Baxarias et al. Parasites & Vectors (2022) 15:134 https://doi.org/10.1186/s13071-022-05251-5 Parasites & Vectors RESEARCH Open Access Use of preventive measures and serological screening tools for Leishmania infantum infection in dogs from Europe Marta Baxarias1, Josep Homedes2, Cristina Mateu2, Charalampos Attipa3 and Laia Solano‑Gallego1* Abstract Background: There are several screening tools for detecting Leishmania infantum infection in dogs and various preventive measures to protect against it. Some studies have investigated them, but not many have described their current use. The aim of this study was to investigate which preventive measures and serological screening tools for L. infantum infection were employed from 2012 to 2018 in dogs from different endemic European countries. Methods: A set of electronic datasheets was completed for each dog from several veterinary centres. Classification of preventive measures included: (1) repellents, (2) vaccines and (3) immunomodulators. Classification of serological tests included the: (1) direct agglutination test (DAT), (2) enzyme-linked immunosorbent assay (ELISA), (3) indirect immunofluorescence (IFI), (4) rapid tests and (5) other assays. Dogs were also classified depending on their risk of exposure and living area. most frequently used were repellents (86.2%) followed by vaccines (39.8%) and ­Leisguard® (15.3%). The different types Results: Information from 3762 dogs was gathered. Preventive measures were applied in 91.5% of dogs and the high-risk group while the low-risk preferred a combination of ­Leisguard® and a repellent (Chi-square test: X2 = 88.41, of repellents (collar and spot-on) were used similarly. A combination of a vaccine and repellents was preferred in the df = 10, P < 0.001). Furthermore, all preventive measures were similarly used through the years except for repellents, which were predicted to have a small increase of use each year. Regarding serological screening tools, the most used were rapid and ELISA tests. Rapid tests, ELISA tests and DAT were used similarly through the years, but a significant change was found in the use of IFI and other assays whose use decreased a little each year. Conclusions: Repellents were the preferred measure, while vaccines and ­Leisguard® were second-line options. Some dogs were not treated by any measures, which highlights the need for dog owner education. Moreover, there seems to be a preference for rapid tests in the clinical setting to detect specific L. infantum antibodies while ELISA or IFI are less often employed. This underlines an increasing problem, as qualitative rapid tests have a variable diagnostic performance limiting the adequate diagnosis of seropositive dogs in endemic areas. Keywords: Leishmaniosis, Canine, Prevention, Screening diagnostic tools, Europe Background Canine leishmaniosis (CanL) caused by the protozoan Leishmania infantum is a zoonotic and endemic dis- ease in the Mediterranean basin [1, 2]. This protozoan *Correspondence: laia.solano@uab.cat 1 Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, is transmitted by the bite of a female phlebotomine sand Universitat Autònoma de Barcelona, Bellaterra, Spain fly following a digenetic life cycle which consists of two Full list of author information is available at the end of the article different phases: (i) a promastigote phase, which is an © The Author(s) 2022. 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The Creative Commons Public Domain Dedication waiver (http://​creat​iveco​ mmons.​org/​publi​cdoma​in/​zero/1.​0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Baxarias et al. Parasites & Vectors (2022) 15:134 Page 2 of 11 ­Letifend® may change the use of the already marketed extracellular and motile form that colonizes the middle and marketing of new preventive measures such as gut of the sand fly, and (ii) an amastigote phase, which is an intracellular and non-motile form that colonizes products. Regarding serological screening tools, several macrophages of infected hosts [3, 4]. There are also other studies have compared their sensitivity and specificity potential routes of transmission such as venereal [5, 6], [18, 26, 27] or the use of different types of samples such transplacental [6, 7] and through blood transfusion [8, as saliva [28]. However, the current use of the differ- 9], which may play a marginal role compared to the vec- ent preventive measures and serological screening tools tor transmission [10]. The dog (Canis lupus familiaris) is available for L. infantum infection is relatively unknown. considered the main domestic reservoir for L. infantum For all these reasons, the aim of this study was to inves- infection in the Mediterranean basin [2, 10], while other tigate the most used serological screening tools and pre- mammals such as wild canids [11], rodents [12] and lago- ventive measures against L. infantum infection in dogs morphs [13] may be able to maintain a wild life cycle. from 2012 to 2018 and how their use changed through The use of preventive measures against L. infantum the years. infection has expanded over the last decades [14]. How- ever, there are still two main ways to prevent