RESUMEN
Bovine tuberculosis is a disease that is widely distributed around the world. Its causative agent, Mycobacterium bovis, has characteristics of a microorganism with clonal multiplication in populations with no evidence of genetic exchange between strains, and, consequently, a group of strains can be identified as descending from a common ancestor. The aim of this study was to investigate the clonal complexes of M. bovis isolated from samples of lesions suggestive of bovine tuberculosis collected from slaughterhouses in various states of Brazil between 2006 and 2012. Ninety samples were analyzed, and it was found that 14.4% belonged to the clonal complex European1 and 81.1% to the clonal complex European2, while 4.65% were not identified as any of the four known complexes.
Asunto(s)
Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Tuberculosis Bovina/epidemiología , Animales , Brasil/epidemiología , Bovinos , Evolución Clonal/genética , Mycobacterium bovis/aislamiento & purificación , Tuberculosis Bovina/microbiologíaRESUMEN
Although Mycobacterium bovis is the major etiological agent of tuberculosis in bovines, it can infect other mammalians. Previously reported cases of tuberculosis caused by M. bovis in cats from the Autonomous City of Buenos Aires (CABA) led to the conclusion that the main source of infection for these felines was the ingestion of raw bovine lungs. Thus, for this study, we collected samples of bovine viscera from butchers' shops of the Greater Buenos Aires (GBA) and the CABA to assess presence and viability of these mycobacteria in bovine lungs (including the lymph nodes) and livers. We analyzed 216 different samples and obtained 5 isolates of M. bovis (4 from lungs and 1 from liver) by culture analysis. We also confirmed the presence of different isolates by polymerase chain reaction, spoligotyping, and MIRU-VNTR assays. The results obtained in this work emphasizes the need of social education for food hygiene, and to change the habit of feeding pets with raw viscera, which carries the risk of epizootic and zoonotic transmission. Moreover, control and eradication programs of bovine tuberculosis should be strengthened and improved.
Asunto(s)
Técnicas de Tipificación Bacteriana/veterinaria , ADN Bacteriano/aislamiento & purificación , Contaminación de Alimentos , Mycobacterium bovis/aislamiento & purificación , Carne Roja/microbiología , Animales , Argentina/epidemiología , Bovinos , Microbiología de Alimentos , Hígado/microbiología , Pulmón/microbiología , Mycobacterium bovis/clasificación , Reacción en Cadena de la Polimerasa/veterinaria , Tuberculosis Bovina/microbiologíaRESUMEN
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: 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 pathogen, evidencing the existence of greater genetic variability among strains than previously contemplated.
Asunto(s)
Bovinos/microbiología , Genoma Bacteriano , Mycobacterium bovis/genética , Animales , Genómica , Genotipo , Mycobacterium bovis/clasificación , Mycobacterium bovis/aislamiento & purificación , Filogenia , UruguayRESUMEN
Bovine tuberculosis (bTB) is a disease caused by Mycobacterium bovis (M. bovis), which affects cattle, animal species and humans. To determinate the genetic structure of strains of M. bovis in mexican cattle, 467 isolates obtained from 2009 to 2010 from different regions of Mexico with known spoligotype were included in the study. The isolates were genotyped by interspersed repeated mycobacterial units-variable number tandem repeats (MIRU-VNTR) obtaining 13 MIRU-VNTR groups. When combining MIRU-VNTR patterns with its spolygotypes, the Hunter genetic discrimination index (HGDI), we obtained 421 genetic patterns distributed in 17 groups. The HGDI for the total loci was 0.99. The locus that presented the higher HGDI was 2461 (0.857), while the locus with the lowest HGDI was 2686 (0.239). When we analyzed our results, using just 6 or 8 MIRU-VNTR we obtained an discriminatory power of 0.8499 and 0.8875 respectively indicating lower HGDI than 12 MIRU-VNTR locus.
