Padronização de ensaio ELISA “in house” para diagnóstico de leishmaniose em equinos e caninos

Abstract

Leishmaniasis are diseases caused by the protozoan Leishmania spp. and are distributed globally. In South America, Brazil concentrates the most significant number of visceral and cutaneous leishmaniasis cases in humans. In the western border of the state of Rio Grande do Sul, Brazil, there are reports about sandflies (the vectors) and the infection in humans, canines, and horses. The domestic dog is the main reservoir in urban areas; however, there is a discussion about horses' participation in the maintenance of the parasite. Several diagnostic techniques are available to identify both the agent and the individual's immune response. The ELISA test (Enzyme-Linked Immunosorbent Assay) allows identifying the immune response through antibody-antigen binding. In 2011, it was defined as a confirmatory test for canine visceral Leishmaniasis (LVC) by the Ministry of Health. Given the importance of Leishmaniasis in dogs in the municipality of Uruguaiana and the need for studies regarding the maintenance of the disease and possible reservoirs, the present work aimed to standardize an in house indirect ELISA protocol to detect an immune response against Leishmania spp. in canine and equine species, as well as the serological evaluation of samples from the municipalities of Uruguaiana and Barra do Quaraí-RS. Initially, protocols for the cultivation of Leishmania in the laboratory were standardized. The strains of Leishmania infantum, a species used as an antigen, were made available by FRIOCRUZ / RJ. Antigen fixation was performed using the parasite's promastigote forms, and the concentration of 10ug/ml demonstrated the best results. The ELISA protocol used for equine and canine species was the same. The test of the dilutions of the primary antibody varied between species: for horses, the concentrations 1/100, 1/200, 1/400, 1/800, and 1/1000 were used; and 1/100, 1/400, and 1/800 for canines. The secondary antibody concentrations used in the tests were: 1/5000 and 1/10000 for horses and canines. After standardization, serological tests were carried out on samples from canines and horses from the municipalities of Barra do Quaraí and Uruguaiana. Testes englobed 51 dogs and 159 horses. Among the 51 canines tested, 17 (33%) were positive in the ELISA technique, 16 of which were also positive in the DPP® immunochromatographic test. Regarding the tested horses, 159 animals, 16 (10%) were positive in the ELISA. With this, it was possible to conclude that it was possible to standardize an indirect ELISA protocol in house to diagnose Leishmaniasis in horses and canines. The presence of positive animals demonstrates that the disease is occurring in the studied population.