The value of in vitro cell culture of granulocytes in the detection of Ehrlichia Valor da cultura celular de granulócito in vitro na detecção de Ehrlichia

Peripheral blood leukocytes from different animals were isolated from whole blood and maintained in Dulbeco’s medium containing homologous serum without antibiotics. After 72 hrs microscopic examination of these cells showed that most animals were infected with Ehrlichia. Observation of thin blood smears from the same animals showed that only two were positive for Ehrlichia. The results of this investigation show that leukocyte culture is superior to the traditional thin blood film method in the detection of Ehrlichia and that asymptomatic carriers are easily detected. The method is inexpensive and does not require specific cell lines although it is necessary to use sterile sera. Key-words: Ehrlichia. Cell Culture. Mononuclear cells. Resumo Leucócitos do sangue periférico de diferentes animais foram isolados do sangue total e mantidos em meio de Dulbeco, contendo soro homólogo sem antibióticos. Após 72 horas, um exame microscópico destas células mostrou que a maioria dos animais era infectada com Ehrlichia. Observação de esfregaços de sangue dos mesmos animais mostrou que apenas dois eram positivos para Ehrlichia. Os resultados desta pesquisa mostraram que a cultura de leucócitos é superior ao método tradicional de película de sangue na detecção de Ehrlichia, e que portadores assintomáticos são facilmente detectados. O método é de baixo custo e não exige linhas de células específicas, embora seja necessário o uso de soro estéril. Palavras-chaves: Ehrlichia. Cultura celular. Células mononucleares. 1. School of Veterinary Medicine, The University of The West Indies, Saint Augustine, Trinidad and Tobago, West Indies. Address to: Dr. Alex Mutani, School of Veterinary Medicine/The University of The West Indies, St. Augustine, Trinidad, West Indies. Tel: 55 1-868-645-2640 Fax: 55 1-868-645-7428. e-mail: [email protected] Recebido para publicação em 10/3/2000. Natural infections of Ehrlichia pose diagnostic problems. In many cases the agents can neither be demonstrated in capillary blood films nor can they be detected in tissue impressions11. For many years, diagnosis of ehrlichiosis depended on clinical signs. This method has considerable limitations since several other pathogens may produce similar findings. Detection of the organism in blood films is a time consuming and laborious process, often resulting in false negatives6. Although serology has been utilized in the diagnosis of ehrlichiosis, it is often associated with problems such as strain variations5 and inter-specific crossreactivity8 12. Belongia et al1 showed that serological methods may produce either false negatives due to use of acute sera or false positives from convalescent sera. The polymerase chain reaction is now a method of choice for diagnosis of ehrlichiosis because of its high predictive value9 10. Unfortunately this method is associated with economic constraints which, in many laboratories with limited resources, would be prohibitive. Since the development of an in vitro method for the cultivation of E. canis by Nyindo et al14 similar reports have been produced by other workers10 11 13 17. In all the published reports some pre-requisites for the successful establishment of Ehrlichia in vitro are mentioned. These include the stage of infection of the animal, whether acute or chronic15, the presence of specific cell lines10 11 12 or the requirement of repeated peritoneal lavage to obtain infected cells17. The results of the present investigation show that: A) in vitro culture of Ehrlichia can be carried out using only the individual host’s peripheral blood leukocytes, hence eliminating the requirement for specific cell lines. B) th is method can detect asymptomat ic carriers.