this infec- Methods tion: (i) physical barriers and insecticides against the Veterinary clinics and cases vector and (ii) immunoprophylaxis. Regarding the vec- Veterinary clinics from Spain (n = 84), Portugal (n = 3), tor, it is recommended to avoid outdoor activities during Italy (n = 17) and Cyprus (n = 2), which implemented at dawn and dusk (when the vector is highly present), to use least two different preventive measures against L. infan- fine mesh nets in windows and to use topical insecticides tum in dogs, were selected for a database search of clini- such as synthetic pyrethroid-based compounds, which cal records by the authors from their contacts and client have both repellent and anti-feeding effects [1, 14, 15]. lists and were contacted to participate. Figure 1 shows Topical insecticides are commercially available in differ- the veterinary clinics that enrolled in the study including ent forms: impregnated collars, spot-on and sprays, each 67 from Spain, 3 from Portugal, 10 from Italy and 1 from of which has different onset and maximum duration [3, Cyprus. These veterinary clinics provided information of and immunomodulators. Domperidone ­(Leisguard®) is 14]. Immunoprophylaxis can be divided into vaccines dogs with the following inclusion criteria: (1) apparently healthy dogs and (2) a previous screening serological test the only marketed immunomodulator for the preven- for the detection of antibodies against L. infantum anti- have been available for dogs in Europe: ­Canileish®, which tion of CanL since 2012 [16]. Two commercial vaccines gen before the initial use of the preventive measures. Letifend®, was first launched in 2011 but is not marketed anymore Study design (withdrawn from the market in 2021), and ­ Each veterinary clinic received a code to access a website which was introduced commercially in 2016 and is cur- with a set of electronic datasheets that allowed easy data rently the only available vaccine in Europe [3, 14, 17]. entry. Once the datasheets were completed, their data Moreover, CanL is a complex infection due to its vari- were automatically uploaded to a common database from able clinical manifestations and a wide spectrum of clini- which the results were analysed. cal signs and laboratory findings, and several diagnostic The online questionnaire permitted gathering relevant techniques are available for its screening and diagnosis clinical data about dog characteristics (sex, weight, age, [17, 18]. Since a vaccine is available in Europe, serologi- breed, risk of exposure and living area) and types of cal screening is mandatory prior to vaccination of dogs serology tests and preventive measures used. Data of pre- [17]. In addition, annual screening of dogs is frequently ventive measures were obtained from 2012 to 2017 while performed in endemic areas to diagnose both dogs pro- data of screening tools were collected from 2012 to 2018. gressing towards disease and subclinical infections [10, 17]. The diagnostic methods used in the clinical setting Case removal include parasitological diagnosis (direct observation of After collection of cases, removal of inadequate cases was the parasite), serological techniques (such as ELISA, IFI performed. A case was defined as inadequate when: (i) it and rapid chromatographic immunoassay) and molecular did not comply with the previously established inclusion techniques (PCR and quantitative PCR) [1, 17, 18]. criteria or (ii) a duplicate case detected. When a dupli- Some studies have investigated the use of preventive cate case was detected, a thorough search was performed measures in L. infantum endemic countries, although to confirm its duplicity as to not lose any information. their focus was the efficacy and safety of those measures Information about the same dog with two different pre- [16, 19, 20] or the veterinary recommendations for their ventive measures and non-overlapping timelines was not use to dog owners [21–25]. In addition, the development defined as a duplicate. Baxarias et al. Parasites & Vectors (2022) 15:134 Page 3 of 11 Fig. 1 Geographical distribution of all participating veterinary clinics from Europe. Spain is marked in red, Portugal in orange, Italy in green and Cyprus in yellow. Black dots represent each enrolled clinic in each country location Preventive measures Dogs that used repellent were classified in three differ- Dogs were classified considering the combined use of ent groups based on type of repellent employed: (i) collar, preventive measures. Eleven groups were considered: (i) (ii) spot-on and (iii) collar + spot-on. lents applied (REP), (iii) only ­Canileish® vaccine (CAN), no preventive measures applied (NON), (ii) only repel- (iv) only ­Letifend® vaccine (LET), (v) only ­Leisguard® Classification of exposure risk and living area ­ anileish® vaccine + repellent (CAN + REP), Dogs were classified in two different groups depending (vii) ­Letifend® vaccine + repellent (LET + REP), (viii) (LEI), (vi) C on their exposure risk to L. infantum infection. High risk ­Leisguard® + repellent (LEI + REP), (ix) ­ Canileish® was considered when dogs lived outdoors or when dogs ® Canileish® that despite living indoors went