Sarcocystis neurona is an apicomplexan parasite that is a major cause of equine protozoal myeloencephalitis (EPM). During intracellular development of S. neurona, many genes are temporally regulated. To better understand gene regulation, it is important to identify and characterize regulatory elements controlling gene expression in S. neurona. To perform this study, it was essential to establish transfection system for this parasite. Hence, the 5 flanking region of the SnSAG1 gene was isolated from a genomic library and used to construct expression plasmids. In transient assays, the reporter molecules -galactosidase (-gal) and yellow fluorescent protein (YFP) were expressed by electroporated S. neurona, thereby confirming the feasibility of performing molecular genetic experiments in this organism. Stable transformation of S. neurona was achieved using a mutant dihydrofolate reductase thymidylate synthase (DHFR-TS) gene of T. gondii that confers resistance to pyrimethamine. This selection system was used to create transgenic S. neurona that stably express -gal and YFP. These transgenic clones were shown to be useful for analyzing growth rate of parasites in-vitro and for assessing drug sensitivities. To uncover possible sequence elements involved in promoter activity, the 5 flanking regions of five S. neurona genes were subjected to comparative analysis. This revealed the presence of a 7-base conserved motif GCGTCTC. Using a dual luciferase assay system, the SnSAG1 promoter was subjected to functional analysis. The motif GAGACGC located between -136 and -129 upstream of the transcription start site was found to be essential for SnSAG1 expression. This motif functions in an orientation dependent manner and was shown to play a role in binding nuclear proteins of S. neurona.

SANTRAUKA PAUKŠČIUOSE APTINKAMŲ NEAPIBŪDINTŲ SARCOCYSTIS GENTIES RŪŠIŲ FILOGENETINIŲ RYŠIŲ TYRIMAS NAUDOJANT DALINES 18S IR 28S rRNR GENŲ SEKAS Petras Prakas Vilniaus Universiteto Ekologijos Institutas, Populiacinės Genetikos Laboratorija, Vilnius, Lietuva. Pagal cistų morfologiją, baltakaktėje žąsyje (Anser albifrons) aptiktos I tipo sarkocistos, didžiojoje antyje (Anas platyrhynchos) rastos II tipo sarkocistos, dviejose baltakaktėse žąsyse ir pilkojoje žąsyje (Anser anser) nustatytos III tipo sarkocistos, varnoje (Corvus cornix) identifikuotos V tipo sarkocistos. Iš paukščių, kaip tarpinių šeimininkų išskirtos sarkosporidijos aprašomos pirmą kartą bei yra neįvardintos rūšys. Baltakaktėje bei pilkojoje žąsyse išskirtos III tipo sarkocistos gali būti tos pačios sarkocistų rūšies parazitavimo skirtingų rūšių tarpiniuose šeimininkuose atvejis. Nustatytos 18S ir 28S rDNR fragmentų sekos pasižymėjo didžiausia homologija Sarcocystis bei Frenkelia gentims. Filogenetinių ryšių medyje sugrupuojamos šiame darbe tiriamos rūšys su Frenkelia microti, Frenkelia glareoli, Sarcocystis muris, Sarcocystis neurona. Rezultatai atskleidė Sarcocystis genties rūšių koevoliuciją su jų galutiniais šeimininkais. Remiantis tyrimo duomenimis, galima teigti, kad Sarcocystidae šeimos filogenetinius ryšius apsprendžia sarkosporidijų gyvybinio ciklo ypatybės, šeimininkų spektras bei parazitų specifiškumas šeimininkui. 18S ir 28S rRNR genų dalinių sekų analizė atskleidžia tirtų Sarcocystis sp. rūšių... [toliau žr. visą tekstą] / SUMMARY INVESTIGATION OF THE PHYLOGENETIC RELATIONSHIPS OF SARCOCYSTIS SPP. FOUND IN BIRDS USING 18S AND 28S rRNA GENE PARTIAL SEQENCES Petras Prakas Institute of Ecology of Vilnius University, Laboratory of Population Genetics, Vilnius, Lithuania Based on cyst morphology, Sarcocystis cysts type I were found in white-fronted goose (Anser albifrons), cysts type II in mallard (Anas platyrhynchos), cyst type III in one grey-lag goose (Anser anser) and two white-fronted geese, cyst type V in hooded crow (Corvus cornix). The sarcocysts isolated from the infected birds as intermediate host have not been previously described and are unnamed. Type III sarcocysts detected in white-fronted and greylag geese may illustrate the