The role of clinically healthy dogs in the transmission of vector-borne diseases in Spain: a nationwide study

The role of clinically healthy dogs in the transmission of vector-borne diseases in Spain: a nationwide study | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The role of clinically healthy dogs in the transmission of vector-borne diseases in Spain: a nationwide study Ana Montoya-Matute, Rocío Checa, Clara Gómez-Velasco, Rosa Gálvez, and 4 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6545534/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background Canine-vector-borne diseases (CVBDs) are a health risk for both dogs and humans. This study sought to determine the role of clinically healthy dogs in transmitting the three main CVBD (Leishmaniosis due to Leishmania infantum , Ehrlichiosis due to Ehrlichia canis and Dirofilariosis due to Dirofilaria immitis ) in Southern Europe. It were reported in all 50 Spanish provinces. Possible associations between seroprevalences and epidemiological variables were also assessed. Methods 11,886 dogs from 609 veterinary clinics were tested using the URANOvet® diagnostic rapid test to detect antibodies against L. infantum and E. canis , and D. immitis antigen. Data were collected regarding sex, age, habitat, clinical signs compatible with each CVBD and the regular use of ectoparasiticides. Results Infection prevalences were L. infantum 17.3% (1915/11,048), D. immitis 3.2% (314/9,938) and E. canis 3.4% (315/9,125). Clinically healthy dogs accounted for 17.6%, 64.7%, and 35.9% of those positive for L. infantum , D. immitis , and E. canis , respectively. Significant differences in the epidemiological variables examined (p < 0.05) were related to positivity for the three pathogens examined including geographic location, habitat, associated clinical signs and use of ectoparasiticides. While a higher seroprevalence of L. infantum and positivity for D. immitis antigen were recorded in older dogs (p < 0.01), male dogs showed a higher seroprevalence of L. infantum (p < 0.01). Conclusions These data indicate that dogs in Spain are at permanent risk of acquiring all of the three CVBD analysed. We recommend that veterinarians should include these main CVBD in their differential diagnoses and, depending on the geographical region, encourage the use of repellents and other prophylactic measures to prevent their transmission by arthropod vectors. These findings highlight the need for early infection detection by routine screening of clinically healthy dogs, as these could be important subclinical carriers. Leishmania infantum Dirofilaria immitis Ehrlichia canis dog vector borne-diseases Spain Figures Figure 1 Figure 2 Figure 3 Figure 4 Background Canine vector-borne diseases (CVBDs) include a wide variety of infectious diseases whose aetiological agents are transmitted by arthropod vectors such as ticks, fleas, lice and Diptera, including mosquitoes and phlebotomine sandflies. Many of these CVBDs are important not only because they cause disease in pets but because they may be zoonotic. Examples of CVBD zoonotic diseases are anaplasmosis, bartonellosis, borreliosis, dirofilariosis, leishmaniosis and thelaziosis, among others [ 1 , 2 , 3 , 4 ]. The distributions of CVBD are determined by the presence of a competent vector and a natural host reservoir (including wild or domestic animals). In recent years, vector arthropods have been described in regions previously considered free of these CVBD and their vectors, mainly because of climate change and ecological factors [ 5 ]. Further, regulations regarding the management of stray and wild animals, together with the movement of pets, could also be having a major impact on the epidemiological situation of vector-borne diseases [ 6 , 7 , 8 ]. In effect, the expansion of endemic areas has been reported for various parasite diseases such as dirofilariosis, babesiosis and leishmaniosis [ 9 , 10 ]. A good example is babesiosis, which has been recently described across central Europe, emerging from previous endemic regions such as the Mediterranean basin and certain regions of Eastern Europe, and expanding into countries like Germany, Poland, and the Netherlands, most likely due to climate change, increased movement of infected hosts, and changes in tick vector distributions [ 11 , 12 ]. The effective control of CVBDs requires a thorough understanding of the pathogens involved, their vectors and their main hosts so that preventive measures can be designed. The present study focuses on three of the main CVBD affecting dogs at our latitudes: leishmaniosis, dirofilariosis and ehrlichiosis. Canine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan Leishmania infantum and transmitted by phlebotomine sandflies in Southern Europe. Dogs are its main peridomestic host. The distribution of L. infantum infection in dogs in Spain is partially associated with the climate conditions of its different regions, which can be more or less suitable for the survival of its sand fly vector (temperatures not lower than 17–18ºC) [ 13 ]. In Spain, L. infantum seroprevalences range from 2–3.3% in northern regions, and from 34.6 to 57.1% in southern and southeastern regions [ 14 ]. A dog infected with L. infantum can show a wide range of clinical signs, such as apathy, weight loss, enlarged lymph nodes, skin lesions (exfoliative dermatitis, ulcerative dermatitis), ocular disorders and chronic kidney disease, among others [ 15 , 16 ]. Some of these clinical signs are non-specific and resemble those produced by other CVBDs in dogs. Moreover, not all infected dogs will develop the disease and endemic areas may feature high numbers of clinical healthy carriers [ 17 ]. Heartworm disease caused by the nematode Dirofilaria immitis is transmitted by mosquitoes of the genera Aedes, Anopheles and Culex , and is endemic in Spain and other southern European countries. As its vector is not too host specific, many mammals can become infected, including humans, in whom the parasite cannot develop to adulthood and complete its life cycle. The overall prevalence of D. immitis infection reported in dogs living in central Spain (Madrid) is 3% [ 18 ], but higher prevalences (> 11%) have been cited in the hyperendemic areas of Canary Islands, Balearic Islands or Salamanca [ 19 , 20 ]. Similarly, in Greece, prevalence variation depending on the geographical region has been described as 0.7–25% [ 21 ]. In contrast, a low prevalence was found in apparently healthy dogs from Croatia (0.4%) [ 22 ]. While heartworm disease is chronic, high parasite burdens may induce severe acute disease. In endemic areas, many dogs can have subclinical infection for several months, even years. In sick dogs, clinical signs include intermittent coughing, exercise intolerance, dyspnea, tachypnea and weight loss. Ehrlichia canis is the aetiological agent of canine monocytic ehrlichiosis (CME). This gram-negative rickettsia infects monocytes and is transmitted by the tick Rhipicephalus sanguineus complex. Therefore, disease distribution is linked to the presence of this hard tick and its habitat range. In Europe, the seroprevalence rate for E. canis infection in dogs ranges from 0.2% in Hungary [ 23 ] and Russia [ 24 ], to 46% and 50% in shelter dogs from Italy [ 25 ] and Portugal [ 26 ], respectively. In Spain, an overall seropositivity rate of 4–5% has been described [ 27 , 28 , 29 ], but higher seroprevalences have been reported in dogs from northeastern Spain [ 30 ] (16.7%) and in shelter dogs from Orense in North West Spain (54.7%) [ 31 ]. CME shows a severity varying from subclinical infection to chronic disease. Its wide variety of clinical signs include pale mucous membranes, fever, splenomegaly, asthenia, weight loss, diarrhoea, haematuria, epistaxis, anaemia and thrombocytopenia. The aim of this nationwide study was to examine the seroprevalences of Leishmania infantum (Li), and Ehrlichia canis (Ec) and prevalence of Dirofilaria immitis (Di) infection in owned dogs across all Spanish provinces. Possible epidemiological variables related to these prevalences were also assessed. Methods Study area The study was carried out in randomly selected veterinary clinics in all Spanish provinces (n = 50), including mainland Spain and the Balearic and Canary Islands. Spain has a wide range of bioclimatic conditions with nine traditionally defined bioclimatic zones in the Iberian Peninsula and Balearic and Canary Islands [ 32 ]. The alpine and subalpine zones in the Eurosiberian region and the cryoro-mediterranean and oro-mediterranean zones in the Mediterranean region are characterized by high altitudes and harsh climate conditions. In contrast, the meso-mediterranean, thermo-mediterranean and supra-mediterranean zones feature milder temperatures. The mesotemperate and supratemperate zones of the Eurosiberian region provide a transitional climate, while the macaronesian bioclimate of the Canary Islands adds a subtropical component. We here assessed the distributions of the vector diseases examined according to this climate and geographical diversity. Study design The dogs included in this study were patients of 609 veterinary clinics randomly selected across the whole country (50 provinces). From these veterinary practices, 11,886 dogs were selected with the help of the veterinarians responsible for each animal. Inclusion criteria were dogs of any breed, sex and age, healthy dogs and/or dogs showing clinical signs suggestive of CVBD such as pale mucous membranes, apathy, anorexia, fever, lameness and skin lesions, among others. A physical exam form was designed to collect information for each dog regarding: sex, age, breed, habitat, travel history, clinical signs, presence of ectoparasites (e.g. fleas, ticks) and use of ectoparasiticides. The study protocol complied with international regulations for biomedical research on animals. Accordingly, a signed informed consent was required from the owners and responsible veterinarians before the clinical examination and blood collection in each dog. Sample and data collection Each dog was thoroughly examined by the practitioner to determine its health status and eligibility for inclusion in our study. Results of the clinical exam and anamnesis were recorded in the clinical examination form. From each dog, approximately 1–3 ml of blood were collected from the cephalic or jugular vein. Serological tests were performed either on fresh samples or on samples stored at 2–8°C for up to one week. Serological testing Blood sampling and further testing was performed by the veterinarians using rapid immunochromatography tests (URANOvet, Barcelona, Spain) in blood, serum or plasma samples: Uranotest Dirofilaria ® (sensitivity 94.4%; specificity 100%) to detect D. immitis antigens, Uranotest Leishmania ® (sensitivity 96.7%; specificity 98.8%) to detect antibodies against L. infantum ; and Uranotest Ehrlichia ® (sensitivity 95%; specificity 94.6%) to detect antibodies against E. canis . Results were recorded in the clinical examination form. Statistical and spatial analysis All statistical tests were performed using the package IBM SPSS Statistics version 25.0 (IBM, New York, USA). A descriptive analysis was performed using standard statistics for qualitative variables (absolute and relative frequencies) and quantitative variables (mean and standard deviation). Seropositivity rates were compared according to age, sex, habitat, clinical signs compatible with the three CVBDs, presence of ectoparasites and use of ectoparasiticides. The Chi-squared test was used to assess associations between CVBD-agent positivity and each epidemiological variable. Figure 4 was prepared using the Spatial Analyst (SAA) application of ArcGis v.10.7.1 GIS (ESRI, Redlands, CA). Each Spanish province was assigned to the bioclimate covering the largest area within the province. When associations proved significant, crude odds ratios (ORs) were obtained for each variable. Significance was set at p < 0.05. Results Signalment of dogs Out of 11,886 dogs enrolled, 5,635 (47.6%) were female (1,649 neutered and 3,986 intact) and 6,200 (52.2%) were male (1,442 neutered and 4758 intact). Age ranged between 2 months and 18 years (mean age: 5.7 years) with four age groups established: 7 years (30.8%; 3,613/11,719). According to the epidemiological data collected: 43.7% (5,164/11,829) lived indoors, 40.8% (4,853/11,829) outdoors and 15.2% (1,812/11,829) both outdoors and indoors. Additionally, 13.7% (1,605/11,748) had an ectoparasite infestation, of which 45.1% (701/1,554) were infested by ticks, 42.6% (662/1,554) by fleas, and 12.3% (191/1,554) by fleas and ticks. 