ORIGINAL

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

Distribución espacial de agentes patógenos transmitidos por vectores en perros en la región del Egeo, Turquía

Kerem Ural,¹ Ph.D, Mehmet Gultekin,¹ Ph.D, Abidin Atasoy,¹ Ph.D, Bulent Ulutas,¹ Ph.D.

¹Adnan Menderes University, Faculty of Veterinary, Department of Internal Medicine, Isikli. Merkez Kampüsü Aytepe Mevkii, 09016, Aydin-Turkey.

*Correspondence: uralkerem@gmail.com

Received: September 2013; Accepted: December 2013.

ABSTRACT

Objective. Assess the spatial distribution of seroprevalence of infection with or exposure to 4 vector-borne 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 Izmir, 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.

RESUMEN

Objetivo. Evaluar la distribución espacial de la seroprevalencia de la infección de 4 agentes patógenos de transmisión por vectores Ehrlichia canis, Anaplasma phagocytophilum, Borrelia burgdorferi y Dirofilaria immitis, en los estados costeros de la región del Egeo con especial referencia a los signos clínicos y las variaciones hematológicas relacionadas con la enfermedad. Materiales y métodos. Se tomaron por conveniencia muestras de sangre de al menos 10 perros en las ciudades Izmir, Aydin, Denizli, Mugla y Manisa. Para la evaluación de las muestras se utilizó un kit de ELISA para la detencción de anticuerpos de las enfermedades del estudio. Resultados. De los 307 perros muestreados, la seroprevalencia más alta fue para E. canis (24.42%), seguido por la coinfección entre E. canis + A. phagocytophilum (10.42%), A. phagocytophilum (7.49%) y D. immitis (2.28%). Sólo 2 casos fueron seropositivos para B. burgdorferi aunque 10 perros fueron coinfectados con más de 2 agentes. En ambos perros infectados con E. canis y coinfectados con E. canis y A. phagocytophilum, se detectó comúnmente anemia, trombocitopenia y leucocitosis, mientras que la trombocitopenia y leucocitosis fueron significativos en perros infectados con A. phagocytophilum o D. immitis , respectivamente. El análisis de varianza mostró diferencias significativas para los promedios de RBC, hemoglobina, hematocrito y valores PLT (p<0.01) entre el grupo control y los otros grupos. Conclusiones. La seropositividad transmitida por vectores patógenos distintos de B. burgdorferi, fue moderada y ampliamente distribuida en los perros que residen en la región del Egeo en Turquía.

Palabras clave: Anaplasma phagocytophilum, Borrelia burgdorferi, Distribución espacial, Dirofilaria immitis, Ehrlichia canis, perros, Turquía (Fuente: CAB).

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, 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

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, 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.

Clinical signs obtained based on presence/absence such as nasal discharge, loss of appetite, weight loss, tick infestation, muscle weakness, bleeding, findings associated with an increased bleeding tendency (dermal petechiae, epistaxis), polyarthritis, lymphadenopathy, neurological, cardiac, and respiratory disorders was calculated among the groups in comparison with the control by use of chi-square test of independence.

This study was summarized partially from a national project completely funded by Adnan Menderes University, Research Projects Funding Unit with Project number VTF-10004/2011.

RESULTS

Animal population enrolled. Dogs (n=137) with naturally occurring infections, were diagnosed by Snap 4Dx test results, of both sexes (63 male, 74 female) were enrolled into 4 different groups. I. group (n = 75) consisted E. canis infected dogs (3 months-15 years of age), II. group involved A. phagocytophilum (n=23) (7 months-15 years of age), III. group E. canis + A. phagocytophilum co-infected dogs (n=32) (3 months-12 years of age) and IV. group D. immitis infected dogs (n=7) (2 to 6 years old). B. burgdorferi-infected (n=2) or co-infected dogs with more than two etiological agents [E. canis + A. phagocytophilum + D. immitis infected dogs n=4, E. canis + Babesia canis infected dogs n=3, E. canis + A. phagocytophilum + Hepatozoon canis infected dogs n=3] were numerically insufficient (totally n=10) for statistical evaluation, therefore left out of inclusion criteria for intragroup comparison.Dogs were identified as negative within Snap 4Dx (n=158) and available physical examination results, were enrolled as healthy control group (Table 1).

