SEROPREVALENCE OF CANINE MONOCYTIC EHRLICHIOSIS IN HUNTING DOGS IN THE AUTONOMOUS PROVINCE OF VOJVODINA, SERBIA

Canine monocytic ehrlichiosis is a bacterial, vector-transmitted infection caused by Ehrlichia canis. Th e pathogen is mainly transmitted by ticks Rhipicephalus sanguineus. Th e disease highest distributionis most widely distributed in tropical and subtropical countries but it is also reported in Mediterranean countries of Europe (Spain, France, Italy, Turkey). Temperate continental climate and presence of these tick species are responsible for the maintenance and spreading of canine monocytic ehrlichiosis within the dog population in our region as well. Since hunting dogs are more exposed to tick bites then pet dogs, ed thisour study was conducted with the aims of determining the seroprevalence and basic epidemiological characteristics of monocytic ehrlichiosis infection in the population of hunting dogs, and comparing the obtained results with the results of other authors. Th is research involved 58 hunting dogs from the region of Autonomous Province of Vojvodina. All dogs were clinically examined and their basic epidemiological characteristics were recorded. Th en, blood samples were collected in order to determine the presence of specifi c G class antibodies against Ehrlichia canis antigens. An indirect immunofl uorescence test manufactured by VMRD, U.S.A., was used. In this study, the seroprevalence of monocytic ehrlichiosis in a population of examined hunting dogs from the region of Vojvodina was 13.79%. Th is rate is similar to the seroprevalence of monocytic ehrlichiosis in the general population of dogs in Vojvodina. 1 * Author for contact: ale@polj.uns.ac.rs Arhiv veterinarske medicine, Vol. 7, No. 1, 11-18, 2014. Potkonjak A. et al.: Seroprevalence of Canine ...


INTRODUCTION
Monocytic ehrlichiosis of dogs is a bacterial, vector borne infection caused by Ehrlichia canis. Species of the genus Ehrlichia are obligatory intracytoplazmatic bacteria of the monocyte-macrophage system. Intracellular development of the bacteria is enabled by the reaction of numerous immunoreactive proteins, which to haveplay a key role in the infection, such as adhesives, regulators of the intake of nutrients and inhibitors of proinfl ammatory cytokines. Th e causative agent E. canis was fi rst detected in 1935 in Algeria. Aft erwards, in 1996, the causative agent of human monocytic ehrlichiosis, E. chaff ensis, was found in dogs, broughtwhich gave more signifi cance to this infection from the aspect of public health. Th e tick Rhipicephalus sanguineus, is the dominant vector for Ehrlichia canis Rhipicephalus sanguineus, but there is evidence on the transmission via Dermacentor variabilis (Johnson et al, 1998), Dermacentor marginatus (Satta et al, 2010), and Ixodes (Wielinga et al, 2006). Th e disease is most widely distributed in tropical and subtropical regions, but it has also been detected in Mediterranean countries of Europe (Spain, France, Italy, Turkey). Some parts of Spain and Italy are considered endemic regions for ehrlichiosis (Trotz-Williams et al, 2003). Temperate continental climate and presence of aforementioned tick species are predisposing factors for the maintenance and spread of monocytic ehrlichiosis in the population of dogs in our region. During the last few years, the disease has been detected in the neighboring countries including Hungary , Romania (Mircean et al, 2012) and Bulgaria (Tsachev et al, 2006). Our previous seroepidemiological study of monocytic ehrlichiosis in the general population of dogs in the region of Vojvodina revealed the seroprevalence rates of 25%was found, and 16% obtained by iELISAand 16% was found in the same population of dogs, by and IIF tests, respectively. (Potkonjak et al, 2013).
Th e aims of this study was obtaining of basic epidemiological characteristics of monocytic ehrlichiosis, establishing of seroprevalence rate of this infection in hunting dogs and comparing the obtained data with the data from the literature on seroprevalence of monocytic ehrlichiosis in general population of dogs.

MATERIAL AND METHODS
Th e research encompassed 58 hunting dogs from the region of Vojvodina. All dogs were clinically examined and all relevant epidemiological data were recorded. Blood samples were taken for blood sera to be isolated. Samples of full blood were taken by aseptic puncture of v. cephalica antebrachii. Blood sera were separated aft er centrifugation at 3000 rpm/min during 10 minutes. Detection of specifi c antibodies of G class against antigen of E. canis was performed using an indirect immunofl uorescence test manufactured by VMRD, USA. Evident presence of the fl uorescence of cytoplasmic bodies (morulas) was considered positive fi nding. Th e absence of fl uorescence was considered negative fi nding. Serum titer values for G class immunoglobulins above 1:50 were considered positive fi nding.