74.3% (8,423/11, 342) of the dogs had been treated with different anti-ectoparasite drugs with acaricidal, repellent and/or insecticidal effects. Among these treated dogs, 81.9% (6,310/7,702) were protected against ticks, fleas, mosquitoes, and sandflies, 17.1% (1,316/7,702) against ticks and fleas, 0.9% (67/7,702) against fleas and 0.1% (9/7,702) against ticks. Leishmania infantum seropositivity Overall seropositivity for L. infantum was 17.3% (1,915/11,048). Seroprevalence data by province and bioclimatic zones are provided in Figure 1 and Figure 4, respectively. Significant differences were observed among Spanish provinces (p<0.01). The highest seroprevalence rates were detected in the southeastern provinces of Córdoba (58.2%; 53/91), Jaen (43.1%; 44/104) and Albacete (40.6%; 69/170), while lowest seroprevalences were found in the Canary Islands (1.3%; 5/387) and the northern provinces of Cantabria (2.2%; 3/136) and A Coruña (2.4%; 4/167) (Figure 1). According to bioclimatic zones (Figure 4), highest seroprevalences were detected in the mesomediterranean (22.9%; 1,354/5,905) and thermomediterranean bioclimates (21.5%; 190/883), followed by medium seroprevalences in the supramediterranean (11.4%; 319/2,789) and mesotemperate (7.5%; 30/402) bioclimates, while lowest seroprevalences were detected in supratemperate (3%; 17/572) and macaronesian bioclimates (1.3%; 5/387) (Table 4). Among the 11,048 dogs tested, 1,915 were seropositive for L. infantum . Notably, 17.5% (336/1,915) of these seropositive dogs showed no clinical signs, suggesting that a substantial proportion could act as clinically healthy carriers of infection. Additionally, 24.5% had only mild and 31.4% moderate clinical signs ( p <0.001) (Table 3). Although L. infantum infection was more frequent in dogs with associated clinical signs (49.3% of the 3,200 subclinical dogs tested positive), a relevant proportion of apparently healthy dogs (4.2%; 332/7,840) also had antibodies against L. infantum . Some significant associations were detected with the epidemiological variables considered (Table 1 and 2). Accordingly, the seroprevalence of L. infantum infection was significantly lower (p<0.001) in dogs under one year of age (7.3%; 87/1,196), while in the other age groups, prevalences ranged from 16.1% to 21.4%. Significant differences were also detected according to sex (p<0.001), with a higher seroprevalence in males (18.8%) compared to females (15.8%). In addition, intact dogs (19.4%) showed a significantly higher percentage of seropositivity compared to neutered dogs (11.7%) (p<0.001). Environmental and preventive factors also play a key role. Hence, seroprevalence was significantly higher in dogs living outdoors (26.8%) (p<0.001) than indoors (9.6%) or both in- and outdoors (14.6%), the risk of infection by L. infantum being 3.4 times greater in outdoor dogs. Further, the proportion of seropositive dogs that were not applied sandfly repellents (e.g., collar or spot-on ) was significantly higher (21.6%) than those subjected to regular preventive measures (13.8%) (p<0.001; OR: 1.73 (95% CI, 1.56-1.92)). Dirofilaria immitis infection Overall positivity for D. immitis antigen was 3.2% (314/9,951). Prevalence data by province are provided in Figure 2 (p<0.001); highest prevalences were noted in the Andalusian province of Cádiz (24.4%; 31/127) and in the Canary Islands, particularly Tenerife (18.6%; 72/387) and Las Palmas (13.3%; 22/165). In 20 Spanish provinces, no dogs tested positive for the D. immitis antigen. By bioclimatic zone (Figure 4), highest prevalences were detected in the Macaronesian bioclimate (13.8%; 97/702), followed by intermediate seroprevalences in the thermomediterranean bioclimate zone (6.7%; 51/763), and lastly, lower seroprevalences in mesomediterranean (2.5%, 127/5120), mesotemperate (2.3%, 9/400) and supratemperate zones (1.2%, 7/584) (Table 4). Out of the 9,951 dogs tested, 314 were positive for D. immitis antigen. More than half of these dogs testing positive for this pathogen showed no clinical signs (64%; 201/314), once again highlighting the potential role of apparently healthy dogs in the transmission and persistence of this disease. Severe clinical signs were observed in only 9% (28/314) of positive dogs ( p <0.001) (Table 3). Although D. immitis infection was more frequently detected in dogs with compatible clinical signs (32.1% of the 352 sick dogs tested positive), a relevant proportion of apparently healthy dogs (2%; 201/9,951) also tested positive. When we considered epidemiological factors, age was found to be significant (p=0.018), with a lower prevalence observed in dogs under one year of age (1.5%) compared to older age groups (3-3.5%). No significant differences were observed by sex (p=0.831). Intact dogs (3.7%) showed a higher prevalence compared to neutered ones (1.7%) (p<0.001). When analysing D. immitis prevalences by habitat, it emerged that only 1.3% indoor dogs tested positive, while this percentage was higher in dogs living outdoors (5%) or both in- and outdoors (3.4%) (p <0.001); the risk of being infected by D. immitis was four times greater in outdoor dogs (OR: 4.02; CI 95%, 2.98-5.43) (Table 2). Preventive measures against D. immitis were also a determining factor. Dogs without prophylactic measures (microfilaricides macrocyclic lactones and insect repellents) showed a higher prevalence (4.3%), compared to dogs subjected to these preventive measures (2.2%) (p<0.001). The risk of infection was twice as high in dogs without any preventive measures (OR: 2.06; CI 95%, 1.63-2.60) (Table 2). Ehrlichia canis seropositivity Overall seropositivity for E. canis was 3.4% (315/9,163), ranging from 0% to 11.3% depending on the province (Figure 3) (p<0.001). Highest seroprevalences were found in the southwestern provinces of Córdoba (11.3%; 7/62), Huelva (11%; 11/100) and Albacete (10%; 12/120), while no seropositive data were recorded in 16 provinces (Figure 3). In terms of bioclimate zones (Figure 4), intermediate seroprevalences were detected in the meso- (5.2%, 242/4,639) and thermomediterranean bioclimates (4.9%; 35/719), and lower seroprevalences were detected in the mesotemperate (0.6%, 2/352), supramediterranean (0.9%, 20/2,136), supratemperate (1%, 8/796), and macaronesian zones (1.5%, 6/408) (Table 4). Of 9,163 dogs examined, 1,161 presented clinical signs associated with E. canis infection (59.2% mild, 28.2% moderate, 12.6% severe). However, of these, 17.4% (202/1,161) tested seropositive for E. canis in the diagnostic test, while, of the 7,988 apparently healthy dogs, 1.4% (n=113) featured antibodies against E. canis . Conversely, among the 315 seropositive dogs for E. canis , 35.9% (113/315) were apparently healthy dogs (Table 3). No significant differences were found between E. canis seroprevalence and age (p=0.150) or sex (p=0.171); however, a higher seroprevalence was observed in non-neutered dogs (3.7%) compared to neutered dogs (2.7%) (p=0.025). Significant differences were also found between E. canis seroprevalence and habitat (p<0.001), the seroprevalence being higher in dogs living outdoors (4.7%) compared to indoors (2.5%) or with mixed access (2.6%). Overall, however, the risk of infection by E. canis was 1.9 times higher in outdoor dogs (OR: 1.922; CI 95%, 1.496-2.470) (Table 2). The link between the presence or absence of ticks on dogs and E. canis infection was also analyzed revealing a higher seropositivity in dogs infested with ticks (9.7%) compared to those without ticks (p< 0.001). Similarly, the seroprevalence was higher in dogs with no tick prevention measures (4.8%) versus those subjected to these measures (2.7%) (p<0.001). The risk of E. canis infection was 1.8 times higher in dogs without tick prevention (OR: 1.85 [1.47–2.31]) (Table 2). Unfortunately, ticks were not identified in the present study due to economic restrictions. Co-infections A significant association was observed between L. infantum and E. canis infection, in that there were 118 coinfected dogs (p<0.001). However, no significant association was found for L. infantum - D. immitis co-infection (44 dogs; p=0.560), or D. immitis - E. canis coinfection (8 dogs; p=0.394). Additionally, six dogs were coinfected with all three pathogens. Discussion This study provides a comprehensive analysis of the seroprevalence of Leishmania infantum , Dirofilaria immitis , and Ehrlichia canis in a large canine population across Spain, highlighting key epidemiological and preventive factors associated with these CVBD. Our findings confirm their widespread presence and underscore the importance of early diagnostics in healthy dogs, given the high proportion of seropositive animals with subclinical infection. This is the first study to determine the prevalence of vector-borne pathogens in dogs across every Spanish province. Previous studies have involved smaller sample sizes and fewer Spanish provinces [ 27 , 28 , 29 , 30 , 33 ]. Our study offers a broader more comprehensive perspective, examining 11,048 dogs across all 50 Spanish provinces. In a recent study including the 17 autonomous communities of Spain, Montoya-Alonso et al. (2020) [ 29 ] found a high seroprevalence of L. infantum (10.36%) and D. immitis (6.25%) in 4,643 dogs. While these authors highlighted the increasing prevalence of these diseases, particularly in northern regions, by including a large number of veterinary practices (n = 609) our study expands these data, covering every province, to provide a more detailed view of regional variation. The results of these studies are difficult to compare because of differences in the diagnostic techniques used, the size and origin of the samples, and the time intervals between them. The serological test chosen here was the immunochromatography test URANOVet ® , which is a rapid diagnostic method used by many veterinarians [ 16 ]. For the present study, owned dogs from all geographical areas were randomly included as they presented to each practice, regardless of clinical suspicion. However, we cannot exclude the possibility that, in some cases, diagnostic tests were more frequently performed on suspect cases, which could introduce a selection bias. The large sample size and wide distribution of practices across the country will reduce the impact of this potential limitation on its overall results. Overall seropositivity rates were 17.3% for L. infantum followed by 3.5% for E. canis and 3.2% for D. immitis , though these rates varied according to geographical location and sample sizes tested, as noted previously [ 28 , 29 ]. For L. infantum , seropositivity indicates exposure or infection but does not necessarily imply clinical disease, as canine leishmaniosis is a chronic disease, and infected dogs may remain seropositive for life. Similarly, seropositivity for E. canis suggests past or current infection, but not necessarily active disease, as antibodies can persist for months or even years after the infection has resolved [ 34 , 35 ]. For D. immitis the situation is comparable; hence if all adult female worms have been killed, the heartworm antigen should be undetectable by 6 months post-treatment [ 36 , 37 ]. It is also important to clarify that the only certainty of diagnosis were the positive cases of D. immitis , as it is the only test that detects the presence of antigen. In contrast, for L. infantum and E. canis , the presence of antibodies does not necessarily mean the presence of disease as it could indicate only exposure to the vector in dogs living in endemic areas. Leishmania infantum seropositivity Numerous studies have mapped the seroprevalence of canine leishmaniosis in Spain [ 14 ]. The present survey includes some previously non-sampled provinces in northern and central Spanish areas (e.g. Cuenca, Huesca, and Teruel) for a more comprehensive understanding of seroprevalence distributions. Overall, the L. infantum seropositivity rate detected in this study was 17.3% (1,915/11,048), that is, higher than that reported by Montoya-Alonso et al. (2020) in 4,634 dogs (10.4%) and by Miró et al. (2022), who reported a seroprevalence of 10.7% for Leishmania spp. in Spain, based on over 98,000 tests conducted between 2016 and 2020 [ 38 ]. Our data are nevertheless comparable to the 15.7% rate obtained in a similar study including 1,100 dogs [ 28 ]. The higher seroprevalence in our study may reflect regional variations, differences in diagnostic methodologies, or increased exposure in certain areas. In general, there was an appreciable increasing trend in the prevalence of L. infantum infection in some areas so far considered non-endemic [ 16 , 39 , 40 , 41 ]. It has been proposed that environmental changes and global warming are having an impact on the geographical distribution of CanL and its vectors all over Europe [ 42 , 43 , 44 ]. In addition, the presence of the parasite in other animals, such as domestic cats and wild animals like hares, further supports the hypothesis of a role of these hosts in its transmission [ 45 ]. The highest prevalence was observed in southeastern provinces (Córdoba, Jaén, and Albacete) and in the meso- and thermomediterranean bioclimatic zones, in line with previous reports [ 46 , 47 ]. The role of climate change in disease distribution is supported by optimal conditions for Phlebotomus perniciosus , its main vector. Recent studies have confirmed the presence of this sand fly in previously non-endemic areas raising concerns about the potential autochthonous transmission of vector-borne pathogens [ 48 , 49 , 50 ]). CanL is a chronic systemic disease whose clinical manifestations vary from subclinical to severe disease. It