DIROFILARIOSIS AND ANGIOSTRONGILOSIS IN PET AND HUNTING DOGS IN NOVI SAD, VOJVODINA, SERBIA

Th e aim of this study is to update the data on the prevalence of Dirofi laria immitis ( D. immitis ) and Dirofi laria repens ( D. repens ) infections in pet dogs, to report a preliminary result of the prevalence of Angiostrongylus vasorum ( A. vasorum ) in hunting dogs, and to assess the presence of concurrent infection with D. immitis and A. vasorum in hunting and pet dogs in Novi Sad. Th e methods used to estimate the prevalence of dirofi laria infections were modifi ed Knott test and detection of antigen of D. immitis . Th e prevalence of A. vasorum was determined using Baermann fecal technique and detection of A. vasorum antigen . Concurrent infection with D. immitis and A. vasorum was assessed only by detection of antigens of each parasite. Overall prevalence values for D. immitis and D. repens were 18.95% (24/143) and 16.32% (27/143), respectively. Th e prevalence of A. vasorum in hunting dogs was 1.96% (1/51). Concurrent infection with D. immitis and A. vasorum did not exist in examined hunting and pet dogs. Further studies with larger number of examined dogs and samples from other region of the country are needed to determine the prevalence of these parasites.


INTRODUCTION
Heartworm diseases caused by Dirofi laria immitis (D. immitis) and angiostrongylosis caused by French heartworm Angiostrongylus vasorum (A. vasorum) are canine parasitic diseases aff ecting mainly the respiratory and cardiovascular system. Both parasites are lungworms having the same primary site of residence -pulmonary arteries, where they can induce severe pathologic alterations. On the other hand, intermediate hosts of these parasites are diff erent, thus defi ning their specifi c seasonal characteristics. Mosquitoes as intermediate hosts for Dirofi laria spp. determine transmission period of the diseases. In case of A. vasorum, a wide range of terrestrial and aquatic gastropods act as obligatory intermediate hosts. Mild and wet climate is suitable for rapid multiplication of these intervertebrate hosts. Both parasites mostly cause chronic diseases with severe clinical complications such as pulmonary thromboembolism, right-sided heart failure and caval syndrome associated with D. immitis infections, whereas verminous pneumonia, right-sided heart failure and bleeding tendencies are associated with A. vasorum. In addition, the diseases are diff erent in their zoonotic potential. D. immitis, capable to form pulmonary nodules in humans, is considered to have a zoonotic potential, while A. vasorum is not a zoonotic agent (Ware, 2011;Morgan and Show, 2010). While canine cardiopulmonary dirofi lariosis and pulmonary angiostrongylosis are diseases of obvious clinical importance, D. repens infection, also known as subcutaneous dirofi lariosis, is less clinically important in dogs (Scott and Vaughn, 1987). Due to the importance of D. repens in humans, it is upon veterinary profession to deal with this agent as well.
Previous study on the prevalence of dirofi lariosis in pet dogs in Novi Sad has shown the increase of D. immitis infection and decrease of infection with D. repens (Spasojević Kosić et al., 2012) as compared with fi rst reports on the prevalence of D. repens infection (Tasić et al., 2008) and mixed infection with both parasites in dogs (Spasojević Kosić et al., 2014). Such signifi cant prevalence rates make the diseases highly important from both epizootical and clinical point of view. Clinical importance of heartworm disease in dogs and zoonotic potential of Dirofi laria spp. prompted us to monitor the prevalence of both parasites among dogs and report data periodically.
Fecal examination technique or sera analysis have been used for studying the prevalence for A. vasorum in dogs also in some surrounding countries including Greece, Hungary, Bulgaria (Papazahariadou et al., 2007;Pantchev et al., 2015). Couple of years ago, the fi rst case of A. vasorum was reported in Posavac hound in Serbia. Moreover, Serbia is abundant with terrestrial and aquatic gastropods proved to be either natural or experimental host for A. vasorum, while climate in Vojvodina off ers suitable conditions for their survival (Simin et al., 2014). From the epizootical point of view, this fi nding is important, yet not suffi cient; thus, further studies are needed to estimate the prevalence of canine pulmonary angiostrongylosis among dogs in Serbia. In regions where this disease is endemic, the true prevalence of the disease is probably underestimated, because most diagnoses of angiostrongylosis are made when infection results in clinical signs (Morgan et al. 2005; Koch and Willesen, 2009). To our knowledge, this is the fi rst report on the prevalence of A. vasorum in dogs in Serbia. Due to the fact that both heartworm diseases and French heartworm can manifest as subclinical conditions (Savić et al., 2012;Simin et al., 2014) or with respiratory signs such as cough and dyspnea, in this study, we investigated the existence of the concurrent infection in some asymptomatic hunting dogs and in pet dogs with clinical signs. Hence, parasitic infestations of respiratory and cardiovascular system should be considered by a clinician when a diff erential diagnosis list is made for patients with respiratory and/or cardiovascular system signs.