frequently for a walk in ® vaccine + ­Leisguard (CAN + LEI), (x) ­ plot of land or forest areas at times when the vector was Letifend® vaccine + ­Leisguard® + repellent vaccine + ­Leisguard + repellent (CAN + LEI + REP) highly present, for example at dawn and dusk. Low risk and (xi) ­ classification included those dogs which lived indoors (LET + LEI + REP). and went only for a walk in urban area or just at times Another classification considered the individual use when the vector was barely present. of each product. These four groups were defined as (i) Another classification depending on living area was repellent group, which included dogs that used repel- also performed. Dogs were classified in three groups: lent alone or in combination with other products (REP, urban area (living in cities or big towns with paved streets and LET + LEI + REP), (ii) ­Canileish®, which included CAN + REP, LET + REP, LEI + REP, CAN + LEI + REP and small green areas), periurban area (city outskirts or Canileish® alone or in combination towns surrounded by large green areas) and rural area dogs that used ­ (small towns or buildings built far away from human and CAN + LEI + REP), (iii) ­Letifend®, which included with other products (CAN, CAN + REP, CAN + LEI settlements like farms, usually agricultural areas and dogs that used ­Letifend® alone or in combination with forests). Leisguard®, which included dogs that used other products (LET, LET + REP and LET + LEI + REP), ­Leisguard® alone or in combination with other prod- and (iv) ­ Screening tools The screening tools were classified in five groups: (i) ucts (LEI, LEI + REP, CAN + LEI, CAN + LEI + REP direct agglutination test (DAT), (ii) enzyme-linked and LET + LEI + REP). immunosorbent assay (ELISA), (iii) indirect immunoflu- orescence (IFI), (iv) rapid tests and (5) other assays. Baxarias et al. Parasites & Vectors (2022) 15:134 Page 4 of 11 Results erinaria SLU was performed in 2018 using ­Leiscan® and Additionally, a screening campaign by Ecuphar vet- Dog characteristics ELISA in house [29] to increase the number of enrolled Dogs from Spain (3603 dogs), Portugal (64 dogs), Italy dogs; therefore, a bias was to be expected. (69 dogs) and Cyprus (26 dogs) were enrolled in this study with a total of 3762 dogs. Dog characteristics such Statistical analysis as sex, age, weight, breed, risk of exposure and living A descriptive study of all collected data was performed. area are displayed in Table 1. The most common breeds Quantitative variables (age, weight) were assessed using a were Yorkshire terrier (7.1%), Labrador retriever (6.7%), non-parametric Mann-Whitney U test when two groups German shepherd (6.2%), Maltese (3.9%), Boxer (3.8%), were compared (high and low risk) while the Kruskal- Golden retriever (3.7%) and French bulldog (3.5%). Wallis H test was used when three groups were com- No statistically significant differences were found pared (living area: urban, periurban or rural). Qualitative between risk of exposure to the vector (low vs. high risk variables (sex, breed, preventive measures and serologi- of exposure) when sex, age and breed were compared. A cal screening tools) were assessed using a Chi-square significant difference (Mann-Whitney test: U = 1,876,996, test. A simple linear regression was calculated to predict Z = – 13.46, n1 = 2613, n2 = 1125, P < 0.0001) was noted the proportion of use for each preventative measure or when weight was compared between groups of risk of serological test based on time (from 2012 to 2017 or from exposure to the vector. Large size dogs (21.9 ± 13.7 kg) 2012 to 2018, respectively). were included in the high-risk group while small size A P-value < 0.05 was considered statistically significant. dogs (15.7 ± 12.6 kg) were included in the low-risk group. The Shapiro-Wilk test was performed to detect normal Quantitative and qualitative characteristics of dogs distribution of quantitative variables. The statistical anal- depending on their living area are listed in Table 2. No ysis was performed using the package Stats for the soft- differences between groups were found when sex and ware R i386 3.5.1 for Windows. Maps were created using breed were compared. In the case of age and weight, the Free and Open Source QGIS 3.10.4 for Windows. dogs living in rural areas were younger than dogs liv- Graphics were plotted using Graphad Prism version 5.00 ing in periurban or urban areas (Kruskal-Wallis H test: for Windows. X2 = 10.73, df = 2, P = 0.005) while dogs living in urban Table 1 Qualitative and quantitative clinical characteristics of the dogs Qualitative clinical characteristics N % (95% CI) Sex Male 2006 53.4 (51.8–55) Female 1753 46.6 (45–48.2) Total 3759 Breed Purebred 2711 72.3 (70.9–73.8) Mixed breed 1037 27.7 (26.2–29.1) Total 3748 Risk of exposure High 2620 69.9 (68.4–71.4) Low 1127 30.1 (28.6–31.6) Total 3747 Living area Urban area 1585 55.5 (53.6–57.3) Periurban area 818 28.6 (27–30.3) Rural area 455 15.9 (14.6–17.3) Total 2858 Quantitative clinical characteristics N Mean (± SD) Minimum Maximum Age (years) 3755 7 (± 3.3) 0.5 18.5 Weight (kg) 3753 20 (± 13.7) 1.4 110 CI confidence intervals, N number of dogs, SD standard deviation