There is little information on the distribution and diversity of Apicomplexan protozoal infections in the tissues of raptors in the United States. Protozoan encephalitis caused by Sarcocystis species and Toxoplasma gondii is being increasingly reported in raptors from various locations in the United States. To better determine the exposure of raptors to these Apicomplexan parasites, we examined breast and heart muscle tissue of raptors from the Carolina Raptor Center for the presence of Sarcocystis species, T. gondii and Neospora caninum via histology, Polymerase Chain Reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP) using DraI and HinfI enzymes (Sarocystis only). Of 187 available HandE stained tissue sections, 33 contained sarcocysts. Nineteen of these slides had a matching DNA sample to compare via PCR. Nine of these 19 were positive for Sarcocystis via ITS PCR. Using ITS PCR, we detected Sarcocystis DNA in 24 of 114 birds (21.1%). Further molecular differentiation using JNB primers showed that 9 of the 24 birds were positive for either S. neurona or S. falcatula. RFLP analysis of these 9 indicated that 4 were S. falcatula samples, and 3 were S. falcatula Arg samples that cut with both enzymes. Our Sarcocystis positive samples were also tested for S. calchasi, S. columbae and Sarcocystis sp. Ex. A. nisus using PCR primers designed for these species. These species are emerging in Europe and have already shown an expansion of their distribution. Two samples (14567 and 15203) suggestive of Sarcocystis sp. Ex. A. nisus were identified, as well as one sample (14567), which suggested the presence of S. columbae. None of these samples were confirmed by sequencing the amplicons and the other 22 samples were all negative for these parasites. Recent reports have demonstrated DNA of S. falcatula in the brain and muscles of great horned owls (Bubo virginianus), golden eagles (Aquila chrysaetos), and bald eagles (Haliaeetus leucocephalus) with encephalitis in rehabilitation centers in Indiana, Minnesota, and Virginia using PCR. DNA of S. calchasi has been found in CNS tissue of several species of birds suffering encephalitis in an aviary in California. Hawks (Accipiter species) are believed to be the source of infection. The prevalence of T. gondii was 18.4% (21 of 114) in these birds by PCR, but none were positive by histopathology. N. caninum prevalence in raptors has been poorly discussed in the literature. This parasite uses canids as the definitive host in its life cycle, and is considered to have a much more restricted host range than T. gondii. Thirty-five of 114 birds (30.7%) were found to be PCR positive for N. caninum, but no tissue cysts of N. caninum were observed in histological sections. Co-infection of 2 or all 3 species was detected in 16 of 114 birds (14%). This study demonstrates that there may be a higher prevalence of S. falcatula in raptors than was previously known, including more, as yet unknown, species of Sarcocystis capable of infecting raptors as intermediate hosts. Our PCR prevalence for T. gondii is similar to the serological prevalence for this parasite in raptors. The high PCR prevalence of N. caninum needs to be confirmed by sequencing the amplicons and the use of additional PCR primers. Information from the present study may help to inform zoos, aviaries and wildlife rehabilitation centers about parasite host diversity and reinforce the importance of preventative measures, such as making sure opossums (S. falcatula and S. falcatula-like), feral cats (T. gondii), and wild raptors (S. calchasi) do not have access to facilities. Insect control should also be emphasized because of their ability to serve as phoretic hosts and carry oocysts/sporocysts into zoos, aviaries, and rehabilitation centers. / Master of Science

Em um trabalho anterior, foi avaliada a variabilidade de Sarcocystis spp. isolados de gambás oriundos do estado do Rio Grande do Sul pesquisando sequências gênicas codificadoras de antígenos de superfície (SAGs). Os resultados indicaram haver linhagens de isolados de Sarcocystis (relacionadas geneticamente a S. falcatula) que trocam genes em prováveis processos de recombinação sexual. A proposta deste estudo foi de conhecer as variações gênicas e relações filogenéticas em Sarcocystis spp. isolados de gambás através da análise de loci gênicos de genoma mitocondrial (CytB), de genoma de apicoplasto (ClpC) e das regiões espaçadoras transcritas internas (ITS-1), comparando a diversidade encontrada nestes grupos com a observada em integrantes da sub-familia Toxoplasmatinae. E também de conhecer a diversidade de genes codificadores de SAG-2, SAG-3 e SAG-4. Neste estudo foram obtidos esporocistos de Sarcocystis spp. através de raspado intestinal e fezes provenientes de gambás do gênero Didelphis, oriundos do Estado de São Paulo, Rio Grande do Sul e Rio Grande do Norte. Os marcadores moleculares CytB, ClpC e ITS-1 revelaram uma ampla variabilidade genotípica e alelos inéditos entre os isolados do Brasil. Os resultados levam a crer que é possível a existência de espécies “híbridas” de Sarcocystis no Brasil, com mistura de genes de ambas as espécies. Em relação aos genes codificadores de SAGs, foi encontrada uma diversidade ainda maior do que trabalho similar realizado. Os resultados sugerem uma possível ocorrência de reassortment e recombinação de alelos em sequências SAGs, contribuindo ainda mais para a geração de variabilidade e consequentemente na configuração antigênica do parasito. Estes resultados demonstram que isolados brasileiros possuem uma composição genética diferente do reportado em demais localidade, o que pode ter reflexos importantes no conhecimento da diversidade das espécies de Sarcocystis que infectam gambás em nosso meio e em toda a epidemiologia das infecções produzidas pelos protozoários deste grupo / A great variability in surface antigens genes (SAGs) of