HАEMATOLOGIC INDICES IN CLINICALLY HEALTHY OUTDOOR DOGS EXPOSED TO VECTOR-BORNE PATHOGENS

Hаematologic indices in clinically healthy outdoor dogs exposed to vector-borne Abstract Haematologic abnormalities possibly associated with exposure to vector-borne pathogens are rarely reported in clinically healthy outdoor dogs. Therefore, we analysed changes in the complete blood count (CBC) of clinically healthy outdoor dogs seroreactive to Anaplasma spp. and Babesia spp., with or without microfilariosis. Stray, other four groups, the frequency ranged between 0.10 and 0.50. Clinically healthy outdoor dogs have multiple CBC abnormalities, consistent with stress and low-level chronic inflammation, but not associated with a previous exposure to Anaplasma spp. or Babesia spp. The presence of microfilaria increases haemolysis in vitro .

other four groups, the frequency ranged between 0.10 and 0.50. Clinically healthy

INTRODUCTION
The epidemiology of canine vector-borne pathogens (VBPs) is commonly investigated in asymptomatic outdoor dogs in Serbia (Gabrielli et al., 2015;Obrenović et al., 2015). Often, the studies included shelter, but also stray and hunting dogs (Spasojević-Kosić et al., 2015). Exposure to various vectors and VBPs is rather common in these dogs because they spend most of the time in a natural environment without any appropriate protection against vectors and parasites. The study of Kovačević Filipović et al. (2018) conducted in Serbia showed that a population of outdoor dogs were a significant source of potential hosts for several VBPs.
Although studies on prevalences of VBPs in clinically healthy dogs are numerous, only some of them assessed the health status of the animals (Spasojević-Kosić et al., 2015). Apart from the possible specific pathogenic effect that is associated with blood cell tropism of VBPs, it is highly possible that frequent exposure to vectors and pathogens causes low-grade tissue damage that could lead to an asymptomatic chronic inflammation (Otranto et al., 2009). Thus, it could be assumed that, as a consequence, haematologic changes consistent with a chronic inflammation, e.g. mild leucocytosis, microcytic anaemia and variable findings in the number of thrombocytes, could be induced.
Our aim was to investigate the frequency of the above-mentioned changes in the complete blood count (CBC) of clinically healthy outdoor dogs that were polymerase chain reaction (PCR) negative but seroreactive to Anaplasma spp., Babesia spp., with and without microfilariosis.

MATERIALS AND METHODS
We used the surplus of venous blood samples collected from clinically healthy stray, hunting and shelter dogs belonging to a larger group formed for our previous study on molecular detection and seroprevalence of Anaplasma phagocytophilum, A. platys, Ehrlichia canis, E. chaffeenses, E. ewingii, Borrelia burgdorferi, Babesia canis, B. gibsoni, and B. vogeli (Kovačević Filipović et al., 2018). Tick/Vector Comprehensive Real PCR Panel Canine (IDEXX Laboratories, Westbrook, Maine) was used for the molecular detection of the previously named pathogens. Seroreactivity was tested with an immunofluorescence antibody test (Megafluo ® Vet, Megacor Diagnostik GmbH, Austria) or SNAP ® 4Dx Plus technology (Kovačević Filipović et al., 2018). Eligible animals were PCR negative to the investigated pathogens and seronegative to Borrelia spp. and Ehrlichia spp. The selected dogs (N=81) were divided into five groups according to their serological status and results of a modified Knott's test (MKT): The venous blood samples in tubes with EDTA, collected for PCR analysis, were also used to determine the CBC (Abacus Junior Vet, Diatron, Vienna, Austria) and to perform MKT as described by Magnis et al. (2013) within two hours of collection.
Statistical analysis included Chi-square test with the Boniferroni correction for multiple comparisons (MedCalc ® software version 16.2.1), where P-value less than 0.05 was considered significant.
In accordance with the European Union declaration 63/2010 and based on the Serbian Animal Welfare Law, we obtained a permission for this study from the Ministry of Agriculture, Forestry and Water Management (number 323-07-03455/2015-05/3) prior to its commencement. Furthermore, we collected the blood samples from shelter and hunting dogs with the informed consent of responsible shelter management personnel or the dog owners.

