Prevalence and Geographic Distribution of Vector-Borne Pathogens in Apparently Healthy Dogs in Croatia

Introduction

Vector-borne pathogens (VBPs) are a group of globally distributed and rapidly spreading pathogens that are transmitted by arthropods, including ticks, fleas, mosquitoes, and phlebotomine sand flies (Otranto et al. 2009).

Protozoans Babesia canis, Leishmania infantum, bacteria Ehrlichia canis, Borrelia burgdorferi sensu lato, Anaplasma phagocytophilum, and the nematode Dirofilaria immitis are among the major VBPs that can infect dogs (Day 2011). The vast majority of these pathogens, except B. canis, are also zoonotic, causing disease in humans, thus representing a great veterinary and public health threat (Alho et al. 2016). It appears that the frequency of some vector-borne diseases is increasing in Europe and that pathogens are circulating more easily (Beugnet and Marié 2009). Important drivers for the emergence and spread of vector-borne parasites include atmospheric and climate changes, habitat changes, deforestation and urbanization, globalization and increased trade, travel, aerial transport by migratory birds, and human movement (Zell 2004, Harrus and Baneth 2005).

As a result of global warming and the rapid spread overall of vector-borne diseases observed in the last decade, there is a need of a constant update of data on disease distribution and prevalence (Gray et al. 2009, Mills et al. 2010). Furthermore, the existence of public kennels for stray dogs, regardless of their health conditions, in areas where VBPs are endemic, contributes to the maintenance of the endemicity of many diseases, e.g., canine leishmaniasis (Otranto et al. 2007). In addition, some arthropod species, particularly ticks, act as vectors of more than one agent and coinfection of individual arthropods can occur (Shaw et al. 2001). The overall factors stated above underscore the importance of periodic surveillance of VBPs. There is little information about the prevalence of many VBPs or the comparative seroprevalence of different pathogens in specific geographical areas (Trotz-Williams and Trees 2003).

Among the abovementioned pathogens, canine babesiosis is the most commonly recognized tick-borne disease by clinicians in Croatia (Matijatko et al. 2009, Barić Rafaj et al. 2013) and at the same time is of worldwide importance. The main vectors for babesiosis, Dermacentor spp. and Rhipicephalus spp., ticks have been recorded in Croatia (see Integrated Consortium on Ticks and Tick-borne disease (ICTTD-3), www.icttd.nl). In another survey, the vectors Ixodes ricinus and Dermacentor reticulatus were identified in public gardens in Zagreb, Croatia (Beck et al. 2010). In the central and southern part of the coastal area (Dalmatia region), canine leishmaniasis (CanL) was recognized as a reemerging disease since 1997 (Živičnjak et al. 1998). Some studies indicate that Phlebotomus neglectus, Phlebotomus perfiliewi, and Phlebotomus tobbi, which are proven vectors of the protozoan agent L. infantum, are found as well in the coastal region of Dalmatia (Miščević et al. 1998, Killick-Kendrick 1999).

Surveys carried out in the Zagreb area have revealed the existence of antibodies to B. burgdorferi sensu lato in dogs (Turk et al. 2000). B. burgdorferi sensu lato DNA was detected in 45% of ticks collected in the Lyme borreliosis (LB) endemic region of the northwest part of continental Croatia (Golubić et al. 1998). In another research study, the tick I. ricinus was confirmed in the same area of Croatia (Rijpkema et al. 1996). In a seroepidemiological study conducted in Slovenia from 1999 to 2006, infection with A. phagocytophilum was confirmed by serology in 70.5% (481/682) of dogs considered to have an infection (Ravnik et al. 2009). The survey among 600 adult dogs detected D. immitis only in littoral Croatia, especially the Istrian peninsula and Dubrovnik-Neretva County (Živičnjak et al. 2007). There is no research on E. canis or canine monocytic ehrlichiosis in Croatia.

With this background, the objectives of the present study were to investigate the seroprevalence and geographic distribution of B. canis, A. phagocytophilum, B. burgdorferi sensu lato, L. infantum, D. immitis, and E. canis in canine populations in Croatia. The significance of several risk factors (age, sex, breed, use, lifestyle, and location) was also included in the investigation.

Materials and Methods

Results

Geographical analysis

Statistical analysis of infection with B. canis showed a significant difference with respect to the sampling area (p < 0.001). Seropositivity to B. canis and A. phagocytophilum was higher in the continental part; with foci of B. canis in the coastal part (Poreč, Dubrovnik), the rate for L. infantum antibodies was significantly higher in the Dubrovnik area compared to other cities (r = 0.166, p = 0.001).

