High discrimination of Mycobacterium bovis isolates in Brazilian herds by spoligotyping.

Bovine tuberculosis is an infectious disease caused by Mycobacterium bovis (M. bovis), that leads to economic losses in infected herds and it is also considered an important zoonosis. The molecular typing methods of M. bovis isolates are fundamental for the bovine tuberculosis surveillance system, and spoligotyping is the standard genotyping technique for this species. Thus, the aim of the present study is to analyze the spatial and cluster distribution of M. bovis strains from several regions of Brazil through molecular typing. Spoligotyping technique was applied on 422 isolates identified as M. bovis, and Ripley's K function was used to perform the spatial and cluster analysis of each identified profile. Forty-three (43) different profiles were identified and spoligotype SB0121 was the most frequent and showed a uniform pattern in the spatial distribution while spoligotypes SB0295, SB1380 and SB1050 formed clusters. In addition, three novel spoligotype profiles (SB2361, SB2362, SB2364) were identified in different herds. In this perspective, it is believed that molecular identification and typing can significantly improve the performance of surveillance systems for bovine tuberculosis in Brazil.

Evaluation of post-mortem diagnostic tests' sensitivity and specificity for bovine tuberculosis using Bayesian latent class analysis.

This study aimed to evaluate the performance of real-time PCR (qPCR), ELISA IDEXX™, and bacterial isolation as post-mortem diagnostic tests in animals with lesions compatible with bovine tuberculosis detected by Brazilian Federal Inspection Service as part of the bovine tuberculosis active surveillance. Bayesian latent class models were used to estimate diagnostic tests' sensitivity, specificity, correlations, predictive values and frequency of infected animals. Samples of tuberculosis-suggestive lesions collected by FIS sanitary inspection routine in slaughterhouses from 11 Brazilian states were analyzed. Isolation was the most sensitive technique, 94.54% (95% Credible Interval (CrI) 90.09%-97.65%), qPCR was 64.69% (95% CrI 54.41%-74.15%) sensitive and ELISA IDEXX™ 26.74% (95% CrI 22.82%-30.97%). Tests' specificities were 98.19% (95% CrI 95.75%-99.45%), 93.49% (95% CrI 79.28%-99.66%), 95.53% (95% CrI 91.71%-98.02%) respectively. Despite its low sensitivity, ELISA IDEXX™ was able to identify positive samples that were not detected by the other techniques. These samples had high probability to be true positives given ELISA's positive predictive value. The correlations between qPCR and isolation were neither biologically nor statistically significant. The low sensitivity of the qPCR is a limiting factor to its use as a post-mortem diagnosis in bovine tuberculosis suggestive lesions. Its use could be recommended in situations of high prevalence, or in parallel association with other tests, such as ELISA IDEXX™. ELISA IDDEX™ should not be used as a unique test, or in substitution of the other tests, for the post-mortem diagnosis of bovine tuberculosis due to its sensitivity.

Comparative study of Mycobacterium bovis primary isolation methods.

For the definitive diagnosis of bovine tuberculosis, isolation of the etiologic agent is required. However, there is no consensus on the best methodology for isolation of Mycobacterium bovis in Brazil. This study evaluated the most used decontaminants and culture media in the country, in order to identify the best combination for the Brazilian samples. Three decontaminants - 2% sodium hydroxide (w/v), 0.75% hexadecylpiridinium chloride (w/v) and 5% sulphuric acid (v/v) and four culture media - 7H11 Middlebrook with additives and OADC supplement "A" (7H11 A), the same media with another supplement trademark (7H11 B), tuberculosis blood agar (B83) and Stonebrink's medium were compared. Regarding the isolation, there were no significant differences between the decontaminants and media combinations, except 7H11A combined to any decontaminant. However, the mean colonies score was significantly greater when the samples were decontaminated with 5% sulphuric acid and inoculated in 7H11 B or SB, without significant difference between them, although colonies appeared earlier on 7H11B than on SB. The trademark of OADC supplement influenced the isolation rate and the number of isolated colonies in Middlebrook 7H11. An incubation time of four weeks was required to detect all positive samples in 7H11 B after decontamination with 5% sulphuric acid but there was an increase in the number of colonies until the sixth week of incubation. Overall, the best strategy for the primary isolation of M. bovis from Brazilian samples was the decontamination with 5% sulphuric acid (final concentration) and inoculation in Middlebrook 7H11 medium formulated with OADC supplement "B".