DOI: 10.1002/vms3.1172 ORIGINAL ARTICLE Serological survey of Leishmania infantum in apparently healthy dogs in different areas of Spain Marta Baxarias1 Cristina Mateu2 Guadalupe Miró3 Laia Solano-Gallego1 1 Departament de Medicina I Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma Abstract de Barcelona, Bellaterra, Spain Background: Canine leishmaniosis caused by Leishmania infantum is an endemic dis- 2 Ecuphar Veterinaria SLU, Sant Cugat del ease in Spain. The dog is considered the main reservoir, and the detection of specific Vallès, Barcelona, Spain 3 Animal Health Department, Veterinary serum antibodies against L. infantum antigen is the most used technique for diag- Faculty, Universidad Complutense de Madrid, nosing this infection. The LEISCAN LEISHMANIA ELISA test is a commercialized Madrid, Spain enzyme-linked immunosorbent assay for the detection and measurement of canine Correspondence anti-Leishmania serum antibodies. Laia Solano-Gallego, Departament de Objectives: The aim of this study was to assess seroprevalence results of apparently Medicina I Cirurgia Animals, Facultat de Veterinària, Universitat Autònoma de healthy dogs in different areas of Spain using LEISCAN. Barcelona, Bellaterra, Spain. Methods: Collection of sera from 5451 apparently healthy dogs was performed Email: laia.solano@uab.cat between 2020 and 2021 in different areas of Spain. Dogs were of adult age (≥12 months), were not previously diagnosed with clinical leishmaniosis or vaccinated against Leishmania and did not present clinical signs compatible with L. infantum infection. LEISCAN was performed following the manufacturer’s protocol. Results: The overall seroprevalence was 5.5%. The highest seroprevalences were found in the Southeast of Spain: Comunidad Valenciana (14%) and Región de Murcia (14%), whereas the lowest seroprevalences were found in Northern Spain: Galicia (1%), Navarra (2%) and Castilla y León (2%) (p-value <0.001). Conclusions: In conclusion, the seroprevalence for L. infantum in apparently healthy dogs in Spain varied from almost no infection to being over 10%. KEYWORDS canine, diagnosis, ELISA, leishmaniosis, seroprevalence 1 INTRODUCTION digestive tract of the vector sand fly and (b) an intracellular and non- motile amastigote that colonizes the monocyte-macrophage system of Canine leishmaniosis caused by the protozoan Leishmania infantum is the vertebrate hosts (Dostálová & Volf, 2012). The dog is considered a zoonotic and endemic disease in Spain (Díaz-Regañón et al., 2020; the main domestic and peridomestic reservoir for L. infantum infection Gálvez et al., 2020; Montoya-Alonso et al., 2020). Leishmania infantum in Spain (Dantas-Torres, 2007; Solano-Gallego et al., 2011), whereas is usually transmitted by the bite of a female phlebotomine sand fly other mammals, such as wild canids (Oleaga et al., 2018), rodents following a digenetic cycle that alternates between two differentiated (Alcover et al., 2020) and lagomorphs (Molina et al., 2012), may be able phases: (a) an extracellular and motile promastigote that colonizes the to maintain a wild cycle. This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made. © 2023 The Authors. Veterinary Medicine and Science published by John Wiley & Sons Ltd. 1980 wileyonlinelibrary.com/journal/vms3 Vet Med Sci. 2023;9:1980–1988. BAXARIAS ET AL . 1981 Moreover, when a dog is infected, the development of clinical ture), record of vital signs (temperature, pulse, heart and respiratory disease depends on the dog’s immune response. Classically, two polar- rates and capillary refill time) and to check all body from head to tail. ized outcomes have been described: (a) a protective T cell-mediated immune response (the dog is infected but does not develop clinical dis- ease) and (b) a non-protective marked humoral immune response with 2.2 Detection of anti-Leishmania antibodies using a reduced or absent T cell mediated immunity (the dog develops clin- LEISCAN ical leishmaniosis) (Toepp & Petersen, 2020). Thus, a wide spectrum of clinical manifestations