Diagnóstico laboratorial de Sarcocystis spp. em bovinos

Abstract

Serological tests are frequently performed to confirm Sarcocystis spp. infection and they also are suitable to evaluate a large number of samples. Molecular tests provide detection and characterization of species. Therefore, the aims of the study were: (1) investigate Sarcocystis spp. infection in cattle hearts through direct microscopic examination, polymerase chain reaction (PCR), restriction fragment length polymorphism (PCR-RFLP) and DNA sequencing (2) detect Sarcocystis spp. infection in cattle through direct microscopic examination, confirm sarcocysts identity by PCR and evaluate and correlate these results with the serological diagnosis of cattle by indirect immunofluorescence (3) compare indirect imunnofluorescence (IFAT) and Dot-Blot method for serological diagnosis of Sarcocystis spp. in experimentally infected mice and detect serological cross-reactions with N. caninum and T. gondii. Chapter 1 of this thesis presents a study that myocardium samples were collected from 314 bovine in an abattoir and analyzed for the occurrence of sarcocysts by light microscopy. Sarcocysts isolated from 134 of these hearts were submitted to DNA extraction and PCR. PCR-amplified DNA fragments were digested with the restriction enzymes BclI and RsaI aiming the differentiation among S. cruzi, S. hirsuta, and S. hominis and the identified Sarcocystis species was confirmed by DNA sequencing. Sarcocysts were detected in all bovine myocardium samples. PCR-RFLP and DNA sequencing resulted in identification of S. cruzi. In the study presented in Chapter 2, myocardium and blood samples from 50 adult beef cattle were collected from a slaughterhouse. Each myocardium was submitted to fresh microscopic examination for investigation of compatible cysts to Sarcocystis spp. Ten sarcocysts were collected of each sample and processed to DNA extraction followed by PCR for molecular diagnosis of Sarcocystis spp. The presence of antibodies against the parasite was tested by IFAT in the blood serum. The frequency of sarcocysts detection by fresh examination was 100% (50/50). Specific antibodies against Sarcocystis spp. were detected in 96% (48/50) and 80% (40/50) of serum samples examined at 1:25 and 1:200 dilutions, respectively. DNA from Sarcocystis spp. was amplified by PCR in 86% (43/50) of the collected cysts. The methods employed allowed to detect the infection by Sarcocystis spp. and RIFI showed good sensitivity compared to fresh microscopic examination. In the study presented in Chapter 3, serological tests (RIFI and Dot-blot) of mice inoculated with Sarcocystis spp. or N. caninum or T. gondii. Dot-Blot showed same specificity and sensibility as IFAT for immunological diagnostic of Sarcocystis spp. in experimentally infected mice and this immunoblot test did not demonstrate serological cross-reactions with N. caninum and T. gondii.