Arthropod-borne infections in travelled dogs in Europe

Pet animal movement is ever increasing within the European Union and in that context canine vectorborne infections gained a considerable importance. Information on these infections in travelled dogs is nevertheless limited. A first prospective study on vector-borne infections was conducted in 106 dogs travelling from Germany to countries in South and South-East Europe. The dogs were screened prior to and consecutively up to three times after travel by haematological (Giemsa-stained buffy coat smears, Knott’s-Test), molecular biological (PCR) as well as serological (IFAT, DiroChek®-ELISA) methods for arthropod-borne infections. Seven animals were seropositive for antibodies against Babesia canis sspp., Leishmania spp. and/or Ehrlichia canis prior to travel to Italy, Spain, France, Croatia, Greece, or Hungary. In the consecutive screening after return there was no increase in the number of seropositive dogs. None was positive in direct methods. The mean duration of the stay was 17 days and 51% of the dogs were prophylactically treated with ectoparasiticidal formulations. Preliminary data from this study on canine vector-borne infections indicate a low risk for infection during a limited single stay in endemic countries.


Introduction
Canine vector-borne infections are a steadily increasing field of veterinary research interest and their diagnosis, treatment and prophylaxis are of considerable importance in small animal medicine [5]. As travel restrictions within the European Union are essentially non-existent, animals are freely transferred from non-endemic regions into countries endemic for vector-borne infections and vice versa. A previous evaluation by questionnaire reported that more than 50% of 5,240 German dogs have crossed the border at least once and travelled e.g., to Italy, France and Spain [7]. Dogs originating from Central Europe can be considered as immunologically naïve to most vector-borne pathogens present in southern Europe and any sojourn to these regions sets dogs at risk of infection. However, recent studies evaluating laboratory data reported low seroprevalence rates (<5.0%) against Babesia canis spp., Leishmania spp. or Ehrlichia canis in travelled dogs [8,10]. This may be indicative for a low exposure to infected vectors during a limited stay, although no information on prophylactic measures, the time or duration of travel was given in these studies [10,24]. So far, no travelled dogs from Germany were tested involving the determination of a pre-and post-travel status on vector-borne infections. In this preliminary study, results on the serological, haematological and molecular biological examination of 106 dogs from Germany with sojourns into countries in the Mediterranean Basin and South-Eastern Europe are presented.

Material and methods
In the spring 2009 and 2010 veterinarians were informed by an advertisement placed in the monthly magazine of the German Veterinarian Society (''Deutsches Tierärzteblatt'') about this prospective study. Interested veterinarians were provided with information on sampling time (e.g., for Knott's-Test) and intervals of sampling. Pet owners presenting their dogs for a routine pre-and post-travel check-up/consultation and willing to participate were asked to fill out questionnaires. The testing (P0) one to two weeks prior to departure aimed at attaining an overview on existing infections, the testing intervals of two to four weeks (P1), of six to eight weeks (P2) and of six months (P3) after travel to detect babesiosis, hepatozoonosis and ehrlichiosis by direct pathogen detection and/or seroconversion (at P1), the prolonged interval of up to 6 months aimed at the detection of patent filarial infections (at P2-P3) and delayed seroconversion in case of Leishmania spp.-infection (at P1-P3) [1,2,9,26,27] (Table 1). Inclusion criteria were at least one pre-and one follow-up canine EDTA-blood sample (P0 and P1) available from routine screening. In total, 287 EDTA-blood samples from 106 dogs belonging to 91 different dog owners could be evaluated. The study was in compliance with local regulations.

