Luciana Medeiros

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.

Identification of Mycobacterium bovis Isolates by a multiplex PCR

Isolates from suggestive bovine tuberculosis lesions were tested by a multiplex polymerase chain reaction (m-PCR) targeting for RvD1Rv2031c and IS6110 sequences, specific for M. bovis and Mycobacterium tuberculosis complex respectively. The m-PCR successfully identified as M. bovis 88.24% of the isolates.

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.

Multiple strains of Mycobacterium bovis revealed by molecular typing in a herd of cattle.

Mycobacterium bovis isolates from an outbreak of bovine tuberculosis in a herd of cattle in Rio de Janeiro, Brazil, were analysed by spoligotyping and variable-number tandem repeat PCR analysis of the mycobacterial interspersed repetitive unit and exact tandem repeats. Molecular typing revealed a high genetic diversity of strains in the herd. The genetic diversity could be explained by the introduction of infected animals from different sources.

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.

In vitro antimicrobial susceptibility of staphylococci isolated from canine pyoderma in Rio de Janeiro, Brazil

The study aimed to determine the etiology and antimicrobial resistance of staphylococci isolated from canine pyoderma. Samples were obtained from dogs with pyoderma and isolates were identified by biochemical reactions and tested for susceptibility to 15 antimicrobials. Thirty nine staphylococci isolates were obtained, and S. pseudintermedius was the most frequent (47.4%). All isolates showed resistance to at least one drug and 77.1% were multiresistant. The most effective drug was oxacillin. The study reports the alarming antimicrobial resistance of Staphylococcus isolated from canine pyoderma samples.

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.

The role of capybaras as carriers of leptospires in periurban and rural areas in the western Amazon

Although leptospirosis has been described in capybaras, usually based on serological evidences, bacterial culture of leptospires has been scarcely reported in this species. The western Amazon is a reportedly endemic area where high seroprevalences have been reported in different species of wildlife, domestic animals and in human beings. The present study aimed at investigating the role of capybaras as carriers of leptospires in periurban and rural areas in the western Amazon region. A total of 44 animals were captured, and 41 blood samples (for serology) and 41 urine samples (for PCR and bacterial culture) were obtained. A total of 18/41 (43.9%) of sera were reactive and titers were generally low, indicating chronic infection. PCR was positive in 13/41 (31.7%) samples, isolates were recovered from urine samples belonging to Icterohaemorrhagiae, Grippotyphosa and Shermani serogroups. A high number of carriers (confirmed by PCR) associated to a tendency for harboring Icterohaemorrhagiae serogroup strains could be noticed. Our results suggest that capybaras are massively infected by leptospires. Analogously to Norway rats, capybaras present chronic infection with low titers and long-term bacterial shedding, and may be acting as reservoirs of this bacterium.

A multidisciplinary approach to diagnose naturally occurring bovine tuberculosis in Brazil

A herd infected naturally with tuberculosis was investigated by different diagnostic methods. Ninety days after a screening test that identified 21 cows as skin test positive, a Comparative Intradermal Tuberculin Test (CITT) was performed in those 21 cows and in 29 other randomly selected skin test negative cows. Milk samples and nasal swabs were collected prior to the CITT for bacteriological culture and PCR, while blood samples were collected for IFN release and antibody responses to MPB70 and MPB83, at three time points post tuberculin injection. Animals positive by CITT were slaughtered and disease confirmation undertaken. Based on the Kappa test, IFN was comparable to the standard tests (culture, PCR and CITT) at all three sampling points. Results from both antibody ELISAs were similar but were not comparable to the standard tests. T-test analysis of the CITT, IFN and ELISAs demonstrated that their performances were not correlated. There is increasing recognition that individually, available diagnostic tests do not detect all infected cattle. Therefore, a comprehensive strategy for the diagnosis of bovine TB should include test results for the detection of both cellular and humoral immune responses where there may be animals at different stages of infection.