular tests, i.e., specific real-time PCR and spoligotyping. These results highlight a non-negligible risk of interference in the bTB diagnosis system and raise concern about the reliability of diagnostic tests used for bTB surveillance. The use of highly specific tests, like the interferon gamma test (IFN-γ) employed in France or new synthetic specific tuberculins for skin testing could alternatively be used to accurately identify M.bovis (or Mycobacterium caprae) infection at ante-mortem examination. At post-mortem diagnosis, the use of specific molecular tools should be considered to accurately distinguish pathogens within the MTBC and to avoid misleading bTB diagnosis.
RESUMO
In France, animal tuberculosis (TB) due to Mycobacterium bovis (M. bovis) affects a multi-host community that include cattle and wildlife species such as wild boars (Sus scrofa), badgers (Meles meles), or wild deer (Cervus elaphus, Capreolus capreolus). The involvement of foxes in the epidemiology of TB is fairly described in countries facing multispecies concerns. After the discovery of grouped cases of TB in foxes in a French TB endemic region, a study was implemented in the core of four TB endemic areas in Dordogne, Charente, Landes (departments of Nouvelle-Aquitaine region), and Côte-d'Or (Burgundy-Franche-Comté region). No infected fox was found in Côte-d'Or (n = 146), where in parallel TB in cattle and other wild species became sparse in the last years. In contrast, in Dordogne, Charente, and Landes, 13 (n = 184), 9 (n = 98) and 7 (n = 140) foxes were found infected by M. bovis, respectively, corresponding to 7.1% (CI95% 3.8-11.8%), 9.2% (4.3-16.7%) and 5.0% (CI95% 2.0-10.0%) prevalence rates, respectively. These infection rates are comparable with those observed in badgers and wild boar in these same three areas (ranging from 9 to 13.2% and 4.3 to 17.9%, respectively), where the number of cattle outbreaks has increased in the last 10-15 years. In each area, the genotypes of foxes' M. bovis isolates were the same as those in local cattle and other wildlife species. None of the infected foxes presented TB-like gross lesions. M. bovis was found in the mesenteric lymph nodes of 28 foxes (68%). For the 12 foxes where retropharyngeal and respiratory lymph nodes were analyzed separately, M. bovis was present in the respiratory lymph nodes of eight individuals. With regard to excretion, appropriate samples were available for 12 infected foxes from Dordogne. M. bovis DNA was detected in the feces of five of these animals, four of which were infected in the mesenteric lymph nodes. Combined with the knowledge on the biology and ecology of foxes, the results of this study suggest that in areas where infection in cattle is still active in France, foxes might play a role of spillover host in the epidemiology of M. bovis.
RESUMO
The Eurasian wild boar (Sus scrofa) is increasingly considered as a relevant actor in the epidemiology of animal tuberculosis (TB). Therefore, monitoring TB in this species is key when establishing comprehensive control schemes for this disease still present in Europe. No data are available on direct and indirect TB diagnostic methods in wild boars in epidemiological contexts where TB is endemic in cattle and detected in wild boars at low prevalence. We aimed to estimate and compare sensitivity and specificity values for bacterial culture, PCR and three commercial ELISAs, i.e. the TB ELISA-VK (using the bPPD antigen), INgezim TB Porcine and IDEXX M. bovis Ab Test (both using the MPB83 and MPB70 antigens), under field conditions in France. We used frequentist methods, with bacteriology as the gold standard, and a Bayesian formulation of the latent class analysis (LCA), without using a gold standard. Submandibular lymph nodes and sera from 495 wild boars hunter-harvested in three endemic areas (Aquitaine region, Côte d'Or region, and Corsica region) were collected between 2014 and 2016. Only eight individuals were positive for M. bovis by bacteriology (1.61%; CI95% 0.70-3.51%). The LCA method provided high specificities (99.2%; CI95% 98.2-99.8% for INgezim TB Porcine and 99.7%; CI95% 98.8-100% for IDEXX M. bovis Ab Test) and sensitivities (78.5%; CI95% 65.1-88.8% for INgezim TB Porcine and 83.9%; CI95% 58.9-97.2% for IDEXX M. bovis Ab Test) for both ELISAs using the MPB83 and MPB70 antigens. Bacterial culture showed limited sensitivity (42.8%; CI95% 19.0-70.6%), estimated as the probability of a positive result in an animal exposed to M. bovis. PCR and ELISA using the bPPD antigens demonstrated high specificities, and sensitivities intermediates between culture and the ELISAs using the MPB83 and MPB70 antigens. These results suggest that ELISA tests using the MPB83 and MPB70 antigens are useful to detect and monitor TB exposure of wild boar populations in field conditions in France.
