Caracterização molecular de Sarcocystis spp. em gambás (Didelphis spp.) e detecção de anticorpos anti-Sarcocystis spp. em equinos das regiões Sudeste e Centro-oeste do Brasil
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2023-02-24
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Universidade Estadual Paulista (Unesp)
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Gambás (Didelphis spp.) são hospedeiros definitivos de Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi e Sarcocystis falcatula. No Brasil, diversos estudos vêm demonstrando a alta frequência de Sarcocystis falcatula-like em esporocistos recuperados dos intestinos de gambás, com grande diversidade nos genes codificadores de antígenos de superfície (SAGs). Neste estudo, a diversidade genética de Sarcocystis spp. oriundos de Didelphis albiventris e Didelphis aurita capturados nos municípios de Campo Grande e São Paulo, foi acessada por meio do sequenciamento de SAG2, SAG3 e SAG4, da primeira região espaçadora interna transcrita (ITS-1) e da subunidade I da citocromo c oxidase (cox1). Adicionalmente, reação de imunofluorescência indireta (RIFI) foi empregada para detecção de anticorpos (IgG) contra S. falcatula-like (Dal-CG23) e S. neurona (SN138) no soro de 342 equinos amostrados nos mesmos municípios. Identificação molecular foi realizada em 16 amostras de DNA obtidas de esporocistos recuperados do intestino de gambás ou merozoítos derivados de cultivo in vitro de Sarcocystis spp. Os fragmentos de ITS-1, cox1, e SAG3 foram clonados, enquanto SAG2 e SAG4 foram sequenciados diretamente dos produtos de PCR. Vinte e sete alelos foram observados em ITS-1, todos filogeneticamente relacionados à S. falcatula-like previamente detectado em aves e/ou em gambás no Brasil. Sarcocystis sp. filogeneticamente relacionado à Sarcocystis rileyi foi evidenciado por cox1 em três gambás. Vinte e um novos alelos SAGs foram descritos, sendo quatro em SAG2 (n=4), 13 em SAG3 (n=13) e quatro em SAG4 (n=7). Nos Imunoensaios utilizando RIFI com ponto de corte de 1:25, anticorpos IgG anti-S. falcatula-like foram detectados em 177/342 amostras de soro equino (51.75%), e anticorpos IgG anti-S. neurona foram detectados em 239/342 amostras (69.88%). Anticorpos IgG contra ambos os isolados foram observados no soro de 132 equinos (38,59%). Ausência de reatividade foi verificada em 16.95% (58/342) animais. A elevada soroprevalência observada neste estudo pode estar relacionada ao baixo ponte de corte utilizado e à presença de gambás infectados com Sarcocystis falcatula-like e Sarcocystis spp. nas áreas onde os equinos foram IX amostrados. Devido à similaridade entre antígenos de superfície detectados nos imunoensaios, relatos de soropositividade contra S. neurona no Brasil podem resultar da exposição dos equinos à outras espécies de Sarcocystis. O papel de outras espécies de Sarcocystis como causa de doença neurológica nos equinos em Brasil permanece incerto.
Opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, several studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Additionally, indirect fluorescent antibody test (IFAT) was used to detect IgG antibodies against Sarcocystis falcatula-like (Dal-CG23) and S. neurona (SN138) in equine sera from 342 horses sampled in the same regions. Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS-1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in birds and/or opossums in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Twenty-one new SAG alelles were described, four in SAG2 (n=4), 13 in SAG3 (n=13) and four in SAG4 (n=7). On IFAT using 1:25 cutoff, IgG antibodies against S. falcatula-like were detected in 177/342 samples (51.75%), whereas IgG antibodies against S. neurona were detected in 239/342 samples (69.88%). IgG antibodies against both isolates were detected in 132 samples (38,59%). No reactivity was observed in 16.95% (58/342) horses. The high seroprevalence observed here may be related to the lower cutoff, and the presence of opossums infected with Sarcocystis falcatula-like and Sarcocystis spp. in the regions where the horses were sampled. Owing to the similarity among antigens targeted in immunoassays, reports on S. neurona-seropositive horses in Brazil might also derived from exposure of horses to other Sarcocystis species. The role of other Sarcocystis species in causing neurological diseases in horses in Brazil remains unclear.
Opossums (Didelphis spp.) are definitive hosts of Sarcocystis neurona, Sarcocystis speeri, Sarcocystis lindsayi and Sarcocystis falcatula. In Brazil, several studies have demonstrated a high frequency of Sarcocystis falcatula-like in sporocysts derived from opossums, and high genetic diversity has been observed in surface antigen-encoding genes (SAGs). In this study, genetic diversity of Sarcocystis spp. derived from Didelphis albiventris and Didelphis aurita from the cities of Campo Grande and São Paulo, was accessed by sequencing SAG2, SAG3, SAG4, the first internal transcribed spacer (ITS-1) and cytochrome c oxidase subunit I (cox1). Additionally, indirect fluorescent antibody test (IFAT) was used to detect IgG antibodies against Sarcocystis falcatula-like (Dal-CG23) and S. neurona (SN138) in equine sera from 342 horses sampled in the same regions. Molecular identification was performed for 16 DNA samples obtained from sporocyst or culture-derived merozoites. The ITS-1, cox1, and SAG3 fragments were cloned, whereas SAG2 and SAG4 were sequenced directly from PCR products. Twenty-seven allele variants were found for ITS-1, all phylogenetically related to S. falcatula-like previously described in birds and/or opossums in Brazil. Sarcocystis sp. phylogenetically related to Sarcocystis rileyi was evidenced by cox1 in three opossums. Twenty-one new SAG alelles were described, four in SAG2 (n=4), 13 in SAG3 (n=13) and four in SAG4 (n=7). On IFAT using 1:25 cutoff, IgG antibodies against S. falcatula-like were detected in 177/342 samples (51.75%), whereas IgG antibodies against S. neurona were detected in 239/342 samples (69.88%). IgG antibodies against both isolates were detected in 132 samples (38,59%). No reactivity was observed in 16.95% (58/342) horses. The high seroprevalence observed here may be related to the lower cutoff, and the presence of opossums infected with Sarcocystis falcatula-like and Sarcocystis spp. in the regions where the horses were sampled. Owing to the similarity among antigens targeted in immunoassays, reports on S. neurona-seropositive horses in Brazil might also derived from exposure of horses to other Sarcocystis species. The role of other Sarcocystis species in causing neurological diseases in horses in Brazil remains unclear.
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Didelphis, Gambá, Patologia animal, Imunoensaio