Diagnosis of tuberculosis in ruminants and camelids: optimization of cell-based and humoral tests
Carmen Casal Comendador defended the PhD Thesis at the Faculty of Veterinary Medicine of the Complutense University of Madrid
January 28th, 2016
Tuberculosis is a bacterial zoonosis that represents an important health problem in humans, domestic animals and wildlife. The most frequently involved species in animal tuberculosis are Mycobacterium bovis and M. caprae. The disease has a major economic impact caused by the decreased production in animals, the commercial restrictions implied, and the costs of eradication campaigns in cattle. In the last years, tuberculosis is gaining relevance in other domestic species i.e. goats, mainly in those countries with a high census of this species. Additionally, tuberculosis in South American camelids is increasingly gaining importance in recent years, llamas (Lama glama) and alpacas (Vicugna pacos) are main species imported into the European Union. Eradication campaigns are based on "test-and-slaughter" strategies which implies the removal of animals that react positively to diagnostic tests. The official diagnostic tests approved in the European Union are the single intradermal tuberculin (SIT) test and the interferon-gamma (IFN-γ) assay, which detect the cell-mediated immune response to the pathogen. Serological assays for the detection of antibodies are also available, commercially and/or experimentally. Despite of the efforts performed for the control and the eradication of the disease, in some areas it is not possible to decrease the prevalence due among others, to the limitations present in the official diagnostic techniques. Therefore, in order to optimize the diagnosis of tuberculosis in ruminants (cattle and goats) and camelids (llamas and alpacas), nine experimental studies divided into five chapters have been performed, each chapter has been designed to achieve the five objectives of this thesis.
Chapter I includes three studies developed for optimizing the performance of the tests currently in use for the diagnosis of tuberculosis in ruminants. The first experimental study was designed to evaluate the effect of the site of inoculation of the bovine PPD on the outcome of the SIT test in OTF and naturally infected cattle. In the absence of infection, the SIT test was not affected by the location of the PPD inoculation, however, in infected animals, the inoculation of bovine PPD in the cervical anterior area increased the probability to obtain a positive result to the SIT test. The second experimental study of this chapter was conducted with the aim to assess the effect of the interpretation criteria applied on the result of the IFN-γ assay performed using the Bovigam kit, on samples collected from OTF and naturally infected cattle. Significant differences in both specificity and sensitivity were recorded according to the interpretation criteria applied. The third experimental study (last work from chapter I), was performed to compare the result of three commercial kits for the detection of IFN-γ assay on samples from naturally infected cattle. Significant differences in the number of animals detected as positive were observed depending on the commercial kit selected.
Chapter II includes the fourth experimental study of this thesis, designed to evaluate the possible interference of M. tuberculosis SO2 vaccine in the diagnosis of tuberculosis in goats. The SIT test and the IFN-y assay were performed with PPDs and alternative antigenic formulations in the same manner as for differentiation of infected animals from those vaccinated with M. bovis BCG. The specificity of the tests that detect the cell-mediated immune response reached 100% whit the antigen formulations.
Chapter III of this thesis aims to study the response to diagnostic tests and the lesions developed in goats experimentally infected with different species of the MTBC. With this purpose, the fifth experimental study was conducted, in order to evaluate the response to the SIT test and the IFN-γ assay and to assess the lesions developed in goats experimentally infected with M. bovis, M. tuberculosis and M. caprae. The course of the infection, the performance of diagnostic tests and the severity of the lesions developed were greatly dependent on the MTBC species involved.
The final chapter (chapter IV) of the present thesis, includes two studies that evaluate the sensitivity and specificity of the SIT test (single and comparative) and an experimental serological assay for the diagnosis of tuberculosis in South American camelids (llamas and alpacas). The sixth experimental study aimed to assess the sensitivity of the tests and was performed in naturally infected alpacas, the single SIT test showed higher sensitivity, regardless of the site of inoculation of the PPDs. Moreover, the sensitivity was higher when the reading was performed 72 hours after the inoculation of the PPDs. The sensitivity obtained with the serological experimental assay was higher when the assay was performed on samples collected 15 days after the SIT test, confirming the existence of a booster effect. The seventh experimental study (last work of this thesis), aimed to assess the specificity of the tests and was performed in Perú, on llamas and alpacas free of tuberculosis. The comparative SIT test was more specific than the single one, regardless of the dose, the site of inoculation of PPD, and the time of reading. The specificity of the experimental serological assay was higher when it was performed with samples collected before the SIT test, furthermore the serial interpretation of the results of both techniques resulted in increased specificity when compared to the one recorded for each test separately.
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