is documented in dogs with leishmaniosis LEISCAN (Ecuphar Veterinaria SLU, Spain) was performed to detect (Solano-Gallego et al., 2011). Furthermore, in endemic regions, such anti-L. infantum antibodies in serum following the manufacturer’s pro- as Spain, there is a high prevalence of L. infantum infection in appar- tocol. Briefly, samples were diluted using the dilution solution included ently healthy dogs (Baxarias, Homedes, et al., 2022; Müller et al., 2022), in the kit and incubated for 10 min at room temperature in 96-well although the prevalence of clinical illness is frequently lower than 10% plates. Then, washes were performed five times with the diluted wash- (Solano-Gallego et al., 2009). ing solution, and afterwards, 100 μL of conjugate were added in each Detection of serum specific antibodies against Leishmania is the well. After incubating the plate for another 5 min at room temper- most frequently used technique for detecting infected dogs (Baxarias, ature, washes were repeated, and 100 μL of substrate were added Homedes, et al., 2022; Maurelli et al., 2020; Paltrinieri et al., 2016). Fur- to each well. Finally, after an incubation of 10 min at room tem- thermore, as an anti-Leishmania vaccine is available in Europe, serologi- perature in the dark, stop reaction solution was added to the plate, cal screening is mandatory prior to vaccination in dogs (Solano-Gallego and the results were read at 450 nm in a spectrophotometer (MB- et al., 2017). Several commercial serological techniques are available, 580 HEALES; Shenzhen Huisong Technology Development Co., Ltd, such as immunochromatographic tests, enzyme-linked immunosorbent Shenzhen, China). assays (ELISA) and immunofluorescent antibody tests (IFI) (Baxarias, LEISCAN results were calculated using the following formula: ratio Homedes, et al., 2022; Maurelli et al., 2020; Miró et al., 2017; Paltrinieri sample = OD sample/OD low control positive. Samples were classified et al., 2016; Solano-Gallego et al., 2014). However, the interpretation following the protocol as positive (when the ratio sample was ≥1.1), of these techniques differs as ELISA and IFI allow the quantification dubious (when the ratio sample was ≥0.9 and <1.1) and negative (when of antibody levels, whereas immunochromatographic tests are only the ratio sample was <0.9). Furthermore, when samples were positive, qualitative techniques and, thus, only give a positive or negative result samples were further classified between low positive (when the ratio (Maurelli et al., 2020; Solano-Gallego et al., 2014). Regarding serolog- sample was ≥1.1 and <2.0) and high positive (when the ratio sample ical quantitative techniques, the difference lies in their interpretation was ≥2.0). as ELISA presents objective results depending on optical density (OD), whereas IFI presents subjective results depending on the operator’s experience (Maurelli et al., 2020; Solano-Gallego et al., 2014). 2.3 Statistical analysis The LEISCAN LEISHMANIA ELISA test is an enzyme immunoassay for the detection and measurement of canine serum anti-Leishmania The statistical analysis was performed using the package Stats for antibodies (Rodríguez-Cortés et al., 2013; Solano-Gallego et al., 2014). the software R i386 3.6.1 for Windows, using t test to compare the Previous studies have evaluated LEISCAN in dogs and obtained good altitudes of the centres between the LEISCAN results (positive or diagnostic sensitivity and specificity (Rodríguez-Cortés et al., 2013; negative) and using chi-square tests to compare seroprevalence and Solano-Gallego et al., 2014). antibody levels between autonomous communities, the different areas The aim of this study was to assess seroprevalence results of of Spain (North, South, East and West) and the type of centre that apparently healthy dogs in different areas of Spain using LEISCAN. collected the samples (veterinary practice or dog shelter). A p-value of <0.05 was considered statistically significant. Maps were created using the Free and Open Source QGIS 3.10.4 for Windows. Information 2 MATERIALS AND METHODS about altitudes of the centres were collected from Google Earth Web (https://earth.google.com/web/). 