FIRST CONFIRMED CLINICAL CASE OF ANAPLASMA PLATYS IN A DOG IN SERBIA ILIĆ BOŽOVIĆ Anja1, RADAKOVIĆ Milena1, SPARIOSU Kristina1, TYRRELL Phyllis2, CHANDRASHEKAR Ramaswamy2, MIŠIĆ Dušan1,3*, KOVAČEVIĆ FILIPOVIĆ Milica1

In September 2018, a four-month-old dog with fever and petechial bleeding came to the internal clinic at the Faculty of Veterinary Medicine University of Belgrade. On hematology analysis, thrombocytopenia and mild anemia were observed. Examination of the blood smear revealed platelet inclusions. The commercial serology test was positive for Anaplasma spp. The dog was treated with doxycycline for 14 days, and after 48 hours from the beginning of the treatment, the symptoms subsided. PCR analysis and sequencing confirmed infection with A. platys.


IntroductIon
Anaplasma (A.) platys is a causative agent of cyclic canine thrombocytopenia (thrombocytotropic anaplasmosis). The disease is geographically distributed on all continents and is potentially zoonotic [1]. It is considered that the vector is Rhipicephalus sanguineus, although, A. platys DNA has also been found in other tick species without the proof of possible transmission. The only clinical sign of A. platys infection in dogs may be severe thrombocytopenia when A. platys inclusions (morulae) can be seen in blood smears. Dogs are mostly asymptomatic, but if symptoms do occur, then pale mucous membranes, fever, petechial hemorrhage, uveitis, and lymphadenopathy could be observed. In the first 15-21 days of infection, thrombocytopenia occurs during which the platelet count may fall below 20 × 10 9 /L. After this phase, the platelet count temporarily returns to normal. This recurs cyclically. In the later stages of the disease, it is increasingly difficult to find A.platys in blood smears [2]. The immune response to A. platys with specific immunoglobulins can be detected by IFA and ELISA. The distinction between A. phagocytophilum and A. platys is possible by molecular methods and by a proprietary, research prototype ELISA SNAP® test (SNAP M-A test) (IDEXX Laboratories, Inc., USA) [3].
Several seroprevalence studies in Serbia have so far found that outdoor dogs were highly exposed to members of the Anaplasmataceae family with a detected prevalence of 20-30% [4][5][6]. However, A. platys has not yet been isolated from dogs on Serbian territory.

case presentatIon
The four-month-old Rottweiler male was brought to the internal clinic at the Faculty of Veterinary Medicine, University of Belgrade, Serbia, in September 2018. The anamnesis described that the dog was depressed for more than 2 weeks. The dog reportedly was regularly vaccinated and treated with an unknown ectoparasiticide after a tick was spotted on it. The dog had a good appetite, but physical examination revealed fever (39.6°C) and petechial bleeding on the abdomen and buccal mucosa. The dog had been normally hydrated and eupnoeic. A blood sample was taken from the cephalic vein in a vacutainer with EDTA (Becton Dickinson, USA) at the day of admission (day 1), and was taken 2 more times; once on Day 7 and then again on Day 30, the last day of treatment. Romanowsky-stained blood smear Day 1) identified A. platys morulae in giant platelets ( Figure 1). Complete blood count (CBC) done immediately upon blood collection revealed moderate normocytic normochromic anemia and severe thrombocytopenia, with a manual count of 12 ×10 9 platelets/L ( Table 1).  test (IDEXX Laboratories, Inc., USA) was positive for antibodies against Anaplasma spp [7,8]. Retrospectively, the SNAP M-A test, which offers additional species specificity, detected specific A. platys antibodies in the serum specimen taken on the day of admission. ELISA SNAP M-A (IDEXX Laboratories, Inc., USA) excluded co-infections with Borrelia spp., Ehrlichia spp. and Dirofilaria spp. The dog was treated with doxycycline (2 mg/kg IM every 24 hours) for 2 weeks. Symptoms of infection resolved completely after 48 hours, and the platelet count increased to normal within 7 days. The SNAP M-A test revealed that the dog was seroreactive against A. platys and seronegative against A. phagocytophylum on presentation, and seronegative against both pathogens on Day 30. The tick was not found on the dog at the time of the examination and thus, the species of the tick could not be confirmed.
The finding of A. platys is not surprising given that it was clear from serological surveys that members of the Anaplasma genus are present amongst the dogs on the territory of Serbia.
The dogs infected in the USA with A. platys are usually asymptomatic, while it appears that those infected in southern Europe most often develop distinct clinical abnormalities [2,9]. Coinfections with other vector-borne pathogens also influence the severity of disease [2,9]. Nonetheless, young dogs, like the one in our presentation, and dogs surviving stressful conditions, appear more susceptible to developing the clinical disease [10][11][12][13]. The dog in this case probably had an underdeveloped immune system and was under stressful conditions related to the change of the owner and consecutive travel. In conclusion, because of the high seroprevalence in dogs suggesting that there is an increased likelihood of exposure and infection with a potentially zoonotic pathogen [14,15], more attention should be given to the diagnosis of anaplasmosis in both human and veterinary medicine in Serbia.

Declaration of conflicting interests
The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article. Phyllis Tyrell and Ramaswamy Chandrashekar are employees of IDEXX Laboratories, Inc. references