Assuntos
Mycobacterium bovis , Sus scrofa/microbiologia , Doenças dos Suínos/microbiologia , Tuberculose/veterinária , Animais , Animais Selvagens/microbiologia , Teorema de Bayes , Ensaio de Imunoadsorção Enzimática/veterinária , Prevalência , Suínos , Doenças dos Suínos/diagnóstico , Doenças dos Suínos/epidemiologia , Tuberculose/diagnóstico , Tuberculose/epidemiologiaRESUMO
Surveillance of bovine tuberculosis (bTB) is partly based on the sanitary inspection of carcasses at the abattoir to detect bTB-like lesions which, in compliance with EU recommendations, are analysed by bacteriology and histopathology to disclose Mycobacterium bovis (or M. caprae) infection. Moreover, since 2012, a PCR method with similar sensitivity and specificity values of histopathology and bacteriology respectively is additionally employed in France, partially compensating for the weaknesses of classical diagnostic methods. We analysed a collection of bTB-like lesions from cattle presenting positive histological results albeit with negative PCR results. We present here the results of these samples, recovered from 292 animals culled between 2013 and 2016, analysed with a second line molecular diagnosis approach that consists in a combination of PCRs targeting the M. tuberculosis-M. avium complexes as well as the Mycobacterium genus and sequencing of hsp65 gene. These molecular analyses disclosed to identify the presence of non-tuberculous bacteria which could be responsible for most of these non-specific TB lesions: non tuberculous mycobacteria (24%) or Actinomycetales (56%) such as Rhodococcus equi (53%); 24% of the samples were negative. M. bovis -or any other MTBC members- was neither detected by molecular methods nor isolated in any of them at the end of the 3 months of culture. In conclusion, these results highlight the lack of specificity of histopathology and the usefulness of a first line PCR with a second line molecular diagnostic test to circumvent it. This diagnostic strategy makes it possible to reduce the number of suspect bTB cases raised at the abattoir or shortening their lock-up periods. By simplifying diagnostic schemes, the use of this tool could improve bTB surveillance and make eradication programs more efficient in the future.
Assuntos
Mycobacterium/genética , Tuberculose Bovina/diagnóstico , Actinomycetales/genética , Actinomycetales/isolamento & purificação , Animais , Proteínas de Bactérias/genética , Bovinos , Chaperonina 60/genética , DNA Bacteriano/metabolismo , RNA Polimerases Dirigidas por DNA/genética , Mycobacterium/isolamento & purificação , Micobactérias não Tuberculosas/genética , Micobactérias não Tuberculosas/isolamento & purificação , Reação em Cadeia da Polimerase , Estudos Retrospectivos , Tuberculose Bovina/microbiologia , Tuberculose Bovina/patologiaRESUMO
Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease affecting livestock, wild animals, and sometimes humans. We report here three draft genome sequences of Mycobacterium bovis strains of spoligotypes SB0821 and SB0134, isolated from wildlife but circulating in wildlife-livestock multihost systems, and SB0121, circulating exclusively in cattle.
RESUMO
Mycobacterium bovis is the etiologic agent of bovine tuberculosis, a chronic infectious disease, affecting livestock, wild animals, and sometimes humans. We report the draft genome sequence of a Mycobacterium bovis strain isolated from wild boar of spoligotype SB0120 (or BCG-like) also present in wildlife-livestock multi-host systems.
RESUMO
The description of the population of M. bovis strains circulating in France from 1978 to 2013 has highlighted the discriminating power of the MLVA among predominant spoligotype groups. In the present study we aimed to characterize clonal groups via MLVA and to better understand the strain's population structure. MLVA was performed with eight MIRU-VNTR loci, most of them defined by the Venomyc European consortium. The discriminatory index of each MLVA loci was calculated for SB0120, SB0134, SB0121 and the "F4-family", the main spoligotype groups in France. Differences in global DI per spoligotype, but also by locus within each spoligotype, were observed, which strongly suggest the clonal complex nature of these major groups. These MLVA results were compared to those of other European countries where strain collections had been characterized (Spain, Portugal, Italy, Northern Ireland and Belgium). Overall, QUB 3232 and ETR D are respectively the most and the least discriminative loci, regardless of the strains geographical origin. However, marked DI differences are observed in the rest of the MIRU-VNTR loci, again highlighting that strain genetic variability in a country depends on the dominant existing clonal complexes. A web application for M. bovis, including spoligotyping and MIRU-VNTR typing data, was developed to allow inter-laboratory comparison of field isolates. In conclusion, combination of typing methods is required for M. bovis optimum discrimination and differentiation of groups of strains. Thus, the loci employed for MLVA in a country should be those which are the most discriminative for the clonal complexes which characterize their M. bovis population.
Assuntos
Repetições Minissatélites/genética , Tipagem Molecular/métodos , Mycobacterium bovis/classificação , Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Animais , Bovinos , Europa (Continente)/epidemiologia , Variação Genética , Genótipo , Mamíferos/microbiologiaRESUMO
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.
Assuntos
Animais Selvagens/microbiologia , Evolução Molecular , Mycobacterium bovis/genética , Tuberculose Bovina/microbiologia , Tuberculose/veterinária , Animais , Técnicas de Tipagem Bacteriana , Bovinos , França/epidemiologia , Variação Genética , Genótipo , Mycobacterium bovis/isolamento & purificação , Sequências de Repetição em Tandem/genética , Tuberculose/epidemiologia , Tuberculose/microbiologia , Tuberculose Bovina/epidemiologia , Tuberculose Bovina/patologiaRESUMO
Mycobacterium bovis isolates from the Iberian Peninsula are dominated by strains with spoligotype patterns deleted for spacer 21. Whole-genome sequencing of three Spanish strains with spacer 21 missing in their spoligotype pattern revealed a series of SNPs and subsequent screening of a selection of these SNPs identified one in gene guaA that is specific to these strains. This group of strains from the Iberian Peninsula missing spoligotype spacer 21 represents a new clonal complex of M. bovis, defined by the SNP profile with a distinct spoligotype signature. We have named this clonal complex European 2 (Eu2) and found that it was present at low frequency in both France and Italy and absent from the British Isles.