Baxarias et al. Parasites & Vectors (2022) 15:134 Page 5 of 11 Table 2 Qualitative and quantitative clinical characteristics of the dogs depending on their living area Qualitative clinical Urban area (N = 1585) Periurban area (N = 818) Rural area (N = 455) P-value characteristics (Chi-square test) N % (95% CI) N % (95% CI) N % (95% CI) Sex Male 842 53.1 (50.6–55.6) 461 56.4 (52.9–59.8) 241 53 (48.3–57.6) 0.284 Female 743 46.9 (44.4–49.4) 357 43.6 (40.2–47.1) 214 47 (42.4–51.7) Breed Purebred 1174 74.1 (71.8–76-2) 576 70.4 (67.2–73.5) 317 69.7 (65.2–73.9) 0.064 Mixed-breed 411 25.9 (23.8–28.2) 242 29.6 (26.5–32.8) 138 30.3 (26.1–34.8) Risk of exposure < 0.001a* High 925 58.4 (55.9–60.8) 676 82.6 (79.9–85.2) 436 95.8 (93.6–97.5) Low 660 41.6 (39.2–44-1) 142 17.4 (14.8–20.1) 19 4.2 (2.5–6.4) Quantitative clinical N Mean (± SD) N Mean (± SD) N Mean (± SD) P-value (Kruskal- characteristics Wallis H test) Age (years) 1585 7.2 (± 3.3) 817 7.1 (± 3.3) 455 6.6 (± 3.1) 0.005b* Weight (kg) 1585 17 (± 13.1) 818 23 (± 13.5) 455 23.9 (± 13.8) < 0.0001c* CI confidence intervals, N number of dogs, SD standard deviation a X2 = 314.67, df = 2, P < 0.001 b X2 = 10.73, df = 2, P = 0.005 c X2 = 176.06, df = 2, P < 0.0001 * P-value < 0.05 (statistically significant) areas were smaller in size than dogs living in rural or periurban areas (Kruskal-Wallis H test: X2 = 176.06, df = 2, P < 0.0001) (Table 2). Moreover, rural area dogs had a higher risk of exposure to L. infantum followed by periurban dogs and finally urban dogs (Chi-square test: X2 = 314.67, df = 2, P < 0.001). Preventive measures General results Preventive measures were applied for 3444 dogs (91.5%) of all the dogs enrolled. Younger dogs (6.9 ± 3.3 years) were more likely to be treated with preventive measures than older dogs (7.7 ± 3.5 years) (Mann-Whitney test: U = 614,890.5, Z = –3.79, n1 = 317, n2 = 3438, P = 0.0002). The individual use of each preventive measure in the 3444 dogs is plotted in Fig. 2. Repellents (alone or in combina- Fig. 2 Proportions of (a) the individual use of each preventive measure followed by vaccines ­(Canileish® or ­Letifend®) tion with other products) were the most used preventive measure, (b) the type of repellent used and (c) the vaccine used. and ­Leisguard® (Fig. 2a). The different types of repellents Preventive measures represented are repellent group (REP), which included dogs that used repellent alone or in combination with ­ anileish® (collar, spot-on or a combination of both) were used simi- vaccine alone or in combination with other products, ­Leisguard® other products, vaccine group (VAC), which included dogs that used group (LEI), which included dogs that used ­Leisguard® alone or (60.8%) was more frequently used than ­Letifend® (39.2%) larly (Fig. 2b) while, in the case of vaccines, C in combination with other products, ­Canileish® group (CAN) and (Fig. 2c). No statistical differences were observed when ­Letifend® group (LET) the individual use of the different preventive measures for ­Canileish®, which was more often used in pure- depending on sex and breed were compared except P = 0.007), ­Letifend® (Mann-Whitney test: U = 1,084,731, bred dogs (Chi-square test: X2 = 9.26, df = 1, P = 0.002) test: U = 900,141.5, Z = – 2.7, n1 = 518, n2 = 3237, Z = -6.42, n1 = 3168, n2 = 587, P < 0.0001) or ­Leisguard® than in mixed-breed dogs. Regarding age, younger dogs were more likely to use repellent (Mann-Whitney Baxarias et al. Parasites & Vectors (2022) 15:134 Page 6 of 11 (Mann-Whitney test: U = 963,611.5, Z = -2.29, n1 = 3184, breed, age and weight. In detail, purebred dogs used more frequently a combination of both collar and dogs were more likely to use ­Canileish® (Mann-Whit- n2 = 571, P = 0.02) than older dogs. As for weight, larger spot-on, while mixed-breed dogs used collars alone ney test: U = 1,213,325, Z = –2.72, n1 = 2846, n2 = 907, more frequently (Chi-square test: X2 = 8.03, df = 2, P = 0.018). Dogs using collars alone were younger ­Leisguard® (Mann-Whitney test: U = 1,043,852.5, P = 0.006) while smaller dogs were more likely to use (6.8 years) than dogs using spot-on alone (7.1 years) Z = -5.56, n1 = 3180, n2 = 573, P < 0.0001). (Kruskal-Wallis H test: X2 = 6.27, df = 2, P = 0.044) Figure 3 shows the combined preventive measures while dogs using spot-on alone were smaller in size used in all the dogs. The most used preventive meas- (14.5 kg) than dogs using collars alone (22.2 kg) or a ure was repellent alone (Fig. 3). When comparing the combination of collar and spot-on (22.5 kg) (Kruskal- proportions of sex, CAN + LEI and CAN + LEI + REP Wallis H test: X2 = 299.11, df = 2, P < 0.0001). presented the highest proportion of females (58.6%) while REP presented the highest proportion of males Preventive measures by risk of exposure (55.9%) (Chi-square test: X2 = 4.78, df = 1, P = 0.029), The use of preventive measures against L. infantum but no other differences were found between the other was similar when risk of exposure was compared groups (Table 3). Regarding breed, only CAN + REP ­Letifend® was used more frequently in the high-risk (91.3% high-risk group and 92.1% low-risk group). was found to have a significantly