Development of a new lateral flow immunochromatographic for the diagnosis of Bovine Tuberculosis

ALONSO MARIA NATALIA; GRIFFA NATANAEL; MARIA LAURA MON; MARTINEZ VIVOT MARCELA; MARIA DE LA PAZ SANTANGELO; ROMANO MARIA ISABEL

Sorrento

Congreso; 18th WAVLD: International Symposium of the World Association of Veterinary Laboratory Diagnosticians; 2017

World Association of Veterinary L boratory. Diagnosticians.

Introduction: Mycobacterium bovis (MB) is a slow-growing mycobacterium that causes Bovine Tuberculosis (BTB) mainly in cattle but has a broad host range and causes disease similar to that caused by M tuberculosis in humans. Tuberculosis is one of the most important zoonotic diseases in the world (Olea-Popelka et al., 2017). In particular, it produces important economic losses in the Argentine agricultural production. This disease is common in developing countries and severe economic losses can occur from livestock deaths, chronic disease and trade restrictions. In some situations, BTB may also be a serious threat to endangered species. Consequently, about 70% of the cattle bred in Latin America are held in areas with high disease prevalence and nearly 17% in areas virtually free from BTB (de Kantor and Ritacco, 2006). Diagnosis of this disease in many cases is difficult and often requires the use of paraclinical methods. Several mycobacterial antigens are involved in the complex interaction with the immune system of the host. Rapid diagnosis of the disease and identification of species is extremely important for proper treatment. Current diagnostic methods are either too slow or lack enough sensitivity or specificity. For this reason, the aim of the present work is to develop a diagnostic tool based on a lateral flow immunochromatography using M bovis specific antigens that allows a fast and efficient diagnosis of the Bovine Tuberculosis. Methods: For the development of the lateral flow immunochromatography (ICFL), different concentrations of the specific antigens ESAT-6, CFP-10, MPB83 of MB conjugated to colloidal gold were evaluated in membranes that were exposed to different bovine sera from experimentally infected animals. In all cases, purified bovine immunoglobulins were used as control test. Subsequently, the validation of the developed device was performed by evaluating divers sera from two different fields that were previously determined by ELISA, a technique routinely used in the laboratory. Further, the necropsy of a positive animal by both techniques, ELISA and ICFL, was performed to confirm the presence of the disease.Results: ESAT-6 and CFP-10 antigens showed nonspecific signals, detected in negative sera at all concentrations evaluated (5, 2 and 1 ug / ul). In contrast, MPB83 was found to be more sensitive and specific at the 1ug / ul concentration. It should be noted that the possible cross-detection in positive sera to bovine Paratuberculosis was also evaluated, without being observed positive signal in any of the sera analyzed. Based on the results obtained, it was decided to use the MBP83 as test line and bovine immunoglobulins as control test for the development of the ICFL.In other to validate the developed test we evaluated 293 sera from one field in Argentina of which 13 were positive by ELISA and 9 of them by ICFL. Subsequently, 164 bovine sera from other field of low prevalence in Argentina were evaluated, 13 were positive by ELISA and 6 by ICFL. One of the animals was sacrificed and necropsy was performed, observing the typical lesions of tuberculosis and confirming the presence of disease.Conclusions: In this work we have developed a new technique that can be used for the specific and rapid diagnosis of bovine Tuberculosis using MPB83, however a smaller number of positive sera is detected by ICFL in comparison to those obtained by ELISA. On the other hand, it should be noted that the diagnostic test proposed does not detect positive sera from bovine Paratuberculosis, a common problem in several developed tests.