Asunto(s)
Bovinos/microbiología , Variación Genética , Repeticiones de Minisatélite , Mycobacterium bovis/genética , Animales , ADN Bacteriano , Humanos , México , Mycobacterium bovis/clasificación , Mycobacterium bovis/aislamiento & purificación , FilogeniaRESUMEN
The purpose of this study was to identify relationships between spoligotypes of M. bovis from cattle in Mexico and those reported in countries with free trade of cattle with Mexico: Australia, Canada, New Zealand and the United States of America. Mexican spoligotypes were obtained from isolates collected from cattle in different parts of the country. Spoligotypes from Canada and New Zealand were obtained from different reports in the literature. Those from the United States were obtained from the database of the National Veterinary Services Laboratory in APHIS-USDA. In order to perform the analysis in a single data set, spoligotypes were all converted to binary data and classified according to www.mbovis.org or www.pasteur-guadeloupe.fr:8081. Epidemiologic information included country and species infected. From 3,198 isolates, 174 different spoligotypes were obtained, 95 were orphans. Ninety one percent of the isolates came from the Unites States (n = 1,609) and Mexico (n = 1,323). Spoligotype SB0265 is shared between Canada and the United States in cattle and wildlife. Six spoligotypes, SB0673, SB0121, SB0145, SB0971, SB0140 and SB1165, were frequent in cattle and wildlife in the United States and cattle in Mexico, suggesting wide exchange of strains. Spoligotype SB0669 was found only in Mexico. Spoligotype SB0140 was the most common in Australia and the sixth in the United States and Mexico. In a phylogenetic analysis, spoligotype SB0140 appears as the oldest spoligotype in the data set, suggesting this as the ancestral spoligotype for all spoligotypes in the five countries. Some spoligotypes are shared by animals and humans, corroborating the zoonotic importance of M. bovis.
Asunto(s)
Comercio , Internacionalidad , Mycobacterium bovis/genética , Animales , Técnicas de Tipificación Bacteriana , Bovinos , México , Mycobacterium bovis/clasificaciónRESUMEN
Accurately identifying Mycobacterium bovis-infected cattle is critical for bovine tuberculosis prevention and control. One method for identifying infected cattle is an ELISA developed by IDEXX laboratories, which detects antibodies to two M. bovis proteins, MPB70 and MPB83. The assay's sensitivity varies by geographic region, with sensitivities of 77%, 45%, and 9% in bovine serum samples from the United Kingdom (n = 126), the United States (n = 146), and Mexico (n = 128), respectively. We hypothesized that geographically-biased sequence variation in mpb70 and mpb83, or in the genes that regulate their expression (sigK and rskA), may explain these differing sensitivities. This hypothesis was tested by comparing the sequences of these four genes in 455 M. bovis strains isolated from cattle in the aforementioned countries. For each gene, a single, common sequence was identified in most genomes of the M. bovis strains collected in all three countries. Twelve of the 455 strains were isolated from infected cattle for which the IDEXX ELISA was also performed. Five of the seven ELISA-positive genomes and three of the five ELISA-negative genomes contained the most common sequence of all four genes. Thus, sequence variation in mpb70, mpb83, sigK, and rskA does not explain the geographic disparities in IDEXX ELISA sensitivity.
Asunto(s)
Proteínas Bacterianas/genética , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Análisis de Secuencia de ADN/métodos , Tuberculosis Bovina/diagnóstico , Animales , Anticuerpos Antibacterianos/metabolismo , Antígenos Bacterianos/genética , Antígenos Bacterianos/inmunología , Proteínas Bacterianas/inmunología , Bovinos , Ensayo de Inmunoadsorción Enzimática , Variación Genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , México , Mycobacterium bovis/inmunología , Sensibilidad y Especificidad , Tuberculosis Bovina/inmunología , Reino Unido , Estados UnidosRESUMEN
Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovis bacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1ß] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.