higher proportion of purebred dogs (77%) when compared to the other pre- group (Chi-square test: X2 = 107.02, df = 1, P < 0.001) ventive measures (44.4%) (Chi-square test: X2 = 16.53, while ­Leisguard® was used more often in the low-risk df = 6, P = 0.011) (Table 3). When comparing their group (Chi-square test: X2 = 54.69, df = 1, P < 0.001). age, LEI was found to be the oldest group (Table 3). Regarding the type of repellents used, the high-risk Regarding weight, LEI + REP and LEI were the groups group had a higher rate of using both types of repel- with smaller dogs and significantly different when lents together (collar and spot-on) while the low-risk compared to the other groups (Kruskal-Wallis H test: group had a higher rate of using collar or spot-on alone X2 = 45.82, df = 10, P < 0.0001) (Table 3). (Chi-square test: X2 = 92.80, df = 2, P < 0.001). Additional file 1: Fig. S1 shows the use of the dif- Most of the preventive measures were more fre- ferent marketed brands of each type of repellent: quently used in the high-risk group except for collar (Additional file 1: Fig. S1a) and spot-on (Addi- LEI + REP and LEI, which were similarly used in both the ­S calibor® collar (70%) and the ­Advantix® spot-on tional file 1: Fig. S1b). The most used products were groups. In fact, LEI + REP and LEI were found to have a significantly higher proportion of use in the low- (61%). Significant differences were found regarding risk of exposure group than other preventive meas- ures (Chi-square test: X2 = 88.41, df = 10, P < 0.0001) (Table 3). On the other hand, LET + REP was found to have the highest proportion of use in the high-risk group and was significantly different when compared to the other groups (Table 3). Preventive measures by living area Preventive measures were applied differently depend- ing on the living area showing a higher rate of use in urban area (93.2%) followed by periurban (91.6%) and rural (87.9%) areas (Chi-square test: X2 = 13.34, df = 2, P = 0.001). The use of collar, spot-on and a combina- tion of both was also compared between urban, per- Fig. 3 Proportions of preventive measures used against L. infantum iruban and rural areas and significant differences were found (Chi-square test: X2 = 194.23, df = 4, P < 0.001) ­ anileish® vaccine + repellent (CAN + REP), in all dogs studied. Preventive measures represented are only ­Letifend® vaccine + repellent (LET + REP), ­Leisguard® + repellent repellents applied (REP), C with a higher use of collar alone in rural and periruban (LEI + REP), no preventive measures applied (NON), only ­Canileish® areas while a combination of both collar and spot-on vaccine applied (CAN), ­Canileish® vaccine + ­Leisguard® + repellent was preferred in urban areas (Fig. 4). (CAN + LEI + REP), only L­ eisguard® applied (LEI), L­ etifend® Furthermore, REP was the preventive measure used vaccine + ­Leisguard® + repellent (LET + LEI + REP), only L­ etifend® at the most similar frequency in all areas with 47.5% of vaccine applied (LET) and ­Canileish® vaccine + ­Leisguard® use in the urban followed by 30.4% in the periurban and (CAN + LEI) 22.1% in the rural areas. CAN + REP and LET + REP Baxarias et al. Parasites & Vectors (2022) 15:134 Page 7 of 11 Table 3 Qualitative and quantitative clinical characteristics of the dogs depending on the preventive measures used Preventive Sex (%, 95% CI) Breed (%, 95% CI) Age (years, Weight (kg, Risk of exposure (%, 95% CI) measures mean ± SD) mean ± SD) Male Female Purebred Mixed-breed High Low NON (N = 318) 50 (44.4–55.6) 50 (44.4–55.6) 69.1 (63.7– 30.9 (25.9– 7.1 (± 3.5) 20 (± 14.3) 71.9 28.1 (23.2–33.4) 74.1) 36.3) (66.6–76.8) REP (N = 1468) 55.9 (53.3– 44.1 (41.5– 71.8 (69.4– 28.2 (25.9– 7 (± 3.4) 18.8 (± 13.2) 66.4 33.6 (31.2–36.1) 58.5) 46.7) 74.1) 30.6) (63.9–68.8) CAN (N = 125) 52 (42.9–61) 48 (39–57.1) 75 (66.4–82.3) 25 (17.7–33.6) 6.5 (± 2.8) 21.8 (± 14.6) 69.4 30.6 (22.7–39.6) (60.4–77.3) LET (N = 28) 53.6 (33.9– 46.4 (27.5– 71.4 (51.3– 28.6 (13.2– 4.4 (± 3.7) 19 (± 10.1) 60.7 39.3 (21.5–59.4) 72.5) 66.1) 86.8) 48.7) (40.6–78.5) LEI (N = 39) 41 (25.6–57.9) 59 (42.1–74.4) 61.5 (44.6– 38.5 (23.4– 8.8 (± 3.3) 11.9 (± 14.1) 43.6 56.4 (39.6–72.2) 76.6) 55.4) (27.8–60.4) CAN + REP 53.8 (50.1– 46.2 (42.5– 77 (73.7–80) 23 (20–26.3) 6.3 (± 3.1) 18 (± 14.7) 72.4 (69–75.6) 27.6 (24.4–31) (N = 719) 57.5) 49.9) LET + REP 51.4 (47.1– 48.6 (44.2– 71.9 (67.9– 28.1 (24.3– 5.8 (± 3.3) 19 (± 13.1) 89.9 10.1 (7.6–13) (N = 527) 55.8) 52.9) 75.7) 32.1) (87.1–92.4) LEI + REP 52.5 (47.7– 47.5 (42.7– 70 (65.5–74.3) 30 (25.7–34.5) 6.1 (± 3.2) 12 (± 13.5) 53.8 (49–58.6) 46.2 (41.4–51) (N = 436) 57.3) 52.3) CAN + LEI 22.2 (2.8–60) 77.8 (40–97.2) 44.4 (13.7– 55.6 (21.2– 4.4 (± 3.5) 10 (± 13.2) 88.9 11.1 (0–48.3) (N = 9) 78.8) 86.3) (51.8–99.7) CAN + LEI + REP 41.4 (28.6– 58.6 (44.9– 75 (61.6–85.6) 25 (14.4–38.4) 6 (± 3) 17.8 (± 15.7) 71.9 (58.5–83) 28.1 (17–41.5) (N = 57) 55.1) 71.4) LET + LEI + REP 53.1 (34.7– 46.9 (29.1– 68.8 (50–83.9) 31.2 (16.1–50) 6 (± 1.2) 22.5 (± 16.8) 78.1 (60–90.7) 21.9 (9.3–40) (N = 32) 70.9) 65.3) P-value P < 0.0001* a P < 0.0001*b P < 0.0001*c P < 0.0001*d P < 0.0001*e CAN only ­Canileish® vaccine, CAN + LEI ­Canileish® vaccine + ­Leisguard®, CAN + LEI + REP ­Canileish® vaccine + ­Leisguard® + repellent, CAN + REP ­Canileish® vaccine + repellent, CI Confidence intervals, LEI only ­Leisguard®, LEI + REP ­Leisguard® + repellent, LET only ­Letifend® vaccine, LET + LEI + REP ­Letifend® vaccine + ­Leisguard® + repellent, LET + REP ­Letifend® vaccine + repellent, N number of dogs, NON no preventive measures applied, REP only repellents applied, SD: standard deviation a Chi-square test: X2 = 39.63, df = 10 b Chi-square test: X2 = 38.72, df = 10 c Kruskal-Wallis H test: X2 = 84.15, df = 10 d Kruskal-Wallis H test: X2 = 45.82, df = 10 e Chi-square test: X2 = 88.41, df = 10 * P-value < 0.05 (statistically significant) were significantly more used in urban areas with 64% and a 78% frequency, respectively (Chi-square test: X2 = 170, df = 20, P < 0.0001). Moreover, LET + REP was signifi- cantly more used in urban areas than CAN + REP (Chi- square test: X2 = 30.35, df = 2, P < 0.001). Preventive measures trends The use of the different products from 2012 to 2017 is plotted in Fig. 5. Repellents were the most used always by Fig. 4 Proportions of the type of repellent used depending on > 80% of the dogs studied (Fig. 5). A significant regres- the living area. Statistical significance was found in the following comparisons: Periurban vs. rural (Chi-square test: X2 = 10.01, df = 2, sion was only found in the use of repellents with an R ­2 P = 0.006) and urban areas (Chi-square test: X2 = 6.07, df = 2, P = 0.04) of 0.75 (Fig. 5). The predicted use of repellents was equal and rural vs. urban areas (Chi-square test: X2 = 26.75, df = 2, P < 0.0001) to -3252.31 + 1.66 of percentage of the use of repellents Baxarias et al. Parasites & Vectors (2022) 15:134 Page 8 of 11 Fig. 5 Proportions of the use of the different products through the years studied (2012–2017). Preventive measures represented Fig. 7 Proportions of the use of the different types of serological alone or in combination with other products, ­Leisguard® group (LEI), are repellent group (REP), which included dogs that used repellent screening tests through the years studied (2012–2018). Screening which included dogs that used L­ eisguard® alone or in combination tools represented are direct agglutination test (DAT), enzyme-linked with other products, ­Canileish® group (CAN), which included dogs immunosorbent assay (ELISA), indirect immunofluorescence that used ­Canileish® alone or in combination with other products, (IFI), rapid tests and other assays. Data in red present a significant ­Letifend® group (LET), which included dogs that used ­Letifend® regression: IFI (F(1,5) = 35.08, P = 0.002) and other (F(1,5) = 9.23, P = 0.0288) alone or in combination with other products, and no preventive measures applied (NON). Data in red present a significant regression: REP (F(1,4) = 12.15, P = 0.0252) use of IFI tests and other tests with an ­R2 of 0.88 and 0.65, respectively. The predicted use of IFI tests was equal to 2066.12—1.02 of percentage of the use of IFI tests when time is measured in years, so the percentage of use of IFI tests decreased 1.02% for each year. The predicted use of other tests was equal to 172.86–0.09 of percentage of the use of other tests when time was measured in years, so the percentage of use of other tests decreased 0.09% for each year. Fig. 6 Proportions of the different types of serological screening Discussion tests. Screening tools represented are the direct agglutination Previous studies have investigated the veterinary rec- test (DAT), enzyme-linked immunosorbent assay (ELISA), indirect ommendations for the use of preventive measures to immunofluorescence (IFI), rapid tests and other assays dog owners in Spain and other European countries and found out that most veterinarians recommend preven- when time was measured in years, so the percentage of tive measures against L. infantum to their clients [21– use of repellents increased 1.66% for each year. 25]. These recommendations can be linked directly to the results of the present study as at least one preventive measure was applied in > 90% of the dogs. Furthermore, Serological screening tools repellents over vaccines or ­Leisguard® [22, 23], which is veterinary recommendations seem to prioritize the use of General results The different types of serological screening tests also highlighted by the results of the present study where employed are shown in Fig. 6 while the different brands and ­Leisguard® were used by < 50% throughout all years a repellent was used in > 80% of the dogs while vaccines of serological screening tests are shown in Additional Idexx) followed by ELISA tests ­(Leiscan®). IFI and DAT file 2: Fig. S2. Rapid tests were the most used (SNAP- studied. As expected, these recommendations are in line with the published guidelines [14], which endorse the use were used in < 10% of the cases (Fig. 6, Additional file 2: cines and ­Leisguard® are described as optional. of repellents in both endemic and fringe areas, while vac- Fig. S2). Regarding repellent brands, a previous study [23] Screening tools trends ­Seresto®, ­Advantix® and ­ Scalibor®. Both the pre- reported that the most frequently recommended