2.1 Dogs Collection of sera from 5451 apparently healthy dogs was performed 3 RESULTS between June of 2020 and June of 2021 by veterinarians from 68 veterinary practices and 12 dog shelters in different areas of Spain The overall seroprevalence and dubious results of the 5451 dogs and (Table 1). The inclusion criteria of dogs enrolled were adult age (≥12 their geographical distribution are shown in Table 1. The highest sero- months), not have been previously diagnosed with clinical leishman- prevalences were found in the Southeast of Spain: Comunidad Valen- iosis nor vaccinated against Leishmania, and the absence of clinical ciana and Región de Murcia, whereas the lowest seroprevalences were signs based on clinical history and a full clinical examination that found in Northern Spain: Galicia, Navarra and Castilla-León (Table 1) includes general appearance (physical body condition, mentation, pos- (Figure 1) (chi-square: χ2 = 88.96, df = 1, p < 0.001). Furthermore, 170 1982 TA B L E 1 Seroprevalence and dubious rates of L. infantum infection in apparently healthy dogs classified by Spanish autonomous community. Number of veterinary Autonomous community Bioclimate (Gálvez practices and dog Mean of the metres of altitude Percentage of Percentage of dubious (number of dogs) et al., 2020) shelters (total) (±SD) seroprevalence (95%CI) results (95%CI) Andalucía (1234)a TM, MM, SM, OM and CM 11 and 2 (13) 468.8 (±287.4) 4.5 (3.4–5.9) 2 (1.3–2.9) Aragón (516) MM, SM, ST and OT 7 and 1 (8) 625.3 (±356) 6.7 (4.7–9.2) 1.6 (0.7–3) Islas Baleares (189) TM and MM 3 and 0 (3) 64.9 (±40.6) 7.4 (4.1–12.1) 3.7 (1.5–7.5) Castilla-La Mancha (18)b MM, SM, OM and CM 2 and 0 (2) 552.2 (±10) 41.2 (18.4–67.1) 5.6 (0.1–27.3) Castilla y León (216) MM, SM, OM and CM 3 and 0 (3) 873.2 (±259.4) 1.9 (0.5–4.8) 2.3 (0.8–5.3) Cataluña (851)a TM, MM, SM, ST and OT 12 and 1 (13) 240.2 (±221.7) 3 (1.9–4.3) 0.8 (0.3–1.7) Comunidad de Madrid (358) MM, SM, OM and CM 6 and 1 (7) 599.4 (±45.9) 5.7 (3.5–8.6) 1.1 (0.3–2.8) Comunidad Valenciana (325) TM, MM, SM, OM and CM 5 and 1 (6) 65.6 (163.9) 13.9 (10.3–18.2) 2.5 (1.1–4.8) Extremadura (472) MM 4 and 0 (4) 298 (±89.8) 3.8 (2.3–6) 0.8 (0.2–2.2) Galicia (235) SM, MT and ST 2 and 1 (3) 38.8 (±16.3) 0.9 (0.1–3.1) 0.4 (0–2.4) Navarra (414) SM, MT and ST 3 and 3 (6) 362 (± 67.9) 2.4 (1.2–4.4) 0.2 (0–1.3) b País Vasco (35) SM, MT and ST 1 and 0 (1) 500 (±0) 5.9 (0.7–19.7) 2.9 (0.1–14.9) Región de Murcia (468) TM, MM, SM, OM and CM 7 and 1 (8) 254.7 (±176.9) 13.7 (10.7–17.3) 3.6 (2.1–5.8) La Rioja (120) SM, MT and ST 2 and 1 (3) 383.3 (±66.7) 2.5 (0.5–7.1) 0 (0–3) Total (5451) 68 and 12 (80) 373.3 (±294) 5.5 (4.9–6.1) 1.6 (1.3–2) Abbreviations: CI; confidence interval; CM, crioro-Mediterranean; MM, meso-Mediterranean; MT, mesotemperate; OM, oro-Mediterranean; OT, orotemperate; SD, standard deviation; SM, supra-Mediterranean; ST, supratemperate; TM, thermo-Mediterranean. a The Spanish autonomous communities from which high numbers of dogs were included were Andalucía and Cataluña. b The significance of results obtained in País Vasco and Castilla-La Mancha remains to be further studied due to the limited number of dogs collected in these regions. BAXARIAS ET AL . BAXARIAS ET AL . 1983 included in the study as it is well known that sick dogs presented higher seroprevalence when compared with apparently healthy dogs (Solano-Gallego et al., 2006). Besides, some regions (País Vasco and Castilla-La Mancha) sampled a limited number of dogs, and thus, the results obtained were not representative of the specific region. Fur- thermore, dubious results were not considered positive results and; therefore, the seroprevalence was lower in all Spanish regions. In addi- tion, it is likely that the differences observed on seroprevalences might also be due to the variable diagnostic performance of previous stud- ies performed (Maurelli et al., 2020; Rodríguez-Cortés et al., 