RESUMO
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.
Assuntos
Alelos , Antígenos de Protozoários/genética , Doenças das Aves/parasitologia , Gambás/parasitologia , Sarcocystis/genética , Sarcocistose/veterinária , Animais , Antígenos de Superfície/genética , Evolução Biológica , Aves , Encéfalo/parasitologia , Brasil , DNA de Protozoário/química , DNA de Protozoário/isolamento & purificação , Variação Genética/genética , Pulmão/parasitologia , Melopsittacus , Meningoencefalite/parasitologia , Meningoencefalite/veterinária , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase/veterinária , Guaxinins/parasitologia , Sarcocystis/classificação , Sarcocystis/imunologia , Sarcocystis/isolamento & purificação , Sarcocistose/parasitologia , Análise de Sequência de DNA/veterináriaRESUMO
Although few species of Sarcocystis are known to use marsupials of the genus Didelphis as definitive host, an extensive diversity of alleles of surface antigen genes (sag2, sag3, and sag4) has been described in samples of didelphid opossums in Brazil. In this work, we studied 25 samples of Sarcocystis derived from gastrointestinal tract of opossums of the genus Didelphis by accessing the variability of sag2, sag3, sag4, gene encoding cytochrome b (cytB) and first internal transcribed spacer (ITS1). Reference samples of Sarcocystis neurona (SN138) and Sarcocystis falcatula (SF1) maintained in cell culture were also analyzed. We found four allele variants of cytB, seven allele variants of ITS1, 10 allele variants of sag2, 13 allele variants of sag3, and 6 allele variants of sag4. None of the sporocyst-derived sequences obtained from Brazilian opossums revealed 100% identity to SN138 at cytB gene, nor to SN138 or SF1 at ITS1 locus. In addition, none of the sag alleles were found identical to either SF1 or SN138 homologous sequences, and a high number of new sag allele types were found other than those previously described in Brazil. Out of ten sag2 alleles, four are novel, while eight out of 13 sag3 alleles are novel and one out of six sag4 alleles is novel. Further studies are needed to clarify if such a vast repertoire of allele variants of Sarcocystis is the consequence of re-assortments driven by sexual exchange, in order to form individuals with highly diverse characteristics, such as pathogenicity, host spectrum, among others or if it only represents allele variants of different species with different biological traits.
Assuntos
DNA de Protozoário/química , Didelphis/parasitologia , Variação Genética , Filogenia , Sarcocystis/classificação , Alelos , Animais , Antígenos de Protozoários/genética , Antígenos de Superfície/genética , Brasil , Citocromos b/genética , DNA Intergênico/genética , DNA de Protozoário/genética , Trato Gastrointestinal/parasitologia , Genótipo , Melopsittacus , Reação em Cadeia da Polimerase , Proteínas de Protozoários/genética , Guaxinins , Sarcocystis/genética , Sarcocistose/parasitologia , Sarcocistose/veterinária , Análise de Sequência de DNARESUMO
South American opossums are the definitive hosts of Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis speeri and Sarcocystis lindsayi. The sporocysts of these species of Sarcocystis are morphologically similar and methods like infectivity and pathogenicity for intermediate hosts (immunodeficient mice and psittacine birds) and molecular tools are used for identification. Opossums are synanthropic wild animals, and widely distributed in Brazilian territory. Previous studies have shown high environmental contamination with S. neurona sporocysts in several Brazilian regions. This paper reviews information on Sarcocystis spp. shed by various opossum species and its occurrence in Brazil.(AU)
Os gambás Sul-americanos são os hospedeiros definitivos de Sarcocystis falcatula, Sarcocystis neurona, Sarcocystis speeri e Sarcocystis lindsayi. Estas espécies de Sarcocystis são morfologicamente similares, mas podem ser distinguidas por sua patogenicidade e infectividade em hospedeiros intermediários (aves e camundongos imunodeficientes) e técnicas moleculares. Os gambás são animais silvestres e sinantrópicos e amplamente distribuídos no território nacional. Estudos anteriores demonstraram uma alta contaminação ambiental com esporocistos de S. neurona em diversas regiões brasileiras. Este artigo revisa informações sobre Sarcocystis spp. excretados por gambás e sua ocorrência no Brasil.(AU)
Assuntos