were The use of the different types of serological screening tests from 2012 to 2018 is displayed in Fig. 7. Rapid tests results with the most used collar being ­Scalibor® while sent study and an additional study [19] showed similar followed by ELISA were the most frequently used tech- niques (Fig. 7). A significant regression was found on the ­Advantix® was the most used spot-on. Interestingly, a Baxarias et al. Parasites & Vectors (2022) 15:134 Page 9 of 11 study performed in north-eastern Spain [22] described a However, ELISA is used more than IFI because IFI’s preference for recommending collars (98% of the veteri- interpretation is subjective and its result depends on narians recommended collars to their clients) over spot- the operator’s experience and skill to interpret the test on (67% of the veterinarians recommended spot-on), while ELISA is interpreted objectively using an ELISA in disagreement with the present results in which there reader to quantify the result [26]. These results high- was no difference between the use of collar or spot-on, light an increasing problem in the clinical setting as although the reason for these results could be related qualitative rapid tests have a good specificity but are to the higher use of collars in periurban and rural areas less sensitive than quantitative laboratory tests such compared to urban areas found in this study. Regarding as IFI and ELISA and therefore rapid tests can misdi- ­Letifend® than ­Canileish®. However, the present study vaccines, Montoya et al. [23] reported a higher use of agnose seropositive cases [10, 17, 18, 34]. It is impor- ­ anileish® was found when tant to remark that rapid tests have a low sensitivity ® differs as a higher use of C in detecting apparently healthy seropositive dogs [26]. compared with L ­ etifend . This discrepancy is due to the This fact is extremely concerning when testing appar- ­Canileish® was still on the market and ­Letifend® was not fact that data on dogs were included from 2012 when ently healthy infected dogs as further investigations will not be performed and therefore infection will not be Interestingly, ­Leisguard® was more frequently adminis- marketed yet [3, 14, 17]. detected. The limitations of the study are that, even as the study of the reasons for this result is that the ­Leisguard® dose tered to smaller dogs [19], as observed in this study. One was expected to collect information from different countries, a limited number of dogs from Portugal, Italy administration is linked to body weight so large dogs and Cyprus were included, so the information received need a high daily dose and therefore a higher expenditure was mainly from Spain. Furthermore, just a small sam- than when being used for small dogs [16]. Another expla- ple of the vast dog population of Spain (> 7.5 million nation is the fact that small size dogs are more prone to registered dogs) [35] was included and the use of pre- adverse effects after vaccination [30, 31]. ventive measures might be overestimated. An association between socioeconomic status of the dog owner and CanL has been previously documented [32]. Owners with a low income cannot afford some Conclusions products and that may affect the disease control and even In conclusion, dog owners in Spain follow the vet- the nutrition and survival of the dog [32]. The presence erinarian’s recommendations for the use of preventive of a backyard at the residence with a predominance of measures against L. infantum infection as endorsed by land and/or vegetation was also associated with CanL measure, while vaccines and ­Leisguard® were second- the published guidelines. Repellents were the preferred [32], which could be a consequence of not only an envi- ronmental factor but also of the smaller use of preven- line options. However, there are still dogs that do not tive measures in periurban and rural areas as described use preventive measures in endemic regions. Regarding in the present study, among other factors. Another study serological screening tools, there seems to be a prefer- from Brazil [33] went further and associated CanL with ence for the use of rapid tests in the clinical setting to not just rural areas (small farms) but also the larger size detect specific L. infantum antibodies while other types of the dogs (usually used as guard dogs) and lack of of tests such ELISA and IFI are less often employed. owner knowledge about CanL. Coincidentally, in this The results of this study reinforce the need to sensi- study, larger dogs were more frequently classified in the tize owners about the importance of protecting dogs high-risk exposure group and living in rural or periurban against the parasite and clinicians about the limitations areas, which could explain its association with CanL. that qualitative serological techniques can present in The use of screening tools was also widespread as the diagnosis of seropositive animals in endemic areas. stated previously by other studies [19, 22–25]. Con- cerning serological tests, rapid tests and ELISA seem CAN: Only ­Canileish® vaccine; CanL: Canine leishmaniosis; CAN + LEI: Abbreviations to be preferred by clinicians in the present study