Bacteriological and molecular detection of Mycobacterium bovis in cattle with inconclusive results to intradermal tuberculin tests.

A dairy herd (77 cows) from Rio de Janeiro, Brazil, with a history of tuberculosis infection was tested by a comparative cervical test (CCT). Seventeen cows were reactive and seven were inconclusive (swelling ≥ 2.0 mm and ≤ 3.9 mm, respectively). All of these 24 cows were slaughtered and necropsied ; samples from lungs and lymph nodes were collected for multiplex polymerase chain reaction (PCR) and culturing. Infection was confirmed in 23/24 (95.8%) of the slaughtered animals (five by culturing, four by PCR, and 14 by both tests). All cows with inconclusive results at CCT were confirmed as infected. Although slaughter of inconclusive reactor cows is not mandatory in many countries, our study provided evidence to support the slaughter of these cows, at least during an outbreak.

Comparison of decontamination methods for primary isolation of Mycobacterium bovis in paucibacillary bovine tissues.

AIMS: To compare three decontamination methods applied to paucibacillary samples for primary isolation of Mycobacterium bovis from suspect lesions. Tuberculosis caused by Myco. bovis is an important infectious disease of cattle in Brazil and also has zoonotic potential. Although a national campaign based on testing and slaughtering cattle has achieved good results, there is a strong need to develop better diagnostic methods to identify cattle with recent infections harbouring few bacilli. METHODS AND RESULTS: A dairy herd (274 adult crossbred cows) located in the state of Rio de Janeiro was tested for tuberculosis with both single intradermal tuberculin test and comparative intradermal tuberculin test. Reactive cows (n=27, 9.8%) were slaughtered and suspect lesions were collected (one sample per cow). Samples considered paucibacillary (based on microscopy) were decontaminated with 0.75% hexadecylpyridinium chloride (HPC), 4% sodium hydroxide (Petroff) or 6% sulphuric acid. Using these methods, 10, five and six, respectively, of the 27 samples yielded positive cultures. Overall, Myco.bovis was isolated from 14 of 24 cows. Although the HPC method resulted in isolation of more Myco.bovis strains than either Petroff or sulphuric acid methods (P=0.015), it did not result in the recovery of Myco.bovis from all samples. However, using both HPC and 6% sulphuric acid methods for decontamination was possible to identify 13 of 14 (92·9%) of infected cows. CONCLUSIONS: At least two methods should be used concurrently for primary isolation of Myco. bovis from bovine tissues, particularly for paucibacillary samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Detection of low numbers of Myco.bovis in tissue is an important goal in optimizing the detection of bovine tuberculosis and should assist in identification of infected cattle, in particular, those with few Myco.bovis bacilli. This was apparently the first study comparing three decontamination methods for the detection of Myco.bovis in paucibacillary samples from naturally infected cattle.

Use of recombinant proteins MPB70 or MPB83 as capture antigens in ELISAs to confirm bovine tuberculosis infections in Brazil.

The objective was to evaluate the use of two indirect IgG-ELISA tests (with recombinant proteins MPB70 or MPB83, respectively, as capture antigens) as confirmatory tests for diagnosis of bovine tuberculosis in a herd of naturally infected dairy cows. Results for ELISA-MPB70 and ELISA-MPB83 were similar (kappa statistic=0.92) on Days 0 (day of intradermal injection with purified protein derivatives, PPD), 7, and 21. The kappa statistic between ELISA and the Comparative Intradermal Tuberculin Test, as well as ELISA sensitivity and specificity (relative to culture or PCR as standards) were: 0.7, 34.4% and 75% on Day 0; 0.25, 53.8% and 66.6% on Day 7; and 0.01, 1.8% and 77.7% on Day 21, respectively. In conclusion, although ELISAs using MPB70 or MPB83 as antigens were not reliable indicators of infection status, especially on Days 7 and 21, they were of potential value as complementary tools to intradermal PPD testing.

Occurrence of false-positive results in three paratuberculosis - ELISAs performed in a tuberculous herd.

The interference of bovine tuberculosis (TB) on the efficacy of paratuberculosis (PTB) diagnostic tests has been evaluated. A group of 32 tuberculous cows identified by both intradermal tests and gamma-interferon assay, 16 of them confirmed by the recovery of M.bovis from tissues, was tested by three different PTB- ELISAs, being two commercials and one in-house. The rest of the adult animals of the herds, totalizing 216 TB-negative animals, were also tested as a control group. Fecal culture for PTB was negative in all animals, but seven (21.8%) tuberculous cows produced false-positive reactions when tested by various PTB-ELISAs, leading to a misdiagnosis. Tuberculosis impairs the specificity of serological tests for paratuberculosis diagnosis and should be considered for the reliability of PTB control programs.

Paratuberculosis: an update: [review]

Paratuberculosis is a chronic enteritis that affects ruminants and is caused by Mycobacterium avium paratuberculosis (Map). The disease is worldwide spread and causes important economic losses. In Brazil, the bacillus was isolated in the south and northeast regions of the country and in Rio de Janeiro, but there are no enough epidemiological studies about its occurrence. Isolation of Map from tissues or fecal samples is 100 percent specific, but Map shows the most fastidious growth of all mycobacteria. Incubation lasts 8 -12 weeks, with a dependency on exogenous mycobactin J. Diagnostic tests based on specific DNA sequences allow fast and secure identification, and PCR has been used to confirm positive culture results and to identify Map in feces, milk and tissues. The most frequently used target sequences are the gene encoding the 16S rRNA, and the insertion element IS900. Serological assays are widely used for the herd diagnosis of the disease. A commercial ELISA with M. phlei pre-adsorption step achieves a specificity of 95.4 percent to 99 percent and a sensitivity of about 45 percent. Until now, there is no effective treatment for ill animals and control programs are based on managing procedures of herds and culling of symptomatic animals. In Brazil, paratuberculosis was recently identified and has been demonstrated even in autochthonous closed herds. Therefore, it is essential to perform an epidemiological national research and to investigate the economic impact of the disease in our herds. These results could promote a control program of paratuberculosis adapted to the Brazilian requirements.

