M.A.B. Moreira; P.A. Lopes; C.A.R. Sultanum; A.S. Ferraz; R. Gonzalez
The canine ehrlichiosis is an endemic disease that affects almost all the regions of Brazil involving the bacterial microorganism Ehrlichia sp. transmitted by the biological vector Rhipicephalus sanguineus, which once infected is able to disseminate the disease for up to 155 days after the separation from its host. The E. canis is the most common among the naturally infected dogs, causing serious clinical disease. The agent presents itself as intracytoplasmic groups (morulae measuring from 3 to 6μm) in the target cells: platelets, monocytes, lymphocytes and neutrophils. After an incubation period from 7 to 21 days, the disease may develop into acute, asymptomatic and chronic phases (Birchard & Sherding, 1998). The diagnosis of canine ehrlichiosis is based on the clinical history, nonspecific laboratory tests (complete blood cell count, serum biochemistry, electrophoretic plasma protein profile), and specific as: serological, through the techniques of indirect immunofluorescence assay, immunoenzymatic assay (ELISA), molecular through the technique of polymerase chain reaction (PCR) and direct examination, through spleen aspiration cytology, lymph nodes and lungs, blood smear and culture (Andereg & Passos, 1999). The indirect immunofluorescent reaction is used to detect serum antibodies. Experimentally, in infected dogs the previous period of antibodies formation starts between 8 and 24 days, although some dogs do not show themselves seropositive until the 28th day post-infection, due to the variation of the individual response. The persistence of the antibody titers in the indirect immunofluorescent reaction for periods longer than 6 months after the treatment may indicate that the agent was not completely eliminated, suggesting that there is also the possibility of a reinfection for the dog (Andereg & Passos, 1999). The ELISA technique, by using purified antigens of a DH 82 cells culture infected with E. canis adhered to sheets of nitrocellulose paper, shows sensitivity and specificity comparable to the IFA. The PCR is sensitive and specific and can be useful to evaluate the elimination of the organisms after antibiotic therapy, and the main advantage is to identify the species of Ehrlichia that caused the infection, however, the method requires appropriate equipment besides the high cost. The detection of recent exposure and active infection may be held comparing paired titers (Andereg & Passos, 1999).
Materials and Methods
To this study, 55 dogs served in the Anhembi Morumbi Veterinary Hospital from September/2002 to May/2008 were selected, showing clinical manifestations and/or hematological changes which suggested canine ehrlichiosis with negative results for the serological method of ELISA-IDEXX®. Subsequently, the IFA was held to compare and evaluate the agreement of the obtained results between the serological techniques.
Among the dog's blood serum samples (n = 55) tested by the ELISA-IDEXX® technique with negative result, when submitted to the IFA method, 31 (56%) showed negative reaction to the presence of anti-Ehrlichia sp. antibodies and 24 (44%) obtained positive reaction.
Discussion and Conclusions
The ELISA technique is a sensitive method, gives fast results, can be used in a lot of situations and is of easy interpretation for the detection of antibodies in the serum (Andereg & Passos, 1999). Despite of Morais et al. (2004) consider ELISA as the Best test to detect the infection "status", providing high sensitivity and specificity, in the present study it appeared as a smaller positivity when compared to the IFA. The anti-Ehrlichia antibodies search through the technique of IFA is highly sensitive and specific, being considered "Gold Standard" (Birchard & Sherding, 1998), however it is subjected to limitations as the subjectivity of the observer, equipped laboratory and specialized labor (Bélanger et al. 2002). The IFA showed a bigger amount of positive results, when compared to the ELISA in the diagnosis of canine ehrlichiosis, indicating a capacity to detect a smaller amount of serum antibodies and that the ELISA cut-off is bigger than the indirect immunofluorescent reaction's one. In conclusion, IFA appeared to be more efficient than ELISA by the snap method in the diagnosis of canine ehrlichiosis, but to its realization appropriate equipment and specialized labor are required. The ELISA technique showed lower efficacy in the detection of anti-E. canis antibodies, however higher easiness of daily use and smaller time to the obtaining of the results. It's good to emphasize how important it is to associate more than one laboratory method to confirm the disease's diagnosis.
1. Andereg PI, Passos LMF. 1999. Erliquiose canina. Clínica Veterinária, n.18, 31-38p;
2. Bélanger M, et al. 2002. Comparas. of serolog. detect. methods for diagn. of E. canis infect. in dogs. Journal. of Clin. Microbiology, n.9, v.40, 3506-3508p;
3. Couto CG. 1998. Doenças riquetsiais, p.138-143. In: Birchard SJ & Sherding RG. Manual Saunders. 1a ed. Roca, São Paulo;
4. Chaichanasiriwithaya W, et al. 1994. Comparas. of PCR with other tests for early diagnosis of canine ehrlichiosis. Journal. of Clin. Microbiology., n.7, vol.32, p.1658-1662;
5. Morais HA, et al. 2004. Diretrizes gerais para diagnóstico e manejo de cães infectados por Ehrlichia spp. Clínica Veterinária. n.48, 2004, p. 28-30;
6. Wen B, et al. 1997. Comparas. of nested PCR with immuno.-antib. assay for detect. of E. canis infect. in dogs treat. with doxyci. Journal of Clin. Microbiology. n.7, vol.35, p.1852-1855.