The 70 kDa heat-shock protein (Hsp70) sequences are considered one of the most conserved proteins in all domains of life from Archaea to eukaryotes. Hammondia heydorni, H. hammondi, Toxoplasma gondii, Neospora hughesi and N. caninum (Hammondia-like organisms) are closely related tissue cyst-forming coccidians that belong to the subfamily Toxoplasmatinae. The phylogenetic reconstruction using cytoplasmic Hsp70 coding genes of Hammondia-like organisms revealed the genetic sequences of T. gondii, Neospora spp. and H. heydorni to possess similar levels of evolutionary distance. In addition, at least 2 distinct genetic groups could be recognized among the H. heydorni isolates. Such results are in agreement with those obtained with internal transcribed spacer-1 rDNA (ITS-1) sequences. In order to compare the nucleotide diversity among different taxonomic levels within Apicomplexa, Hsp70 coding sequences of the following apicomplexan organisms were included in this study: Cryptosporidium, Theileria, Babesia, Plasmodium and Cyclospora. Such analysis revealed the Hammondia-like organism to be the lowest divergent group when compared to other groups within the phylum Apicomplexa. In conclusion, the Hsp70 coding sequences proved to be a valuable genetic marker for phylogenetic reconstruction and may constitute a good candidate to be used with other genes for species phylogeny within this group of organisms.

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Neospora caninum is one of the most important causes of abortion in dairy cattle worldwide. The distribution of N. caninum in tissues of adult cattle is unknown and the parasite has not been demonstrated histologically in tissues of cows. In the present study the distribution of N. caninum in different tissues of adult cattle was evaluated by bioassays in dogs. Seventeen dogs (2-3 month-old) were fed different tissues of 4 naturally exposed adult cattle (indirect fluorescent antibody test N. caninum titer ≥ 400): 5 were fed with masseter; 5 with heart, 3 with liver, 4 with brain, and 3 pups were used as non-infected control. Two dogs fed masseter, 2 fed heart, 1 fed liver, and 3 fed brain shed oocysts, and all dogs presented no seroconvertion to N. caninum during the observation period of 4 weeks. The oocysts were confirmed as N. caninum based on the detection of N. caninum-specific DNA by PCR and sequencing. The results indicate that dogs can be infected by N. caninum with different tissues of infected cattle.

Sarcocystis neurona and Sarcocystis falcatula are very similar species of Apicomplexan protozoa that use marsupials of the genus Didelphis as definitive hosts. These mammals can serve as definitive hosts not only for these two parasites, but for other Sarcocystis such as Sarcocystis speeri and Sarcocystis lindsayi. Sarcocystis shed by opossums (with the exception of S. neurona) can cause disease in a great variety of birds, being commonly associated with acute pulmonary sarcocystosis in zoos. S. neurona is the most commonly associated parasite with the equine protozoal myeloencephalitis in horses. Herein we assessed the variability of Sarcocystis spp. isolated from opossums of the state of Rio Grande do Sul, Brazil, by sequencing fragments of genes coding for glycosylphosphatidylinositol-anchored surface antigens (termed surface antigen or SAG), SAG2, SAG3 and SAG4. Two genetic groups were identified, one of them related to S. falcatula and the other related to S. neurona. Various allelic combinations of SAG2, SAG3 and SAG4 occur among S. falcatula related isolates and strong evidences suggest that such isolates may exchange high divergent alleles in possible sexual recombination processes. Regarding the group S. neurona-like (isolates G37 and G38), none of the individuals in this group share alleles with individuals of the other group. Comparing G37 and G38 strains and North American strains of S. neurona, four polymorphisms were identified at SAG-3, five at SAG-2 and three at SAG-4. Gene sequences of locus SAG-3 from isolates G37 and G38 differed from the other sequences by an insertion 81bp long. This insertion contains several dinucleotide repeats of AT, resembling a microsatellite locus and has already been detected in SAG3 sequences of S. neurona from North America. When aligned against North American strains of S. neurona, G37 and G38 isolates have a deletion of 8 nucleotides within this intron which indicate that S. neurona strains of South America are divergent from that of North America. From the results obtained so far, we have shown extensive variability in surface antigens coding sequences among Sarcocystis eliminated by mammals of the genus Didelphis spp. In addition, such divergent alleles may be exchanged in possible sexual recombination processes between different isolates of S. falcatula related isolate. The evolutionary relationships within S. falcatula related isolates will be best clarified after markers less subjected to selection pressures are analyzed in conjunction with surface antigen genes. These results may have a striking impact on the knowledge of the Sarcocystis species that infect opossums in Brazil and also in the epidemiology of the infections caused by these protozoans.

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