case of polyhostal nature of sarcocysts when the same-species parasites infesting intermediate hosts of different species. The obtained 18S and 28S ribosomal RNA partial gene sequences showed the highest homology for the genera Sarcocystis and Frenkelia. In the tree of phylogenetic relationships, the species involved in this study were grouped with Frenkelia microti, Frenkelia glareoli, Sarcocystis muris and Sarcocystis neurona. Results show co-evolution of Sarcocystis spp. with the final host. Based on data obtained it could be stated that phylogenetic relationships of Sarcocystidae family are influenced by peculiarity of life cycle, hosts spectrum and host specificity. Analysis of the partial sequences of 18S and 28S ribosomal RNA revealed the phylogenetic and... [to full text]

Diss. (sammanfattning) Uppsala : Sveriges lantbruksuniv. / Härtill 4 uppsatser.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / "May 2004." Typescript. Includes bibliographical references (leaves 30-40). Also issued on the Internet.

Thesis (M.S.)--University of Missouri--Columbia, 2004. / Typescript. Includes bibliographical references (leaves 30-40). Also issued on the Internet.

Encephalitozoon cuniculi, Toxoplasma gondii and Sarcocystis neurona are intracellular parasites that infect a wide range of mammalian host species including domestic cats. The prevalence of antibodies to these parasites in cats was examined using an indirect immunofluorescence antibody assay. E. cuniculi targets the kidneys of rabbits but the prevalence of disease in cats is unknown. Chronic kidney disease (CKD) is a common cause of illness in cats. T. gondii is a widespread parasite of cats; however, it is not considered a major causative agent of CKD. The first hypothesis was that E. cuniculi and T. gondii are unrecognized causes of chronic kidney disease in domestic cats. Serum and plasma samples were examined for protozoal antibodies from 232 feline patients at the VMRCVM Teaching Hospital. Thirty-six of the 232 samples met the IRIS criteria for CKD. Antibodies to E. cuniculi were found in 15 samples, 4 of which came from cats with CKD. Antibodies to T. gondii were found in 63 samples; 10 cats of the 63 had CKD. These were not significantly different from cats with no CKD and the null hypothesis was rejected. Domestic cats, armadillos, raccoons and skunks are intermediate hosts (IH) for S. neurona while opossums are the definitive host (DH). The seroprevalence of S. neurona was examined in domestic cats from Virginia and Pennsylvania. The second hypothesis was that domestic cats are important IH for S. neurona transmission. A low seroprevalence was found in 32 of the 441 cats and the null hypothesis was rejected. / Master of Science in Life Sciences

Didelphis virginiana (gambá) é hospedeiro definitivo do Sarcocystis falcatula, Sarcocystis neurona e Sarcocystis speeri. Os esporocistos de S. neurona, S. falcatula e S. speeri são similares morfologicamente, mas podem ser distinguidos por sua patogenicidade e infectividade em aves e camundongos imunodeficientes e métodos moleculares. Porém, há uma considerável controvérsia a respeito da identificação das espécies de Sarcocystis de gambás. A heterogeneidade genética das espécies de Sarcocystis dificulta a identificação definitiva destes parasitos. Utilizou-se D. aurita e D. albiventris (gambás) mortos provenientes de Zoológicos e Centros de Triagens do Estado de São Paulo. Após usar o método de centrífugo-flutuação em solução de sacarose, purificou-se 19 (31,25%) das 25 amostras positivas em duplicatas para realização de PCR e ensaios biológicos utilizando periquitos australianos (Melopsittacus undulatus) como modelo biológico do S. falcatula e camundongos Balb/c nude como modelo experimental para S. neurona. As amostras de esporocistos foram identificadas por PCR e sequenciamento de nucleotídeos de fragmentos codificadores de gene de proteína de superfície 2 (SAG-2) como S. falcatula. Os resultados das inoculações experimentais em periquitos australianos também demonstraram