Prevalence of canine tick-borne haemoparasites in three Divisional Secretariat Divisions (Rambewa, Tirappane, and Galenbidunuwewa) in the Anuradhapura district, Sri Lanka

Introduction and Objectives: Ticks act as the vector for transmission of many parasitic pathogens. Several tick-borne haemoparasite infections are widespread among canines in tropical countries. However, information is scarce regarding canine tick-borne infections in Sri Lanka. The present study was therefore carried out to identify the canine tick-borne haemoparasite species and thereby assess their prevalence in three Divisional Secretariat divisions in the Anuradhapura district. Materials and methods: Blood samples were collected from dogs in three Divisional Secretariat (DS) divisions namely, Rambewa, Tirappane, and Galenbindunuwewa in the Anuradhapura district, Sri Lanka. From each blood sample, two thin smears were prepared and stained with Giemsa. Stained slides were subsequently examined with a light microscope to detect haemoparasites. Results: Out of 319 blood samples, 139 were positive for haemoparasites. Of the positives, 51.61% were from Rambewa, 40.47% were from Galenbindunuwewa and 38.88% were from Tirappane. Three haemoprotozoan species, Hepatozoon canis, Babesia gibsoni, Babesia canis and one bacterial species, Ehrlichia canis were morphologically identified in the present study. Conclusions: Comparatively, the prevalence of ehrlichiosis (20.06%) and babesiosis (16.29%) were high in three DS divisions compared to the prevalence of H. canis infection (1.56%). The prevalence of the haemoprotozoan infections was significantly different among thee DS divisions


Introduction
Canine tick-borne diseases are a major health problem among dogs in tropical and subtropical countries. Ticks have the capability to successfully transmit disease agents such as viruses, bacteria andprotozoa. 1 Babesiosis, ehrlichiosis and hepatozoonosis are some common tick-borne parasitic infections in dogs. 2,3 These infections can occur in dogs due to the transmission of haemoparasites by several tick species or by the same species. 4 For example, the main vector of B. canisvogeli, H. canis, and E. canis is the brown dog tick (Rhipicephalus sanguineus). 5 Babesiosis is caused by intraerythrocytic piroplasms of the genus Babesia. It is a common disease among domestic and wild canines. 6 Ehrlichia is an alphaproteobacterium belonging to the family Ehrlichiaceae. E. canis and E. ewingii cause tropical canine pancytopenia and canine granulocytic ehrlichiosis respectively. 7  Previous studies have determined the prevalence of canine haemoparasites in Sri Lanka. H. canis was first identified in 1961 in Sri Lanka. 9 In 2017, cases of acute hepatozoonosis, characterized by neurological symptoms, ataxia or paresis, emaciation and anaemia was detected. 10 An early study has determined the prevalence of canine ehrlichiosis in Sri Lanka based on conventional examination of stained blood smears. 11 A study carried out to determine the prevalence of canine vector-borne diseases in the Western Province identified the prevalence of both single and mixed infections among dogs. 12 However, there is a relative paucity of studies on the prevalence of canine tick-borne infections in Sri Lanka. In the present study, our objectives were to identify the canine tick-borne haemoparasite species and assess their prevalence in three Divisional Secretariat divisions in the Anuradhapura district.

Study area
This study was conducted in three Divisional Secretariat (DS) divisions: Rambewa, Tirappane, and Galenbindunuwewa in the Anuradhapura district, Sri Lanka ( Figure 1).

Sample collection
The sample size was calculated using the Creative Research Systems survey software (http://www.surveysystem.com/sscalc.htm). The calculated sample size was 375 (dog population was about 14,698 and the confidence interval was 95%). Blood samples were collected from April 2014 to July 2014 at the prophylactic anti-rabies vaccination and sterilization centers. Blood samples (1-2 ml) were collected into ethylenediaminetetraacetic acid (EDTA) tubes. Samples were stored at 4 °C and dispatched to the Department of Parasitology, Faculty of Medicine, University of Peradeniya, Sri Lanka to carry out the laboratory investigations.