D. immitis antigen was present only in the Dubrovnik region, while B. burgdorferi sensu lato was in the continental part (Varaždin region), as well as the coastal part (Rijeka and Dubrovnik) and E. canis was only in Rijeka and the island of Krk. Kernel density analysis indicated the highest density of dogs positive for B. canis to be in the eastern and western part of Croatia (Fig. 1).

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FIG. 1.

Spatial distribution of vector-borne pathogens in dogs in Croatia.

Discussion

This is the most complete and comprehensive survey of VBP seropositivity that has been conducted in Croatia. Our results confirmed that 25.74% (95% CI 21.70–30.12) of dogs were seropositive for one of six VBPs.

The highest seroprevalence against B. canis in our study is in agreement with seroprevalences reported for dogs from other European countries. Thus, the prevalence of B. canis in France was from 14.1% to 20.0%, while in Western Romania it was 19.8% (Mas 1990, Cabannes et al. 2002, Imre et al. 2013). In other European countries the prevalence was 7.3% and 13% in Albania, 5.7% in Hungary, and from 0.8% to 17% in Italy (Traldi et al. 1988, Trotz-Williams and Trees 2003, Hornok et al. 2006, Lazri et al. 2008, Hamel et al. 2009). The highest seroprevalence (average 34%) was obtained by Cassini et al. (2009), with a reduction in the prevalence in central to northern Italy. The authors found in the central regions of Italy the seroprevalence to be 52–57%. The highest recorded seroprevalence is probably overestimated, considering that titers were generally low and cross-reaction is commonly reported for IFAT (Taylor et al. 2007).

Related to our research, it is reasonable to assume that the sensitivity and specificity of serological tests is not 100%, therefore further validation of serological tests used in our study in field conditions is needed to obtain a true prevalence of the investigated diseases in Croatia. In addition, evaluation of population dynamics of ticks responsible for the dispersal of pathogens should be performed in the future. The seroprevalence obtained in the present study indicates the majority of Croatia to be an endemic region. A total of 21 species of hard tick were registered to occur in Croatia (Krčmar 2012). The continental Croatian area is populated mainly by Dermacentor reticulatus and I. ricinus (Dobec et al. 2009, Beck et al. 2010, Krčmar 2012).

Antibodies to B. canis were confirmed in the majority of Croatia to be related to the tick D. reticulatus. An expanding area of D. reticulatus distribution has been observed in central Europe up to the Baltic countries and in the Netherlands, Belgium, Germany, Hungary, and the southwest of England (Matjila et al. 2005, Sreter et al. 2005, Dautel et al. 2006). Probably the region of Croatia is also included due to the expanding area of D. reticulatus distribution. Generally, for tick-borne diseases, the emergence of clinical cases and detection of seropositivity correspond to the distribution and activity of arthropod vectors. Empirically, clinical cases of tick-borne diseases in Croatia occur at the end of winter until the end of spring, and also in autumn due to favorable conditions for Ixodid tick activity, although during mild winters and rainy summers tick-borne diseases are common as well.

The dynamics of spreading of canine babesiosis in Europe has markedly changed in the last decade. However, the prevalence rates may vary considerably depending on the presence and density of potential arthropod vectors and hosts (Hamel et al. 2011). The cause of these changes is probably due to the effect of global warming, shifting use of the landscape, the increase of wild animal populations, spreading of vectors by wild birds and animals, and the change of habitat structure of wildlife (Leschnik et al. 2008). At the same time, the occurrence of B. canis in asymptomatic dogs is very important, because these animals may serve as reservoirs if moved to nonendemic regions (Beck et al. 2009).

Our results confirm that seroprevalence for B. canis is dependent on use/lifestyle, and it was, respectively, significantly higher in hunting/mixed dogs (p < 0.001) and shelter/outdoor dogs (p < 0.006), compared to companion/indoor animals. When it comes to dogs living in shelters, they are often outdoors and, thus, have a higher risk of exposure to ticks. They are also provided with limited healthcare management and are consequently more exposed to the vectors and the agents these vectors transmit (Cassini et al. 2009). At the same time, shelter dogs, as dogs of promiscuous origin, kennelled in an environment with a high animal and a high tick density, are significantly more at risk to become infected with tick pathogens (Pennisi et al. 2012).

