Diagnóstico sorológico de erliquiose canina com antígeno brasileiro de Ehrlichia canis
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Data
2007-06-01
Autores
Aguiar, Daniel Moura
Saito, Taís Berelli
Hagiwara, Mitika Kuribayashi
Machado, Rosângela Zacarias [UNESP]
Labruna, Marcelo Bahia
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Editor
Universidade Federal de Santa Maria (UFSM)
Resumo
O presente trabalho relata o isolamento de Ehrlichia canis em cultivo de células DH82 e posterior padronização da Reação de Imunofluorescência Indireta (RIFI). Leucócitos de uma cadela experimentalmente infectada com o isolado Jaboticabal de E. canis foram inoculados em cultivo de células DH82. A inoculação foi monitorada após a segunda semana, a cada 5-6 dias, através de exames citológicos e pela amplificação de um fragmento do gene dsb de Ehrlichia pela Reação em Cadeia pela Polimerase (PCR) para confirmação da infecção. A cultura apresentou-se positiva aos 27 dias pós-inoculação pela PCR e aos 28 dias pela citologia. No 33o dia pós-inoculação, observou-se 20% de células infectadas e, aos 53 dias, 60% de infecção. Atualmente, o isolado encontra-se estabelecido em células DH82, com várias passagens atingindo 90-100% de células infectadas entre 7-10 dias após a inoculação. Após o seqüenciamento do produto de PCR, o isolado apresentou-se 100% similar à seqüência correspondente de E. canis depositada no GenBank. As células infectadas foram utilizadas como antígeno para a padronização da RIFI para detecção da infecção em cães.
The present study describes a successful isolation of Ehrlichia canis and its establishment in DH82 cells, followed by the development of an Indirect Fluorescent Antibodies Test (IFAT). Leukocytes collected from an experimentally infected dog with the Jaboticabal strain of E. canis were used to inoculate a DH82 cell monolayer. Two weeks later, the inoculated culture was checked for infectivity, every 5-6 days by both cytological staining and PCR, targeting a fragment of the dsb gene. The cell culture showed to be infected by Ehrlichia on day 27 by PCR and on day 28 by cytological staining. By the day 33, the infection rate reached 20% and on day 53, 60%. Currently, the isolate is established in DH82 cells, with several passages reaching 90-100% of infected cells, within 7 to 10 days post inoculation. After sequencing, the amplicon was identical to other E. canis corresponding sequences available in the GenBank. DH82 infected cells were used to standardize an IFAT for the diagnosis of canine ehrlichiosis.
The present study describes a successful isolation of Ehrlichia canis and its establishment in DH82 cells, followed by the development of an Indirect Fluorescent Antibodies Test (IFAT). Leukocytes collected from an experimentally infected dog with the Jaboticabal strain of E. canis were used to inoculate a DH82 cell monolayer. Two weeks later, the inoculated culture was checked for infectivity, every 5-6 days by both cytological staining and PCR, targeting a fragment of the dsb gene. The cell culture showed to be infected by Ehrlichia on day 27 by PCR and on day 28 by cytological staining. By the day 33, the infection rate reached 20% and on day 53, 60%. Currently, the isolate is established in DH82 cells, with several passages reaching 90-100% of infected cells, within 7 to 10 days post inoculation. After sequencing, the amplicon was identical to other E. canis corresponding sequences available in the GenBank. DH82 infected cells were used to standardize an IFAT for the diagnosis of canine ehrlichiosis.
Descrição
Palavras-chave
Ehrlichia canis, Isolation, DH82, Immunofluorescence, Serology, Ehrlichia canis, Isolamento, DH82, Imunofluorescência, Sorologia
Como citar
Ciência Rural. Universidade Federal de Santa Maria (UFSM), v. 37, n. 3, p. 796-802, 2007.