should be noted that the individual immune response of the dog and virulence of the L. infantum isolate are important contributing factors [ 51 ]. In this study, 29% of the tested dogs showed clinical signs compatible with CanL. However, among the 1,915 seropositive dogs, 17.5% showed no clinical signs, underlining the significant presence of clinically healthy dogs that could be potential reservoirs of this infection. These results highlight the difficulty in diagnosing CanL mainly in endemic areas where a large number of infected dogs do not develop clinical signs and remain infected over long periods [ 52 ]. This relevance of clinically healthy infected dogs is supported by the results of a recent study by Baxarias et al. (2023) who reported a 5.5% overall seroprevalence in apparently healthy dogs across Spain, with regional variations ranging from 1–14% [ 53 ]. Similarly, Müller et al. (2022) found that 64.6% of seropositive stray dogs in Madrid did not have clinical signs, further emphasizing that clinically healthy carrier dogs play a significant role in the spread and persistence of infection [ 54 ]. Notwithstanding, to adequately control CanL, an annual serological analysis is recommended, along with screening diagnostics before pets travel to or from endemic areas and the proper use of prophylactic measures [ 16 , 51 ]. In effect, the LeishVet guidelines strongly recommend a quantitative serological test and a good check-up (CBC, biochemical profile and urinalysis) to establish antibody levels before treatment [ 15 , 51 , 55 ]. The Europe-wide study by Miró et al. (2022) supports these recommendations, showing that increased screening and preventive measures have contributed to a decline in seropositivity rates for Leishmania spp. across Europe. The findings of these studies underscore the need for continued surveillance and standardized diagnostic procedures to minimize zoonotic risks and ensure effective pet health management [ 38 ]. In the present study, differences were detected in L. infantum seropositivity according to animal age such that rates were significantly higher in middle-aged dogs (3–7 years), in agreement with previous reports [ 54 , 56 ]. However, other authors argue that L. infantum infection in dogs shows a bimodal distribution pattern with two peaks of higher seroprevalences (in dogs under 3 years and older than 8 years). This could be related to an immature immune system in these dogs, making them more vulnerable to infection [ 17 , 57 ]. No significant impact of sex on seroprevalence has been reported [ 14 , 56 ]. However, we observed a higher seropositivity rate in males and non-neutered dogs. This could be explained by the association between gender and owner preferences for different dog uses. For example, males are more often chosen as guard dogs, and these are more likely to be outdoors for longer periods [ 56 ]. Other studies have found a higher prevalence of L. infantum in males, and evidence suggests this could be attributed to a higher mortality of females, in which pregnancy and the nursing period are relevant factors to be considered [ 58 ]. Moreover, oestrus has been associated with a diminished immune response [ 59 , 60 ]. The main risk factor associated with L. infantum infection is the presence of sand flies, which is directly related to geographical area, weather variables, habitat, the presence of other reservoirs and whether preventive measures are used against the vector [ 13 , 14 ]. Here, it was observed that seropositivity was notably higher in dogs living outdoors, the risk of testing positive being 2.86 times higher and related to greater exposure to the vector. This, along with a habitat in an endemic area could increase the likelihood of infection [ 14 , 57 ]. However, other studies have not found differences related to habitat [ 40 , 56 ]. When the use of ectoparasiticides was considered, it emerged that L. infantum seroprevalence was significantly higher in animals not subjected to prophylactic measures, confirming the repellent and/or insecticidal efficacy of pyrethroids against sandflies [ 13 , 51 , 61 ]. Dirofilaria immitis infection Overall seropositivity toward D. immitis in our study was 3.2%, ranging from 0 to 24.4%. This wide prevalence range is determined by climate factors like high temperatures and humidity especially in irrigated crop areas providing moist habitats which are favourable for mosquito breeding [ 62 , 63 ]. Simón et al. (2014) developed a predictive model for D. immitis , identifying high-risk areas across several regions of Spain, including Andalusia, Extremadura, Castilla-La Mancha, Murcia, Valencia, Aragón and Cataluña, as well as the Balearic and Canary islands [ 62 ]. However, our findings suggest a lower prevalence in southeastern regions, such as Murcia and parts of Andalusia, as well as the Canary Islands. This decline may be attributed to the implementation of preventive measures and an awareness of this disease on the part of pet owners in recent years [ 10 , 20 , 29 ]. In contrast, a higher prevalence has been detected in northern regions of our country, such as Soria and Navarra [ 29 , 41 ]. This is likely the consequence of scarce preventive measures, as these regions were considered non-endemic until now. Another explanation could be a land use change towards more irrigated areas providing adequate conditions for the mosquito’s life cycle [ 62 , 63 ]. Heartworm disease is chronic in dogs, and its clinical signs develop gradually. Initially, the disease is often subclinical, but over time, it progresses to present clinical signs such as moderate to severe dyspnoea, weakness, exercise intolerance, and syncope. In our study, a significant proportion of clinically healthy dogs tested positive; specifically, 64% (201/314) of positive dogs showed no clinical signs, highlighting the potential role of clinically healthy dogs in infection transmission. Severe clinical signs were observed in only 9% (28/314) of infected dogs. This suggests that D. immitis infection can pass unnoticed in many dogs, which may act as silent reservoirs of the parasite, contributing to ongoing transmission in endemic areas. In line with our results, Alho et al. (2018) reported in Portugal a prevalence of D. immitis infection of 4% in apparently healthy dogs, and one of 9% in dogs with clinical suspicion of heartworm disease [ 64 ]. These findings emphasize the need for routine diagnostic testing of dogs living in or travelling to endemic areas of dirofilariosis, as clinical signs may not always be apparent. Moreover, infected dogs may not show signs until the disease has reached an advanced stage. In endemic areas, early infection detection through annual testing is crucial to prevent severe complications such as heart failure and pulmonary hypertension [ 65 ]. Given the significant role that clinically healthy dogs play in the persistence and transmission of D. immitis , we would stress the importance of and early diagnosis and prevention as essential to control the spread of this zoonotic disease. We here found that prevalence was significantly associated with age, being lower in dogs younger than one year. This was expected as the estimated prepatent period is 120–180 days [ 66 ]. No significant differences were detected in relation to sex. In contrast, a higher prevalence was observed in non-neutered dogs, as observed with L. infantum seropositivity rates. Additionally, indoor dogs showed a lower infection risk likely due to reduced exposure to the mosquito vector. However, it must be taken into account that other prophylactic measures are necessary, such as the use of nets and repellents since, as confirmed by Montoya-Alonso et al. (2014) in that cats in the Barcelona metropolitan area without outdoor access showed an 11.5% prevalence of D. immitis infection [ 67 ]. Preventive measures (macrocyclic lactones and ectoparasiticides) have proved effective in controlling this infection in endemic areas such as Canary Islands, where its prevalence has been significantly reduced in the past decades from up to 60% to less than 20% [ 10 , 19 , 29 , 63 , 68 ]. Similar data were obtained in our study, in which the prevalence of D. immitis infection was significantly higher in dogs not subjected to prophylactic measures as described previously [ 33 , 69 ]. Ehrlichia canis seropositivity The seroprevalence of E. canis obtained in this survey was slightly lower than those recorded in previous CVBD surveys of dogs in Spain [ 28 , 29 ], although similar to the data obtained in the recent large-scale study by Miró et al. (2022), including 39,526 dogs across Europe, in which the seroprevalence for E. canis in Spain was reported at 3.1% [ 38 ]. The overall distribution pattern, however, was similar and significantly higher in southern and eastern areas of the Iberian Peninsula. This was expected as the prevalence of E. canis infection is greater in areas where the presence of the vector ( R. sanguineus ) is well established [ 70 , 71 ]. This vector’s distribution across Spain influences E. canis seroprevalence. Environmental conditions, such as temperature and humidity, further modulate tick distribution, affecting the epidemiology of canine monocytic ehrlichiosis. In northern regions, competition with Ixodes and Dermacentor species reduces R. sanguineus infestations, whereas in central and southern Spain, R. sanguineus remains the dominant tick species affecting dogs [ 70 , 71 , 72 ]. CME may manifest as three disease stages: i) an acute stage involving different clinical signs that may resolve without treatment, ii) a subclinical stage characterized by the absence of clinical signs, although thrombocytopenia may be observed, and iii) a chronic stage, which may be fatal for sick dogs. The subclinical stage may persist for months or years, making diagnosis difficult [ 34 , 73 , 74 ]. Our findings are in line with these observations in that a significant proportion (35.9%) of seropositive dogs were clinically healthy, reinforcing the importance of routine screening for early detection and management of E. canis infection. Similar trends have been reported in other endemic areas, highlighting the silent role of subclinically infected dogs in maintaining the pathogen in canine populations [ 35 , 75 ]. Regarding epidemiological factors, according to prior results, CME may appear at any age and affect both sexes [ 35 ]. However, some studies have detected a higher seroprevalence in young dogs (under one year) [ 28 ], while others have noted higher seropositivity rates in older animals [ 29 , 33 ]. The latter may be attributed to a higher probability of exposure to E. canis as a dog age rather than to an increase in susceptibility with age [ 35 ]. No significant sex predisposition was detected to E. canis infection, although a higher seropositivity was observed in non-sterilized dogs, attributable to greater exposure to vectors due to behavioural characteristics [ 76 ]. As for the other two vector-borne pathogens, the seroprevalence of E. canis infection was found higher in animals housed outdoors, as they are exposed for a longer period to the vector. In addition, it was noted here that seroprevalence was higher in those animals that had ticks when clinically examined by the veterinarian. Further, an effect was observed of the antiparasite drugs used to control ticks in dogs, as dogs treated with tick repellents and acaricides showed a lower prevalence of infection. In agreement with global studies, E. canis prevalence is higher in dogs not undergoing preventative measures against ticks. In Russia, a study found a low seroprevalence (0.2%), with no positive cases recorded among dogs receiving prophylactic treatment [ 24 ]. Similarly, in another study conducted in Italy, a significantly higher seroprevalence was observed in dogs living in public shelters than private ones, where they were regularly treated against tick infestations [ 25 , 61 ]. These findings highlight the need for consistent tick control strategies to mitigate CME transmission. Co-infections are common in between CVBD due to exposure to competent vectors, a lack of protection against ectoparasites, and a longer exposure time (e.g. more time spent outdoors). The results of the present survey revealed a higher prevalence of clinical CanL and CME as observed previously [ 29 , 77 , 78 ]. Hence, if a CVBD is diagnosed by the veterinarian, the rest of endemic CVBD in the geographic area where the dog is living or has travelled will need to be assessed as well because, as seen in the present study, clinical signs are not always observed in seropositive animals. Conclusions The results of the present study indicate that dogs in Spain are at risk of acquiring any of the three main vector-borne diseases examined: leishmaniosis, dirofilariosis and ehrlichiosis. The implications of this are that veterinarians in different regions should include these CVBDs in their differential diagnoses depending on the specific geographical area. In addition to recommending the use of repellents and other prophylactic measures to reduce vector transmission, early detection through annual routine diagnostic testing is crucial, even in clinically healthy dogs. Identifying subclinical infections enables timely intervention, mitigates disease progression, improves treatment outcome, and ultimately contributes to the broader goal of controlling vector-borne diseases in both animals and humans. Abbreviations CanL canine leishmaniosis CI confidence interval CME canine monocytic ehrlichiosis CVBD canine vector-borne diseases Di Dirofilaria immitis Ec Ehrlichia canis Li Leishmania infantum OR odds ratio Declarations Acknowledgements This publication was sponsored by ELANCO. The authors are indebted to the veterinarians participating in this study. The authors also acknowledge the work of Mireia Campas and the Commercial and Technical Team from ELANCO Spain for selecting the veterinary clinics and helping with the data collection. Funding This paper has been sponsored by Elanco Animal Health in the framework of the CVBD ® World Forum Symposium. Availability of data and material All data generated or analyzed during this study are included in this published article. Authorsʼ contributions GM and AM designed the survey. GM drafted the first version of the manuscript and finalized the manuscript. AM performed the statistical analysis of data, constructed the tables, drafted the first version of the manuscript and finalized the manuscript. RC, CGV, IM, JS and JPB collected the data reviewed and finalized the manuscript. RG prepared the figures and reviewed and finalized the manuscript. GM reviewed and finalized the manuscript. All authors read and approved the final version of the manuscript. 