Carla Dray Marassi

Potential application of new diagnostic methods for controlling bovine Tuberculosis in Brazil

Bovine tuberculosis, a chronic infection in cattle caused by Mycobacterium bovis, remains an economic and public health problem for several countries. Due to its economic impact on international trade, contagious nature, and implications for human health, global programs to eradicate the disease were implemented worldwide. Those programs are based on slaughtering PPD-reactive animals. Despite the National Programs in Brazil, complete eradication has not been achieved, and the disease remains, albeit at a lower prevalence. The central purpose of this review is to address diagnostic tests for tuberculosis. Considering the course of the infection in cattle, at least two tests, ideally complementary to one another, may be necessary for an adequate diagnosis: the first based on the cellular response, and the second capable of identifying anergic animals by detection of specific anti-M.bovis antibodies.

Interference of intradermal tuberculin tests on the serodiagnosis of paratuberculosis in cattle

In this experiment 63 animals from a paratuberculosis (PTB) and tuberculosis-free herd were tested by Intradermal Tuberculin Tests (ITT) and blood samples were collected before PPD inoculation and on days 3, 15, 30, 60 and 90 post-inoculation (p.i.). Sera were tested for PTB-specific antibodies by ELISA-PPA and confirmed by a commercial ELISA. Three (4.76%) animals were positive by ELISA-PPA and five (7.93%) in the commercial ELISA, between days 30 and 90 p.i. These results suggest that ITT can interfere in the reliability of ELISAs and that serological testing for PTB should be avoided for 90d after PPD inoculation.

The use of MPB70 and MPB83 to distinguish between bovine tuberculosis and paratuberculosis

In order to demonstrate the potential to distinguish paratuberculosis (PTB) from bovine tuberculosis infection (TB), ELISAs with M. bovis-specific MPB70 or MPB83 as capture antigens were developed and tested on two groups of cattle: Group A comprised 23 animals positive for Mycobacterium avium paratuberculosis (Map) and TB free. Group B comprised 48 animals from a Map free herd during the previous 5 years, but confirmed as tuberculous by positive results on PPD testing and M. bovis culture. Results demonstrated a significant difference (p<0.01) between reactivity of sera from these groups, encouraging the study of purified proteins to differentiate between both diseases.

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.

The use of a Gamma-Interferon assay to confirm a diagnosis of bovine tuberculosis in Brazil.

Control of bovine tuberculosis (TB) is currently based on slaughter of cattle deemed positive on the basis of tuberculin testing; although the test has been broadly used for several years, there are some disadvantages such as the need for holding animals for 72 h. Besides it, test can lack both sensitivity and specificity depending on the interpretation and the site of the PPD inoculation. Therefore, the objective of the current study was to evaluate the use of the Gamma-Interferon (IFN) assay as a confirmatory test in Brazil. A Comparative Intradermal Tuberculin Test (CITT) was performed in 50 cows from a dairy herd known to be infected with TB. Blood samples for IFN testing were collected concurrent with the CITT, as well as seven and 21 days later. At 30 days after the CITT, all cattle deemed reactive to this test were slaughtered and samples were processed by both bacteriological culture and PCR. The sensitivity of IFN as a diagnostic tool was 91.4%, whereas specificity was 86.7%. When applied seven or 21 days after injection of PPD, sensitivity was 74.3 and 71.4%, respectively, whereas specificity was 86.7 and 80%. There was no significant difference between the reliability of the IFN assay and the other standards, on all three days of sampling. Therefore, we concluded that the IFN assay could be effectively used as a confirmatory test seven or 21 days after injection of PPD.

The use of MPB70-ELISA for the diagnosis of caprine tuberculosis in Brazil.

After one clinical case that evidenced the outbreak, a complete screening by intradermal tuberculin test was performed in one goat herd in Brazil. The herd was composed by 500 animals and 83 of them (16.6%) showed to be reactive to the comparative double cervical intradermal test. Four months after the test, all the 83 reactive animals were slaughtered and blood samples were collected from 45 of them, for serological assays. From those 45, 32 were randomly chosen for necropsy and histopathological and bacteriological procedures were conducted. Histopathology evidenced at least one characteristic lesion of tuberculosis in each animal, with typical granulommas where acid-fast bacilli (AFB) could be observed. Bacteriology was positive for Mycobacterium bovis in 22 samples (68.7%), therefore confirming the etiology of the outbreak. Sera of 45 animals plus 20 other from a certified free tuberculosis farm were tested in an ELISA using the recombinant M.bovis protein MPB70 as capture antigens. From those, 43 were reactive to the test, with high ODs results, considering a cut-off point established by ROC curve analyzing results (cut-off = 0.8; mean = 0.55; range: 0.157–1.357). These results suggest that MPB70-ELISA can be considered as a reliable tool to diagnose tuberculosis in goat herds, since this assay was capable to correctly detect 95.6% of the animals here examined.

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.

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.

Assessing the histopathology to depict the different stages of bovine tuberculosis infection in a naturally infected herd

The standard method for detection of bovine tuberculosis (TB) is the single intradermal tuberculin test (SITT). Nevertheless, current studies suggest that a single test is not enough to detect all cattle infected by TB, particularly when animals present different stages of infection. A dairy herd comprised of 270 cows was studied and 15 were reactive to SITT plus nine inconclusive animals. Blood samples (for IFN and ELISA) were collected from these 24 cows. At 30 days after injection of PPD, all the cows that were reactive to any of the employed tests were slaughtered, and tissues were processed by Bacteriology, Histopathology (HP) and PCR. According to HP 33.4% of the animals were positive, 45.8% inconclusive and 20.8% were negative. The inconclusive samples came from IFN positive animals, signalizing recent infection. Regarding the animals that were negative to HP, all of them were identified by IFN while ELISA was negative. Immune responses are different in recent and advanced infections, what supports the identification between chronically or recently infected animals. This multidisciplinary approach is mandatory for the interpretation of the various tools that are frequently employed for the diagnosis of TB and mainly to identify all infected animals.

A complementary diagnosis of naturally occurring tuberculosis in water buffaloes (Bubalus bubalis) in Rio de Janeiro using a MPB70-ELISA, Brazil.

As tuberculosis is still a worldwide infection and buffalo breeding represents an important economic activity in various countries, the purpose of this study was to employ an enzyme-linked immunosorbent assay (ELISA) using MPB70 as a capture antigen for the diagnosis of naturally occurring tuberculosis in water buffaloes in Brazil. After the introduction of newly acquired cattle onto a tuberculosis (TB) free farm, an outbreak of TB was recorded in a mixed herd comprising water buffaloes (21) and cattle (46). The entire herd was tested by intradermal tuberculin injection (ITT) and positive animals were slaughtered and tested by culture, polymerase chain reaction (PCR), and ELISA. From the 21 buffaloes sampled, three were reactive by ITT. All the three had positive culture and ELISA, while PCR was positive in two of them. Besides that, one ITT-negative buffalo was slaughtered and presented positive results by both culture and ELISA, and was considered as anergic. Although there were only few animals, those findings demonstrate the diagnostic usefulness of an MPB70-ELISA to correctly detect Mycobacterium bovis tuberculosis in water buffaloes.