Multilocus characterization of Sarcocystis falcatula-related organisms isolated in Brazil supports genetic admixture of high diverse SAG alleles among the isolates.

In a previous study in Brazil, six isolates of Sarcocystis spp. recovered from budgerigars fed sporocysts excreted by opossums of the genus Didelphis were characterized by means of sequencing fragments of gene coding cytochrome B (CYTB), internal transcribed spacer 1 (ITS1), and surface antigen genes (SAG2, SAG3 and SAG4). The isolates shared identical ITS1 and CYTB sequences, but differed at SAG2, SAG3 and SAG4: three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in three multilocus genotypes (MLGs) (MLG1, MLG2, and MLG3). At ITS1 and CYTB, all the isolates from budgerigars were identical to the Sarcocystis falcatula-like isolate 59-2016-RS-BR that was detected in a barefaced ibis (Phimosus infuscatus) causing necrotizing meningoencephalitis in Brazil. At ITS1 locus, all the above isolates were clearly distinct from Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis lindsayi, and Sarcocystis speeri, the four known species of Sarcocystis that use opossums of the genus Didelphis as definitive hosts. Here, we replicated the experiment above to identify additional MLGs or other species of Sarcocystis. Fifteen budgerigars were experimentally infected with sporocysts of Sarcocystis spp. from 12 opossums of the genus Didelphis. All the birds died 9-19 days after infection and tissue samples containing merozoites and schizonts of Sarcocystis spp. were recovered. Fractions of sequences coding for 18S ribosomal RNA gene (18S), CYTB, ITS1, SAG2, SAG3 and SAG4 were PCR amplified and sequenced from the infected lungs. In addition, fractions of 18S, SAG2, SAG3 and SAG4 were sequenced from the isolate 59-2016-RS-BR and fractions of 18S were sequenced from the six isolates from budgerigars described above. From the results, all the isolates shared identical 18S, ITS1 and CYTB sequences. Among the 15 new isolates from budgerigars, three allele variants of SAG2, 3 allele variants of SAG3 and 2 allele variants of SAG4 were encountered in five MLGs, of which four were novel (MLG1, MLG4, MLG5, MLG6 and MLG7). Isolate 59-2016-RS-BR was assigned to an eighth MLG (MLG8). Molecular data pointed that Sarcocystis assigned to MLGs 1 to 8 are variants of the same species, but the SAG-based trees of the isolates conflicted, which supports genetic admixture among them. The sarcocystinae studied have high diversity of SAG alleles per locus and the correlation of such an abundant variety of SAG alleles to host specificity and pathogenicity needs to be assessed. Remains to be elucidated if the parasites studied here and S. falcatula are variants of the same species that have diverged to the point of possessing differences at ITS1 level, but that are still capable of exchanging genes.

Detection of Hammondia heydorni and related coccidia (Neospora caninum and Toxoplasma gondii) in goats slaughtered in Bahia, Brazil.

Hammondia heydorni is a coccidian parasite with an obligatory two host life cycle, with dogs and foxes as definitive hosts, and a number of intermediate hosts, including goats. While infection by this parasite seems to be unassociated with any clinical signs, infection by the closely related parasites Neospora caninum and Toxoplasma gondii can result in abortion, stillbirths and low yielding in caprine herds. The aim of this work was to investigate the frequency of goats infected with H. heydorni using a nested PCR, specific to Toxoplasmatinae internal transcribed spacer 1 (ITS1) of the rDNA, followed by sequencing of the purified PCR fragments. The same molecular techniques were used to determine the frequencies of N. caninum and T. gondii-infected animals. A total frequency of 13.72% (14/102) was obtained for Toxoplasmatinae DNA in goat tissues. After sequencing the PCR products from all positive tissues, a frequency of 3.92% (4/102), 1.96% (2/102) and 7.84% (8/102) were obtained for H. heydorni, N. caninum and T. gondii, respectively. All sequences shared 98-100% identity with sequences from other strains of these coccidia present in GenBank. To the authors' knowledge, this is the first report of H. heydorni DNA in tissues from naturally infected intermediate hosts.