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Pathogens. 2021;10 5; doi: 10.3390/pathogens10050507 . https://www.ncbi.nlm.nih.gov/pubmed/33922459 . Simón L, Afonin A, López-Díez LI, González-Miguel J, Morchón R, Carretón E, et al. Geo-environmental model for the prediction of potential transmission risk of Dirofilaria in an area with dry climate and extensive irrigated crops. The case of Spain. Vet Parasitol. 2014;200 3–4:257 – 64; doi: 10.1016/j.vetpar.2013.12.027 . https://www.ncbi.nlm.nih.gov/pubmed/24456900 . Morchón R, Carretón E, González-Miguel J, Mellado-Hernández I. Heartworm Disease (Dirofilaria immitis) and Their Vectors in Europe - New Distribution Trends. Front Physiol. 2012;3:196; doi: 10.3389/fphys.2012.00196 . https://www.ncbi.nlm.nih.gov/pubmed/22701433 . Alho AM, Meireles J, Schnyder M, Cardoso L, Belo S, Deplazes P, et al. Dirofilaria immitis and Angiostrongylus vasorum: The current situation of two major canine heartworms in Portugal. Vet Parasitol. 2018;252:120–6; doi: 10.1016/j.vetpar.2018.01.008 . https://www.ncbi.nlm.nih.gov/pubmed/29559132 . Society AH: American Heartworm Society Canine Guidelines for the Prevention, Diagnosis, and Management of Heartworm ( Dirofilaria immitis ) Infection in Dogs. 2024. Kotani T, Powers KG. Developmental stages of Dirofilaria immitis in the dog. Am J Vet Res. 1982;43 12:2199–206. https://www.ncbi.nlm.nih.gov/pubmed/7165165 . Montoya-Alonso JA, Carretón E, García-Guasch L, Expósito J, Armario B, Morchón R, et al. First epidemiological report of feline heartworm infection in the Barcelona metropolitan area (Spain). Parasit Vectors. 2014;7:506; doi: 10.1186/s13071-014-0506-6 . https://www.ncbi.nlm.nih.gov/pubmed/25387458 . Montoya-Alonso JA, Carretón E, Corbera JA, Juste MC, Mellado I, Morchón R, et al. Current prevalence of Dirofilaria immitis in dogs, cats and humans from the island of Gran Canaria, Spain. Vet Parasitol. 2011;176 4:291–4; doi: 10.1016/j.vetpar.2011.01.011 . https://www.ncbi.nlm.nih.gov/pubmed/21310532 . Bowman DD, Charles SD, Arther RG, Settje T. Laboratory Evaluation of the Efficacy of 10% Imidacloprid + 2.5% Moxidectin Topical Solution (Advantage® Multi, Advocate®) for the Treatment of Dirofilaria immitis Circulating Microfilariae in Dogs. Parasitol Res. 2015;114 Suppl 1:S165-74; doi: 10.1007/s00436-015-4522-z . https://www.ncbi.nlm.nih.gov/pubmed/26152417 . Estrada-Peña A, Roura X, Sainz A, Miró G, Solano-Gallego L. Species of ticks and carried pathogens in owned dogs in Spain: Results of a one-year national survey. Ticks Tick Borne Dis. 2017;8 4:443–52; doi: 10.1016/j.ttbdis.2017.02.001 . https://www.ncbi.nlm.nih.gov/pubmed/28188085 . Balmori-de la Puente A, Rodríguez-Escolar I, Collado-Cuadrado M, Infante González-Mohino E, Vieira Lista MC, Hernández-Lambraño RE, et al. Transmission risk of vector-borne bacterial diseases (Anaplasma spp. and Ehrlichia canis) in Spain and Portugal. BMC Vet Res. 2024;20 1:526; doi: 10.1186/s12917-024-04383-3 . https://www.ncbi.nlm.nih.gov/pubmed/39593089 . Barandika JF, Olmeda SA, Casado-Nistal MA, Hurtado A, Juste RA, Valcárcel F, et al. Differences in questing tick species distribution between Atlantic and continental climate regions in Spain. J Med Entomol. 2011;48 1:13–9; doi: 10.1603/me10079 . https://www.ncbi.nlm.nih.gov/pubmed/21337943 . Harrus S, Kass PH, Klement E, Waner T. Canine monocytic ehrlichiosis: a retrospective study of 100 cases, and an epidemiological investigation of prognostic indicators for the disease. Vet Rec. 1997;141 14:360–3; doi: 10.1136/vr.141.14.360 . https://www.ncbi.nlm.nih.gov/pubmed/9351183 . Mylonakis ME, Koutinas AF, Breitschwerdt EB, Hegarty BC, Billinis CD, Leontides LS, et al. Chronic canine ehrlichiosis (Ehrlichia canis): a retrospective study of 19 natural cases. J Am Anim Hosp Assoc. 2004;40 3:174–84; doi: 10.5326/0400174 . https://www.ncbi.nlm.nih.gov/pubmed/15131097 . Morelli S, Diakou A, Frangipane di Regalbono A, Colombo M, Simonato G, Di Cesare A, et al. Use of In-Clinic Diagnostic Kits for the Detection of Seropositivity to. Pathogens. 2023;12 5; doi: 10.3390/pathogens12050696 . https://www.ncbi.nlm.nih.gov/pubmed/37242366 . Costa LM, Rembeck K, Ribeiro MF, Beelitz P, Pfister K, Passos LM. Sero-prevalence and risk indicators for canine ehrlichiosis in three rural areas of Brazil. Vet J. 2007;174 3:673–6; doi: 10.1016/j.tvjl.2006.11.002 . https://www.ncbi.nlm.nih.gov/pubmed/17204439 . Attipa C, Solano-Gallego L, Papasouliotis K, Soutter F, Morris D, Helps C, et al. Association between canine leishmaniosis and Ehrlichia canis co-infection: a prospective case-control study. Parasit Vectors. 2018;11 1:184; doi: 10.1186/s13071-018-2717-8 . https://www.ncbi.nlm.nih.gov/pubmed/29554932 . Attipa C, Solano-Gallego L, Leutenegger CM, Papasouliotis K, Soutter F, Balzer J, et al. Associations between clinical canine leishmaniosis and multiple vector-borne co-infections: a case-control serological study. BMC Vet Res. 2019;15 1:331; doi: 10.1186/s12917-019-2083-6 . https://www.ncbi.nlm.nih.gov/pubmed/31533745 . Tables Table 1 to 4 are available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files MontoyaMatute2025Tablesdef.docx Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6545534","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":451435074,"identity":"65fb2988-c7db-44a5-9f97-ca1c0303a069","order_by":0,"name":"Ana Montoya-Matute","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Ana","middleName":"","lastName":"Montoya-Matute","suffix":""},{"id":451435075,"identity":"6c9cc581-670d-452f-95f8-24e5232f9972","order_by":1,"name":"Rocío Checa","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Rocío","middleName":"","lastName":"Checa","suffix":""},{"id":451435077,"identity":"22511881-d73e-4766-8244-698259dab1aa","order_by":2,"name":"Clara Gómez-Velasco","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Clara","middleName":"","lastName":"Gómez-Velasco","suffix":""},{"id":451435082,"identity":"8f4846e2-f5c7-43fb-b0ff-d43bdf012763","order_by":3,"name":"Rosa Gálvez","email":"","orcid":"","institution":"Universidad Autónoma de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Rosa","middleName":"","lastName":"Gálvez","suffix":""},{"id":451435083,"identity":"27a19631-4e53-4b4b-a966-0c76441cf747","order_by":4,"name":"Isabel Mendoza","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Isabel","middleName":"","lastName":"Mendoza","suffix":""},{"id":451435084,"identity":"0a3f04b7-40e3-4790-85f7-ef159c67d562","order_by":5,"name":"Juliana Sarquis","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Juliana","middleName":"","lastName":"Sarquis","suffix":""},{"id":451435085,"identity":"bf37ba20-f484-45fd-b51a-88548f395871","order_by":6,"name":"Juan P. Barrera","email":"","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":false,"prefix":"","firstName":"Juan","middleName":"P.","lastName":"Barrera","suffix":""},{"id":451435087,"identity":"06ce4a15-2b88-4a76-bc28-6e01958a1029","order_by":7,"name":"Guadalupe Miró","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAmElEQVRIiWNgGAWjYFACxgYGhgrStZwh3aI2UlTLhx1u+/Bx3mF53Qbmwx+I0mJ4O7F55sxthw23HWBLkyBOy+zEZmbebYcTzA7wmBHnMIiWOSAt/J+Jc5i8NEhLA9gWBuIcZgDUwjjjWLrhtsNsZsRpkZ+d/pjhQ421vNnx5sfEOczgAIzFTJR6kC0NxKocBaNgFIyCkQsAa4At2znQeOQAAAAASUVORK5CYII=","orcid":"","institution":"Universidad Complutense de Madrid","correspondingAuthor":true,"prefix":"","firstName":"Guadalupe","middleName":"","lastName":"Miró","suffix":""}],"badges":[],"createdAt":"2025-04-28 08:23:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6545534/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6545534/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":82100942,"identity":"a85e0875-4b9a-47da-8fb7-c5bf5ea7c307","added_by":"auto","created_at":"2025-05-06 18:46:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1854357,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eLeishmania infantum\u003c/em\u003e seropositivity results for each Spanish province.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/0eefa2dac3efe21c1aecb6b4.png"},{"id":82100643,"identity":"e99dd153-d80e-485c-a286-ae1350b1dd3b","added_by":"auto","created_at":"2025-05-06 18:38:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":1827066,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eDirofilaria immitis\u003c/em\u003e positivity results for each Spanish province.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/22eb176c126587c3f0add603.png"},{"id":82100648,"identity":"539e6403-811b-4bd5-a12e-c1048f5585fb","added_by":"auto","created_at":"2025-05-06 18:38:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1845698,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cem\u003eEhrlichia canis\u003c/em\u003e seropositivity results for each Spanish province.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/b2384401980e9c47c0e0b065.png"},{"id":82100645,"identity":"1dea4b2a-cfc6-44ba-ae13-0f31fabbe173","added_by":"auto","created_at":"2025-05-06 18:38:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":94877,"visible":true,"origin":"","legend":"\u003cp\u003eCVBD pathogen seropositivity results according to bioclimate zone.\u003c/p\u003e","description":"","filename":"OnlineFigure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/3ecea572f5ca84e46baf188f.png"},{"id":88614989,"identity":"790f4e18-8300-43a0-b0bc-9b99cd8d779f","added_by":"auto","created_at":"2025-08-08 10:32:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4011344,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/06784add-c4e1-4b7a-a3c7-e4d4790b9518.pdf"},{"id":82100642,"identity":"dbf75799-b490-491c-85f0-2e9dcd8217ab","added_by":"auto","created_at":"2025-05-06 18:38:56","extension":"docx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":30113,"visible":true,"origin":"","legend":"","description":"","filename":"MontoyaMatute2025Tablesdef.docx","url":"https://assets-eu.researchsquare.com/files/rs-6545534/v1/6e2faa5ad54a0a0b08132757.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"The role of clinically healthy dogs in the transmission of vector-borne diseases in Spain: a nationwide study","fulltext":[{"header":"Background","content":"\u003cp\u003eCanine vector-borne diseases (CVBDs) include a wide variety of infectious diseases whose aetiological agents are transmitted by arthropod vectors such as ticks, fleas, lice and Diptera, including mosquitoes and phlebotomine sandflies. Many of these CVBDs are important not only because they cause disease in pets but because they may be zoonotic. Examples of CVBD zoonotic diseases are anaplasmosis, bartonellosis, borreliosis, dirofilariosis, leishmaniosis and thelaziosis, among others [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe distributions of CVBD are determined by the presence of a competent vector and a natural host reservoir (including wild or domestic animals). In recent years, vector arthropods have been described in regions previously considered free of these CVBD and their vectors, mainly because of climate change and ecological factors [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. Further, regulations regarding the management of stray and wild animals, together with the movement of pets, could also be having a major impact on the epidemiological situation of vector-borne diseases [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. In effect, the expansion of endemic areas has been reported for various parasite diseases such as dirofilariosis, babesiosis and leishmaniosis [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e, \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. A good example is babesiosis, which has been recently described across central Europe, emerging from previous endemic regions such as the Mediterranean basin and certain regions of Eastern Europe, and expanding into countries like Germany, Poland, and the Netherlands, most likely due to climate change, increased movement of infected hosts, and changes in tick vector distributions [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe effective control of CVBDs requires a thorough understanding of the pathogens involved, their vectors and their main hosts so that preventive measures can be designed. The present study focuses on three of the main CVBD affecting dogs at our latitudes: leishmaniosis, dirofilariosis and ehrlichiosis.