2013; Solano-Gallego et al., 2014). Previous serosurveys performed in dogs from other countries have documented different results (Table 2). In a study performed in North- F I G U R E 1 Geographical distribution of L. infantum seroprevalence in apparently healthy dogs in Spain: (1) Galicia, (2) western Iran (Barati et al., 2015), a higher seroprevalence in asymp- Castilla y León, (3) Comunidad de Madrid, (4) La Rioja, (5) Navarra, (6) tomatic dogs (23%) was observed when compared to previous studies Aragón, (7) Cataluña, (8) Islas Baleares, (9) Comunidad Valenciana, (17%) performed in the dog population in the same area (Moshfe et al., (10) Región de Murcia, (11) Andalucía, (12) Extremadura. N/A, not 2008). On the other hand, in a serosurvey performed in asymptomatic applicable. dogs in Kosovo, an overall seroprevalence of 4% was observed (Xhekaj et al., 2023), which was lower than the seroprevalence detected in both (56.7%) of the seropositive dogs were considered low positive, whereas healthy and sick dogs in previous years (18%) (Xhekaj et al., 2020). In 130 (43.3%) were high positive. No differences in antibody levels were the present study, a similar result to Kosovo (Xhekaj et al., 2020, 2023) observed among dogs from different Spanish regions (p > 0.05). was observed as the overall seroprevalence (5.5%) was lower than the The majority of centres that collected samples were veterinary prac- previous seroprevalences (around 10%) found in the Spanish dog popu- tices (68/80, 85%), whereas only a few were dog shelters (12/80; lation (Díaz-Regañón et al., 2020; Gálvez et al., 2020; Miró et al., 2022; 15%). The geographical distribution of centres is shown in Table 1. Montoya-Alonso et al., 2020). The number of veterinary practices and dog shelters was similar in all In terms of specific investigated regions, previous serological sur- regions studied (p > 0.05). Practitioners collected samples from 4733 veys in Spain have documented similar results, detecting lower sero- apparently healthy dogs (86.8% of the total), whereas dog shelters col- prevalences in the North of Spain and higher in the Southeast (Díaz- lected 718 samples (13.2%). The majority of dog shelter samples were Regañón et al., 2020; Gálvez et al., 2020; Montoya-Alonso et al., 2020) collected in Andalucía (147, 20.5%) and Navarra (137, 19.1%). Further- which is also the nearest region to the Mediterranean. However, the more, there was a higher proportion of dogs sampled in dog shelters results found in Islas Baleares are lower than expected (7.4%) when in Northern Spain (243, 28.1%) when compared to the Southeast of compared to previous studies that found seroprevalences of around Spain (111, 13.7%) (chi-square: χ2 = 52.14, df = 1, p < 0.001). No differ- 20% (Baxarias, Viñals, et al., 2022; Gálvez et al., 2020; Montoya- ences in seroprevalence were detected between dogs from veterinary Alonso et al., 2020; Solano-Gallego et al., 2001, 2006). This could be practices and dogs from shelters (p > 0.05). explained with the same reasons as the lower overall seroprevalence: The mean altitude depending on the geographical distribution of the only sampling apparently healthy dogs, not considering dubious results veterinary practices is also shown in Table 1. The highest altitudes were as positive and the test performed. Furthermore, the samples were col- found in Castilla-León (over 800 m) and Aragón (over 600 m), whereas lected all year around in the present study, whereas in previous studies, the lowest altitudes were found in Galicia (under 40 m), Islas Baleares all the samples were collected in a specific time of the year (Baxarias, (under 70 m) and Comunidad Valenciana (a under 70 m) (Table 1). Viñals, et al., 2022; Solano-Gallego et al., 2001) or, conversely, were No differences in altitude were detected when comparing between collected for various years (Gálvez et al., 2020; Montoya-Alonso et al., seropositive and seronegative dogs (p > 0.05). 