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).

Serological findings. All related data involving spatial distribution of provinces was shown in figures 1 and 2.

Anaplasmosis. An overall prevalence of 7.49% was detected, besides 10.42% presented both E. canis and A. phagocytophilum antibodies. The highest prevalence of samples with antibodies to A. phagocytophilum were reported from Aydin province (n=10); samples from Izmir also were frequently found to be positive (n=8). None of the dogs tested positive for A. phagocytophilum were co-infected with B. burgdorferi.

Lyme borreliosis. B. burgdorferi antibodies were detected solely in 2 cases in KuSadası, Aydın.

Ehrlichiosis. The overall prevalence for E. canis was 24.42% of 307 dogs. Antibodies to E. canis were detected most often in dogs in Aydin (n=33), followed by 22 and 15 cases in Izmir and Mugla provinces, respectively.

Dirofilariosis. The highest percentage of D. immitis antigen-positive samples, was obtained from Aydin (n=4) and Izmir (n=2).

Taking into account the probable transmission via the bite of ticks for ehrlichial organisms, active tick infestation was evident among 82.60%, 81.25%, and 86.66% of dogs infected with A. phagocytophilum mixed E. canis and A. phagocytophilum, and E. canis, respectively. Cases were deemed individually, 5 dogs showed corneal and gingival hemorrhage (Figures. 3 a-b), 4 of dogs infected with E. canis presented distal limb edema (probably related to deep vein thrombosis) (Figure 3c-d), whereas some of the A. phagocytophilum infected dogs showed icterus (Figure 4a), lameness in association with polyarthritis and central nervous system signs (Figure 4b). Dogs infected with E. canis or A. phagocytophilum presented anemia (Figure 4c), hyperemia (Figure 4d) and bleeding tendency.

DISCUSSION

Canine granulocytic anaplasmosis has been reported worlwide, possessing parallel 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 A. phagocytophilum may remain clinically healthy. This may be briefly explained with the frequent distribution of anaplasmosis in endemic locations even in the absence of a clinical illness (6, 7). Furthermore A. phagocytophilum infection may be self-limiting in dogs (3). As positive titers might be indicating previous exposure, the implications for the discovery of seropositive dogs does not involve active infection all the time. Risk factors for A. phagocytophilum infected dogs involved season of the year, mostly between April and July (3, 8). Although the data was not shown in detail, in our study in agreement with the latter authors, the vast majority of the dogs infected with A. phagocytophilum and/or E. canis were detected in warmer months (between May and July in Aegean region)

In a relatively recent study determining the seroprevalence of anti-B. burgdorferi antibodies in a population of Turkish dogs by use of both enzyme-linked protein A/G assay and novel flow-cytometry-based borreliacidal antibody test, 23.2 and 27.75% seropositivities, respectively, were detected (9). In another study performed in Aydın and Izmir provinces, in Turkey IgG antibodies were detected by use of ELISA among 49 dogs out of 140 with 35% seropositivity (10).

Because A. phagocytophilum shares a vector and reservoir host system with B. burgdorferi, the geographic distribution of cases of HGA parallels that of Lyme borreliosis (11). Seropositivity for B. burgdorferi was similar geographically to that for A. phagocytophilum, especially in a study evaluating the spatial distribution of seroprevalence for 4 vector-borne pathogens, A. phagocytophilum, B. burgdorferi, E. canis and D. immitis, across the western coastal states of the United States (1). In the present study among involved dogs a seropositivity of 7.49% was detected for A. phagocytophilum whereas only 2 dogs were seropositive to B. burgdorferi. This is surprising given the shared tick vectors (Ixodes spp.) and mammalian reservoirs for both A. phagocytophilum and B. burgdorferi. The lack of travel history to A. phagocytophilum or B. burgdorferi seropositive dogs and low seroprevalence rates suggested that disease transmission may be inefficient in Aegean Region, probably due to climate or other environmental conditions, despite the presence of I. ricinus. This specificity is of considerable concern in areas with low seroprevalence for B. burgdorferi, moreover unexposed dogs may be misclassi�ed (1).