The results of other researchers confirm our results on hunting and kennel dogs compared to companion animals (Solano-Gallego et al. 2008, Imre et al. 2013). The survey conducted in Western Romania confirmed that hunting lifestyle is a major risk for acquiring B. canis infection (Imre et al. 2013). Our results from the Mediterranean part, particularly Poreč and the island of Krk, also confirmed this hypothesis. The reason could be that in the survey a large proportion of hunting dogs were included. These dogs are often involved in hunting in the continental part of Croatia and, thus, have greater contact with ticks. The available evidence shows that the Rhipicephalus sanguineus tick is abundant in coastal and insular Croatia (Mikačić 1965, Tovornik and Vesenjak-Hirjan 1988). It is important to note that in the Croatian coastal area the presence of pathogen Hepatozoon canis (Vojta et al. 2009) and its vector R. sanguineus was identified. Therefore, we may assume that the protozoan parasite B. vogeli is also present in this area, since both B. vogeli and H. canis are transmitted through the same vector R. sanguineus.

Our data also showed that significant differences (p < 0.05) were present in the seroprevalence of infected animals with regard to age. Seropositivity was significantly higher in dogs older than 1 year. Similar results were obtained by some investigations (Yamane et al. 1994), while another survey indicated that different age groups did not differ significantly from each other (Imre et al. 2013). The increase in the prevalence of seropositive dogs with age could be related to the cumulative increase of the exposure period to arthropod vectors over the years. Our results also confirmed that the seroprevalence for B. canis was breed dependent (OR = 0.59; 95% CI 0.37–0.94; p < 0.026). Additional analyses have shown that purebred dogs have a 1.78 times greater chance of developing disease than crossbred dogs. The higher prevalence of antibodies to B. canis was present in Posavac hound (11/24) and Alpine Dachsbracke (10/34) purebreds.

Similar results were obtained in other studies as well (Bizzeti et al. 1997, Adaszek et al. 2011). Authors have found that breed was associated with the seropositive reaction to B. canis, but more often in purebred dogs than in crossbred dogs. Some authors have observed that the prevalence of antibodies to B.canis was significantly higher among German Shepherds and Komondors, while in another survey B. gibsoni was typically associated with American Pit Bull Terriers. At the same time, there are surveys that show breed to not be associated with seropositive reactions to B. canis in the dog populations tested (Yamane et al. 1994, Costa-Júnior et al. 2009, Imre et al. 2013).

It is difficult to biologically interpret purebred dogs as risk factors for babesiosis. However, in our research, hunting dogs were mainly purebred dogs and probably more likely to be infected with VBPs. In addition, in our survey dogs from shelters were living mostly outdoors, unlike companion dogs that are living mainly indoors. Adaszek et al. (2011) have considered that despite the statistical significance, babesiosis is not connected with predispositions of particular breeds, but with the dogs' living conditions and the nature of their work (Bourdoiseau 2006).

In our survey, no significant gender predisposition to disease was found, in contrast to another investigation that observed babesiosis more frequently in male animals (Adaszek et al. 2011). There is clear evidence that B. canis prevalence is different among areas (p < 0.001) and Kernel density analysis indicated two areas with the highest density of dogs positive for B. canis in the western part of Croatia around Rijeka and in the eastern part around Đakovo (Fig. 1). Observed differences could be due to vector density and duration of the hunting season, which should be further investigated.

This study provides the first serological evidence for canine exposure to A. phagocytophilum. The seroprevalence of antibodies was 6.21% with a geographical distribution of seropositive cases homogeneous throughout the country. This could be explained by the ubiquitous character of I. ricinus, the main tick vector of A. phagocytophilum in Europe (Strle 2004). In addition, research studies indicate that migrating birds may be important in the dispersal of A. phagocytophilum infected I. ricinus in Europe (de la Fuente et al. 2005, Skoracki et al. 2006). In the investigation conducted in Hungary, circulating antibodies to A. phagocytophilum showed it to be the most prevalent pathogen, after babesiosis, with a positive antibody titer frequency of 7.9% (Farkas et al. 2014). Our study confirmed that antibodies against A. phagocytophilum were not age, breed, sex, use/lifestyle, or location dependent (p > 0.05). A similar result has been reported in Hungary (Farkas et al. 2014).