2020). These results highlight the need to use preventive measures against L. infantum in any region of Spain (Baxarias, Homedes, et al., 2022; Miró et al., 2017) as well as to perform an annual health check-up 4 DISCUSSION and serology for the detection of anti-Leishmania antibodies in dogs liv- ing in L. infantum-endemic countries (Miró et al., 2017; Solano-Gallego The seroprevalence of L. infantum infection in dogs in Spain has been et al., 2017). previously investigated, and seroprevalences of around 10% between Regarding the antibody levels of the seropositive dogs, surprisingly, 2011 and 2020 have been described (Table 2) (Díaz-Regañón et al., there were similar percentages of low (57%) and high (43%) posi- 2020; Gálvez et al., 2020; Miró et al., 2022; Montoya-Alonso et al., tive dogs. Apparently healthy dogs usually present low anti-Leishmania 2020). The seroprevalence in the present study (5.5%) is lower than antibody levels, whereas dogs with clinical leishmaniosis present high expected. However, the reason could be explained by the inclusion antibody levels (Solano-Gallego et al., 2006, 2017). However, these criteria as only apparently healthy dogs with no clinical signs were percentages could be also affected by not considering dubious results 1984 TA B L E 2 Seroprevalence rates of L. infantum infection in dogs in different countries with similar macroclimates. Macroclimate (European Environment Number Percentage of seroprevalence Country Agency, 2017) of dogs Type of dogs (serological test) (%) References Albania Mediterranean and alpine 602 Dogs with and without clinical signs 5.1 (IFI) Hamel et al. (2016) 308 Dogs with and without clinical signs 3.2 (ELISA) Myrseli et al. (2016) Algeria Mediterranean and desertic 4812 Symptomatic and asymptomatic dogs 21.2 (ELISA, IFI, DAT, rapid test) Touhami et al. (2023) 227 Dogs with and without clinical signs 35.7 (IFI, rapid test) Medkour et al. (2020) Bosnia and Mediterranean, continental and alpine 180 No data 16.7 (IFI) Colella et al. (2019) Herzegovina 172 No data 11 (ELISA) Miró et al. (2022) Croatia Mediterranean and alpine 200 Asymptomatic dogs 8 (IFI) Zivicnjak et al. (2007) 1761 No data 7 (ELISA) Miró et al. (2022) 435 Apparently healthy dogs 1.4 (rapid test) Mrljak et al. (2017) Cyprus Mediterranean 278 Symptomatic and asymptomatic dogs 1.1 (IFI) Beyhan et al. (2016) 281 Symptomatic and asymptomatic dogs 3.6 (IFI) Çanakçı et al. (2016) Egypt Mediterranean and desertic 450 Asymptomatic dogs 21.3 (ELISA) Selim et al. (2021) France Mediterranean, Atlantic, continental 80 Symptomatic and asymptomatic dogs 6.2 (IFI) Davoust et al. (2013) and alpine 5307 No data 8.7 (ELISA) Miró et al. (2022) Greece Mediterranean 2620 Asymptomatic dogs 20 (ELISA, IFI) Athanasiou et al. (2012) 9568 No data 18.5 (ELISA) Miró et al. (2022) Iran Alpine, anatolian and steppic 508 Asymptomatic dogs 23.4 (DAT) Barati et al. (2015) 384 Symptomatic and asymptomatic dogs 17.4 (DAT) Moshfe et al. (2008) Israel Mediterranean and desertic 60 Dogs with and without clinical signs 37 (ELISA) Baneth et al. (2020) Italy Mediterranean and continental 90,532 No data 11.9 (ELISA) Miró et al. (2022) 13,292 Dogs with and without clinical signs 6.7 (IFI) Rombolà et al. (2021) Kosovo Mediterranean, continental and alpine 125 Dogs with and without clinical signs 18.4 (ELISA) Xhekaj et al. (2020) 285 Asymptomatic dogs 4.2 (ELISA) Xhekaj et al. (2023) Malta Mediterranean 289 No data 15.9 (ELISA) Miró et al. (2022) Montenegro Mediterranean and alpine 1500 Dogs with suspected leishmaniosis 83 (IFI) Andric et al. (2013) Morocco Mediterranean and desertic 1514 No data 15.8 (rapid test) El Berbri et al. (2020) 3900 Dogs with and without clinical signs 14.9 (ELISA, IFI, rapid test El-Mouhdi et al. (2022) Portugal Mediterranean and Atlantic 1860 Dogs with and without clinical signs 12.5 (DAT) Almeida et al. (2022) 1329 No data 13.8 (ELISA) Miró et al. (2022) (Continues) BAXARIAS ET AL . BAXARIAS ET AL . 