The value of in vitro cell culture of granulocytes in the detection of 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: mutani@excite.com 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.

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 impressions 11 .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 negatives 6 .Although serology has been utilized in the diagnosis of ehrlichiosis, it is often associated with problems such as strain variations 5 and inter-specific crossreactivity 8 12 .Belongia et al 1 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 value 9 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 al 14 similar reports have been produced by other workers 10 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 chronic 15 , the presence of specific cell lines 10 11 12 or the requirement of repeated peritoneal lavage to obtain infected cells 17 .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) this method can detect asymptomatic carriers.

MATERIAL AND METHODS
Thin blood smears were prepared from six dogs, ten cattle, two horses, and one goat.The animals were of different ages and originated from different areas in Trinidad.Their previous exposure to ixodid ticks was not available.Apart from the dogs, which were brought to the veterinary clinic for various reasons, all other animals were apparently healthy (Table 1).The smears were stained with Wrights-Giemsa stain and examined .Thereafter, the plasma and leukocytes were aseptically collected into Leighton tubes, allowing 2ml per tube.The tubes were incubated at 37°C for 48hr under atmospheric conditions.The plasma was then discarded and the tubes washed with Dulbeco's medium without antibiotics.Two mililitres of Dulbeco's medium containing 20% homologous serum without antibiotics was then added to each tube.The tubes were subsequently fed with the same medium between 48 and 72 hrs.At the end of two and four days post-incubation the cover slips were removed, stained with Wrights-Giemsa stain and examined under immersion oil under a Nikon Optiphot-2 photomicroscope.

RESULTS
Figure 1 shows the appearance of ehrlichial organisms as seen in bovine peripheral blood smear.
Although blood smears from some animals were negative, (Table 1) results from cell culture revealed that all animals tested were infected with Ehrlichia.
The organisms could be detected in mononuclear cells as early as 2 days post-incubation.Initially the organisms appeared as small and monomorphic bodies which changed into polymorphic bodies by the fourth day post-incubation (Figures 2 and 3).  Figure 4 shows a canine mononuclear granulocyte on the fourth day of culture.Several bodies are evident, displacing the host cell's nucleus to the periphery.After five days of incubation most of the host cells had disintegrated and several bodies could be seen in the extracellular space (Figure 5).

DISCUSSION
The morphological appearance of the organisms both in peripheral blood smears and in culture conformed to those originally reported by Ristic and Huxsoll 16 .In the present investigation it was noted that blood smears from the majority of animals tested were negative; only becoming positive after culture.These results suggest that parasitaemias in the originally negative animals were too low to be detected by the usual blood smear techniques.These observations support those of Dawson et al 6 .In addition Carmichael and Fiennes 3 and Carter et al 4 reported that in low parasitaemias, small ehrlichial bodies and azurophilic granules could be confused with each other, thus contributing to the difficulty in microscopic diagnosis using blood smears.The present results show that cell culture amplifies the numbers of ehrlichial bodies in a reasonably short time and do not reveal the species identity of the ehrlichial organisms.However, these results suggest that Ehrlichia may be widespread in many animal species in Trinidad.Several species of Ehrlichia are known to exist 16 ; being transmitted by various species of ticks present in a particular locality.In Trinidad and Tobago tick genera which have been reported include Amblyomma, Anocentor, Boophilus, Haemaphysalis, Ixodes and Rhipicephalus 7 .Although ticks may cross host species, the question whether Ehrlichia can cross host species remains to be resolved.The present results have shown that many apparently healthy animals may be carriers of Ehrlichia and that these asymptomatic cases can be detected by a relatively simple culture technique.

Table 1 -
Species and clinical status of animals tested.
under immersion oil.From each animal 7.5ml of blood was drawn aseptically into sterile monoject heparinized tubes (Sherwood Medical, St. Louis, Missouri, U.S.A.).The tubes were left in a vertical position at room temperature for various times depending on the erythrocyte sedimentation rate of the species (canine: 1 h.; bovine: 24 hr.; equine: 30 m. and caprine: 24 hr.) as reported byBenjamin 2