RESULTS AND DISCUSSION
Our study focuses on the changes in CBC in dogs with different serological status against Anaplasma spp. and Babesia spp., with or without the presence of microfilaria.
First, we analysed total leukocyte number. Over all five dog groups, leucocytosis ( Figure 1A) and increased granulocyte counts ( Figure 1B) had a frequency of 0.35 and 0.44, respectively. Lymphopenia was present with frequency of 0.22, while lymphocytosis (frequency 0.05) was a rather rare finding ( Figure 1C). The alterations in the number of monocytes and part of eosinophils (denoted jointly as medium (MID) cells) were minimal, i.e., the frequency of decreased count was 0.10, while an increased count was encountered in only one dog ( Figure 1D). This finding is in accordance with a previous study in hunting dogs seropositive to VBPs (Spasojević-Kosić et al., 2015). Our result showing an increased granulocyte count in combination with lymphopenia probably reflects exposure to different environmental stressors in addition to the evident exposure to ticks and tick-borne pathogens. We suppose that some of the environmental stressors for outdoor dogs could be quantitative and/or qualitative nutrition deficiencies, exposure to bad weather conditions, parasite load and even a lack of appropriate social relationships (Uetake et al., 2016). However, it is clear thatthe presence of Anaplasma spp., Babesia spp. and microfilaria is not a major stressor in the population of outdoor dogs, as the change in neutrophil and lymphocyte number is unequivocally present in all our investigated groups.
When all five groups were analysed together, the decreased number of red blood cells (RBC) was also a rare finding, as illustrated with frequency of 0.07 (Figure 2A).
A similar situation was noticed for haemoglobin (HGB), as concentrations below the reference range occurred with a frequency of 0.05 (Figure2B). Anaemia among all dogs occurred at a frequency of 0.28, using the haematocrit (HTC) value below 0.37 as the cut-off ( Figure 2C). This finding together with the microcytosis frequency of 0.74 ( Figure 3A) may indicate anaemia due to chronic inflammation was present in the population of investigated dogs. The other possibility is a nutritional deficiency, which at present cannot be proved.
Among all investigated dogs, increased mean cell haemoglobin concentration (MCHC) was present with the frequency of 0.37. Nevertheless, it was the only parameter that showed a significant difference between the groups ( Figure 3B). The prevalence of dogs that had an increased MCHC was higher in the SR-M group (0.80) than in the SR-A (0.10), SR-B group (0.24), SR-AB group (0.24) and SN group (0.50). This finding is consistent with the study of Milanović et al. (2017), which demonstrated that in dogs with acute phase reaction due to B. canis infection, haemolysis was more pronounced when the dogs were concurrently infected with Dirofilaria immitis. Furthermore, there is also a possibility that blood sampling potentiates some degree of haemolysis in erythrocytes that already have an increased osmotic fragility due to microfilariosis. Finally, thrombocytopenia was encountered in one third (0.33) of all the dogs ( Figure  4), pointing to the fact that thrombocytopenia is not more frequent among dogs exposed to Anaplasma spp. and Babesia spp. than among non-exposed dogs. Since thrombocytopenia is the most frequent haematologic abnormality in veterinary medicine (Zimmerman, 2000), this finding was not unexpected, but its true nature remains unknown.  Still, we cannot rule out the possibility the dogs were infected with agents not investigated in our study. For example, golden jackals in the wilderness in Serbia, apart from being reservoirs of Anaplasma spp. and Babesia spp. (Sukara et al., 2018), can also be infected with Brucella spp. and Leishmania spp. (Ćirović et al., 2014), and probably a number of other pathogens that could trigger subclinical inflammatory response. Furthermore, in general, ticks in Serbia are infected with Rickettsia spp., Canididatus Neoehrlichia mikurensis and Hepatozoon canis (Potkonjak et al., 2016), all of which could be transmitted to dogs, and induce, at least, mild CBC abnormalities, similar to those found in our study. More complex analysis of the dogs' microenvironment could identify the exact stressors. Additionally, thorough investigation of the ectoparasites and endoparasites present in the dogs as well as possible co-infections and comorbidities could give amore precise information about the dogs' overall health and the reasons for the observed CBC changes.