que as amostras continham esporocistos de Sarcocystis falcatula de alta patogenicidade e infectividade. Quinze animais inoculados vieram a óbito 10 dias pós-inoculação. As análises histopatológicas revelaram lesões severas principalmente em pulmão e fígado. Entretanto, não foram encontradas lesões em camundongos Balb/c nude inoculados com duas dessas amostras de esporocistos. Este trabalho demonstrou a importância desta enfermidade no Estado de São Paulo. / Didelphis virginiana (opossum of North America) is a definitive host of Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. Sporocysts of S. neurona, S. falcatula and S. speeri are morphologically similar but can be distinguished by its pathogenicity and infectivity in immunodeficient mice and birds and molecular methods. However, there is considerable controversy regarding the identification of Sarcocystis species from opossums. The genetic heterogeneity of species of Sarcocystis hinders definitive identification of these parasites. We used dead D. aurita and D. albiventris (opossum) from Zoos and Trials Centers of São Paulo. After using the centrifugal flotation in sucrose solution, nineteen (31.25%) of 25 positive samples were purified in duplicate for PCR and biological assays using budgerigars (Melopsittacus undulatus) as a biological model of S. falcatula and Balb/c nude as a model for S. neurona. Samples of sporocysts were identified by PCR and sequencing of gene fragments encoding surface protein 2 (SAG-2) as S. falcatula. The results of experimental inoculations in budgerigars also showed that the samples contained high pathogenic and infective Sarcocystis falcatula sporocists. Fifteen infected animals died 10 days post-inoculation. The histopathological analysis showed severe lesions mainly in lung and liver. However, no lesions were found in Balb/c nude inoculated with two samples of sporocysts. This study demonstrated the importance of this disease in the state of São Paulo.

Didelphis virginiana (gambá) é hospedeiro definitivo do Sarcocystis falcatula, Sarcocystis neurona e Sarcocystis speeri. Os esporocistos de S. neurona, S. falcatula e S. speeri são similares morfologicamente, mas podem ser distinguidos por sua patogenicidade e infectividade em aves e camundongos imunodeficientes e métodos moleculares. Porém, há uma considerável controvérsia a respeito da identificação das espécies de Sarcocystis de gambás. A heterogeneidade genética das espécies de Sarcocystis dificulta a identificação definitiva destes parasitos. Utilizou-se D. aurita e D. albiventris (gambás) mortos provenientes de Zoológicos e Centros de Triagens do Estado de São Paulo. Após usar o método de centrífugo-flutuação em solução de sacarose, purificou-se 19 (31,25%) das 25 amostras positivas em duplicatas para realização de PCR e ensaios biológicos utilizando periquitos australianos (Melopsittacus undulatus) como modelo biológico do S. falcatula e camundongos Balb/c nude como modelo experimental para S. neurona. As amostras de esporocistos foram identificadas por PCR e sequenciamento de nucleotídeos de fragmentos codificadores de gene de proteína de superfície 2 (SAG-2) como S. falcatula. Os resultados das inoculações experimentais em periquitos australianos também demonstraram que as amostras continham esporocistos de Sarcocystis falcatula de alta patogenicidade e infectividade. Quinze animais inoculados vieram a óbito 10 dias pós-inoculação. As análises histopatológicas revelaram lesões severas principalmente em pulmão e fígado. Entretanto, não foram encontradas lesões em camundongos Balb/c nude inoculados com duas dessas amostras de esporocistos. Este trabalho demonstrou a importância desta enfermidade no Estado de São Paulo. / Didelphis virginiana (opossum of North America) is a definitive host of Sarcocystis falcatula, Sarcocystis neurona and Sarcocystis speeri. Sporocysts of S. neurona, S. falcatula and S. speeri are morphologically similar but can be distinguished by its pathogenicity and infectivity in immunodeficient mice and birds and