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
Este estudo objetivou pesquisar a epidemiologia da erliquiose canina no Nordeste do Brasil, com especial atenção na identificação da espécie de Ehrlichia envolvida nas infecções caninas e vetoriais detectadas. Para isso foram coletadas amostras de 472 cães domiciliados nos distritos sanitários de Cajazeiras e Itapuã. A prevalência de anticorpos anti-E. canis, pela imunofluorescência indireta (título ≥1:80), em cães foi de 35,6% (168/472). Os animais soropositivos foram analisados por uma nested-PCR para identificação da espécie de Ehrlichia responsável pela infecção. Dentre os positivos, 58 (34,5%) cães foram PCR-positivos para E. canis. Foram coletados e classificados os carrapatos em 32 cães. A nested‑PCR de Rhipicephalus sanguineus resultou em 21,9% (7/32) de infecção por E. canis. A nested-PCR de amostras de sangue de cães e Rhipicephalus sanguineus para E. chaffeensis e E. ewingii foi negativa, não havendo amplificação de fragmento de DNA.(AU)
This study investigated the epidemiology of canine ehrlichiosis in Northeastern Brazil, focusing the identification of the Ehrlichia species and vectors involved. Samples were collected from 472 domestic dogs residing in the health districts of Cajazeiras and Itapuã of Salvador city. The average prevalence of antibodies reactive to E. canis by immunofluorescent antibody test (IFAT) (titer ≥1:80) was 35.6% (168/472). Blood samples from the E. canis-seropositive animals were tested by nested PCR in order to identify the Ehrlichia species responsible for the infection. Among the seropositives, 58 (34.5%) were found to be PCR-positive for E. canis. Ticks were found in 32 dogs. Nested-PCR analysis showed that 21.9% (7/32) of the Rhipicephalus sanguineus were infected by E. canis. In both dogs and Rhipicephalus sanguineus, nested-PCR for E. ewingii and E. chaffeensis was negative, with no amplification of DNA fragment.(AU)
Assuntos
Animais , Ehrlichiose/epidemiologia , Ehrlichia canis , Cães/parasitologia , Técnica Indireta de Fluorescência para Anticorpo/métodos , Reação em Cadeia da Polimerase/métodos , Rhipicephalus sanguineus/parasitologiaRESUMO
Hammondia heydorni is a coccidian parasite with an obligatory two host life cycle, with dogs and foxes as definitive hosts, and a number of intermediate hosts, including goats. While infection by this parasite seems to be unassociated with any clinical signs, infection by the closely related parasites Neospora caninum and Toxoplasma gondii can result in abortion, stillbirths and low yielding in caprine herds. The aim of this work was to investigate the frequency of goats infected with H. heydorni using a nested PCR, specific to Toxoplasmatinae internal transcribed spacer 1 (ITS1) of the rDNA, followed by sequencing of the purified PCR fragments. The same molecular techniques were used to determine the frequencies of N. caninum and T. gondii-infected animals. A total frequency of 13.72% (14/102) was obtained for Toxoplasmatinae DNA in goat tissues. After sequencing the PCR products from all positive tissues, a frequency of 3.92% (4/102), 1.96% (2/102) and 7.84% (8/102) were obtained for H. heydorni, N. caninum and T. gondii, respectively. All sequences shared 98-100% identity with sequences from other strains of these coccidia present in GenBank. To the authors' knowledge, this is the first report of H. heydorni DNA in tissues from naturally infected intermediate hosts.