Seroprevalence of ehrlichia canis antibodies among dogs in Turkey

Abstract

Canine monocytic ehrlichiosis (CME) is a potentially fatal tickborne disease caused by the rickettsia Ehrlichia canis. Its principal vector is the brown dog tick Rhipicephalus sanguineus. The distribution of CME is directly related to the distribution of this vector, and most cases occur during the warm season when the tick is active (Harrus and others 1997, Leib and Monroe 1997). The disease has been reported in Asia, Africa, Europe and America. Serosurveys conducted in Egypt, Israel, Germany and France have revealed 33, 30,25·1 and 9 to 12 per cent E canis antibody seroprevalence, respectively (Davoust 1994, Botros and others 1995, Baneth and others 1996, Gothe 1998).

The pathogenesis of CME involves an incubation period of eight to 20 days, followed by acute, subclinical and, in some cases, chronic phases (Waner and others 1997). The disease may manifest in a wide variety of clinical signs, of which fever, anorexia, weight loss, depression, lymphadenomegaly, splenomegaly and platelet-related bleeding are predominant (Harrus and others 1997, Leib and Monroe 1997, Kontos and Athanasiou 1998). Most cases of CME are diagnosed by serological testing. Antibodies against E canis are detected circulating in the serum of infected animals by the indirect immunofluorescent antibody (IFA) test, dot-ELISA and Western blot immunoassay (Waner and others 1996, Harrus and others 1997).

The aim of this study was to investigate the seroprevalence of E canis antibodies among dogs in Turkey, and to characterise the first 20 serologically confirmed clinical cases of CME that were admitted to the Faculty of Veterinary Medicine in Bursa, Turkey.

Blood was taken from 284 dogs (164 males and 120 females, comprising 124 crossbred dogs, 133 pure breeds [ 19 different breeds] and 27 unknown breeds) from three different geographical and climatic regions in Turkey: 143 samples from Bursa and 38 samples from Balikesir in the Marmara region, 32 samples from Izmir in the Agean region, and 27 samples from Şanliurfa, 26 samples from Adana and 18 samples from Antalya in southern Turkey. In Bursa, all blood samples were collected from dogs that were presented to the small animal clinic of the Faculty of Veterinary Medicine, University of Uludağ, Bursa, for a general health check and vaccination, or due to illness. The samples from the other cities were collected from clinically healthy kennel dogs. Samples were collected between October 1997 and November 1998.

Serum samples were separated off by centrifugation for serological testing, and kept frozen at -20°C until shipped in an iced container to the Laboratory of Infectious Diseases of the School of Veterinary Medicine, the Hebrew University of Jerusalem, in Israel. Serology was performed using the IFA test as described by Ristic and others (1972). The test was performed on slides prepared with acetone-fixed heavily infected cells (dog macrophage cell line DH82), with the local Israeli isolate of E canis (Keysary and others 1996). Two-fold serum dilutions (5 μl) were added to wells, and slides were incubated in a humidified chamber at 37°C for 30 minutes. Known positive and negative controls were used on each slide. The slides were then washed gently with phosphate buffered saline (PBS), air dried and 5 μl of rabbit anti-dog immunoglobulin G-fluorescein isothiocyanate conjugate solution (Sigma) 1:20 in PBS, was added to each well. After further incubation at 37°C for 30 minutes, the slides were washed, air dried and examined under a fluorescence microscope. A titre of 1:40 or greater was considered to be positive. In addition, the clinical and clinical pathological findings of 20 dogs suffering from clinical CME (confirmed by serology) that were admitted to the University of Uludağ, Bursa, were reviewed. Blood samples with an anticoagulant ethylenediamine tetra-acetic acid were taken from the 20 dogs, and complete blood counts were performed by an autoanalyser (Serono). The dogs were treated with 10 mg/kg doxycycline (Tetradox; Fako), once daily for 21 days, or 5 mg/kg enrofloxacin (Baytril; Bayer) every 12 hours for 21 days.