\u003c/p\u003e \u003cp\u003eCanine leishmaniosis (CanL) is a zoonotic disease caused by the protozoan \u003cem\u003eLeishmania infantum\u003c/em\u003e and transmitted by phlebotomine sandflies in Southern Europe. Dogs are its main peridomestic host. The distribution of \u003cem\u003eL. infantum\u003c/em\u003e infection in dogs in Spain is partially associated with the climate conditions of its different regions, which can be more or less suitable for the survival of its sand fly vector (temperatures not lower than 17\u0026ndash;18\u0026ordm;C) [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In Spain, \u003cem\u003eL. infantum\u003c/em\u003e seroprevalences range from 2\u0026ndash;3.3% in northern regions, and from 34.6 to 57.1% in southern and southeastern regions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. A dog infected with \u003cem\u003eL. infantum\u003c/em\u003e can show a wide range of clinical signs, such as apathy, weight loss, enlarged lymph nodes, skin lesions (exfoliative dermatitis, ulcerative dermatitis), ocular disorders and chronic kidney disease, among others [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Some of these clinical signs are non-specific and resemble those produced by other CVBDs in dogs. Moreover, not all infected dogs will develop the disease and endemic areas may feature high numbers of clinical healthy carriers [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeartworm disease caused by the nematode \u003cem\u003eDirofilaria immitis\u003c/em\u003e is transmitted by mosquitoes of the genera \u003cem\u003eAedes, Anopheles\u003c/em\u003e and \u003cem\u003eCulex\u003c/em\u003e, and is endemic in Spain and other southern European countries. As its vector is not too host specific, many mammals can become infected, including humans, in whom the parasite cannot develop to adulthood and complete its life cycle. The overall prevalence of \u003cem\u003eD. immitis\u003c/em\u003e infection reported in dogs living in central Spain (Madrid) is 3% [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], but higher prevalences (\u0026gt;\u0026thinsp;11%) have been cited in the hyperendemic areas of Canary Islands, Balearic Islands or Salamanca [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Similarly, in Greece, prevalence variation depending on the geographical region has been described as 0.7\u0026ndash;25% [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e]. In contrast, a low prevalence was found in apparently healthy dogs from Croatia (0.4%) [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. While heartworm disease is chronic, high parasite burdens may induce severe acute disease. In endemic areas, many dogs can have subclinical infection for several months, even years. In sick dogs, clinical signs include intermittent coughing, exercise intolerance, dyspnea, tachypnea and weight loss.\u003c/p\u003e \u003cp\u003e \u003cem\u003eEhrlichia canis\u003c/em\u003e is the aetiological agent of canine monocytic ehrlichiosis (CME). This gram-negative rickettsia infects monocytes and is transmitted by the tick \u003cem\u003eRhipicephalus sanguineus\u003c/em\u003e complex. Therefore, disease distribution is linked to the presence of this hard tick and its habitat range. In Europe, the seroprevalence rate for \u003cem\u003eE. canis\u003c/em\u003e infection in dogs ranges from 0.2% in Hungary [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and Russia [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], to 46% and 50% in shelter dogs from Italy [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e] and Portugal [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], respectively. In Spain, an overall seropositivity rate of 4\u0026ndash;5% has been described [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], but higher seroprevalences have been reported in dogs from northeastern Spain [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e] (16.7%) and in shelter dogs from Orense in North West Spain (54.7%) [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. CME shows a severity varying from subclinical infection to chronic disease. Its wide variety of clinical signs include pale mucous membranes, fever, splenomegaly, asthenia, weight loss, diarrhoea, haematuria, epistaxis, anaemia and thrombocytopenia.\u003c/p\u003e \u003cp\u003eThe aim of this nationwide study was to examine the seroprevalences of \u003cem\u003eLeishmania infantum\u003c/em\u003e (Li), and \u003cem\u003eEhrlichia canis\u003c/em\u003e (Ec) and prevalence of \u003cem\u003eDirofilaria immitis\u003c/em\u003e (Di) infection in owned dogs across all Spanish provinces. Possible epidemiological variables related to these prevalences were also assessed.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy area\u003c/h2\u003e \u003cp\u003eThe study was carried out in randomly selected veterinary clinics in all Spanish provinces (n\u0026thinsp;=\u0026thinsp;50), including mainland Spain and the Balearic and Canary Islands.\u003c/p\u003e \u003cp\u003eSpain has a wide range of bioclimatic conditions with nine traditionally defined bioclimatic zones in the Iberian Peninsula and Balearic and Canary Islands [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. The alpine and subalpine zones in the Eurosiberian region and the cryoro-mediterranean and oro-mediterranean zones in the Mediterranean region are characterized by high altitudes and harsh climate conditions. In contrast, the meso-mediterranean, thermo-mediterranean and supra-mediterranean zones feature milder temperatures. The mesotemperate and supratemperate zones of the Eurosiberian region provide a transitional climate, while the macaronesian bioclimate of the Canary Islands adds a subtropical component.\u003c/p\u003e \u003cp\u003e We here assessed the distributions of the vector diseases examined according to this climate and geographical diversity.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eStudy design\u003c/h3\u003e\n\u003cp\u003eThe dogs included in this study were patients of 609 veterinary clinics randomly selected across the whole country (50 provinces). From these veterinary practices, 11,886 dogs were selected with the help of the veterinarians responsible for each animal. Inclusion criteria were dogs of any breed, sex and age, healthy dogs and/or dogs showing clinical signs suggestive of CVBD such as pale mucous membranes, apathy, anorexia, fever, lameness and skin lesions, among others.\u003c/p\u003e \u003cp\u003eA physical exam form was designed to collect information for each dog regarding: sex, age, breed, habitat, travel history, clinical signs, presence of ectoparasites (e.g. fleas, ticks) and use of ectoparasiticides.\u003c/p\u003e \u003cp\u003eThe study protocol complied with international regulations for biomedical research on animals. Accordingly, a signed informed consent was required from the owners and responsible veterinarians before the clinical examination and blood collection in each dog.\u003c/p\u003e\n\u003ch3\u003eSample and data collection\u003c/h3\u003e\n\u003cp\u003eEach dog was thoroughly examined by the practitioner to determine its health status and eligibility for inclusion in our study. Results of the clinical exam and anamnesis were recorded in the clinical examination form.\u003c/p\u003e \u003cp\u003eFrom each dog, approximately 1\u0026ndash;3 ml of blood were collected from the cephalic or jugular vein. Serological tests were performed either on fresh samples or on samples stored at 2\u0026ndash;8\u0026deg;C for up to one week.\u003c/p\u003e\n\u003ch3\u003eSerological testing\u003c/h3\u003e\n\u003cp\u003eBlood sampling and further testing was performed by the veterinarians using rapid immunochromatography tests (URANOvet, Barcelona, Spain) in blood, serum or plasma samples: Uranotest \u003cem\u003eDirofilaria\u003c/em\u003e\u0026reg; (sensitivity 94.4%; specificity 100%) to detect \u003cem\u003eD. immitis\u003c/em\u003e antigens, Uranotest \u003cem\u003eLeishmania\u003c/em\u003e\u0026reg; (sensitivity 96.7%; specificity 98.8%) to detect antibodies against \u003cem\u003eL. infantum\u003c/em\u003e; and Uranotest \u003cem\u003eEhrlichia\u003c/em\u003e\u0026reg; (sensitivity 95%; specificity 94.6%) to detect antibodies against \u003cem\u003eE. canis\u003c/em\u003e. Results were recorded in the clinical examination form.\u003c/p\u003e\n\u003ch3\u003eStatistical and spatial analysis\u003c/h3\u003e\n\u003cp\u003eAll statistical tests were performed using the package IBM SPSS Statistics version 25.0 (IBM, New York, USA). A descriptive analysis was performed using standard statistics for qualitative variables (absolute and relative frequencies) and quantitative variables (mean and standard deviation). Seropositivity rates were compared according to age, sex, habitat, clinical signs compatible with the three CVBDs, presence of ectoparasites and use of ectoparasiticides. The Chi-squared test was used to assess associations between CVBD-agent positivity and each epidemiological variable. Figure\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e4\u003c/span\u003e was prepared using the Spatial Analyst (SAA) application of ArcGis v.10.7.1 GIS (ESRI, Redlands, CA). Each Spanish province was assigned to the bioclimate covering the largest area within the province. When associations proved significant, crude odds ratios (ORs) were obtained for each variable. Significance was set at \u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.05.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eSignalment of dogs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOut of 11,886 dogs enrolled, 5,635 (47.6%) were female (1,649 neutered and 3,986 intact) and 6,200 (52.2%) were male (1,442 neutered and 4758 intact). Age ranged between 2 months and 18 years (mean age: 5.7 years) with four age groups established: \u0026lt; 1 year (10.9%; 1,283/11,719), 1-3 years (22.8%; 2,673/11,719), 3-7 years (35.4%; 4,150/11,719) and \u0026gt; 7 years (30.8%; 3,613/11,719). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eAccording to the epidemiological data collected: 43.7% (5,164/11,829) lived indoors, 40.8% (4,853/11,829) outdoors and 15.2% (1,812/11,829) both outdoors and indoors. Additionally, 13.7% (1,605/11,748) had an ectoparasite infestation, of which 45.1% (701/1,554) were infested by ticks, 42.6% (662/1,554) by fleas, and 12.3% (191/1,554) by fleas and ticks. 74.3% (8,423/11, 342) of the dogs had been treated with different anti-ectoparasite drugs with acaricidal, repellent and/or insecticidal effects. Among these treated dogs, 81.9% (6,310/7,702) were protected against ticks, fleas, mosquitoes, and sandflies, 17.1% (1,316/7,702) against ticks and fleas, 0.9% (67/7,702) against fleas and 0.1% (9/7,702) against ticks.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eLeishmania infantum seropositivity\u0026nbsp;\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall seropositivity for \u003cem\u003eL. infantum\u003c/em\u003e was 17.3% (1,915/11,048). Seroprevalence data by province and bioclimatic zones are provided in Figure 1 and Figure 4, respectively. \u0026nbsp;Significant differences were observed among Spanish provinces (p\u0026lt;0.01). The highest seroprevalence rates were detected in the southeastern provinces of C\u0026oacute;rdoba (58.2%; 53/91), Jaen (43.1%; 44/104) and Albacete (40.6%; 69/170), while lowest seroprevalences were found in the Canary Islands (1.3%; 5/387) and the northern provinces of Cantabria (2.2%; 3/136) and A Coru\u0026ntilde;a (2.4%; 4/167) (Figure 1). According to bioclimatic zones (Figure 4), highest seroprevalences were detected in the mesomediterranean (22.9%; 1,354/5,905) and thermomediterranean bioclimates (21.5%; 190/883), followed by medium seroprevalences in the supramediterranean (11.4%; 319/2,789) and mesotemperate (7.5%; 30/402) bioclimates, while lowest seroprevalences were detected in supratemperate (3%; 17/572) and macaronesian bioclimates (1.3%; 5/387) (Table 4).