as previously reported [19, 22–25]. Rapid tests (56.7%) Canileish® vaccine + ­Leisguard®; CAN + LEI + REP: Canileish® vac‑ are being used more in the clinical setting probably cine + ­Leisguard® + repellent; CAN + REP: Canileish® vaccine + repellent; IFI: Indirect immunofluorescence; LEI: Only ­Leisguard®; LEI + REP: Leis‑ DAT: Direct agglutination test; ELISA: Enzyme-linked immunosorbent assay; because of their fast results, low price and easy perfor- mance, while other types of tests such ELISA (34.1%) guard® + repellent; LET: Only L­ etifend® vaccine; LET + LEI + REP: Letifend® and IFI (7.4%) are employed less because of increased vaccine + ­Leisguard® + repellent; LET + REP: Letifend® vaccine + repellent; NON: No preventive measures applied; PCR: Polymerase chain reaction; REP: time of performance and mainly because they need Only repellents applied. to be conducted in laboratories by trained personnel. Baxarias et al. Parasites & Vectors (2022) 15:134 Page 10 of 11 Supplementary Information Funding This work was supported by Ecuphar veterinaria SLU. The online version contains supplementary material available at https://​doi.​ org/​10.​1186/​s13071-​022-​05251-5. Availability of data and materials The datasets analysed during the current study are available from the cor‑ Additional file 1: Figure S1. Proportions of a) the use of collar marketed responding author on reasonable request. brands and b) the use of spot-on marketed brands in all dogs studied. Additional file 2: Figure S2. Proportions of the different brands of sero‑ Declarations logical screening tests. Ethics approval and consent to participate No ethics approval was needed for this study as only retrospective information Acknowledgements was collected. Signed informed consent about information used was obtained the 16th ­CVBD® World Forum Symposium. The paper has been sponsored by Elanco Animal Health in the framework of from veterinarians. The authors would like to thank the veterinary practices that participated Consent for publication in the study: Abros Hospital Veterinario (Ourense, Spain), Albeitar Clínica Not applicable. Veterinaria (Zaragoza, Spain), La Alcayna Clínica Veterinaria (Murcia, Spain), Amanecer (Balearic Islands, Spain), Amantea Clínica Veterinaria (Badajoz, Competing interests Ecuphar veterinaria SLU which markets both ­Leisguard® and ­Leiscan®. Spain), Ambulatorio Veterinario Andolina di Gesù (Palermo, Italy), Ambulato‑ JH and CM have financial competing interests as they receive a salary from rio Veterinario Fortunati (Turin, Italy), Ambulatorio Veterinario Primo Amico (Naples, Italy), Ambulatorio Veterinario Spiezia (Naples, Italy), Animalex Clínica Veterinaria (Huelva, Spain), Animax Clínica Veterinaria (Badajoz, Spain), Aragó Author details 1 Hospital Veterinari (Balearic Islands, Spain), Arboleda Hospital Veterinario Departament de Medicina i Cirurgia Animals, Facultat de Veterinària, Uni‑ (Cádiz, Spain), L’Arca Hospital Veterinari (Barcelona, Spain), Argentona Serveis versitat Autònoma de Barcelona, Bellaterra, Spain. 2 Ecuphar Veterinaria SLU, Veterinaris (Barcelona, Spain), Asis Clínica Veterinaria (Ciudad Real, Spain), Barcelona, Spain. 3 Department of Tropical Disease Biology, Liverpool School Bembibre Clínica Veterinaria (León, Spain), Bofarull Veterinaris (Tarragona, of Tropical Medicine, Liverpool, UK. Spain), Cabomanga Clínica Veterinaria (Murcia, Spain), Catalunya Hospital Veterinari (Barcelona, Spain), Centelles Centre Veterinari (Barcelona, Spain), Received: 21 January 2022 Accepted: 21 March 2022 Centro Veterinario Lombardo (Catania, Italy), Clínica Veterinaria Bufalini Gian‑ nini (Viterbo, Italy), Clínica Veterinaria Dogvet (Naples, Italy), Clínica Veterinaria Palermovet (Palermo, Italy), Clínica Veterinaria San Lorenzo (Palermo, Italy), Colon (Albacete, Spain), La Corraliza Clínica Veterinaria (Navarre, Spain), Coso Clínica Veterinaria (Zaragoza, Spain), Cyvets Veterinary Centre Paphos References Cyprus (Cyprus), Del Pla Clínica Veterinaria (Lleida, Spain), Doctor Can Clínica 1. Ribeiro RR, Suzan M, Michalick M, Eduardo M, Cheim C, Jean F, et al. 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Cantos-Barreda A, Escribano D, Bernal LJ, Cerón JJ, Martínez-Subiela S. Quantification of anti-Leishmania antibodies in saliva of dogs. Vet Parasi‑ Ready to submit your research ? Choose BMC and benefit from: tol. 2017;242:54–8. 29. Solano-Gallego L, Di Filippo L, Ordeix L, Planellas M, Roura X, Altet L, et al. • fast, convenient online submission Early reduction of Leishmania infantum-specific antibodies and blood parasitemia during treatment in dogs with moderate or severe disease. • thorough peer review by experienced researchers in your field Parasit Vectors. 2016;9:235. • rapid publication on acceptance 30. Moore GE, Guptill LF, Ward MP, Glickman NW, Faunt KK, Lewis HB, et al. • support for research data, including large and complex data types Adverse events diagnosed within three days of vaccine administration in dogs. J Am Vet Med Assoc. 2005;227:1102–8. • gold Open Access which fosters wider collaboration and increased citations 31. Tizard IR. 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