Asunto(s)
Quimiocinas/metabolismo , Macrófagos/inmunología , Viabilidad Microbiana/inmunología , Mycobacterium bovis/clasificación , Línea Celular , Quimiocina CCL3/metabolismo , Quimiocina CCL4/metabolismo , Citocinas/metabolismo , Humanos , Interleucina-1/metabolismo , Interleucina-6/metabolismo , Macrófagos/clasificación , Macrófagos/efectos de los fármacos , Mycobacterium bovis/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Vacunas de Productos InactivadosRESUMEN
Control eradication campaigns of bovine tuberculosis based on the «test and slaughter¼ approach were successful in many countries and regions; however, in some areas the infection persists and one of the main reasons is Mycobacterium bovis infection in wild life species. Argentina has applied the same approach since 1999, achieving progress in dairy cattle herds. Nonetheless, the wildlife role has never been investigated. The objective of this study was to determine if wildlife from the Santa Fe dairy area is infected with M. bovis. Wildlife species having a positive tuberculin skin test were captured in five dairy farms. Ninety five wildlife mammals were captured; M. bovis was recovered from 7 possums (Didelphys albiventris), from one fox (Lycolapex gimnocercus) and from one rat (Rattus norvegicus). None of the animals exhibited macroscopic lesions. The most frequently isolated M. bovis spoligotypes were types 34 (4 isolates) and 12 (3 isolates). Spoligotype 34 is the most frequently isolated type in Argentine cattle. The role of D. albiventris as spillover host of M. bovis is discussed in this study.
Asunto(s)
Animales Salvajes/microbiología , Industria Lechera , Reservorios de Enfermedades/microbiología , Mycobacterium bovis/aislamiento & purificación , Animales , Argentina/epidemiología , Técnicas de Tipificación Bacteriana , Bovinos/microbiología , Femenino , Zorros/microbiología , Mycobacterium bovis/clasificación , Zarigüeyas/microbiología , Ratas/microbiología , Tuberculosis Bovina/epidemiología , Tuberculosis Bovina/microbiología , Tuberculosis Bovina/transmisiónRESUMEN
Tuberculosis has great public health impact with high rates of mortality and the only prophylactic measure for it is the Mycobacterium bovisbacillus Calmette-Guérin (BCG) vaccine. The present study evaluated the release of cytokines [interleukin (IL)-1, tumour necrosis factor and IL-6] and chemokines [macrophage inflammatory protein (MIP)-1α and MIP-1β] by THP-1 derived macrophages infected with BCG vaccine obtained by growing mycobacteria in Viscondessa de Moraes Institute medium medium (oral) or Sauton medium (intradermic) to compare the effects of live and heat-killed (HK) mycobacteria. Because BCG has been reported to lose viability during the lyophilisation process and during storage, we examined whether exposing BCG to different temperatures also triggers differences in the expression of some important cytokines and chemokines of the immune response. Interestingly, we observed that HK mycobacteria stimulated cytokine and chemokine production in a different pattern from that observed with live mycobacteria.
Asunto(s)
Humanos , Quimiocinas , Macrófagos/inmunología , Viabilidad Microbiana/inmunología , Mycobacterium bovis/clasificación , Línea Celular , Citocinas , Interleucina-1 , Macrófagos/clasificación , Macrófagos/efectos de los fármacos , Mycobacterium bovis/inmunología , Factor de Necrosis Tumoral alfa , Vacunas de Productos InactivadosRESUMEN
Panama remains free of zoonotic tuberculosis caused by Mycobacterium bovis. However, DNA fingerprinting of 7 M. bovis isolates from a 2013 bovine tuberculosis outbreak indicated minimal homology with strains previously circulating in Panama. M. bovis dispersion into Panama highlights the need for enhanced genotype testing to track zoonotic infections.
Asunto(s)
Mycobacterium bovis/clasificación , Tuberculosis Bovina/epidemiología , Animales , Bovinos , Genotipo , Historia del Siglo XXI , Repeticiones de Minisatélite , Tipificación Molecular , Mycobacterium bovis/genética , Panamá/epidemiología , Filogenia , Polimorfismo Genético , Tuberculosis Bovina/historiaRESUMEN
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.
