Frequency and Clinical Epidemiology of Canine Monocytic Ehrlichiosis in Dogs Infested with Ticks from Sinaloa, Mexico

Ehrlichia canis is a rickettsial intracellular obligate bacterial pathogen and agent of canine monocytic ehrlichiosis. The prevalence of this disease in veterinary medicine can vary depending on the diagnostic method used and the geographic location. One hundred and fifty-two canine blood samples from six veterinary clinics and two shelters from Sinaloa State (Mexico) were analyzed in this study. All animals were suspected of having Canine Monocytic Ehrlichiosis (CME). The diagnostic methods used were the ELISA (Snap4Dx, IDEXX) together with blood smear and platelet count. From all dogs blood samples analyzed, 74.3% were positive to E. canis by ELISA and 40.1% were positive by blood smear. The sensitivity and specificity observed in the ELISA test were 78.8% and 86.7%. In addition, thrombocytopenia was presented in 87.6% of positive dogs. The predominant clinical manifestations observed were fever, anorexia, depression, lethargy, and petechiae. Consequently, this is the first report in which the morulae were visualized in the blood samples, and E. canis-specific antibodies were detected in dogs from Sinaloa, Northwest of Mexico.


Introduction
Ehrlichia canis is the causative agent of canine monocytic ehrlichiosis (CME). Moreover, CME is an emerging disease in veterinary medicine, and E. canis has been considered in the last decade as a potential zoonotic pathogen [1,2]. It is a worldwide disease transmitted by a tick bite. The competent vector for its transmission is the Ixodidae ticks Rhipicephalus sanguineus and Dermacentor variabilis [3]. In dogs, the CME is a multiphase disorder that progresses in three stages: acute, subclinical, and chronic. Each phase is characterized by several clinical and hematologic abnormalities. Thrombocytopenia is a common finding in E. canis infected dogs and many clinicians tend to use it as an indication for antibiotic treatment, and it is observed in 84% of the cases and its severity varies in the different disease phases [4]. During the subclinical stage a moderate thrombocytopenia is observed, while the chronic phase is characterized by severe leukopenia and anemia. In this stage dogs show other complications such as hypocellular marrow, suppressed splenic sequestration, decreased life of platelets, and an increase of circulating migration-factor platelet inhibitor [3,4]. The relationship between the magnitude of thrombocytopenia and prevalence of E. canis has been established in countries such as Brazil in 2004 where 84.1% of infected dogs showed thrombocytopenia [3]. Taken together, more data is necessary to determine the environmental factors and infected vector prevalence in Mexico due to high incidence of CME in this region of the country. The purpose of this study was to evaluate the frequency and clinical manifestations of dogs from Sinaloa, Mexico, with clinical suspicion of CME by ELISA (SNAP4Dx) and blood smears and with a history of tick infestation.

Samples and Serological
Analysis. Samples were collected between March 2006 and July 2007, and 152 blood samples from dogs were collected in six veterinary clinics and two shelters from Sinaloa State, Mexico. Blood samples were chosen by dogs (males and females) with tick infestation and clinical signs suggesting CME (fever, anorexia, lethargy, depression, petechiae, bruising of the skin or prone to bleeding in mucous membranes, and epistaxis). Three ml of blood sample in EDTA tubes was obtained from each dog by the radial vein. All samples were processed using two techniques to detect E. canis specific IgG antibodies, the IDDEX ELISA kit and the Snap test. Samples were processed according to the manufacturer's recommendations. In addition, blood smears were done immediately after the blood was drawn to detect forms of E. canis morulae in monocytes, using Wright's stain. Finally platelet count was performed by an Automatic Hematology Analyzer (IDEXX QBC VetAutoread). All counts less than 200,000 platelets/ L were considered as thrombocytopenia [4].

Discussion
The results obtained in this study were similar to those observed in 2002, in which the authors compared five serodiagnostic methods for E. canis and found 79.2% positive samples using the same ELISA techniques as the one utilized in our study [6]. In Mexico (2000) a national study reported a 33% of seroprevalence for E. canis using the same diagnostic method as those presented in here [7]. Contrary to what was found in this study, our results showed the highest ehrlichiosis case. In Yucatan, Mexico, a 44.1% seroprevalence for CME has been reported using the same diagnostic method. Yucatan is in a climate zone similar to that of Sinaloa. Taken together, our study shows a frequency for CME similar if not higher than that observed in other regions of the country, as well as in other countries of Center and South America, which suggests the increasing importance of this disease in the canine population. E. canis has been described as a potential zoonotic pathogen for humans [5], and therefore our findings and current studies suggest its importance as human pathogen in Latin American countries.
A positive result for E. canis by ELISA indicates that the dog was or is exposed to this bacterial pathogen and does not necessarily imply that there is a latent infection. On the other hand, when using the ELISA test, cross-reactivity with other Ehrlichia species may occur since it is not specific for E. canis. Antibodies against this pathogen decrease approximately 6 to 9 months after infection. This together with the presence of subclinical infection in canids causes difficulties in the diagnosis of the disease by the clinicians. Consequently, there is a need of tools to support the diagnosis of the disease with hematological tests to evaluate thrombocytopenia, since this sign implies bacterial growth and its effect on antibodyproducing organs.
Previous E. canis experimental studies [3,4] reported thrombocytopenia in 89% of the infected dogs. In addition, thrombocytopenia was described in every stage of infection, and it persisted during subclinical stages of the disease. The morulae are easily identified by light microscopy during the acute phase of infection (4-5 days after exposure) but difficult to detect when the disease progressed undetected. Some studies reported that only 4% of the blood samples studied showed morulae in blood smears [8][9][10]. In this case 40.1% were found positive, which indicates that the search for the morulae in peripheral blood was done in the acute phase [11]. Moreover, dogs in endemic areas may have high IgG titers specific to E. canis without clinical signs and this can cause false positive serological tests [3], reinforcing the necessity for more specific test to be used in veterinary medicine.

Conclusions
In this study we have introduced the presence of the Canine Monocytic Ehrlichiosis in Sinaloa, Mexico. Dogs infested with ticks showed high frequency for E. canis in dogs which suggests the classification of this region of Mexico as an endemic area for the disease. Based on our findings and previous studies done in Mexico and other Latin American countries, we suggest that, in the absence of molecular methodologies for the diagnostics of CME, a serology test to detect E. canis-specific antibodies plus the presence of clinical manifestations and thrombocytopenia is a basic tool for its diagnosis in endemic areas.