Spatial distribution of vector borne disease agents in dogs in Aegean region , Turkey

Objective. Assess the spatial distribution of seroprevalence of infection with or exposure to 4 vectorborne pathogens Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi and Dirofilaria immitis, across the coastal states of the Aegean region with special reference to clinical signs and haematological variances related to disease condition. Materials and methods. A convenience sample, targeting blood from at least 10 pet dogs from İzmir, Aydin, Denizli, Mugla and Manisa cities involved was evaluated using a canine point-of-care ELISA kit. Results. Out of 307 dogs tested the overall seroprevalence was highest for E. canis (24.42%), followed by E. canis + A. phagocytophilum co-infection (10.42%), A. phagocytophilum (7.49%) and D. immitis (2.28%). Only 2 cases were seropositive to B. burgdorferi albeit 10 dogs were co-infected with more than 2 agents. For both dogs infected with E. canis and co-infected with E. canis and A. phagocytophilum, anemia, thrombocytopenia and leukocytosis, were more commonly detected, whereas thrombocytopenia and leukocytosis were significant finding in dogs infected with A. phagocytophilum or D. immitis, respectively. Variance analysis showed significant differences for mean RBC, Hb, PCV and PLT values (p<0.01) among control group and other groups. Conclusions. Seropositivity for vector-borne pathogens other than B. burgdorferi, is moderately to widely distributed in dogs residing in the Aegean region in Turkey.


INTRODUCCIÓN
L a s e n f e r m e d a d e s i n f e c c i o s a s c a n i n a s transmitidas por vectores son problemas emergentes en la medicina veterinaria, además del potencial zoonótico de lo anterior los agentes causantes son de importancia para la salud humana.El diagnóstico de presunción, las aplicaciones de terapia y la prevención de estas infecciones es dependiente de la identificación de la inmunopatología de las enfermedades relevantes.A pesar de que se han explorado avances en prospección en el diagnóstico molecular y la epidemiología de estas infecciones y sus vectores, el principio de conocimiento de la patología y las reflexiones inmunológicos de las enfermedades han quedado rezagadas (1,2).

INTRODUCTION
The canine vector-borne infectious diseases are emerging problems in veterinary medicine, besides the zoonotic potential of the latter causative agents may carry of importance for human health.Tentative diagnosis, therapy applications and prevention of those infections is dependent upon identification of immunopathology of relevant diseases.Despite prospecting advances have been explored in molecular diagnosis and epidemiology of these infections and their vectors, principle pathology knowledge and immunological reflections of the diseases has lagged behind (1,2).
The vector-borne canine disease caused by Dirofilaria immitis, Borrelia burgdorferi, Ehrlichia canis, and Anaplasma phagocytophilum are frequently observed worldwide.Infection with the latter agents may be controlled in partly through vector avoidance and relevant preventive measures, however morbidity and mortality to those diseases continue to occur in dogs.Albeit the usage of acaricides and insecticides may be insufficient for breaking the enzootic transmission cycles.Within the better knowledge of the pathogenesis of the latter agents for animal and human health, there is clearly a need for additional data on the natural occurence and therefore the prevalence of these infections (1,2).
Diagnosis, preventive measurements, and therapy of vector borne diseases among dogs are effective even if the extent of infection in particular geographic areas is taken into consideration (1).The real seroprevalences of selected vector borne pathogens in the present study has, in general, been lacking among dogs in Turkey, as limited seroprevalence for each pathogen was evaluated individually and separetely within limited populations in local areas.Therefore cited references from Turkey is lacking through Science Citation Index journals.In this project according to the authors previous experiences and observations Eagean region of Turkey and relevant provinces were sampled, Ural -Spatial distribution of vector borne disease agents in dogs Por lo tanto, las referencias citadas de Turquía son pocas en la revistas del Science Citation Index.En este proyecto de acuerdo con las experiencias anteriores de los autores se tomaron muestras y observaciones de la región del Egeo en Turquía y las provincias relevantes, ya que en estos lugares se ha conocido previamente resultados positivos para E. canis o D. immitis, mientras que la pregunta verdadera son las tasas de seroprevalencia para B. burgdorferi y A. phagocytophilum.
as the latter locations were previously known to have E. canis or D. immitis positivity, whereas the real question was the seroprevalence rates for B. burgdorferi and A. phagocytophilum.
Because dogs in the Aegean region of Turkey are not routinely tested by veterinary surgeons in clinic for vector-borne agents, informative data points were needed for this region.The purpose of the present study was to assess the spatial distribution of seroprevalence of infection with or exposure to 4 vector-borne pathogens such as E. canis, A. phagocytophilum, B. burgdorferi and D. immitis in 5 out of 8 provinces in Aegean region of Turkey, with special reference to clinical signs and hematological variances related to disease condition.