molecular methods. However, there is considerable controversy regarding the identification of Sarcocystis species from opossums. The genetic heterogeneity of species of Sarcocystis hinders definitive identification of these parasites. We used dead D. aurita and D. albiventris (opossum) from Zoos and Trials Centers of São Paulo. After using the centrifugal flotation in sucrose solution, nineteen (31.25%) of 25 positive samples were purified in duplicate for PCR and biological assays using budgerigars (Melopsittacus undulatus) as a biological model of S. falcatula and Balb/c nude as a model for S. neurona. Samples of sporocysts were identified by PCR and sequencing of gene fragments encoding surface protein 2 (SAG-2) as S. falcatula. The results of experimental inoculations in budgerigars also showed that the samples contained high pathogenic and infective Sarcocystis falcatula sporocists. Fifteen infected animals died 10 days post-inoculation. The histopathological analysis showed severe lesions mainly in lung and liver. However, no lesions were found in Balb/c nude inoculated with two samples of sporocysts. This study demonstrated the importance of this disease in the state of São Paulo.

Infections by protozoa of the Sarcocystidae family have worldwide distribution and are common in ruminants, causing important economic losses. This study evaluated Sarcocystis spp., Toxoplasma gondii and Neospora caninum infections in sheep from Southwest region of Rio Grande do Sul, Brazil. Myocardium samples of 80 sheep raised on extensive system were collected. Tissue cysts were detected by direct examination and the presence of the agents was also confirmed by PCR. Macroscopic evaluation did not reveal changes, but direct microscopic examination showed cysts in 76.2% (61/80) of samples and all cysts were morphologically similar with Sarcocystis tenella or Sarcocystis arieticanis. PCR detected Sarcocystis spp. DNA in 21.2% (17/80) of samples tested and T. gondii DNA in 15% (12/80). In 6.2% (5/80) DNA of both protozoan were detected. Presence of N. caninum nucleic acids was not observed in the samples tested. All PCR-positive samples (23.7% - 19/80) were also positive by direct examination (microscopic cysts). Thus, a high occurrence of microscopic tissue cysts in sheep from the Southwest region of the state of Rio Grande do Sul was detected. Although PCR did not show a good sensitivity to identify the causative agents of these cysts, was possible to verify the presence of Sarcocystis spp. and T. gondii in cardiac muscle samples of the ovine. This may be a risk factor for animal and human contamination, not only through consumption, but also through handling the carcasses of these animals. Equine protozoal myeloencephalitis is a neurologic disease of horses, most often caused by the Sarcocystis neurona. However, the role of horses in the life cycle this parasite is not completely understood. This study attempts to elucidate the role of horses as intermediate hosts in S. neurona cycle, through occurrence of cysts in these animals and determine the antibodies frequency for Sarcocystis spp., Neospora spp. and T. gondii in slaughtered horses. Were collected 197 serum and heart samples of equines. None of the myocardium samples were detected tissue cysts, nucleic acids or histopathological changes associated to Sarcocystis spp. In antibodies detection, 146 (74.1%) serum samples were positive for studied protozoa. Antibodies against Sarcocystis spp. were detected in 36% (71/197), to Neospora spp. in 39.1% (77/197) and to T. gondii in 47,2% (93/197). Thus, the failure in detect tissue cysts, associated with antibodies anti-Sarcocystis spp. detection, increases the role of horses as accidental hosts in the cycle this protozoan, declining your participation in the epidemiology of Sarcocystis infection. / As infecções causadas por protozoários da família Sarcocystidae têm distribuição mundial e são comuns em ruminantes, causando perdas econômicas importantes. Este estudo avaliou infecções de Sarcocystis