In Europe, A. phagocytophilum is transmitted by the tick I. ricinus, whereas A. platys could be transmitted by R. sanguineus, the brown dog tick (Dantas-Tores 2008, Chomel 2011). In the continental part of Croatia Varaždin region has confirmed A. phagocytophilum and B. burgdorferi sensu lato. Both are transmitted by ticks of the genus Ixodes (Parola and Raoult 2001).

The available evidence shows that the R. sanguineus tick is abundant in the coastal Croatia. We had identified E. canis and Anaplasma spp. infection along the Croatian coast. Recent research confirmed that the A. phagocytophilum analyte could cross-react with the Anaplasma platys-infected dog sera samples by the SNAP 4Dx ELISA (Bowman et al. 2009, Chandrashekar et al. 2010, Pantchev 2010). We assume that the parasite A. platys could also be present in the region, since both E. canis and A. platys are transmitted by the same tick vector species R. sanguineus. Therefore, more specific diagnostic methods are required to distinguish A. phagocytophilum from A. platys because of serological cross-reactivity. It is important to keep in mind that positive serological results presented in this study might be due to either evidence of prior exposure to the corresponding pathogen at some point and some location in the dog's history or on-going infection. Serological testing detects basically chronic or inconspicuous infections and is limited by a reduced ability to identify acute infections (Menn et al. 2010).

Antibodies against B. burgdorferi sensu lato were detected in three dogs (0.67%), one dog in the continental part (Varaždin region) and two dogs in the coastal part in coinfection with other vector borne diseases (Rijeka and Dubrovnik regions). In Europe, at least six different genospecies that belong to the B. burgdorferi sensu lato complex have been found: Borrelia afzelii, Borrelia garinii, B. burgdorferi sensu stricto, Borrelia valaisiana, Borrelia lusitaniae, and Borrelia spielmanii (Derdakova and Lenčakova 2005, Richter et al. 2006). In another study from Croatia, Rijpkema et al. (1996) identified four genomic groups of B. burgdorferi sensu lato (Borrelia afzelii, Borrelia garinii, group VS116, and B. burgdorferi sensu stricto) in I. ricinus ticks collected in a LB endemic region.

In Croatia, antibodies to B. burgdorferi sensu lato were detected in six (5%) dogs from the Zagreb area (Turk et al. 2000). Our findings suggest that exposure to B. burgdorferi sensu lato exists in dogs in Croatia not only in an endemic region (Varaždin region) but also in Rijeka and Dubrovnik region. Our results may also indicate that LB foci are restricted to small areas, but due to the small number of dogs further studies on Borrelia prevalence are necessary to confirm this hypothesis. Very low seroprevalence, similar to our result, was found in some countries such as Spain (Miró et al. 2013), France (Pantchev et al. 2009), Romania (Mircean et al. 2012), and Hungary (Farkas et al. 2014). The differences in seroprevalence rates could arise from variability in tick densities or the proportion of infected ticks (Kybicova et al. 2009, Farkas et al. 2014). Some authors reported a higher prevalence of LB in older dogs, while others claim the same for younger dogs (<1 year) (Amusategui et al. 2008, Miró et al. 2013, Farkas et al. 2014).

CanL caused by L. infantum is a widespread endemic disease in the Mediterranean basin, and its seroprevalence ranges from 10% up to 53.1% in some foci in Italy (Brandonisio et al. 1992, Fisa et al. 1999, Miró et al. 2012). In the southern parts of Croatia, CanL was recognized as a problem for the first time in the first part of the 20th century (Tartaglia 1937). In our research, seropositivity toward L. infantum was detected in Dubrovnik regions in dogs from shelters (1.38%), while antibodies were not detected in dogs from Split and Zadar regions. For the coastal city of Split and in the hinterland of Split regions, another research has confirmed seroprevalence ranging from 0% to 42.85%, depending on the location (Živičnjak et al. 2005).

In central and southern parts of Dalmatia, pathologic and parasitologic analysis confirmed a case of visceral leishmaniasis (L. infantum) in a gray wolf (Canis lupus) (Beck et al. 2008). Such differences between studies could be attributable to the different population analyzed, the sampling season, arthropod vector distribution and density, the increased exposure to phlebotomines due to spending more time outdoors, as well as the diagnostic technique used (Miró et al. 2013). Generally, leishmaniasis is endemic/enzootic only in the middle and south Dalmatia area, and phlebotomine sandy flies are active during the warmest period of the year. Because of the chronic course of the disease, according to our clinical experience with dogs living in a nonenzootic area, exposure to sand flies happened typically 1, 2, or 3 years before clinical signs appeared.