\u003c/p\u003e\n\u003cp\u003eAmong the 11,048 dogs tested, 1,915 were seropositive for \u003cem\u003eL. infantum\u003c/em\u003e. Notably, 17.5% (336/1,915) of these seropositive dogs showed no clinical signs, suggesting that a substantial proportion could act as clinically healthy carriers of infection. Additionally, 24.5% had only mild and 31.4% moderate clinical signs (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001) (Table 3). Although \u003cem\u003eL. infantum\u003c/em\u003e infection was more frequent in dogs with associated clinical signs (49.3% of the 3,200 subclinical dogs tested positive), a relevant proportion of apparently healthy dogs (4.2%; 332/7,840) also had antibodies against \u003cem\u003eL. infantum\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSome significant associations were detected with the epidemiological variables considered (Table 1 and 2). \u0026nbsp;Accordingly, the seroprevalence of \u003cem\u003eL. infantum\u003c/em\u003e infection was significantly lower (p\u0026lt;0.001) in dogs under one year of age (7.3%; 87/1,196), while in the other age groups, prevalences ranged from 16.1% to 21.4%. Significant differences were also detected according to sex (p\u0026lt;0.001), with a higher seroprevalence in males (18.8%) compared to females (15.8%). In addition, intact dogs (19.4%) showed a significantly higher percentage of seropositivity compared to neutered dogs (11.7%) (p\u0026lt;0.001).\u003c/p\u003e\n\u003cp\u003eEnvironmental and preventive factors also play a key role. Hence, seroprevalence was significantly higher in dogs living outdoors (26.8%) (p\u0026lt;0.001) than indoors (9.6%) or both in- and outdoors (14.6%), the risk of infection by \u003cem\u003eL. infantum\u003c/em\u003e being 3.4 times greater in outdoor dogs. Further, the proportion of seropositive dogs that were not applied sandfly repellents (e.g., collar or \u003cem\u003espot-on\u003c/em\u003e) was significantly higher (21.6%) than those subjected to regular preventive measures (13.8%) (p\u0026lt;0.001; OR: 1.73 (95% CI, 1.56-1.92)).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eDirofilaria immitis infection\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall positivity for \u003cem\u003eD. immitis\u003c/em\u003e antigen was 3.2% (314/9,951). Prevalence data by province are provided in Figure 2 (p\u0026lt;0.001); highest prevalences were noted in the Andalusian province of C\u0026aacute;diz (24.4%; 31/127) and in the Canary Islands, particularly Tenerife (18.6%; 72/387) and Las Palmas (13.3%; 22/165). In 20 Spanish provinces, no dogs tested positive for the \u003cem\u003eD. immitis\u0026nbsp;\u003c/em\u003eantigen. By bioclimatic zone (Figure 4), highest prevalences were detected in the Macaronesian bioclimate (13.8%; 97/702), followed by intermediate seroprevalences in the thermomediterranean bioclimate zone (6.7%; 51/763), and lastly, lower seroprevalences in mesomediterranean (2.5%, 127/5120), mesotemperate (2.3%, 9/400) and supratemperate zones (1.2%, 7/584) (Table 4).\u003c/p\u003e\n\u003cp\u003eOut of the 9,951 dogs tested, 314 were positive for \u003cem\u003eD. immitis\u0026nbsp;\u003c/em\u003eantigen. More than half of these dogs testing positive for this pathogen showed no clinical signs (64%; 201/314), once again highlighting the potential role of apparently healthy dogs in the transmission and persistence of this disease. Severe clinical signs were observed in only 9% (28/314) of positive dogs (\u003cem\u003ep\u003c/em\u003e \u0026lt;0.001) (Table 3). Although \u003cem\u003eD. immitis\u003c/em\u003e infection was more frequently detected in dogs with compatible clinical signs (32.1% of the 352 sick dogs tested positive), a relevant proportion of apparently healthy dogs (2%; 201/9,951) also tested positive.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen we considered epidemiological factors, age was found to be significant (p=0.018), with a lower prevalence observed in dogs under one year of age (1.5%) compared to older age groups (3-3.5%). No significant differences were observed by sex (p=0.831). Intact dogs (3.7%) showed a higher prevalence compared to neutered ones (1.7%) (p\u0026lt;0.001).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eWhen analysing \u003cem\u003eD. immitis\u003c/em\u003e prevalences by habitat, it emerged that only 1.3% indoor dogs tested positive, while this percentage was higher in dogs living outdoors (5%) or both in- and outdoors (3.4%) (p \u0026lt;0.001); the risk of being infected by \u003cem\u003eD. immitis\u003c/em\u003e was four times greater in outdoor dogs (OR: 4.02; CI 95%, 2.98-5.43) (Table 2). Preventive measures against \u003cem\u003eD. immitis\u0026nbsp;\u003c/em\u003ewere also a determining factor. Dogs without prophylactic measures (microfilaricides macrocyclic lactones and insect repellents) showed a higher prevalence (4.3%), compared to dogs subjected to these preventive measures (2.2%) (p\u0026lt;0.001). The risk of infection was twice as high in dogs without any preventive measures (OR: 2.06; CI 95%, 1.63-2.60) (Table 2).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eEhrlichia canis seropositivity\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOverall seropositivity for \u003cem\u003eE. canis\u003c/em\u003e was 3.4% (315/9,163), ranging from 0% to 11.3% depending on the province (Figure 3) (p\u0026lt;0.001). Highest seroprevalences were found in the southwestern provinces of C\u0026oacute;rdoba (11.3%; 7/62), Huelva (11%; 11/100) and Albacete (10%; 12/120), while no seropositive data were recorded in 16 provinces (Figure 3). In terms of bioclimate zones (Figure 4), intermediate seroprevalences were detected in the meso- (5.2%, 242/4,639) and thermomediterranean bioclimates (4.9%; 35/719), and lower seroprevalences were detected in the mesotemperate (0.6%, 2/352), supramediterranean (0.9%, 20/2,136), supratemperate (1%, 8/796), and macaronesian zones (1.5%, 6/408) (Table 4).\u003c/p\u003e\n\u003cp\u003eOf 9,163 dogs examined, 1,161 presented clinical signs associated with \u003cem\u003eE. canis\u0026nbsp;\u003c/em\u003einfection (59.2% mild, 28.2% moderate, 12.6% severe). However, of these, 17.4% (202/1,161) tested seropositive for \u003cem\u003eE. canis\u003c/em\u003e in the diagnostic test, while, of the 7,988 apparently healthy dogs, 1.4% (n=113) featured antibodies against \u003cem\u003eE. canis\u003c/em\u003e. Conversely, among the 315 seropositive dogs for \u003cem\u003eE. canis\u003c/em\u003e, 35.9% (113/315) were apparently healthy dogs (Table 3).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eNo significant differences were found between\u003cem\u003e\u0026nbsp;E. canis\u003c/em\u003e seroprevalence and age (p=0.150) or sex (p=0.171); however, a higher seroprevalence was observed in non-neutered dogs (3.7%) compared to neutered dogs (2.7%) (p=0.025). Significant differences were also found between \u003cem\u003eE. canis\u003c/em\u003e seroprevalence and habitat (p\u0026lt;0.001), the seroprevalence being higher in dogs living outdoors (4.7%) compared to indoors (2.5%) or with mixed access (2.6%). Overall, however, the risk of infection by \u003cem\u003eE. canis\u003c/em\u003e was 1.9 times higher in outdoor dogs (OR: 1.922; CI 95%, 1.496-2.470) (Table 2).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe link between the presence or absence of ticks on dogs and \u003cem\u003eE. canis\u003c/em\u003e infection was also analyzed revealing a higher seropositivity in dogs infested with ticks (9.7%) compared to those without ticks (p\u0026lt; 0.001). Similarly, the seroprevalence was higher in dogs with no tick prevention measures (4.8%) versus those subjected to these measures (2.7%) (p\u0026lt;0.001). The risk of \u003cem\u003eE. canis\u003c/em\u003e infection was 1.8 times higher in dogs without tick prevention (OR: 1.85 [1.47\u0026ndash;2.31]) (Table 2). Unfortunately, ticks were not identified in the present study due to economic restrictions.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e\u003cem\u003eCo-infections\u003c/em\u003e\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA significant association was observed between \u003cem\u003eL. infantum\u0026nbsp;\u003c/em\u003eand\u003cem\u003e\u0026nbsp;E. canis\u003c/em\u003e infection, in that there were 118 coinfected dogs (p\u0026lt;0.001). However, no significant association was found for \u003cem\u003eL. infantum\u003c/em\u003e-\u003cem\u003eD. immitis\u003c/em\u003e co-infection (44 dogs; p=0.560), or \u003cem\u003eD. immitis\u003c/em\u003e-\u003cem\u003eE. canis\u003c/em\u003e coinfection (8 dogs; p=0.394). Additionally, six dogs were coinfected with all three pathogens.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study provides a comprehensive analysis of the seroprevalence of \u003cem\u003eLeishmania infantum\u003c/em\u003e, \u003cem\u003eDirofilaria immitis\u003c/em\u003e, and \u003cem\u003eEhrlichia canis\u003c/em\u003e in a large canine population across Spain, highlighting key epidemiological and preventive factors associated with these CVBD. Our findings confirm their widespread presence and underscore the importance of early diagnostics in healthy dogs, given the high proportion of seropositive animals with subclinical infection.\u003c/p\u003e \u003cp\u003eThis is the first study to determine the prevalence of vector-borne pathogens in dogs across every Spanish province. Previous studies have involved smaller sample sizes and fewer Spanish provinces [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Our study offers a broader more comprehensive perspective, examining 11,048 dogs across all 50 Spanish provinces. In a recent study including the 17 autonomous communities of Spain, Montoya-Alonso et al. (2020) [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e] found a high seroprevalence of \u003cem\u003eL. infantum\u003c/em\u003e (10.36%) and \u003cem\u003eD. immitis\u003c/em\u003e (6.25%) in 4,643 dogs. While these authors highlighted the increasing prevalence of these diseases, particularly in northern regions, by including a large number of veterinary practices (n\u0026thinsp;=\u0026thinsp;609) our study expands these data, covering every province, to provide a more detailed view of regional variation. The results of these studies are difficult to compare because of differences in the diagnostic techniques used, the size and origin of the samples, and the time intervals between them. The serological test chosen here was the immunochromatography test URANOVet\u003csup\u003e\u0026reg;\u003c/sup\u003e, which is a rapid diagnostic method used by many veterinarians [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. For the present study, owned dogs from all geographical areas were randomly included as they presented to each practice, regardless of clinical suspicion. However, we cannot exclude the possibility that, in some cases, diagnostic tests were more frequently performed on suspect cases, which could introduce a selection bias. The large sample size and wide distribution of practices across the country will reduce the impact of this potential limitation on its overall results.\u003c/p\u003e \u003cp\u003eOverall seropositivity rates were 17.3% for \u003cem\u003eL. infantum\u003c/em\u003e followed by 3.5% for \u003cem\u003eE. canis\u003c/em\u003e and 3.2% for \u003cem\u003eD. immitis\u003c/em\u003e, though these rates varied according to geographical location and sample sizes tested, as noted previously [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. For \u003cem\u003eL. infantum\u003c/em\u003e, seropositivity indicates exposure or infection but does not necessarily imply clinical disease, as canine leishmaniosis is a chronic disease, and infected dogs may remain seropositive for life. Similarly, seropositivity for \u003cem\u003eE. canis\u003c/em\u003e suggests past or current infection, but not necessarily active disease, as antibodies can persist for months or even years after the infection has resolved [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. For \u003cem\u003eD. immitis\u003c/em\u003e the situation is comparable; hence if all adult female worms have been killed, the heartworm antigen should be undetectable by 6 months post-treatment [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. It is also important to clarify that the only certainty of diagnosis were the positive cases of \u003cem\u003eD. immitis\u003c/em\u003e, as it is the only test that detects the presence of antigen. In contrast, for \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eE. canis\u003c/em\u003e, the presence of antibodies does not necessarily mean the presence of disease as it could indicate only exposure to the vector in dogs living in endemic areas.