MATERIAL AND METHODS
Inclusion criteria and sampling.The present study was enrolled among 307 dogs reffered to the Department of Internal Medicine, Faculty of Veterinary, Adnan Menderes University and privately owned small animal clinics in Aegean region (Aydin, Denizli, İzmir, Manisa and Muğla provinces).One hundred fourty nine diseased dogs, referred with histories of at least one of the clinical signs involving anorexia, weight loss, fever, generalized lymphadenopathy, splenomegalia, muscle weakness, bleeding, spontaneous rhinorrhagia, distal limb edema, dyspnoea, and/ or polyarthritis were evaluated using a canine point-of-care ELISA kit for diagnosis of naturally occuring vector borne diseases (Snap 4Dx).Out of 307 dogs, 158 of them (also subjected to a canine point-of-care ELISA kit and found to be negative against all 4 vector borne agents) were also involved and enrolled as control group.The study protocol was approved by the institutional laboratory animals ethics committee of Adnan Menderes University (HADYEK No: B.30.2.A DU.0.00.00.00/050.04/2010/017) and informed written consent was obtained from all of the dogs owners prior to enrollment of the dogs participated in study (n=307, at the age of 3 months to 15 years, 157 male, 150 female).
Haematological examination.Blood samples were withdrawn from vena cephalica antebrachii into anticoagulated (EDTA) and nonanticoagulated tubes.Complete blood counts were performed before within Abacus Junior Vet hematology analyzer.
Serological examinations.Serological analyses were performed for differential diagnoses of some selected diseases appeared to have influence on the study and may have the potential effect on the clinical signs.Each sample belonging to diseased and healthy dogs, was tested by use of an ELISA kit (SNAP 4Dx, IDEXX Laboratories, Examen hematológico.Se tomaron muestras de vena cefálica antebraquial en tubos con anticoagulante (EDTA) y sin anticoagulante.Se realizaron conteos sanguíneos completos con el analizador hematológico Abacus Junior Vet.

RESULTADOS
Población animal incluida.Los perros (n=137) con infecciones naturales fueron diagnosticados mediante los resultados de la prueba Snap 4Dx, USA) relevant to the protocol listed in the product insert, and as described previously (1).Aforementioned canine point-of-care ELISA kit detects antibodies occuring against to immunodominant proteins of E. canis (p30 and p30-1), immunodominant protein of A. phagocytophilum (msp2) and the C6 peptide of B. burgdorferi, besides detects circulating carbohydrate antigen of D. immitis (1).The 4 test results were recorded in an Excel spreadsheet.

Statistical analysis.
All the data distribution analysis for normality assumptions (Shapiro-Wilk test) and homogeneity of variance (Levene test) were evaluated.Kruskal-Wallis test was used for the data variance is not homogeneous or do not dissolve normally.Mann-Whitney U test with Bonferroni correction was used for Post hoc multiple comparisons and p<0.05 was considered significant.Results were given as mean ± standard error.
Haematological findings.Among hematological findings evaluated, relevant changes were shown in table 2. For both dogs infected with E. canis and co-infected with A. phagocytophilum, anemia, thrombocytopenia and leukocytosis, were more commonly detected, whereas thrombocytopenia was a significant finding in dogs infected with A. phagocytophilum.In dogs infected with D. immitis there was a remarkable leukocytosis.
Haematological variables obtained were shown in table 3. Variance analysis showed significant differences for mean RBC and PLT values (p<0.01)among control group and other groups.Statistically significant differences were observed for mean values of Hb and PCV among control group and others, besides between group III and IV.Variance analysis showed significant differences for mean MCV (p<0.05)[among control group and groups II and IV] and MCHC values (p<0.01)[among group II and other groups] (Table 2).

Canine granulocytic anaplasmosis has been rep o rt ed w o rl w i d e, p os s es s i ng p aral l el geographic distribution of human cases (3).
Relatively novel study suggested that A. phagocytophilum infections may be seen in humans from different climatic regions among Turkey.In the latter study it was detected that both the seroprevalence of A. phagocytophilum and probably co-infections of A. phagocytophilum and B. burgdorferi are higher in areas with more suitable habitats for Ixodes ricinus (4).Indeed longitudinal information relevant to I. ricinus among European countries are lacking, and it was suggested that higher incidence of the latter agent could have an efficacy on increased seroprevalence in dogs (5).
The detected A. phagocytophilum ELISA seroprevalence of 7.49% in dogs is not comparable, as relevant published data is lacking.The tested dogs of the present study lived most of their life in Aegean region; no seroprevalence data from this region were available among dogs at the time of testing.It should be kept in mind that the vast majority of dogs naturally infected with  Ural -Spatial distribution of vector borne disease agents in dogs europeos, y se sugirió que la mayor incidencia del último agente pudo haber sido efectiva en el aumento de la seroprevalencia en perros (5).