A Paratuberculose é uma enterite crônica que afeta ruminantes, causada por Mycobacterium avium paratuberculosis (Map). A doença é cosmopolita e determina importantes perdas econômicas. No Brasil, o agente foi isolado nas regiões Sul e Nordeste além do Rio de Janeiro, mas não há estudos epidemiológicos suficientes sobre sua ocorrência. O isolamento de Map dos tecidos ou amostras fecais é 100 por cento especifico, mas Map apresenta o crescimento mais fastidioso dentre todas as micobactérias, com período de incubação de até 8 -12 semanas, dependente de fonte exógena de micobactina J. Os testes diagnósticos baseados em seqüências especificas de DNA permitem uma identificação rápida e segura, e PCR tem sido usado para confirmar resultados de cultura positiva e para identificar Map em fezes, leite ou tecidos. O alvo mais frequentemente utilizado é o gene que codifica para o 16S rRNA, e o elemento de inserção IS900. Testes sorológicos são amplamente utilizados para o diagnóstico de rebanho. Um ELISA comercial com uma etapa de pré-adsorção com M. phlei alcança uma especificidade de 95,4 por cento a 99 por cento e uma sensibilidade de cerca de 45 por cento. Até agora, não existe tratamento efetivo para animais doentes e os programas de controle são baseados em medidas de manejo e descarte dos animais sintomáticos. No Brasil, a paratuberculose foi recentemente demonstrada mesmo em rebanhos fechados e autóctones. Assim, é essencial realizar-se um inquérito epidemiológico nacional e investigar o impacto econômico da enfermidade em nossos rebanhos. Estes resultados poderiam ser utilizados para um programa de controle da paratuberculose adaptado ás necessidades de nosso país.

Paratuberculosis: an update

Paratuberculosis is a chronic enteritis that affects ruminants and is caused by Mycobacterium avium paratuberculosis (Map). The disease is worldwide spread and causes important economic losses. In Brazil, the bacillus was isolated in the south and northeast regions of the country and in Rio de Janeiro, but there are no enough epidemiological studies about its occurrence. Isolation of Map from tissues or fecal samples is 100% specific, but Map shows the most fastidious growth of all mycobacteria. Incubation lasts 8 -12 weeks, with a dependency on exogenous mycobactin J. Diagnostic tests based on specific DNA sequences allow fast and secure identification, and PCR has been used to confirm positive culture results and to identify Map in feces, milk and tissues. The most frequently used target sequences are the gene encoding the 16S rRNA, and the insertion element IS900. Serological assays are widely used for the herd diagnosis of the disease. A commercial ELISA with M. phlei pre-adsorption step achieves a specificity of 95.4% to 99% and a sensitivity of about 45%. Until now, there is no effective treatment for ill animals and control programs are based on managing procedures of herds and culling of symptomatic animals. In Brazil, paratuberculosis was recently identified and has been demonstrated even in autochthonous closed herds. Therefore, it is essential to perform an epidemiological national research and to investigate the economic impact of the disease in our herds. These results could promote a control program of paratuberculosis adapted to the Brazilian requirements.

A Paratuberculose é uma enterite crônica que afeta ruminantes, causada por Mycobacterium avium paratuberculosis (Map). A doença é cosmopolita e determina importantes perdas econômicas. No Brasil, o agente foi isolado nas regiões Sul e Nordeste além do Rio de Janeiro, mas não há estudos epidemiológicos suficientes sobre sua ocorrência. O isolamento de Map dos tecidos ou amostras fecais é 100% especifico, mas Map apresenta o crescimento mais fastidioso dentre todas as micobactérias, com período de incubação de até 8 -12 semanas, dependente de fonte exógena de micobactina J. Os testes diagnósticos baseados em seqüências especificas de DNA permitem uma identificação rápida e segura, e PCR tem sido usado para confirmar resultados de cultura positiva e para identificar Map em fezes, leite ou tecidos. O alvo mais frequentemente utilizado é o gene que codifica para o 16S rRNA, e o elemento de inserção IS900. Testes sorológicos são amplamente utilizados para o diagnóstico de rebanho. Um ELISA comercial com uma etapa de pré-adsorção com M. phlei alcança uma especificidade de 95,4% a 99% e uma sensibilidade de cerca de 45%. Até agora, não existe tratamento efetivo para animais doentes e os programas de controle são baseados em medidas de manejo e descarte dos animais sintomáticos. No Brasil, a paratuberculose foi recentemente demonstrada mesmo em rebanhos fechados e autóctones. Assim, é essencial realizar-se um inquérito epidemiológico nacional e investigar o impacto econômico da enfermidade em nossos rebanhos. Estes resultados poderiam ser utilizados para um programa de controle da paratuberculose adaptado ás necessidades de nosso país.