1985 as positive dogs and the test performed. Other tests with higher sensi- Montoya-Alonso et al. (2020) Martín-Sánchez et al. (2009) Solano-Gallego et al. (2006) tivities (Solano-Gallego et al., 2014) could detect a proportion of these Díaz-Regañón et al. (2020) Dumitrache et al. (2016) dubious results as very low seropositive dogs increasing the number of Bouattour et al. (2021) Gálvez et al. (2010) Gálvez et al. (2020) Kotnik et al. (2021) low positive dogs of the study. Ozbel et al. (2000) Miró et al. (2013) Miró et al. (2022) Interestingly, it has been described that owned dogs usually have References a lower risk of infection than dogs living in dog shelters or kennels (Rombolà et al., 2021), which could be associated to environmental fac- tors such as living outdoors, although it has also been described the opposite, being dogs living in kennels less likely to present L. infantum infection (Tamponi et al., 2021). In the present study, no differences in Percentage of seroprevalence seroprevalence were detected between owned dogs from veterinary practices and dogs from shelters. However, a higher percentage of sam- ples from dogs living in dog shelters was observed in Northern Spain, (serological test) (%) which was also the region with the lowest seroprevalences and could 15.7 (rapid test) 10.4 (rapid test) be affecting the overall seroprevalence of dog shelters. Another impor- 5.3 (IFI, DAT) 9 (rapid test) 10.7 (ELISA) 3.7 (ELISA) 1.9 (ELISA) 30 (ELISA) 58.3 (IFI) tant factor could be that owned dogs are more frequently tested in the 10.1 (IFI) 8.1 (IFI) 13 (IFI) clinical setting (for clinical suspicion or annual health check-up) than dogs living in dog shelters (usually only sampled at kennel admittance) (Rombolà et al., 2021). In endemic areas, it is appropriate to screen dogs for L. infantum antibodies at least every 6–12 months (Solano-Gallego Abbreviations: DAT, direct agglutination test; ELISA, enzyme-linked immunosorbent assay; IFI, indirect immunofluorescence assay. et al., 2009, 2017). suggestive of vector-borne diseases Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Dogs with and without clinical signs Regarding the vector importance over seroprevalences, the distri- bution and density of sand flies in a region affect directly the transmis- sion of L. infantum and the rate of infected dogs, and thus, changes in the vector distribution can determine changes in the seroprevalence Asymptomatic dogs Asymptomatic dogs of a region (Ballart et al., 2014; Díaz-Sáez et al., 2021). For example, in recent studies, phlebotomine sand flies have been described to be Type of dogs able to maintain L. infantum infection in regions above 1300 m (Díaz- No data No data Sáez et al., 2021), which are higher altitudes than previously reported and, therefore, could be able to infect animal populations that were not previously affected by L. infantum (Alten et al., 2016; Hartemink et al., 2011). However, in the present study, all sampled areas were under 1000 m of altitude, and no differences were detected among Number of dogs 317 80 465 556 1076 1739 439 1100 98,737 4643 466 490 different altitudes. Another factor that could also impact vector den- sity and species is the bioclimate of the region and its climate changes (Ballart et al., 2014; Semenza & Suk, 2018). Spain presents several bioclimates such as supratemperate and mesotemperate in the North Mediterranean, black sea and anatolian Macroclimate (European Environment Mediterranean, continental and alpine and meso-Mediterranean and thermo-Mediterranean in the South- east (Ballart et al., 2014; Gálvez et al., 2020). For example, Ballart et al. (2014) reported that Phlebotomus perniciosus is mainly present in Mediterranean and desertic Mediterranean and Atlantic meso-Mediterranean and supra-Mediterranean bioclimates, whereas Phlebotomus ariasi preferred coline, subalpine and montane biocli- mates. In the present study, higher seroprevalences were detected in Agency, 2017) the Southeast of Spain (meso-Mediterranean bioclimate) and could be related to higher densities of sand flies in this region as previously reported (Ballart et al., 2014). (Continued) Dog populations of countries that have a Mediterranean macrocli- mate in some regions present a similar seropositivity to L. infantum with a range from 4% to 20% (Table 2). For example, Spain, Por- tugal, France and Italy have similar seroprevalence rates of around TA B L E 2 Country Slovenia 10%. Interestingly, Greece, which is the country that only presents Tunisia Turkey Spain a Mediterranean macroclimate, reports higher seroprevalence rates of 20% (Table 2). 