Sarcocystis falcatula-like derived from opossum in Northeastern Brazil: In vitro propagation in avian cells, molecular characterization and bioassay in birds

Abstract

Most reported isolates of Sarcocystis spp. derived from Brazilian opossums ( Didelphis sp.) have genetic characteristics distinct from the known species of Sarcocystis , but behave similarly as Sarcocystis falcatula , as they are infective to budgerigars. In previous studies, these Brazilian isolates, classified as Sarcocystis falcatula -like, were originated from South and Southeast regions of Brazil. In the current work, we aimed to culture and to perform multilocus sequence analysis of Sarcocystis sp. derived from a Brazilian opossum ( D. aurita / D. marsupialis ) that inhabited the city of Salvador, Bahia, in the Northeast of Brazil. The parasite was isolated in Vero cells, referred here as Sarco-BA1, and propagated in avian cells (DF-1). Molecular analysis of Sarco-BA1 revealed that the nucleotide sequence of the internal transcribed spacer 1 (ITS1) of the rDNA was identical to all isolates (n = 19) of Sarcocystis spp. reported in two studies from South and Southeast regions of the country. Two budgerigars were inoculated with 10 and 1000 sporocysts of Sarco-BA1, respectively, and developed acute sarcocystosis, showing that the parasite behaves like S. falcatula . It was interesting to observe that Sarco-BA1 had almost identical ITS1 and SAG sequences to all 16 isolates of S. falcatula -like recently described in Magellanic penguins ( Spheniscus magellanicus ) rescued on the coast of Espírito Santo state, Brazil. Our results suggest that Sarco-BA1 and S. falcatula -like may represent a single species of Sarcocystis . Propagation of the parasite in a permanent avian cell line significantly improved the yield of merozoites in cell culture. To our knowledge, this is the first molecular study and in vitro isolation of S. falcatula -like derived from Northeastern Brazil. Studies are under way to determine the infectivity of Sarco-BA1 to other animal species, as well as to investigate serological cross-reactivity among Sarco-BA1, S. neurona and related species.

Reactivity of Horse Sera to Antigens Derived From Sarcocystis falcatula–Like and Sarcocystis neurona

“…Antigens for IFATs and for WB consisted of merozoites of a North American strain of S. neurona (SN-138) ( 30 ) and merozoites from a South American strain of S. falcatula –like ( 27 ). S. neurona merozoites were propagated in Vero cells supplemented with RPMI-1650 + l -glutamine (Invitrogen/Gibco®, Carlsbad, USA), 1% antibiotic–antimycotic (100 units/mL of penicillin, 100 μg/mL of streptomycin, and 0.25 μg/mL of amphotericin B) (Gibco®, Carlsbad, USA), and 5% of inactivate bovine serum (Invitrogen/Gibco®, Auckland, NZ), at 37°C in a humidified incubator containing 5% CO 2 .…”

“…However, these isolates were genetically distinct from both S. falcatula and S. neurona and, for this reason, classified as S. falcatula-like (20,21,(27)(28)(29). The Brazilian isolates that were submitted for sequencing of SAG genes possessed a high allelic variation in their coding genes for SAG2, SAG3, and SAG4, which seems to represent genetic recombination between S. neurona, S. falcatula, or other unidentified Sarcocsytis species (19)(20)(21)(27)(28)(29). Based on this peculiar scenario in Brazil, we hypothesized that Brazilian horses may be exposed and seroconvert to other species of Sarcocystis shed by opossums, besides S. neurona.…”

“…Based on this peculiar scenario in Brazil, we hypothesized that Brazilian horses may be exposed and seroconvert to other species of Sarcocystis shed by opossums, besides S. neurona. To address this hypothesis, we tested horse sera using antigen from a North American strain of S. neurona and antigen derived from a recently isolate of S. falcatula-like, which has been propagated in an avian cell line (27).…”

First molecular characterization of Sarcocystis neurona causing meningoencephalitis in a domestic cat in Brazil

“…DNA amplification of Sarcocystis spp. cytochrome c oxidase subunit I (nPCR-COX1) was performed using primers designed by Gondim et al (2019). Complete internal transcribed spacer 1 was nested PCR amplified using the primer set described by Soares et al (2011).…”

“…Complete internal transcribed spacer 1 was nested PCR amplified using the primer set described by Soares et al (2011). Finally, genetic sequences of SAG2, SAG3, and SAG4 were nested PCR amplified using the primers as designed in Monteiro et al (2013), Valadas et al (2016), and Gondim et al (2019). Primers used to amplify genetic sequences of Sarcocystis spp.…”