MATERIAL AND METHODS
In the period from 2010 to 2016, pet dogs from Novi Sad were tested for dirofi laria infections. Th e research included 190 privately owned pet dogs. At the moment of testing, the dogs were at least 7 months old, exposed minimally to one mosquito season (in Serbia it is from April to October), and without history of treatment with macrocyclic lactones. All animals were clinically examined and blood samples were taken from all dogs to the purpose of parasitological examination. Th e parasitological examination consisted of wet blood smears, modifi ed Knott test and antigen testing. Techniques for detecting circulating microfi lariae included microscopic examination of fresh blood smears and modifi ed Knott test. Detection and enumeration of circulating microfi lariae (mf) of both D. immitis and D. repens were carried out using modifi ed Knott test (Bazzochi et al., 2008). Morphological characteristics of microfi lariae such as length, width, cephalic and caudal ends, were assessed in order to diff erentiate microfi lariae of two Dirofi laria species (Genchi et al., 2007). Detection of circulating D. immitis antigens was carried out using commercial kit (SNAP Heartworm RT Test, IDEXX Veterinary Diagnostics) according to manufacturer's instruction.
In order to detect infestation with A. vasorum in hunting dogs, Baermann fecal examination method and antigen detection (Angio Detect Test, IDEXX Laboratories) were used. For the detection of the A. vasorum larvae, modifi ed Baermann test was performed (Zajac and Conboy, 2006). A total of 51 hunting dogs were examined for A. vasorum infestation by both Baermann fecal examination and antigen detection. Fecal examinations by Baerman test in dogs were done fi rst. Th e sera of these dogs were frozen and later tested for A. vasorum antigen.
Th e number of hunting dogs examined for concurrent infections with heartworm and French heartworm was 37, and the analyses were done by anti-gen detections for both parasites (SNAP Heartworm RT Test, Idexx Veterinary Diagnostics and Angio Detect Test, Idexx Laboratories). Twelve pet dogs with respiratory clinical signs (mainly cough and dyspnea) were also evaluated for the infestation with both D. immitis and A.vasorum using antigen detection for each parasite.

RESULTS AND DISCUSSION
In this study as well as in our previous studies (Spasojević Kosić (11/190) In order to compare the prevalence reported in this study with the prevalence of dirofi lariosis in the previous studies, it is necessary to take into consideration the methods used for the diagnosing of D. immitis and D repens infection. Having in mind this fact, we can compare our new results with our previously published results, and with the study of Tasić et al. (2008). In this study, we observed an overall prevalence for D. repens of 16.32% (31/190), which is lower than that from our previous study (18.88%, 27/143 dogs) (Spasojević Kosić et al., 2014) and lower than that from the fi rst report of D. repens in dogs in Novi Sad (Tasić et al., 2008). Th e prevalence of D. immitis in dogs is 18.95% (36/190), which is higher than that reported in our previous study (16.78%, 24/143 dogs). Th e prevalence of D. immitis is particularly increased in this year, which could be explained by the fact that the majority of dogs examined in this year were those with respiratory clinical signs. For the period 2009-2013, the prevalence of dirofi lariosis in Vojvodina was reported by Savić et al. (Savić et al., 2014.); however, in this study, the diff erentiation of microfi lariae has not been done and no conclusion can be made on the prevalence of D. immitis and D. repens. In the study of Savić et al. (2015), the prevalence of dirofi lariosis, being 15.29%, was established for the period of 2 years of study, and in 92.3% of positive samples, D. immitis were determined by PCR.
In order to diagnose A. vasorum, Baermann fecal examination technique was used because the test is useful when larvae are being shed. In hunting dogs examined by this test, A. vasorum larvae have been detected in one dog (Simin et al., 2014) making the prevalence for A. vasorum of 1.96%. Prevalence of 1.1% was found in Greece based on fecal examination of 281 samples (Papazahariadou et al., 2007), while the prevalence determined in 1247 dogs from Hungary ranged from 1.36% to 2.73% (depending on sera analyses) . In 167 sera of dogs from Bulgaria, no positive fi ndings were recorded (Pantchev et al., 2015). Overall estimated prevalence in this region of Europe in not so high, but the number of examined dogs was higher as compared with our study population. Having in mind that some dogs might not be shedding larvae at the moment of examination, we wanted to increase a detection of infestation with A. vasorum by using the detection of the parasite's antigen. However, the antigen of A. vasorum was confi rmed in none of these animals, not even in the dog in which the larvae of the parasite were detected. Th e possible explanation for this result could be the formation of antigen-antibody complexes, which inhibit detection of antigen in a sample as it has been previously shown with D. immitis detection (Matsumura et al., 1986;Brunner et al., 1988) and with A. vasorum detection by rapid device   Further studies are needed to determine the actual prevalence of A. vasorum in dogs in Serbia. Studies like these, aimed at emphasizing the need for specifi c diagnosis and prevention in dogs, could be useful for clinical practice. With regard to proven heartworm disease and A. vasorum fi nding in Serbia, use of specifi c diagnostic procedures for detection of these two parasites in dogs with signs indicating respiratory disease is essential. Th is approach would provide reliable results of clinical studies on dogs.

CONCLUSION
Th is study revealed the prevalence rates of 13.16% and 10.53% for single infections with D. immitis and D. repens, respectively, whereas the prevalence for mixed infections with both Dirofi laria species in pet dogs in Novi Sad was 5.79%. Prevalence of D. immitis in hunting dogs was 5.40%. According to the results of Baermann fecal examination method, the prevalence of A. vasorum in hunting dogs from Novi Sad was 1.96%. Concurrent infection with D. immitis and A. vasorum was established in neither hunting nor pet dogs in Novi Sad, Vojvodina, Serbia.