1986 BAXARIAS ET AL . This study presents some limitations. First, there were some Span- Saenz Clínica Veterinaria (Aragón, Spain), Faunivet Clínica Veterinaria ish regions with a limited number of sampled dogs which could not be (Comunidad Valenciana, Spain), Francisco J Ramos Clínica Veteri- truly representative of that region. Second, there was access to limited naria (Extremadura, Spain), Fundación Benjamin Menhert (Andalucía, information about the sampled dogs, and therefore, statistical analysis Spain), Galápago Clínica Veterinaria (Andalucía, Spain), Germanias of dog characteristics such as age or breed was not possible to perform. Clínica Veterinaria (Comunidad Valenciana, Spain), Hospital Clínico Finally, LEISCAN protocol classified some samples as dubious and rec- Veterinario de la Facultad de Veterinaria de la Universidad Com- ommended to repeat the test in 6 months which was not possible in this plutense de Madrid (UCM) (Comunidad de Madrid, Spain), Huellas study. Even when repeating the test with the same sample, the major- Clínica Veterinaria (Galicia, Spain), Infantes Clínica Veterinaria (Castilla ity of samples presented a second dubious result which could not be y León, Spain), Jaca Clínica Veterinaria (Aragón, Spain), Jara Clínica further classified as positive and negative. Veterinaria (Andalucía, Spain), La Casa Morada Clínica Veterinaria In conclusion, the seroprevalence for L. infantum as detected by the (Andalucía, Spain), La Corraliza Clínica Veterinaria (Navarra, Spain), ELISA technique used in apparently healthy dogs in Spain varied from La Reserva Protectora (Andalucía, Spain), Las Cigueñas Clínica Veteri- almost no infection in the Northern areas of Spain to being over 10% in naria (La Rioja, Spain), Lazareto (Navarra, Spain), Los Dolores Clínica the Southeast close to the Mediterranean basin. These results highlight Veterinaria (Región de Murcia, Spain), Malaka Clínica Veterinaria the need to use preventive measures against L. infantum in any region (Andalucía, Spain), Maresme Hospital Veterinari (Cataluña, Spain), of Spain and to perform an annual check-up that includes a quantitative Mataró Serveis Veterinaris (Cataluña, Spain), Miman’s Hospital Veteri- serological test. nari (Cataluña, Spain), Mimascota Clínica Veterinaria (Castilla y León, Spain), Mirovet Clínica Veterinaria (Castilla-La Mancha, Spain), Mister- AUTHOR CONTRIBUTIONS can Clínica Veterinaria (Andalucía, Spain), Mivet Hospital Veterinari Conceptualization; data curation; formal analysis; investigation; methodol- Manacor (Islas Baleares, Spain), Modepran Protectora (Comunidad ogy; project administration; writing – original draft; writing – review and Valenciana, Spain), Montaña del Sol Clínica Veterinaria (Andalucía, editing: Marta Baxarias. Funding acquisition; methodology; project admin- Spain), Monte Aloia Protectora (Galicia, Spain), Myvet Clínica Vet- istration; resources; supervision: Cristina Mateu. Investigation; resources; erinaria (Aragón, Spain), Navalvet Clínica Veterinaria (Comunidad de writing – review and editing: Guadalupe Miró. Conceptualization; fund- Madrid, Spain), Navia Clínica Veterinaria (Galicia, Spain), Nutrizoo ing acquisition; project administration; resources; supervision; validation; Clínica Veterinaria (Región de Murcia, Spain), Otto Can Coria Clínica writing – review and editing: Laia Solano Gallego. Veterinaria (Extremadura, Spain), Otto Can Moraleja Clínica Veteri- naria (Extremadura, Spain), Pacífico Clínica Veterinaria (Comunidad ACKNOWLEDGEMENTS de Madrid, Spain), Pawspatas Clínica Veterinaria (Región de Murcia, This work was founded by Ecuphar Veterinaria SLU. The authors