Caracterização biológica, morfológica e molecular de sarcocystis sp. Isolado de didelphis sp. no nordeste do Brasil

Abstract

TAVARES, ALINE S., Biological, morphological and molecular characterization of Sarcocystis sp. isolated from Didelphis sp. in the Northeast of Brazil. Salvador, Bahia, 2021. 95p. Thesis (Doctorate in Animal Science in the Tropics) - School of Veterinary Medicine and Animal Science - Federal University of Bahia, 2021. The present study aimed to in vitro isolate Sarcocystis spp. from Brazilian opossums to genetically compare the obtained isolates with those species described in North America, to reproduce their life cycles and to characterize the parasitic stages. Budgerigars (Melopsittacus undulatus) and opossums were used in bioassays. Sarcocystis sp. sporocysts were obtained from an opossum’s carcass and used for in vitro isolation of the parasite in a permanent avian cell line (DF-1). The isolated strain was named Sarco-BA1 and DNA from the cultured parasite was used for PCR and multilocus sequencing. Bioassay in nine budgerigars were conducted by oral inoculation of sporocysts and subcutaneous injection of merozoites. In the molecular analysis, the nucleotide sequence of the internal transcript 1 (ITS-1) of the rDNA was identical to ITS-1 sequences of the isolates (n = 19) of Sarcocystis spp. reported in two studies conducted in the south and southeast regions of the country. Two budgerigars that were orally inoculated with sporocysts (10 and 1000) developed acute sarcocystosis, showing that Sarco-BA1 is pathogenic for the employed bird species. Sequences of ITS-1 and for genes coding for three surface antigens (SAG2, SAG3 and SAG4) were almost identical to 16 isolates of S. falcatula-like described in Magellanic penguins (Spheniscus magellanicus) rescued on the coast of Espírito Santo state, Brazil. These findings suggest that Sarco-BA1 and S. falcatula-like represent a single species of Sarcocystis. In order to produce S. falcatula-like tissue cysts for morphological evaluation, budgerigars were inoculated with different doses of sporocysts that had been stored for approximately 12 months at 4°C. However, no cysts or signs of infection were observed in the inoculated animals, what is highly suggestive that sporocysts had lost their infectivity. Morphological analysis of merozoites and sporocysts were conducted by transmission electron microscopy (TEM). Ultrastructural details observed in the two studied parasitic stages were the same as described for Sarcocystis neurona and S. falcatula. The sporocysts examined by TEM showed degenerated sporozoites, as well as detachment of their outer wall. xi These findings strengths the suspicion that the prolonged storage of sporocysts led to the loss of their infectivity. The current work is the first in vitro isolation, morphological, biological and genetic analysis of S. falcatula-like from northeastern Brazil. Genetic data obtained here indicate that Brazilian isolates of S. falcatula-like possess similar genetic characteristics, i.e., high level of homology ITS-1 and high degree of variation for the genes that code for surface antigens (SAG); in contrast, Brazilian isolates of S. falcatula-like have significant genetic differences when compared to North American isolates of S. falcatula and S. neurona, as well as to other species of Sarcocystis identified in the Americas.