RESULTS
Th e examination of 58 hunting dogs' blood sera using the indirect immunofl uorescence test 58 blood sera samples from hunting dogs,revealed presence of antibodies of G class against E. canis antigen in 8 samplesE. canis antigene was found, while the remaining 50 samples were seronegative to E. canis. Th e seroprevalence of monocytic ehrlichiosis in the population of examined hunting dogs from the region of Vojvodina was 13.79%. Th e positive fi nding of IF test for E. canis antigen is shown in Figure 1. Th e age of seropositive dogs was between 1.5 and 6 years, and of the investigated population included the following breeds: Posavic hound, English setter, German hunting terrier, German shorthaired pointer, German wirehaired pointer and brac dachshund. Antibodies of G class against E. canis antigen were found in three females and fi ve males. For the prevention of ectoparasites, the following ectoparasiticides were used in positive dogs: Neostomosan sampoules, Frontline and Ectanon, Taktik and Baygon pulver. Th e basic epidemiological characteristics found in seropositive dogs to E. canis antigens are shown in Table 1.

DISCUSSION
Because of the climate changes and the geographical spread of the most important vector, Rhipicephalus sanguineus tick, proportional increase in prevalence of monocytic ehrlichiosis in dogs is found. Besides the tropical and subtropical regions, this tick species can also be found in the regions with temperate continental climate, including the region of our country (Christova et al, 2003;Parola et al, 2001).
Hornok et al performed molecular research on the presence of E. canis in ticks from the south region of Hungary and indicated the signifi cance of wild animals as reservoirs of this infection. During the summer 2011, 348 ticks were collected from red foxes (Vulpus vulpus), golden jackals (Canis aureus) and German shepherd. Th e researchers established that Dermacentor marginatus and Dermacentor reticulatus prefer dogs as hosts, which was not the case before. Dermacentor reticulatus and Ixodes canisuga were most commonly found species in red foxes and golden jackals. In our research, in the nymphs of D. marginatus and larvae of I. canisuga the presence of E. canis was established in the nymphs of D. marginatus and larvae of I. canisuga, which indicates potential signifi cance of foxes as a reservoir of infection in the wilderness (Hornok et al, 2013). An experimental transmission of E. canis from grey foxes (Urocyon cinereoargenteus ) to Beale dogs, with Rhipicephalus sanguineus as vector (Amyx et al, 1973)confi rmed that foxes could be a potential reservoir in the wilderness. A research performed in Italy in 1995 revealed a seroprevalence rate of 72% in 154 examined stray dogs from the territory of Imola city in Emilia -Romagna region (Baldelli et al, 1995). During the following decade in Northern Italy, the researchers have found seroprevalence rates of 2.9% 8% in central region and 9.7% in southern Italy (Solano-Gallego et al, 2006). Seroepidemiological study of E. canis infection in 120 dogs with owners in northern Bulgaria in 2006, showed a total prevalence of 37.5% whilst the highest prevalence of 60% was established in a coastal city of Varna. In this research, IF test was used, also (Tsachev et al, 2006). In Former Yugoslav Republic of Macedonia in 2011, sporadic E. canis infection was found in a female of Samoyed breed, 11 years old, using ELISA test. In the same dog, Leishmania infantum was identifi ed by IF test. In this case, the authors state that the coinfection has contributed to the deterioration of clinical fi ndings (Atanaskova et al, 2012). Ina study done in 2012 in Romania, 1146 blood serum samples of dogs were analyzed and an E. canis seroprevalence of 2.1% was established (Mircean et al, 2012).
In previous seroepidemiological research on monocytic ehrlichiosis in general population of dogs in the region of Vojvodina, seroprevalence rates of 25% and 16% were established by iELISA and IF test, respectively (Potkonjak et al, 2013). In this research, the established seroprevalence of monocytic ehrlichiosis in the population of hunting dogs in the region of Vojvodina was 13.79%. Th is seroprevalence rate for E. canis is in accordance with the data reported by other authors. In spite of general opinion that hunting dogs are more exposed to tick bites, the results of this research demonstrated that the seroprevalence of monocytic ehrlichiosis in hunting dogs was similar to that in the general population of dogs in the same region of Vojvodina. To the purpose of a more effi cient monitoring of monocytic ehrlichiosis in dogs in the region of Vojvodina, further and more detailed acarological and epidemiological research is needed in both dog population and the population of wild animals. In the context of the signifi cance of wild animals as reservoirs of infection, we consider the investigation of wild animals and hunting dogs in hunting regions highly important.

CONCLUSION
Th e results obtained in this study demonstrated that seroprevalence of monocytic ehrlichiosis in the population of examined hunting dogs in the region of Vojvodina reached 13.79%. Th e signifi cance of these results is refl ected by the fact that the prevalence is very similar to prevalence of this infectionthat among the general population of dogs in the region of Vojvodina (Potkonjak et al, 2013).