spp., Toxoplasma gondii e Neospora caninum em ovinos da região sudoeste do Rio Grande do Sul, Brasil. Foram coletadas amostras de miocárdio de 80 ovelhas criadas em sistema extensivo. Cistos de tecido foram detectados por exame direto e a presença dos agentes também foi confirmada por PCR. A avaliação macroscópica não revelou alterações, mas o exame microscópico direto mostrou cistos em 76,2% (61/80) das amostras e todos os cistos eram morfologicamente semelhantes a Sarcocystis tenella ou Sarcocystis arieticanis. A PCR detectou DNA de Sarcocystis spp. em 21,2% (17/80) das amostras testadas e DNA de T. gondii em 15% (12/80). Em 6,2% (5/80) foram detectados DNA de ambas os protozoários. Todas as amostras de PCR positivas (23,7% - 19/80) também foram positivas pelo exame direto (cistos microscópicos). Assim, uma alta ocorrência de cistos teciduais microscópicos em ovinos da região do sudoeste do estado do Rio Grande do Sul foi detectada. Apesar de o PCR não ter mostrado boa sensibilidade para identificar os agentes causadores destes cistos, foi possível verificar a presença de Sarcocystis spp. e T. gondii em amostras do músculo cardíaco de ovinos. Isso pode ser um fator de risco para animais e contaminação humana, não só através do consumo, mas também através de manipulação das carcaças desses animais. A Mieloencefalite Equina por Protozoário (MEP) é uma enfermidade neurológica que acomete equinos e possui como principal agente o protozoário Sarcocystis neurona. Porém o papel dos equinos no ciclo deste protozoário não está completamente esclarecido. O objetivo deste trabalho foi elucidar o papel dos equinos como hospedeiros intermediários no ciclo do protozoário S. neurona, através da ocorrência de cistos nestes animais e determinar a frequência de detecção de anticorpos anti- Sarcocystis spp., Neospora spp. e T. gondii em equinos abatidos em frigorífico no Estado do Rio Grande do Sul, Brasil. Foram coletadas 197 amostras de soro e coração de equinos. Das amostras pesquisadas, não houve detecção de cistos, ácidos nucléicos ou alterações histopatológicas relacionadas ao Sarcocystis spp. Relacionado com a detecção de anticorpos, 146 (74,1%) foram positivos para pelo menos um dos protozoários pesquisados. Anticorpos para Sarcocystis spp. foram encontrados em 36% (71/197), para Neospora spp. em 39,1% (77/197) e para T. gondii em 47,2% (93/197). Assim a não detecção de cistos teciduais, associado com a detecção de anticorpos anti-Sarcocystis spp., reforça a participação dos equinos como hospedeiros acidentais no ciclo deste protozoário, minimizando a participação destes animais na epidemiologia da infecção por Sarcocystis spp.

Equine protozoal myeloencephalitis (EPM) is a progressive neurological disease of horses caused by Sarcocystis neurona. Two projects were conducted to identify factors involved in the development of EPM. The first study explored a possible genetic susceptibility to EPM by attempting a genome-wide association study (GWAS) on formalin-fixed, paraffin-embedded (FFPE) tissue from 24 definitively-positive EPM horses. DNA extracted from tissues older than 14 months was inadequate for SNP analysis on the Illumina Equine SNP50 BeadChip probably due to degradation and formalin cross-linking. Results were inconclusive as analysis was not possible with the small sample set. The second study evaluated an artificial infection method in creating a reliable equine EPM model. Five horses were injected intravenously at 4 time points with autologous blood incubated with 1,000,000S. neurona merozoites. Challenged horses progressively developed mild to moderate clinical signs and had detectable S. neurona serum antibodies on day 42 post challenge. Horses appeared to have produced a Th1 immune response and cleared the infection by the conclusion of the study on day 89. No histopathological evidence of S. neurona infection was found within central nervous system tissue. This artificial infection method was not effective in replicating the severe clinical EPM seen in natural infections.