Entomological surveys carried out in Dalmatia revealed sand fly species of the Phlebotomus genus, among which three belonged to the Larroussius subgenus, being proven to be L. infantum vectors (Phlebotomus tobbi, Phlebotomus Neglectus, and Phlebotomus perfi liewi) (Bosnić et al. 2006). Recent entomological studies evidenced two competent Leishmania vectors, P. neglectus and P. tobbi; the first was prevalent (75.9%), being also the species much more associated with habitats where dogs are present. Moreover, the prevalence of P. neglectus females feeding on human blood reached 30% (Živičnjak et al. 2011). Possibly due to global warming, cases of canine leishmaniasis have been reported in foci outside the Mediterranean countries, for example, autochthonous cases in Hungary and Germany (Mencke 2011, Tanczos et al. 2012).

The seroprevalence of D. immitis was 0.46% with a focal region in the southern part of the coast (Dubrovnik). The results from the coastal part of Croatia, particularly the Istrian Peninsula, may be somewhat unexpected. Namely, we did not find circulating D. immitis antigen in two locations (Poreč, Pula) in the Istrian Peninsula. A possible explanation for this is the small number of dogs in our survey, but also the possibility that the ELISA test can give rise to false negatives in dogs with low heartworm burdens or in blood samples from dogs infected only by male worms (Atkins 2003).

Other studies confirmed the presence of D. immitis in the Istrian Peninsula (Jurić et al. 2007, Holler et al. 2010). Even in the former Yugoslavia, in Croatia between 1987 and 1989 several cases of canine dirofilariasis were reported, but were not considered autochthonous (Brglez and Senk 1987, Genchi at al. 2001). Živičnjak et al. (2007) confirmed the existence and spreading of D. immitis in dogs in the area of Dubrovnik and the Istrian Peninsula. One of the reasons for spreading of dirofilariasis in dogs in Europe is the fact that the mosquito of the Aedes albopictus genus has been spreading aggressively along the coasts of the Istrian peninsula and toward its hinterland (Klobučar et al. 2006, Živičnjak et al. 2006, Holler et al. 2010). Another reason may be the occurrence and spreading of A. albopictus in Italy and implications for its introduction into other parts of Europe (Knudsen et al. 1996).

Canine monocytic ehrlichiosis is a widespread tick-borne infection in the world and is the only Ehrlichia species that has been isolated in dogs from Europe (Keysary et al. 1996, Aguirre et al. 2004). In the present study we found an overall seroprevalence of 0.46%. The low prevalence may be explained by several factors, such as the absence of E. canis, rare exposure of pet dogs to R. sanguineus, or inefficient transmission of E. canis by R. sanguineus in colder parts of Croatia (Gary et al. 2006).

In the areas where VBPs are endemic, coinfection is a frequent event in dogs, especially in the environment in which the vector population density is high. At the same time, Solano-Gallego et al. (2016) suggest that coinfection with Babesia spp. is not well documented and rarely reported in dogs. In our survey, coinfection was found in 12 animals (2.76%), of which 10 were seropositive for two pathogens. The coinfection was mostly with B. canis and A. phagocytophilum (1.61%). In general, coinfections are present in continental and coastal parts of Croatia, particularly in the region of Dubrovnik. Namely, dog populations from coastal areas of the Mediterranean show high infection rates with various VBPs. In addition, our results show that coinfections were more frequently found in shelter dogs, especially in the Dubrovnik region. This is consistent with other studies (Pennisi et al. 2012).

In Hungary, coinfection was found in eight dogs (0.61%), of which seven were seropositive for two pathogens, and one dog was serologically positive for three pathogens (Farkas et al. 2014). In the Mediterranean region, there have been many reports of coinfections with L. infantum and E. canis (Trotz-Williams and Trees 2003), while in our study we had only two coinfections with L. infantum and E. canis.

Conclusions

This is the first comprehensive survey of VBP seropositivity conducted in Croatia. The incidence and geographic distribution indicate that dogs in Croatia are at risk of developing infections with VBPs. Humans are also susceptible to many of these VBPs. The results of this study demonstrate that B. canis is the most prevalent among the studied VBPs. Coinfections are present in the continental and coastal parts of Croatia, which is very important for the clinical management of canine patients. This study will be useful for veterinary and public health authorities with the increased importance of VBPs in Croatia.