Laboratory methods
Giemsa-stained buffy coat smears were performed for the detection of pathogens in the blood and Knott's-Test for the detection of blood-borne microfilariae. The DiroChek Ò Canine/Feline Antigen Test Kit (Synbiotics Corp., Missouri, USA) was used for screening for D. immitis-antigen. DNA was extracted from 200 ll EDTA-blood with the Qiagen DNA MiniKit (Qiagen, Hilden, Germany) following manufacturer's instructions. Quality and quantity were tested with a spectrophotometer (NanoDrop Ò ND-1000, Erlangen, Germany). Conventional PCRs were applied for the detection of Babesia spp.-, H. canis-and filarial-DNA (in case of positive Knott's-Test) according to previously published protocols [3,11,23]. Real-time PCRs were used for the detection of Leishmania spp.-and E. canis-DNA [15,Silaghi et al.,unpublished]. Each PCR run involved a positive control and a negative control (PCR-clean water). Plasma for IFAT was collected by centrifugation of EDTA-blood samples. Antibodies against B. canis spp. and E. canis were tested applying commercial IFAT-tests (MegaCor, Hörbranz, Austria) and an in-house Leishmania spp.-IFAT was used for the detection of anti-Leishmania spp.-antibodies [13]. Titres of 1:64 were considered as seroreactive to B. canis spp. and Leishmania spp. and of 1:40 to E. canis.

Animal data and questionnaire
Participating pet owners were asked to fill in the provided questionnaires prior to and after travel to obtain individual animal data and information on travel destination, duration of the stay, prophylactic measures applied against arthropod vectors, on tick infestation on their dogs observed during holidays and to record any additional ectoparasiticidal treatment bought at travel destination.

Laboratory results
Pre-travel screening (P0) One hundred and six canine EDTA-blood samples were submitted for pre-travel screening in a mean of 7 days (1-47 days) prior to departure. None of the dogs was microfilaremic in Knott's-Test or positive for D. immitis-antigen in DiroChekÒ-ELISA. All Giemsa-stained buffy coat smears were negative for parasites of the blood. Six dogs had antibodies against B. canis spp. (3·), Leishmania spp. (2·) and E. canis (1·) in IFAT, another animal was seropositive for both B. canis spp.-and Leishmania spp.-antibodies ( Table 2). One animal positive for B. canis spp.-antibodies was vaccinated against canine babesiosis according to information given by the owner. All seropositive animals had travelled previously with their owners.
Post-travel screening (P1-P3) One hundred and six samples were available for the first post-travel screening between four and 373 days (mean 38 days) after return. None of the samples was positive for Table 1. Pre-and post-travel screening panel for arthropod-borne infections.

Method
Timepoint of testing vector-borne infections in direct methods. Dogs with antibodies against Leishmania spp (3·) and one seroreactive against E. canis were again tested positive. In two dogs, B. canis spp. IFAT was negative. A total of 61 samples were submitted between 34 and 423 days (mean 171 days) after return for P2 screening. There were neither microfilariae nor antigen of D. immitis detectable. Two animals were again seropositive for antibodies against Leishmania spp. while no sample was submitted from the third seroreactive dog. Only 16 samples out of 106 were sent in for a third post-travel examination (P3) between 193 and 493 days (mean 322 days) after the return to Germany. All were negative in Knott's-Test and DiroChekÒ-ELISA. The two samples positive in P2 were again seropositive for Leishmania spp.-antibodies (Table 2).

Animal data
The 44 male dogs and 62 bitches (106 dogs) in this study were between 0.5 and 14.5 years old (mean 5.1 years, standard deviation (SD) 3.4). Forty-four dogs of breed (26 different breeds), 31 mixed breed dogs and 31 dogs without specified breed were enrolled. Sixteen dogs were taken abroad previously, 90 dogs had not travelled across the German border. The travel duration was given for 98 dogs and varied between 3 and 62 days (mean 17 days, SD 8.6); 14 dogs were taken abroad for up to one week, 26 between 8 and 14 days, 40 between 15 days and 3 weeks and 18 for more than 3 weeks. The main travel period in 2009 was between May and September (89 dogs) and between July and September in 2010 (17 dogs). The travel destinations are summarized in Table 3 other medication and no information was given in 41 (38.7%) cases. Tick infestation was observed by the owners on 7 treated (3· AdvantixÒ, Bayer; 3· ScaliborÒ, Intervet; 1· FrontlineÒ, Merial) and 3 untreated animals. Only 10 pet owners (11.0%) bought additional ectoparasiticidal products (5·; ScaliborÒ, Intervet; 4·; AdvantixÒ, Bayer; 1· FrontlineÒ, Merial) during holidays and seven (7.7%) pet owners bought other products.