Obtained data available in the present study indicated areas where Lyme disease does not appear to be present, suggesting that strong emphasis on prevalence researches of other areas may be required for affected dogs, possibly by use of a more speci�c test, as Western blot (1). Only scarcity and limited information is available in Turkey relevant to sero-epidemiology of CME, in reality the overall prevalence is underestimated. A prevalence rate of 20.8 % was obtained among 284 dogs in 3 different regions in Turkey (12). In the latter study among cities involved Adana (65.3%) and Izmir (40.6%) showed highest prevalences. In previous years, frequently clinical CME cases (13), were reported among dogs in Turkey, whereas among one of the first molecular studies E. canis antibodies was detected by use of IFA and dot-ELISA techniques, with 67.8% and 53.3% seropositivity rates, respectively (14). In later years in another molecular study 3 out of 12 dogs were seropositive to CME (15). In a recent study in Diyarbakir, Snap 3Dx rapid test kit analysis resulted in 4.8 % seropositivity among 82 dogs (16). Among 4 dogs with CME, 3 were female and only 1 dog was male, and 3 of them were at the age of 4 to 7 years (16). In our study most of the dogs with CME were at the age of 3 months to 15 years of age with no age or sex predisposition.

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, with the highest regional rate observed in Aydin (44%) and Izmir (29.33%). Taking into account that brown dog tick thrives near territories inhabited by many stray or kennel dogs, including shelters and some multiple-dog facilities in Aegean Region, it should not be unwise to suggest that E. canis is endemic in this area. Further evaluation of specific environments in which seropositive dogs residing would be helpful for interpretation of suggested data and probably for taking control measures and for developing management strategies. Besides, additional information about the distribution of R. sanguineus might be useful for evaluating risks of other probable pathogens transmitted by the latter tick, as reported previously (1). The prevalence of CME may be related to the distribution of the vector, R. sanguineus, which occurs mainly in tropical and subtropical regions. The responsible agent causing canine granulocytic ehrlichiosis in Europe has been determined by nucleotide sequencing of the 16S rRNA gene to be identical to the agent of human granulocytic ehrlichiosis (17). The vector of this pathogen in Europe is I. ricinus and its reservoir -wild and domestic animals-. In accordance, it was reported that ticks of the genera Ixodes, Rhipicephalus and other relevant ones were widespread throughout Anatolia in Turkey (19).

Interestingly among E. canis infected dogs (n=75), 45.82% were residing in Aydin city and to those of most of them were living in coastal areas (Kusadasi, Didim etc.). According to the history obtained, owned dogs enrolled in the present study did not resided outside the city borders almost their live, therefore the question raised was the real prevalence of the etiological agents, except Aydin city in Eagean region. The high prevalence of CME in the current study may be explained within several factors involving the presence of E. canis in ticks from Aegean Region, frequent exposure of pet dogs to R. sanguineus, and sufficient transmission of E. canis by the latter tick in warmer climates.

Antigenic testing for Dirofilariosis mostly encountered following the last 15 years among Turkish studies, whereas researhes involving solely one type of diagnostic method (microfilaria or antigenic examination) are the vast majority. Turkish studies detected seropositivity within the rates from (0 to 46.22%) (20-27). Taking into account the latter serosurveys, tha vast majority of them involved limited populations and according to the authors’ knowledge none of them reported clinical signs related to disease condition. In a prior serosurvey involving 380 stray dogs from Istanbul and Izmir, a seroprevalence rate of 1.52% was detected in Ístanbul 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 anectodal evidence that prevalence of 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.

Positive serological results may be 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 area-specific 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.

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