\u003c/p\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003eLeishmania infantum seropositivity\u003c/h2\u003e \u003cp\u003eNumerous studies have mapped the seroprevalence of canine leishmaniosis in Spain [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. The present survey includes some previously non-sampled provinces in northern and central Spanish areas (e.g. Cuenca, Huesca, and Teruel) for a more comprehensive understanding of seroprevalence distributions. Overall, the \u003cem\u003eL. infantum\u003c/em\u003e seropositivity rate detected in this study was 17.3% (1,915/11,048), that is, higher than that reported by Montoya-Alonso et al. (2020) in 4,634 dogs (10.4%) and by Mir\u0026oacute; et al. (2022), who reported a seroprevalence of 10.7% for \u003cem\u003eLeishmania\u003c/em\u003e spp. in Spain, based on over 98,000 tests conducted between 2016 and 2020 [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Our data are nevertheless comparable to the 15.7% rate obtained in a similar study including 1,100 dogs [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The higher seroprevalence in our study may reflect regional variations, differences in diagnostic methodologies, or increased exposure in certain areas. In general, there was an appreciable increasing trend in the prevalence of \u003cem\u003eL. infantum\u003c/em\u003e infection in some areas so far considered non-endemic [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. It has been proposed that environmental changes and global warming are having an impact on the geographical distribution of CanL and its vectors all over Europe [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. In addition, the presence of the parasite in other animals, such as domestic cats and wild animals like hares, further supports the hypothesis of a role of these hosts in its transmission [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. The highest prevalence was observed in southeastern provinces (C\u0026oacute;rdoba, Ja\u0026eacute;n, and Albacete) and in the meso- and thermomediterranean bioclimatic zones, in line with previous reports [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. The role of climate change in disease distribution is supported by optimal conditions for \u003cem\u003ePhlebotomus perniciosus\u003c/em\u003e, its main vector. Recent studies have confirmed the presence of this sand fly in previously non-endemic areas raising concerns about the potential autochthonous transmission of vector-borne pathogens [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]).\u003c/p\u003e \u003cp\u003eCanL is a chronic systemic disease whose clinical manifestations vary from subclinical to severe disease. It should be noted that the individual immune response of the dog and virulence of the \u003cem\u003eL. infantum\u003c/em\u003e isolate are important contributing factors [\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In this study, 29% of the tested dogs showed clinical signs compatible with CanL. However, among the 1,915 seropositive dogs, 17.5% showed no clinical signs, underlining the significant presence of clinically healthy dogs that could be potential reservoirs of this infection. These results highlight the difficulty in diagnosing CanL mainly in endemic areas where a large number of infected dogs do not develop clinical signs and remain infected over long periods [\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis relevance of clinically healthy infected dogs is supported by the results of a recent study by Baxarias et al. (2023) who reported a 5.5% overall seroprevalence in apparently healthy dogs across Spain, with regional variations ranging from 1\u0026ndash;14% [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. Similarly, M\u0026uuml;ller et al. (2022) found that 64.6% of seropositive stray dogs in Madrid did not have clinical signs, further emphasizing that clinically healthy carrier dogs play a significant role in the spread and persistence of infection [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Notwithstanding, to adequately control CanL, an annual serological analysis is recommended, along with screening diagnostics before pets travel to or from endemic areas and the proper use of prophylactic measures [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In effect, the LeishVet guidelines strongly recommend a quantitative serological test and a good check-up (CBC, biochemical profile and urinalysis) to establish antibody levels before treatment [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. The Europe-wide study by Mir\u0026oacute; et al. (2022) supports these recommendations, showing that increased screening and preventive measures have contributed to a decline in seropositivity rates for \u003cem\u003eLeishmania\u003c/em\u003e spp. across Europe. The findings of these studies underscore the need for continued surveillance and standardized diagnostic procedures to minimize zoonotic risks and ensure effective pet health management [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the present study, differences were detected in \u003cem\u003eL. infantum\u003c/em\u003e seropositivity according to animal age such that rates were significantly higher in middle-aged dogs (3\u0026ndash;7 years), in agreement with previous reports [\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, other authors argue that \u003cem\u003eL. infantum\u003c/em\u003e infection in dogs shows a bimodal distribution pattern with two peaks of higher seroprevalences (in dogs under 3 years and older than 8 years). This could be related to an immature immune system in these dogs, making them more vulnerable to infection [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. No significant impact of sex on seroprevalence has been reported [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. However, we observed a higher seropositivity rate in males and non-neutered dogs. This could be explained by the association between gender and owner preferences for different dog uses. For example, males are more often chosen as guard dogs, and these are more likely to be outdoors for longer periods [\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. Other studies have found a higher prevalence of \u003cem\u003eL. infantum\u003c/em\u003e in males, and evidence suggests this could be attributed to a higher mortality of females, in which pregnancy and the nursing period are relevant factors to be considered [\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e]. Moreover, oestrus has been associated with a diminished immune response [\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e, \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe main risk factor associated with \u003cem\u003eL. infantum\u003c/em\u003e infection is the presence of sand flies, which is directly related to geographical area, weather variables, habitat, the presence of other reservoirs and whether preventive measures are used against the vector [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. Here, it was observed that seropositivity was notably higher in dogs living outdoors, the risk of testing positive being 2.86 times higher and related to greater exposure to the vector. This, along with a habitat in an endemic area could increase the likelihood of infection [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. However, other studies have not found differences related to habitat [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e]. When the use of ectoparasiticides was considered, it emerged that \u003cem\u003eL. infantum\u003c/em\u003e seroprevalence was significantly higher in animals not subjected to prophylactic measures, confirming the repellent and/or insecticidal efficacy of pyrethroids against sandflies [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003eDirofilaria immitis infection\u003c/h2\u003e \u003cp\u003eOverall seropositivity toward \u003cem\u003eD. immitis\u003c/em\u003e in our study was 3.2%, ranging from 0 to 24.4%. This wide prevalence range is determined by climate factors like high temperatures and humidity especially in irrigated crop areas providing moist habitats which are favourable for mosquito breeding [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Sim\u0026oacute;n et al. (2014) developed a predictive model for \u003cem\u003eD. immitis\u003c/em\u003e, identifying high-risk areas across several regions of Spain, including Andalusia, Extremadura, Castilla-La Mancha, Murcia, Valencia, Arag\u0026oacute;n and Catalu\u0026ntilde;a, as well as the Balearic and Canary islands [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e]. However, our findings suggest a lower prevalence in southeastern regions, such as Murcia and parts of Andalusia, as well as the Canary Islands. This decline may be attributed to the implementation of preventive measures and an awareness of this disease on the part of pet owners in recent years [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In contrast, a higher prevalence has been detected in northern regions of our country, such as Soria and Navarra [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e]. This is likely the consequence of scarce preventive measures, as these regions were considered non-endemic until now. Another explanation could be a land use change towards more irrigated areas providing adequate conditions for the mosquito\u0026rsquo;s life cycle [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eHeartworm disease is chronic in dogs, and its clinical signs develop gradually. Initially, the disease is often subclinical, but over time, it progresses to present clinical signs such as moderate to severe dyspnoea, weakness, exercise intolerance, and syncope. In our study, a significant proportion of clinically healthy dogs tested positive; specifically, 64% (201/314) of positive dogs showed no clinical signs, highlighting the potential role of clinically healthy dogs in infection transmission. Severe clinical signs were observed in only 9% (28/314) of infected dogs. This suggests that \u003cem\u003eD. immitis\u003c/em\u003e infection can pass unnoticed in many dogs, which may act as silent reservoirs of the parasite, contributing to ongoing transmission in endemic areas.\u003c/p\u003e \u003cp\u003eIn line with our results, Alho et al. (2018) reported in Portugal a prevalence of \u003cem\u003eD. immitis\u003c/em\u003e infection of 4% in apparently healthy dogs, and one of 9% in dogs with clinical suspicion of heartworm disease [\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e]. These findings emphasize the need for routine diagnostic testing of dogs living in or travelling to endemic areas of dirofilariosis, as clinical signs may not always be apparent. Moreover, infected dogs may not show signs until the disease has reached an advanced stage. In endemic areas, early infection detection through annual testing is crucial to prevent severe complications such as heart failure and pulmonary hypertension [\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e]. Given the significant role that clinically healthy dogs play in the persistence and transmission of \u003cem\u003eD. immitis\u003c/em\u003e, we would stress the importance of and early diagnosis and prevention as essential to control the spread of this zoonotic disease.\u003c/p\u003e \u003cp\u003eWe here found that prevalence was significantly associated with age, being lower in dogs younger than one year. This was expected as the estimated prepatent period is 120\u0026ndash;180 days [\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e]. No significant differences were detected in relation to sex. In contrast, a higher prevalence was observed in non-neutered dogs, as observed with \u003cem\u003eL. infantum\u003c/em\u003e seropositivity rates. Additionally, indoor dogs showed a lower infection risk likely due to reduced exposure to the mosquito vector. However, it must be taken into account that other prophylactic measures are necessary, such as the use of nets and repellents since, as confirmed by Montoya-Alonso et al. (2014) in that cats in the Barcelona metropolitan area without outdoor access showed an 11.5% prevalence of \u003cem\u003eD. immitis\u003c/em\u003e infection [\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e]. Preventive measures (macrocyclic lactones and ectoparasiticides) have proved effective in controlling this infection in endemic areas such as Canary Islands, where its prevalence has been significantly reduced in the past decades from up to 60% to less than 20% [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR68\" class=\"CitationRef\"\u003e68\u003c/span\u003e]. Similar data were obtained in our study, in which the prevalence of \u003cem\u003eD. immitis\u003c/em\u003e infection was significantly higher in dogs not subjected to prophylactic measures as described previously [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e, \u003cspan citationid=\"CR69\" class=\"CitationRef\"\u003e69\u003c/span\u003e].