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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.

SUBMITTER: Gondim LFP

PROVIDER: S-EPMC6732708 | biostudies-literature | 2019 Dec

REPOSITORIES: biostudies-literature

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

Gondim Luís F P LFP Soares Rodrigo M RM Tavares Aline S AS Borges-Silva Waléria W de Jesus Rogério F RF Llano Horwald A B HAB Gondim Leane Q LQ

International journal for parasitology. Parasites and wildlife 20190830

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 multilo ...[more]

PMID: 31516824

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Project description:The genus Sarcocystis and the species Toxoplasma gondii are the most prevalent sarcocystid organisms found in birds. Molecular phylogenies based on the first internal transcribed spacer of the ribosomal coding DNA (ITS1) have been widely used to identify them. Here, pectoral muscles from 400 wild birds from Brazil were screened by means of molecular methods using nested PCR, and Sanger sequencing yielded amplicons. A pan-sarcocystid ITS1-directed nested PCR revealed 28 birds infected by Sarcocystis falcatula (ten Piciformes, eight Psittaciformes, five Columbiformes, two Accipitriformes, one Anseriformes, one Passeriformes and one Strigiformes); one infected by Sarcocystis halieti (one Accipitriformes); nine infected by unknown or undescribed Sarcocystis (six Passeriformes, one Piciformes, one Cathartiformes and one Cuculiformes); and six harboring Toxoplasma gondii DNA (three Pelecaniformes, two Falconiformes and one Columbiformes). Samples harboring S. falcatula-related ITS1 sequences were further characterized by means of PCR and sequencing of genetic sequences of three surface antigen coding genes (SAGs). From this, 10 new allelic combinations of SAGs (SAG2, SAG3 and SAG4) were identified, in addition to 11 SAG allelic combinations already found in Brazil. Samples with S. falcatula-unrelated ITS1 sequences were further characterized by means of PCR and sequencing of cytochrome c oxidase subunit I coding sequences (CO1) and 18S ribosomal DNA gene (18S rDNA). This study was the first extensive survey of wild birds in Brazil for Sarcocystidae species. It provides the first molecular evidence of natural S. falcatula infection in 14 species, including in the order Piciformes, and shows the high genetic diversity of S. falcatula in intermediate hosts in South America. Evidence of occurrence of at least three non-described species of Sarcocystis was also presented in this study. This survey corroborated the ubiquity of T. gondii infection but revealed surprisingly low prevalence of this parasite (1.5%).

Project description:Sarcocystis neurona and Sarcocystis falcatula are protozoan parasites endemic to the Americas. The former is the major cause of equine protozoal myeloencephalitis, and the latter is associated with pulmonary sarcocystosis in birds. The opossum Didelphis virginiana is the definitive host of these parasites in North America. Four Didelphis species are found in Brazil, and in most reports in this country, Sarcocystis species shed by opossums have been classified as S. falcatula-like. It is unknown whether reports on S. neurona-seropositive horses in Brazil are also derived from exposure of horses to S. falcatula-like. The aim of this study was to test the sera reactivity of 409 horses in Brazil using antigens derived from a Brazilian strain of S. falcatula-like (Sarco-BA1) and from a North American strain of S. neurona (SN138). Samples were examined by immunofluorescent antibody tests (IFATs) at start dilutions of 1:20, and a selected number of samples was tested by Western blot (WB). Sera from 43/409 (10.5%) horses were reactive to S. falcatula-like and 70 of 409 (17.1%) were reactive to S. neurona antigen; sera from 25 animals (6.1%) were positive for both parasites by IFAT. A poor agreement was observed between the two employed IFATs (κ = 0.364), indicating that horses were exposed to more than one Sarcocystis species. Horse sera evaluated by WB consisted of four sera reactive to S. falcatula-like by IFAT, six sera positive to S. neurona by IFAT, two sera that tested negative to both parasites by IFAT, and a negative control horse serum from New Zealand. Proteins in the range of 16 and 30 kDa were recognized by part of IFAT-positive sera using both antigen preparations. We concluded that Brazilian horses are exposed to distinct Sarcocystis species that generate different serological responses in exposed animals. Antigens in the range of 16 and 30 kDa are probably homologous in the two parasites. Exposure of the tested horses to other Sarcocystis species, such as Sarcocystis lindsayi, Sarcocystis speeri, and Sarcocystis fayeri, or Sarcocystis bertrami cannot be excluded in the current study.

Project description:Abstract Poster session 1, September 21, 2022, 12:30 PM - 1:30 PM Introduction Protothecosis is an emergent disease caused by members of the genus Prototheca. Most such infections probably occurred by traumática inoculation into subcutaneous tissues. Objectives It is to report a case of human cutaneous protothecosis identified in the state of Maranhão, northeast Brazil. Case report: 75-year-old patient, Merchant, from the municipality of São Luís Island, Northeastern Brazil. He sought care referring to an erythematous and painful lesion on the left arm that started 6 months before the treatment. On examination, he presented an infiltrative, hyperemic lesion with burning pain throughout the upper limb (Fig. 1). The patient reported that a week before the onset of the condition, he suffered trauma on the arm, with a laceration in the skin, while cleaning a sewage system with clay pipes. During the healing process, he noticed a hyperemic, slightly pruritic lesion measuring 2 cm which did not improve. He sought medical assistance at the dermatological service, who suspected dermatophytosis, initiating treatment with terbinafine (250 mg, once a day), evolving with worsening of the lesion. A lesion biopsy was indicated, to histopathological examination, which showed circular, moniliform structures, diagnosed as protothecosis (Fig. 2). Treatment with itraconazole (200 mg/day) was started, with no therapeutic response and the lesion spread throughout the patient's left upper limb. Submitted to a new investigation with biopsy for direct research and culture for fungi, being identified Prototheca Wicherhamii, by Maldi-Tof®, with sensitivity to itraconazole and ampfotericin B. PCR amplification of the genetic material obtained in the clinical isolate was performed with purification of its product, and sequencing showed genetic similarity of 97,46% with Prototheca Wickerhamii. The sequence obtained was deposited in Genbank under number MZ409514. In the absence of therapeutic response to itraconazole (400 mg/day), and significant worsening of the lesion, with presentation of a secondary infection caused by Staphylococcus haemoliticus, treatment with Clindamycin (900 mg/day for 10 days) and Liposomal Amphotericin B (4 mg/kg/day for 45 days) were performed. After suspension of Liposomal Amphotericin B, the lesions recurred in 15 days, and voriconazole (200 mg 12/12 h) was prescribed for 6 months, with complete regression of the lesions. Currently, he is free of injuries, having been followed up every 6 months. Conclusion Rare disease caused by chlorophyllous algae may be surprising due to the severity and lack of response to antifungals that show sensitivity in vitro.

Dataset Information

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

Publications

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

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