Microscopic examination
From each blood sample, 2-3 thin smears were prepared and stained with Giemsa according to the method described in a previous study. 13 Smears were examined with a light microscope (Carl Zeiss ™ PrimoStar ™ , Germany) under oil immersion (×1000) to identify the haemoparasites. H. canis was identified by observing the gamonts with an ellipsoidal, brick-like shape inside the cytoplasm of the neutrophils. 14 In the erythrocytes, B. canis was identified by the morphology of merozoites described in a previous study. 15 Detection of B. gibsoni in the infected erythrocytes was carried out according to the morphological features described by Lempereur et al.(2014). 16 E. canis was identified by the round shape and purple colour morulae in the cytoplasm of lymphocytes. 17

Statistical analysis
Chi square test was conducted in R 18 to identify whether the prevalence of the haemoprotozoan infections was significantly different among the three DS divisions.

Sample collection
All the dogs included in this study were females with an average age of 3 years and were asymptomatic. A total of 319 blood samples were collected from Rambewa (124), Tirappane (108) and Galenbindunuwewa (87) DS divisions. Of those, 312 were obtained from stray dogs and 7 were from pet dogs.
In Rambewa DS division, the number of mixed infections was high (

Discussion
In this study, the prevalence and distribution of canine vector-borne infections in three DS divisions in the Anuradhapura district were investigated. There were 43.88% dogs positive for haemoparasites indicating infections caused by this group of parasites are common among dogs in the localities examined. Single infections were found in 37.93% of dogs. This is higher than a previous report on dogs in the Western Province (29.15%). 11 Mixed infections were detected in 5.95% of dogs examined which agrees with the previous study done by Kumara et al. in 2013. 12 Mixed infections with only two pathogens were observed in 5.6% dogs. Similar results have been documented in the Western Province of Sri Lanka. 11 This study reports a high prevalence of E. canis infections (20.06%) compared to the previously reported prevalence of 14% in dogs in the Western Province. 12 The prevalence of B. canis (1.25%) and B. gibsoni (15.04%) reported in this study were also higher than previously reported in the Western Province. 11 The prevalence of H. canis (1.56%) was lower than in the Western Province (8.57%). 12 A study conducted in Nigeria has shown a high rate of mixed infection (37%) that was caused by the same haemoparasite species identified in this sutdy. 19 E. canis infection can predispose dogs to opportunistic pathogens, such as B. canis and H. canis. 5 Having multiple tick-transmitted infections in dogs is associated with severe and fatal conditions. 5 The Brown dog tick (R. sanguineus) is the main vector transmitting the pathogens found in mixed infections. 20 The prevalence of this tick species is common among domestic animals in the dry zone. 21 It can therefore be suggested that any control measures taken to eradicate this tick species can reduce more than one type of haemoparasitic infection found in dogs. All the haemoparasite infections identified are known to be asymptomatic. [22][23][24][25] However, in some dog breeds, B. gibsoni infection can be symptomatic. 26 Differences in the distribution of vector-borne diseases are largely determined by the geographical distribution and local density of their arthropod vectors. Furthermore, environmental changes have a direct impact on the geographical distribution of arthropods, size of their population and their ability to act as a vector to transmit diseases. 27,28 Future studies are required to examine the putative tick vectors and their population characteristics. Results obtained during this study would be useful for the accurate design of such investigations. We would suggest implementation of coordinated control and prevention programs of canine tick-borne diseases with the help of veterinary and public authorities. Prophylactic measures, including the use of acaricides and insecticides can be used to control the vectors. 29 These control measures are necessary to prevent any possible zoonotic transmission.

Limitations of the study
Blood samples were collected only at the prophylactic anti-rabies vaccination and sterilization centers. However, the expected numbers of dogs were not brought to these centers. As a result, the number of samples collected are less than the calculated sample size. Any background information about these dogs was not considered. Haemoparasite species were identified using morphological characteristics. Species identities should be further confirmed using molecular techniques.

Conclusions
This study was carried out on tick-borne canine haemoparasitic infections in three DS divisions in Anuradhapura district, Sri Lanka. Comparatively, the prevalence of ehrlichiosis and babesiosis was high in the three DS divisions. The prevalence of H. canis was low. Co-infections among dogs suggest that the same vector may be involved in transmitting different infections.