would Spain), Perrikus (Comunidad de Madrid, Spain), Pratdesaba Veterinaris like to thank the technicians of Ecuphar Veterinaria SLU that contacted Malla (Cataluña, Spain), Provença Clínica Veterinaria (Cataluña, Spain), all the shelters and the veterinary practices that participated in the Quipar Clínica Veterinaria (Región de Murcia, Spain), Rio Veral Clínica study: Albaitari Clínica Veterinaria (País Vasco, Spain), Albayda Centro Veterinaria (Aragón, Spain), Safracan Clínica Veterinaria (Región de Veterinario (Andalucía, Spain), Alta Paterna Clínica Veterinaria (Comu- Murcia, Spain), San Bernardo Clínica Veterinaria (Comunidad Valen- nidad Valenciana, Spain), Rioja Centro Veterinario (La Rioja, Spain), Ani- ciana, Spain), San Francisco Hospital Veterinari (Andalucía, Spain), malex Clínica Veterinaria (Andalucía, Spain), Animalia Servet (Castilla y Santa Perpètua Clínica Veterinaria (Cataluña, Spain), Sevilla Clínica León, Spain), Animals! Serveis Veterinaris (Islas Baleares, Spain), Belén Veterinaria (Andalucía, Spain), Teruel Clínica Veterinaria (Aragón, Carasa Clínica Veterinaria (Navarra, Spain), Bestioles Centre Veterinari Spain), Urgell Hospital Veterinari (Cataluña, Spain), Utrizoo Clínica (Cataluña, Spain), Bichos Clínica Veterinaria (Comunidad de Madrid, Veterinaria (Aragón, Spain), Veteralia Movet (Cataluña, Spain), Vic- Spain), Bocairent Clínica Veterinaria (Comunidad Valenciana, Spain), tor Serena Clínica Veterinaria (Región de Murcia, Spain), Viladecans Buztintxuri Clínica Veterinaria (Navarra, Spain), Caballero Clínica Vet- Clínica Veterinaria (Cataluña, Spain), Vilazoo Clínica Veterinaria (Islas erinaria (Cataluña, Spain), Caminando con ellos Protectora (Región Baleares, Spain) and Zeus Clínica Veterinaria (Región de Murcia, Spain). de Murcia, Spain), Centre d’Atenció d’Animals Domèstics (CAAD) del Maresme (Cataluña, Spain), Centro Comarcal de Protección Animal CONFLICT OF INTEREST STATEMENT Ribera Baja Navarra (Navarra, Spain), Centro de Acogida de Animales CM has financial competing interests as they receive a salary from (CAA) de Logroño (La Rioja, Spain), Centro de Protección Animal Ecuphar Veterinaria SLU which markets and LEISCAN. (CPA) de Zaragoza (Aragón, Spain), Centro de Protección Animal (CPA) del Gobierno de Navarra (Navarra, Spain), Ciempozuelos Clínica Vet- FUNDING INFORMATION erinaria (Comunidad de Madrid, Spain), Clinivex Clínica Veterinaria Ecuphar veterinaria SLU (Extremadura, Spain), CVR Clínica Veterinaria (Aragón, Spain), Del Pla Clínica Veterinaria (Cataluña, Spain), Desvern Clínica Veterinaria DATA AVAILABILITY STATEMENT (Cataluña, Spain), Dingo Clínica Veterinaria (Región de Murcia, Spain), The data is available from the corresponding author on reasonable El Acebo Clínica Veterinaria (Comunidad de Madrid, Spain), Elena request. BAXARIAS ET AL . 1987 ETHICS STATEMENT Baxarias, M., Viñals, J., Fernández, A. Á., Alcover, M. M., & Solano-Gallego, Study authorization was obtained from the Spanish authority, Agencia L. (2022). Detection of specific antibodies against Leishmania infantum in canine serum and oral transudate using an in‑house ELISA. Parasites Española de Medicamentos y Productos Sanitarios (AEMPS) with the & Vectors, 15, 164. authorization number 008/EPA-2383ESP. Beyhan, Y. E., Celebi, B., Ergene, O., & Mungan, M. (2016). Seroprevalance of leishmaniasis in dogs from Hatay and Burdur provinces of Turkey and Northern Cyprus. Turkiye Parazitolojii Dergisi, 40, 9–12. CONSENT TO PARTICIPATE Bouattour, A., Amri, A., Belkhiria, J. A., Rhim, A., Fezaa, O., Gantier, J.-C., & Consent was obtained from the owner or the tutor of the dog(s) to M’ghirbi, Y. (2021). Canine leishmaniosis in Tunisia: Growing prevalence, collect the sample and perform LEISCAN. larger zones of infection. PLoS Neglected Tropical Diseases, 15, 1–17. Canakci, T., Kurtdede, A., Pasa, S., Toz Ozensoy, S., & Ozbel, Y. (2016). Seroprevalence of canine leishmaniasis in Northern Cyprus. Turkiye ORCID Parazitolojii Dergisi, 40, 117–120. 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