\u003c/p\u003e \u003cp\u003e \u003cb\u003eEhrlichia canis\u003c/b\u003e \u003cb\u003eseropositivity\u003c/b\u003e\u003c/p\u003e \u003cp\u003eThe seroprevalence of \u003cem\u003eE. canis\u003c/em\u003e obtained in this survey was slightly lower than those recorded in previous CVBD surveys of dogs in Spain [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], although similar to the data obtained in the recent large-scale study by Mir\u0026oacute; et al. (2022), including 39,526 dogs across Europe, in which the seroprevalence for \u003cem\u003eE. canis\u003c/em\u003e in Spain was reported at 3.1% [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. The overall distribution pattern, however, was similar and significantly higher in southern and eastern areas of the Iberian Peninsula. This was expected as the prevalence of \u003cem\u003eE. canis\u003c/em\u003e infection is greater in areas where the presence of the vector (\u003cem\u003eR. sanguineus\u003c/em\u003e) is well established [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e]. This vector\u0026rsquo;s distribution across Spain influences \u003cem\u003eE. canis\u003c/em\u003e seroprevalence. Environmental conditions, such as temperature and humidity, further modulate tick distribution, affecting the epidemiology of canine monocytic ehrlichiosis. In northern regions, competition with \u003cem\u003eIxodes\u003c/em\u003e and \u003cem\u003eDermacentor\u003c/em\u003e species reduces \u003cem\u003eR. sanguineus\u003c/em\u003e infestations, whereas in central and southern Spain, \u003cem\u003eR. sanguineus\u003c/em\u003e remains the dominant tick species affecting dogs [\u003cspan citationid=\"CR70\" class=\"CitationRef\"\u003e70\u003c/span\u003e, \u003cspan citationid=\"CR71\" class=\"CitationRef\"\u003e71\u003c/span\u003e, \u003cspan citationid=\"CR72\" class=\"CitationRef\"\u003e72\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eCME may manifest as three disease stages: i) an acute stage involving different clinical signs that may resolve without treatment, ii) a subclinical stage characterized by the absence of clinical signs, although thrombocytopenia may be observed, and iii) a chronic stage, which may be fatal for sick dogs. The subclinical stage may persist for months or years, making diagnosis difficult [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR73\" class=\"CitationRef\"\u003e73\u003c/span\u003e, \u003cspan citationid=\"CR74\" class=\"CitationRef\"\u003e74\u003c/span\u003e]. Our findings are in line with these observations in that a significant proportion (35.9%) of seropositive dogs were clinically healthy, reinforcing the importance of routine screening for early detection and management of \u003cem\u003eE. canis\u003c/em\u003e infection. Similar trends have been reported in other endemic areas, highlighting the silent role of subclinically infected dogs in maintaining the pathogen in canine populations [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e, \u003cspan citationid=\"CR75\" class=\"CitationRef\"\u003e75\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eRegarding epidemiological factors, according to prior results, CME may appear at any age and affect both sexes [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. However, some studies have detected a higher seroprevalence in young dogs (under one year) [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], while others have noted higher seropositivity rates in older animals [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. The latter may be attributed to a higher probability of exposure to \u003cem\u003eE. canis\u003c/em\u003e as a dog age rather than to an increase in susceptibility with age [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. No significant sex predisposition was detected to \u003cem\u003eE. canis\u003c/em\u003e infection, although a higher seropositivity was observed in non-sterilized dogs, attributable to greater exposure to vectors due to behavioural characteristics [\u003cspan citationid=\"CR76\" class=\"CitationRef\"\u003e76\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAs for the other two vector-borne pathogens, the seroprevalence of \u003cem\u003eE. canis\u003c/em\u003e infection was found higher in animals housed outdoors, as they are exposed for a longer period to the vector. In addition, it was noted here that seroprevalence was higher in those animals that had ticks when clinically examined by the veterinarian. Further, an effect was observed of the antiparasite drugs used to control ticks in dogs, as dogs treated with tick repellents and acaricides showed a lower prevalence of infection. In agreement with global studies, \u003cem\u003eE. canis\u003c/em\u003e prevalence is higher in dogs not undergoing preventative measures against ticks. In Russia, a study found a low seroprevalence (0.2%), with no positive cases recorded among dogs receiving prophylactic treatment [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. Similarly, in another study conducted in Italy, a significantly higher seroprevalence was observed in dogs living in public shelters than private ones, where they were regularly treated against tick infestations [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. These findings highlight the need for consistent tick control strategies to mitigate CME transmission.\u003c/p\u003e \u003cp\u003eCo-infections are common in between CVBD due to exposure to competent vectors, a lack of protection against ectoparasites, and a longer exposure time (e.g. more time spent outdoors). The results of the present survey revealed a higher prevalence of clinical CanL and CME as observed previously [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR77\" class=\"CitationRef\"\u003e77\u003c/span\u003e, \u003cspan citationid=\"CR78\" class=\"CitationRef\"\u003e78\u003c/span\u003e]. Hence, if a CVBD is diagnosed by the veterinarian, the rest of endemic CVBD in the geographic area where the dog is living or has travelled will need to be assessed as well because, as seen in the present study, clinical signs are not always observed in seropositive animals.\u003c/p\u003e \u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe results of the present study indicate that dogs in Spain are at risk of acquiring any of the three main vector-borne diseases examined: leishmaniosis, dirofilariosis and ehrlichiosis. The implications of this are that veterinarians in different regions should include these CVBDs in their differential diagnoses depending on the specific geographical area. In addition to recommending the use of repellents and other prophylactic measures to reduce vector transmission, early detection through annual routine diagnostic testing is crucial, even in clinically healthy dogs. Identifying subclinical infections enables timely intervention, mitigates disease progression, improves treatment outcome, and ultimately contributes to the broader goal of controlling vector-borne diseases in both animals and humans.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCanL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecanine leishmaniosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003econfidence interval\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecanine monocytic ehrlichiosis\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCVBD\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ecanine vector-borne diseases\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eDirofilaria immitis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eEc\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eEhrlichia canis\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLi\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e \u003cem\u003eLeishmania infantum\u003c/em\u003e \u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eOR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eodds ratio\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis publication was sponsored by ELANCO. The authors are indebted to the veterinarians participating in this study. The authors also acknowledge the work of Mireia Campas and the Commercial and Technical Team from ELANCO Spain for selecting the veterinary clinics and helping with the data collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis paper has been sponsored by Elanco Animal Health in the framework of the CVBD\u003csup\u003e\u0026reg;\u003c/sup\u003e World Forum Symposium.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and material\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data generated or analyzed during this study are included in this published article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthorsʼ contributions\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGM and AM designed the survey. GM drafted the first version of the manuscript and finalized the manuscript. AM performed the statistical analysis of data, constructed the tables, drafted the first version of the manuscript and finalized the manuscript. RC, CGV, IM, JS and JPB collected the data reviewed and finalized the manuscript. RG prepared the figures and reviewed and finalized the manuscript. GM reviewed and finalized the manuscript. All authors read and approved the final version of the manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study was carried out in accordance with the international guidelines for the Care and Use of Experimental Animals and Spanish Legislation (RD 53/2013).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll authors give their consent for publication\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor details\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBeugnet F, Mari\u0026eacute; JL. 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BMC Vet Res. 2019;15 1:331; doi: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1186/s12917-019-2083-6\u003c/span\u003e\u003cspan address=\"10.1186/s12917-019-2083-6\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/pubmed/31533745\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/pubmed/31533745\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003cp\u003eTable 1 to 4 are available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Leishmania infantum, Dirofilaria immitis, Ehrlichia canis, dog, vector borne-diseases, Spain","lastPublishedDoi":"10.21203/rs.3.rs-6545534/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6545534/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eCanine-vector-borne diseases (CVBDs) are a health risk for both dogs and humans. This study sought to determine the role of clinically healthy dogs in transmitting the three main CVBD (Leishmaniosis due to \u003cem\u003eLeishmania infantum\u003c/em\u003e, Ehrlichiosis due to \u003cem\u003eEhrlichia canis\u003c/em\u003e and Dirofilariosis due to \u003cem\u003eDirofilaria immitis\u003c/em\u003e) in Southern Europe. It were reported in all 50 Spanish provinces. Possible associations between seroprevalences and epidemiological variables were also assessed.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003e11,886 dogs from 609 veterinary clinics were tested using the URANOvet\u0026reg; diagnostic rapid test to detect antibodies against \u003cem\u003eL. infantum\u003c/em\u003e and \u003cem\u003eE. canis\u003c/em\u003e, and \u003cem\u003eD. immitis\u003c/em\u003e antigen. Data were collected regarding sex, age, habitat, clinical signs compatible with each CVBD and the regular use of ectoparasiticides.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eInfection prevalences were \u003cem\u003eL. infantum\u003c/em\u003e 17.3% (1915/11,048), \u003cem\u003eD. immitis\u003c/em\u003e 3.2% (314/9,938) and \u003cem\u003eE. canis\u003c/em\u003e 3.4% (315/9,125). Clinically healthy dogs accounted for 17.6%, 64.7%, and 35.9% of those positive for \u003cem\u003eL. infantum\u003c/em\u003e, \u003cem\u003eD. immitis\u003c/em\u003e, and \u003cem\u003eE. canis\u003c/em\u003e, respectively. Significant differences in the epidemiological variables examined (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) were related to positivity for the three pathogens examined including geographic location, habitat, associated clinical signs and use of ectoparasiticides. While a higher seroprevalence of \u003cem\u003eL. infantum\u003c/em\u003e and positivity for \u003cem\u003eD. immitis\u003c/em\u003e antigen were recorded in older dogs (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01), male dogs showed a higher seroprevalence of \u003cem\u003eL. infantum\u003c/em\u003e (p\u0026thinsp;\u0026lt;\u0026thinsp;0.01).\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eThese data indicate that dogs in Spain are at permanent risk of acquiring all of the three CVBD analysed. We recommend that veterinarians should include these main CVBD in their differential diagnoses and, depending on the geographical region, encourage the use of repellents and other prophylactic measures to prevent their transmission by arthropod vectors. These findings highlight the need for early infection detection by routine screening of clinically healthy dogs, as these could be important subclinical carriers.\u003c/p\u003e","manuscriptTitle":"The role of clinically healthy dogs in the transmission of vector-borne diseases in Spain: a nationwide study","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-06 18:38:51","doi":"10.21203/rs.3.rs-6545534/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6e0a1d4-2911-48c7-8cfa-9c3190233367","owner":[],"postedDate":"May 6th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-08-08T10:24:03+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-06 18:38:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6545534","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6545534","identity":"rs-6545534","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}