Discussion
Canine populations from the Mediterranean Basin show high prevalence rates of vector-borne infections [24,29]. Consequently, this topic attracted an increasing interest in veterinary medicine [5]. It is therefore noteworthy that in this study none of the dogs was infected with arthropod-borne infections due to  the recent stay in South and South-East Europe. In total, seven dogs were seroreactive to Leishmania spp., E. canis and/or B. canis spp. at P0 prior to travel and all of these animals had been taken along on holidays previously. None of the animals were positive in Giemsa-stained buffy coat smears, Knott's-Test, Dir-oChekÒ-ELISA or PCR. The overall seroprevalence rate of 6.6% in the 106 dogs is comparable to data presented in retrospective studies in dogs with travel history [8,10]. A publication from the Netherlands based on questionnaire and diagnostic data identified a chance of 0.23% for individual travelling dogs for attaining a Leishmania-infection [28]. In a study from the UK, dogs with travel history and positive for leishmaniosis had spent at least six months in Spain, while the average stay in the present study was only 17 days [25]. The risk of acquiring a vector-borne infection is dependent on the presence of infected vectors, their seasonal activity, the duration of exposure, the presence of suitable (reservoir-)hosts and prophylactic treatment. As this study encompassed the summer holiday period in Germany, the time of travel did not necessarily coincide with peak activity of potential vectors. While the brown dog tick is active all year round in the subtropical areas of the Mediterranean, other vectors, e.g., phlebotomine sandflies, may show bimodal activity patterns in spring and autumn or primarily in the summer month, such as mosquitoes [4,12,26]. This impacts the chance of a potential pathogen transmission. The low number of examined blood samples may also bias the results of the study. Moreover, the diagnosis of vector-borne infections can vary considerably depending on the applied technique as well as of the course of clinical disease [17,18]. Nevertheless, all animals were so far considered as healthy and it can be expected that dog owners would have sent additional EDTA-blood samples of their dogs in case of impaired health status. The results are in clear contrast to laboratory data from over 15 000 dogs imported into Germany published over the last ten years. Seroprevalence rates against E. canis, Leishmania spp. or B. canis spp. range from 10.6 to 18.5%, cumulative prevalence rates for microfilariae and D. immitis-antigen are 2.6% and 1.8% in over 10 000 dogs tested and 2.1% of 7500 dogs carry gamonts of H. canis in the blood [16,22,24]. Recommendations on prophylactic treatment are primarily based on data from studies in local dog populations from endemic countries [16,29] Interestingly, of the 54 (51.0%) prophylactically treated dogs in this study, 50 (92.6%) were treated with actives (28· ScaliborÒ, Intervet; 19· AdvantixÒ, Bayer; 3· ExspotÒ, Essex) registered for the use against Phlebotomus spp., giving evidence that veterinarians and pet owners are aware of this important vector and rely on formulations with anti-feeding effect. It has been proven that the prevalence of vector-borne infections increases with consecutive transmission seasons and with the age of dogs [6,14]. A study in Italy conducted over two transmission seasons with more than 600 dogs presented an increase in Leishmania-antibody seroprevalence from 5% to more than 25% in untreated dogs while the rate in the deltamethrin-treated group remained at around 3% [14]. Moreover, the prophylactic treatment with imidacloprid/permethrin also significantly reduced the risk of infection in treated dogs exposed to high infection pressure [19][20][21]. Further studies should evaluate the impact of repeated travel in dogs to assess a cumulative risk for infection.

Conclusion
These first preliminary results on vector-borne infections in travelled dogs present evidence for a low individual risk for infection during a single limited stay abroad. None of the prophylactically treated as well as untreated dogs attained an infection during the average stay of 17 days. Future studies should evaluate the impact on repeated sojourns in a larger cohort of animals.