Asunto(s)
ADN Bacteriano/análisis , Inspección de Alimentos/métodos , Carne/microbiología , Tipificación Molecular/veterinaria , Mycobacterium bovis/aislamiento & purificación , Tuberculosis Bovina/microbiología , Tuberculosis Ganglionar/veterinaria , Mataderos , Animales , Brasil , Bovinos , ADN Bacteriano/aislamiento & purificación , ADN Bacteriano/metabolismo , Eficiencia , Cabeza , Límite de Detección , Pulmón/química , Pulmón/microbiología , Ganglios Linfáticos/química , Ganglios Linfáticos/microbiología , Carne/análisis , Tipificación Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/veterinaria , Mycobacterium bovis/clasificación , Cuello , Reacción en Cadena en Tiempo Real de la Polimerasa/veterinaria , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/veterinaria , Tórax , Tuberculosis Bovina/fisiopatología , Tuberculosis Bovina/prevención & control , Tuberculosis Ganglionar/etiologíaRESUMEN
In order to identify the genetic characteristics of the strains of mycobacteria circulating in the Estado de México, one of the states with the lowest prevalence of tuberculosis in Mexico, spoligotyping and 12-loci MIRU-VNTR typing were used to genotype tuberculosis clinical isolates. The average age of the 183 patients analyzed was 50 (± 17) years, drug resistance was noted in 57 (31%) and multidrug resistance in 22 (12%) individuals. The results from the isolates recovered showed that 80% were located in four major Euro-American lineages: Haarlem (17%), LAM (15%), T (20%) and X (29%). Other lineages found in lower proportions were: EAI, S, Beijing, West African, Turkey, Vole and Bovis. Eighteen isolates were orphans. Only 57 isolates were grouped in nine clusters and the SIT119 (X1) showed the highest number of members (23). The LAM lineage showed an increased risk for development of drug resistance (RR=4, IC: 95%: 1.05-14.2, p = 0.03). Despite the important prevalence of four major lineages found and the diversity of strains circulating in the population, we found the presence of one of the largest populations of isolates clustered to the X lineage in a setting from a Latin American country.
Asunto(s)
Mycobacterium bovis/genética , Mycobacterium tuberculosis/genética , Tuberculosis Pulmonar/epidemiología , Tuberculosis Pulmonar/microbiología , ADN Bacteriano/genética , Farmacorresistencia Bacteriana Múltiple , Femenino , Variación Genética , Humanos , Masculino , México/epidemiología , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Tipificación Molecular , Mycobacterium bovis/clasificación , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/aislamiento & purificación , PrevalenciaRESUMEN
We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169 C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.
Asunto(s)
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Amidohidrolasas/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/diagnósticoRESUMEN
Mycobacterium bovis is the main causative agent of animal tuberculosis (TB) and it may cause TB in humans. Molecular typing of M. bovis isolates provides precise epidemiological data on issues of inter- or intra-herd transmission and wildlife reservoirs. Techniques used for typing M. bovis have evolved over the last 2 decades, and PCR-based methods such as spoligotyping and mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) have been extensively used. These techniques can provide epidemiological information about isolates of M. Bovis that may help control bovine TB by indicating possible links between diseased animals, detecting and sampling outbreaks, and even demonstrating cases of laboratory cross-contamination between samples. This review will focus on techniques used for the molecular typing of M. bovis and discuss their general aspects and applications.
Asunto(s)
Tipificación Molecular/métodos , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Animales , Humanos , Epidemiología Molecular/métodos , Tuberculosis/microbiología , Tuberculosis/veterinariaRESUMEN
Bovine tuberculosis is a major infectious disease of the cattle. In this study, 85 M. bovis isolates from 162 lymph nodes, obtained from a herd of cattle on a farm in southern Brazil, were evaluated using spoligotyping and VNTR. The strains were grouped into five clusters and five orphans, showing a heterogenic genetic profile, what could represent diverse geographic origins of the introduced cows and/or the frequent movement of cattle between different properties.