B e c a u s e A . p h a g o c y t o p h i l u m
Las alteraciones hematológicas más comúnmente detectadas fueron anemia, trombocitopenia y leucocitosis en perros infectados con E. canis y coinfectados con A. phagocytophilum, mientras que la trombocitopenia fue un hallazgo significativo en los perros infectados con A. phagocytophilum.Curiosamente, algunos de los perros infectados con E. canis presentaron edema de miembro distal (probablemente relacionado con trombosis venosa profunda), similarmente a lo reportado en otros lugares (17,18).Las variables hematológicas mostraron diferencias significativas para los valores promedio de RBC, hemoglobina, hematocrito y PLT (p<0.01)entre el grupo de control y los demás grupos.Aunque no es comparable debido a la naturaleza y situación diferente de las enfermedades unexposed dogs may be misclassified (1).(17,18).Hematological variables presented significant differences for mean RBC, Hb, PCV and PLT values (p<0.01)among control group and other groups.Although not comparable due to the different nature and status of the diseases involved, it may be suggested that the before mentioned hematological variables showed discrepancy in contrast to healthy controls.
The disease agent for which we observed the highest prevalence on a regional level was E. canis.Regarding enrolled population 24.42% of dogs were positive (solely monoinfection with CME) for E. canis on regional pattern, Ural -Spatial distribution of vector borne disease agents in dogs involucradas, puede sugerirse que las variables hematológicas mencionadas anteriormente mostraron una discrepancia en contraste con los controles sanos.

Figure. 1 .
Figure. 1. Snap 4Dx test results relevant to distribution of etiological agents among total study population

Figure 2 .
Figure 2. Spatial distribution of vector borne agents according to cities enrolled; a) E.canis, b) A. phagocytophilum, c) D. immitis and d) E.canis+ A. phagocytophilum seropositive cases.

Figure. 3 .
Figure. 3. E. canis seropositive clinical cases showing bleeding tendency a) corneal, b) gingival hemorrhage, c) and d) distal limb oedema due to probable deep vein thrombosis.

Figure 4 .
Figure 4. A. phagocytophilum seropositive cases a) generalized icterus, b) a Rottweiler presenting anemia and central nervous signs, c) a Terrier with anemia and d) Irish Setter with ocular hyperemia.

Table 1 .
Demographic data among groups.

Table 2 .
haematological abnormalities of dogs infected with E. canis/A.phagocytophilum and D. immitis

Table 3 .
Descriptive statistics relevant to leukocyte and erthrocyte parameters.
a (±0.08 (1)) prior serosurvey involving 380 stray dogs from Istanbul and Izmir, a seroprevalence rate of 1.52% was detected in İstanbul whereas no seropositivity was determined among dogs in Izmir(28).In Aydin city, where the present authors residing and working, microfilaremia was detected with a rate of 13.9% among 158 dogs(25).In the present research solely 7 dogs presented D. immitis antigens.This may be partly explained as a reflection of vector availability.The low prevalence of D. immitis found in this study confirmed a n e c t o d a l e v i d e n c e t h a t p r e va l e n c e o f Dirofilariasis is indeed not very high in Aegean Region, Turkey.Furthermore it has also been well recognized that mosquito control programmes are well adapted in this region, which might have influence on Dirofilariasis seroprevalence.P o s i t i v e s e r o l o g i c a l r e s u l t s m a y b e attributable to prior exposure and not necessarily disease condition, indeed they may forward clinicians to consider further clinical and diagnostic evaluation of dogs for underlying subclinical illness probably requiring treatment.Serologic test results also may be helpful for determining areaspecific disease prevalence, with contribution to understanding distribution of disease in both human and animal patients(1).Albeit to the present authors' experience the seroprevalence studies might possess more scientific value in case of invoving expression of clinical signs.The diseases are managed differently, and all may have complications.Antibacterial chemotherapy and preventive methods, involving vaccination against B. burgdorferi, are currently available for all 4 diseases (1), examined in the present study.