Genotyping of Toxoplasma gondii samples isolated from ostriches (Struthio camelus) from slaughterhouse, and from commercial farms in the State of São Paulo

Abstract

Toxoplasma gondii is a parasite with high importance to animal production and public health, involving fetal alterations and abortion in humans and animals, being an important opportunistic pathogen in immunocompromised patients too. It has a populational structure with predominant lineages: type I, II and III. The transmission of the parasite by food has been characterized as high risk to the human health. The world consumption and commercialization of meat products from ostriches have shown high increase, but with low studies related to toxoplasmosis, representing a potential risk of infection by food for man. In this way, soil will be collected two times per year, in one year, in the commercial farms that supply animals to the slaughter in the slaughterhouse from Amparo-SP. In the slaugherhouse, blood, brain and heart samples of the same ostriches will be collected. After to obtain the serum samples, these samples will be submitted to a serological test, modified agglutination test, with formalin-fixed antigen (MAT-AF). Tissue samples from seropositive animals will be macerated and digested by pepsin action, and bioassayed in mice for the isolation of the parasite. The research of oocysts in the soil will be performed by centrifuge-flutuation method in Sheater's sugar fluctuation solution. The parasite load will be determined by real-time PCR, using SYBR Green system. Genotyping of the samples isolated from ostriches and soil will be performed by multiplex-PCR, nested-PCR and the restriction fragment length polymorphism (RFLP-PCR) in 12 loci (SAG1, 5'-3'SAG2, aSAG2, SAG3, BTUb, GRA6, c22-8, c29-2, L358, PK1, Apico and CS3), besides the association of the virulence obtained in bioassay with the profiles obtained in ROPD18Del and ROP18UPS markers, and sequencing of the samples that present amplification with different profiles. The results of the genotyping will be analyzed by the determination of the phylogeny in the SplitsTree program. Thus, this study is aimed to isolate T.gondii of tissues from ostriches slaughtered for human consumption, and in soil from commercial farms; to compare the genotyping profiles obtained in RFLP-PCR for 12 genetic markers and perform the phylogenetic analysis of the isolates to determine the phylogenetic relationship among these. (AU)