Asunto(s)
Tipificación Molecular , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Tuberculosis Bovina/microbiología , Animales , Brasil/epidemiología , Bovinos , Análisis por Conglomerados , ADN Bacteriano/genética , Genotipo , Ganglios Linfáticos/microbiología , Mycobacterium bovis/aislamiento & purificación , Tuberculosis Bovina/epidemiologíaRESUMEN
Despite control and eradication efforts, bovine tuberculosis continues to be identified at low levels among cattle in the United States. We evaluated possible external sources of infection by characterizing the genetic relatedness of bovine tuberculosis from a national database of reported infections, comparing strains circulating among US cattle with those of imported cattle, and farmed and wild cervids. Farmed cervids maintained a genetically distinct Mycobacterium bovis strain, and cattle occasionally became infected with this strain. In contrast, wild cervids acted as an epidemiologically distinct group, instead hosting many of the same strains found in cattle, and the data did not show a clear transmission direction. Cattle from Mexico hosted a higher overall richness of strains than US cattle, and many of those strains were found in both US and Mexican cattle. However, these two populations appeared to be well-mixed with respect to their M. bovis lineages, and higher resolution data is necessary to infer the direction of recent transmission. Overall patterns of both host and geographic distributions were highly variable among strains, suggesting that different sources or transmission mechanisms are contributing to maintaining different strains.
Asunto(s)
Mycobacterium bovis/clasificación , Mycobacterium bovis/fisiología , Tuberculosis Bovina/epidemiología , Tuberculosis Bovina/transmisión , Animales , Bovinos , Evolución Molecular , Interacciones Huésped-Patógeno , México/epidemiología , Filogenia , Filogeografía , Tuberculosis Bovina/microbiología , Estados Unidos/epidemiologíaRESUMEN
We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.
Asunto(s)
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Amidohidrolasas/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/diagnósticoRESUMEN
We evaluated a multiplex-PCR to differentiate Mycobacterium bovis from M. tuberculosis Complex (MTC) by one step amplification based on simultaneous detection of pncA 169C > G change in M. bovis and the IS6110 present in MTC species. Our findings showed the proposed multiplex-PCR is a very useful tool for complementation in differentiating M. bovis from other cultured MTC species.
Asunto(s)
Técnicas Bacteriológicas/métodos , Técnicas de Diagnóstico Molecular/métodos , Reacción en Cadena de la Polimerasa Multiplex/métodos , Mycobacterium bovis/aislamiento & purificación , Mycobacterium tuberculosis/aislamiento & purificación , Tuberculosis/microbiología , Amidohidrolasas/genética , Elementos Transponibles de ADN , ADN Bacteriano/genética , Mycobacterium bovis/clasificación , Mycobacterium bovis/genética , Mycobacterium tuberculosis/clasificación , Mycobacterium tuberculosis/genética , Tuberculosis/diagnósticoRESUMEN
This study aimed to develop and validate real-time PCR for the diagnosis of Mycobacterium bovis isolates. Two hundred and seventy-four M. bovis isolates and 156 M. tuberculosis isolates were tested. Both qPCRs amplified all of the 274 M. bovis samples, but none of the 156 M. tuberculosis samples. The qPCR for PE-PGRS 20 had 91% efficiency and a detection limit of 0.32 ng (sensitivity and specificity for qPCR "Mbovis.100" were 99.64 and 100%, respectively). The qPCR for RD4 had 100% efficiency, and a detection limit of 4 pg (diagnostic sensitivity and specificity were 100 and 100%. The qPCR tests were performed using 4 extraction sets, 3 qPCR kits, and with a range of equipment; yet, all combinations produced similar results in a diagnostic test, demonstrating the robustness of this method. The techniques proved to be efficient, robust, sensitive, and specific for the diagnosis of M. bovis.