Animais , Didelphis/parasitologia , Gambás/parasitologia , Contagem de Ovos de Parasitas/veterinária , Sarcocystis/parasitologia , Sarcocystis/patogenicidade , BrasilRESUMO
South American opossums are the definitive hosts of Sarcocystis neurona, Sarcocystis falcatula, Sarcocystis speeri and Sarcocystis lindsayi. The sporocysts of these species of Sarcocystis are morphologically similar and methods like infectivity and pathogenicity for intermediate hosts (immunodeficient mice and psittacine birds) and molecular tools are used for identification. Opossums are synanthropic wild animals, and widely distributed in Brazilian territory. Previous studies have shown high environmental contamination with S. neurona sporocysts in several Brazilian regions. This paper reviews information on Sarcocystis spp. shed by various opossum species and its occurrence in Brazil.(AU)
Os gambás Sul-americanos são os hospedeiros definitivos de Sarcocystis falcatula, Sarcocystis neurona, Sarcocystis speeri e Sarcocystis lindsayi. Estas espécies de Sarcocystis são morfologicamente similares, mas podem ser distinguidas por sua patogenicidade e infectividade em hospedeiros intermediários (aves e camundongos imunodeficientes) e técnicas moleculares. Os gambás são animais silvestres e sinantrópicos e amplamente distribuídos no território nacional. Estudos anteriores demonstraram uma alta contaminação ambiental com esporocistos de S. neurona em diversas regiões brasileiras. Este artigo revisa informações sobre Sarcocystis spp. excretados por gambás e sua ocorrência no Brasil.(AU)
Assuntos
Animais , Sarcocystis , Gambás/parasitologia , Contagem de Ovos de Parasitas/veterinária , Brasil , Didelphis/parasitologiaRESUMO
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.
Assuntos
Antígenos de Protozoários/genética , Antígenos de Superfície/metabolismo , Sarcocystis/genética , Sarcocistose/veterinária , Animais , Antígenos de Superfície/genética , Biodiversidade , Brasil/epidemiologia , Variação Genética , Marsupiais , Filogenia , Reprodução , Sarcocistose/epidemiologia , Sarcocistose/parasitologiaRESUMO
Este estudo avaliou o grau de infecção por Sarcocystis spp. em marsupiais do gênero Didelphis spp., conhecidospopularmente por gambás, no município de Bagé, na região sul do estado do Rio Grande do Sul (RS). Este parasito é oagente causador da Mieloencefalite Protozoária Equina (MEP), sendo que neste município estão localizados importantescriatórios de cavalos Puro-Sangue Inglês, demonstrando a importância deste estudo na região. O gambá atua comohospedeiro definitivo do parasito e elimina oocistos juntamente com as fezes. Os equinos infectam-se ao ingerir alimentoscontaminados por fezes de gambás. Para o estudo, foram analisados 19 marsupiais da espécie Didelphis albiventris.Foram realizadas raspagens das três secções do intestino delgado dos gambás para avaliação de lesões macroscópicase histopatológicas, como também para o isolamento e identificação de Sarcocystis spp. Na avaliação histopatológica,observou-se a presença de células inflamatórias, infiltrado de eosinófilos e áreas hemorrágicas, caracterizando enterite.Nos 19 (100%) animais necropsiados e examinados, foi observada a presença de Sarcocystis spp., e em um (5,3%) animalfoi isolado Sarcocystis neurona.
RESUMO
Este estudo avaliou o grau de infecção por Sarcocystis spp. em marsupiais do gênero Didelphis spp., conhecidospopularmente por gambás, no município de Bagé, na região sul do estado do Rio Grande do Sul (RS). Este parasito é oagente causador da Mieloencefalite Protozoária Equina (MEP), sendo que neste município estão localizados importantescriatórios de cavalos Puro-Sangue Inglês, demonstrando a importância deste estudo na região. O gambá atua comohospedeiro definitivo do parasito e elimina oocistos juntamente com as fezes. Os equinos infectam-se ao ingerir alimentoscontaminados por fezes de gambás. Para o estudo, foram analisados 19 marsupiais da espécie Didelphis albiventris.Foram realizadas raspagens das três secções do intestino delgado dos gambás para avaliação de lesões macroscópicase histopatológicas, como também para o isolamento e identificação de Sarcocystis spp. Na avaliação histopatológica,observou-se a presença de células inflamatórias, infiltrado de eosinófilos e áreas hemorrágicas, caracterizando enterite.Nos 19 (100%) animais necropsiados e examinados, foi observada a presença de Sarcocystis spp., e em um (5,3%) animalfoi isolado Sarcocystis neurona.