Sarcocystis spp. sporocysts was found in a previous study, through the intestinal scraping technique of opossums from the south region of Brazil. The results indicated probable sexual recombination between lineages of Sarcocystis isolates (genetically related to Sarcocystis falcatula). The aim of this research is the study of genetic variability and phylogenetic relationships among Sarcocystis spp. isolates from captured opossums in Brazil, by the analysis of molecular markers less subjected to selective pressure, such as DNA barcode, for this purpose, genetic loci from mitochondrial (CytB) and apicoplast genome (ClpC), and internal transcribed spacer regions (ITS-1) were applied and compare the results with the diversity found within members of the Toxoplasmatinae sub-family. The diversity among the SAG-2, SAG-3 and SAG-4 genes was also studied. The Sarcocystis sporocysts were obtained from opposum’s intestinal scraping and faeces from São Paulo State, Rio Grande do Sul State and Rio Grande do Norte State, Brazil. The molecular markers CytB, ClpC and ITS-1 revealed a vast genotypic variability and novel alleles among the Brazilian isolates. The results indicate possible existence of hybrid species of Sarcocystis in Brazil, with a gene mixture from both species. Regarding the SAG genes, an even greater variability was found than previously reported. The results also suggest the occurrence of reassortment and allele recombination of SAG sequences, contributing even more to the variability and thus in the parasites antigenic configuration. In this study, the results demonstrate that isolates from Brazil have a different genetic composition than reported in other locations, which may have important impacts in the knowledge of the diversity of Sarcocystis species shed by opossums in our environment and in all of the epidemiology of infections of protozoa of this particular group

Em um trabalho anterior, foi avaliada a variabilidade de Sarcocystis spp. isolados de gambás oriundos do estado do Rio Grande do Sul pesquisando sequências gênicas codificadoras de antígenos de superfície (SAGs). Os resultados indicaram haver linhagens de isolados de Sarcocystis (relacionadas geneticamente a S. falcatula) que trocam genes em prováveis processos de recombinação sexual. A proposta deste estudo foi de conhecer as variações gênicas e relações filogenéticas em Sarcocystis spp. isolados de gambás através da análise de loci gênicos de genoma mitocondrial (CytB), de genoma de apicoplasto (ClpC) e das regiões espaçadoras transcritas internas (ITS-1), comparando a diversidade encontrada nestes grupos com a observada em integrantes da sub-familia Toxoplasmatinae. E também de conhecer a diversidade de genes codificadores de SAG-2, SAG-3 e SAG-4. Neste estudo foram obtidos esporocistos de Sarcocystis spp. através de raspado intestinal e fezes provenientes de gambás do gênero Didelphis, oriundos do Estado de São Paulo, Rio Grande do Sul e Rio Grande do Norte. Os marcadores moleculares CytB, ClpC e ITS-1 revelaram uma ampla variabilidade genotípica e alelos inéditos entre os isolados do Brasil. Os resultados levam a crer que é possível a existência de espécies “híbridas” de Sarcocystis no Brasil, com mistura de genes de ambas as espécies. Em relação aos genes codificadores de SAGs, foi encontrada uma diversidade ainda maior do que trabalho similar realizado. Os resultados sugerem uma possível ocorrência de reassortment e recombinação de alelos em sequências SAGs, contribuindo ainda mais para a geração de variabilidade e consequentemente na configuração antigênica do parasito. Estes resultados demonstram que isolados brasileiros possuem uma composição genética diferente do reportado em demais localidade, o que pode ter reflexos importantes no conhecimento da diversidade das espécies de Sarcocystis que infectam gambás em nosso meio e em toda a epidemiologia das infecções produzidas pelos protozoários deste grupo / A great variability in surface antigens genes (SAGs) of Sarcocystis spp. sporocysts was found in a previous study, through the intestinal scraping technique of opossums from the south region of Brazil. The results indicated probable sexual recombination between lineages of Sarcocystis isolates (genetically related to Sarcocystis falcatula). The aim of this research is the study of genetic variability and phylogenetic relationships among Sarcocystis spp. isolates from captured opossums in Brazil, by the analysis of molecular markers less subjected to selective pressure, such as DNA barcode, for this purpose, genetic loci from mitochondrial (CytB) and apicoplast genome (ClpC), and internal transcribed spacer regions (ITS-1) were applied and compare the results with the diversity found within members of the Toxoplasmatinae sub-family. The diversity among the SAG-2, SAG-3 and SAG-4 genes was also studied. The Sarcocystis sporocysts were obtained from opposum’s intestinal scraping and faeces from São Paulo State, Rio Grande do Sul State and Rio Grande do Norte State, Brazil. The molecular markers CytB, ClpC and ITS-1 revealed a vast genotypic variability and novel alleles among the Brazilian isolates. The results indicate possible existence of hybrid species of Sarcocystis in Brazil, with a gene mixture from both species. Regarding the SAG genes, an even greater variability was found than previously reported. The results also suggest the occurrence of reassortment and allele recombination of SAG sequences, contributing even more to the variability and thus in the parasites antigenic configuration. In this study, the results demonstrate that isolates from Brazil have a different genetic composition than reported in other locations, which may have important impacts in the knowledge of the diversity of Sarcocystis species shed by opossums in our environment and in all of the epidemiology of infections of protozoa of this particular group