Seroprevalence of 12 serovars of pathogenic Leptospira in red foxes (Vulpes vulpes) in Poland

Seroprevalence of 12 serovars of pathogenic Leptospira in red foxes (Vulpes vulpes) in Poland Background: Leptospira spp. infect humans and a wide range of domestic and wild animals, but certain species such as small rodents and red foxes (Vulpes vulpes) play a particular role as reservoirs and transmission of leptospirosis as they easily adapt to many habitats including human environments. To investigate the significance of red foxes in the epidemiology of leptospirosis in Poland, a seroprevalence survey was conducted. During the 2014–2015 hunt- ing season, blood samples of 2134 red foxes originating from the central-eastern part of Poland were collected. Serum samples were tested by a microscopic agglutination test for the presence of specific antibodies to Leptospira serovars Icterohaemorrhagiae, Grippotyphosa, Sejroe, Tarassovi, Pomona, Canicola, Hardjo, Ballum, Australis, Bataviae, Saxkoebing and Poi. Results: Antibodies to at least one serovar were detected in 561 sera (26.3%). The highest seroprevalence was found in the Subcarpathia (41.6%) and Warmia-Masuria (40.3%) provinces. Antibodies were mainly directed against serovars Poi (12.4%), Saxkoebing (11.3%), and Sejroe (6.0%). Conclusions: Exposure of red foxes to certain Leptospira serovars seems to be common in central and eastern Poland. In addition, the high prevalence of antibodies against Leptospira spp. in foxes may indicate a potential risk of infection for humans and other species coming into contact with these animals. Keywords: Leptospirosis, Prevalence, Red fox, Serology, Vulpes vulpes, Zoonosis Background rodents. In addition to rodents, other wild animal species Leptospirosis caused by pathogenic spirochetes of such as the red fox (Vulpes vulpes) may act as a reservoir the genus Leptospira is an important but sometimes [2]. The bacteria are occasionally transmitted through direct contact with mammal hosts, but the majority are neglected infection that affects people and animals usually transmitted via contact with contaminated soil worldwide. Leptospirosis is a re-emerging major public and water [3], where leptospires’ survival outside the health problem in many countries and is one of the most host is favoured by warm moist conditions [4]. The red widespread zoonoses. It is an excellent example validat- fox lives throughout Europe, mainly inhabiting forests, ing the “One Health” approach, where the relationship meadows, coastal dunes and urbanized areas [5]. The between humans, animals and ecosystems needs to be Polish hunting statistics for 2015 indicate that the pop considered in order to better understand and manage a - disease [1]. Some serovars of Leptospira can chronically ulation of red foxes in Poland is 190,000–200,000 indi- infect domestic and wild animals and in particular small viduals, with a tendency to remain stable [6]. Red foxes prey upon small rodents, among other animals and the red fox may transmit leptospirosis to humans. A recent *Correspondence: jaca@piwet.pulawy.pl study indicate that small mammals might be an impor- Swine Diseases Department, National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland tant source of human leptospirosis as both rodents and Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 2 of 9 humans share infections caused by Leptospira spp. from titre was defined as the highest dilution where ≥ 50% of the same serogroups [7]. The aim of the present study the antigen suspension added to the tested serum was was to determine the seroprevalence for Leptospira spp. agglutinated. When agglutination was observed, the rel- in red foxes from central and eastern Poland. evant sera were end-point tested using twofold dilutions ranging from 1:100 to 1:25,600. The quality control of the MAT was performed by using Methods certified reference Leptospira strains and anti-Leptospira Sample collection and study area rabbit antisera (Veterinary Sciences Division, AFBI, Blood samples from red foxes (n = 2134) were collected OIE Leptospira Reference Laboratories, Belfast, and the during the 2014–2015 hunting seasons in Poland. Blood WHO/FAO and National Collaborating Centre for Refer- was taken from the thoracic cavity or heart of animals ence and Research on Leptospirosis, Royal Tropical Insti- culled primarily through the rabies monitoring pro- tute (KIT), Amsterdam, the Netherlands). Testing of the gram. Sex and geographic location were recorded and age samples was conducted at the National Reference Labo- was determined by the degree of dentine surface wear ratory of Leptospirosis, National Veterinary Research and tooth eruption (juveniles: < 1  year; mature > 1  year) Institute in Pulawy, Poland using an accredited method (Table  1). The samples originated from 134 counties according to PN/EN ISO/IEC 17025-2005. of nine provinces of Poland and were mainly collected from the central and eastern (49–55°N, 17–23°E) parts of the country (Fig.  1). Blood samples were centrifuged Statistical analysis at 4500  g for 30  min and serum stored at − 20  °C until Statistical analysis was used to study the impact of the analysis. season, sex, age, region and population density of foxes on Leptospira seroprevalence. It was based on logistic regression models to describe the influence of several Microscopic agglutination test variables X , X , …, X on the dichotomous variable Y: Serum samples were tested by a microscopic agglutina- 1 2 n tion test (MAT) using a range of 12 Leptospira serovars (β + β x ) 0 i i i=1 representative of 10 serogroups found in Europe: Ictero- e P(Y = 1|x , x , . . . , x ) = 1 2 n n haemorrhagiae (RGA strain, representing the Ictero- β + β x ( 0 i i) i=1 1 + e haemorrhagiae serogroup), Grippotyphosa (Moskva V strain, Grippotyphosa serogroup), Sejroe (M84 strain, where β is the regression coefficient for i = 0, …, n, χ are Sejroe serogroup), Tarassovi (Perepelicyn strain, Tarass- i i independent variables (measurable or qualitative) for ovi serogroup), Pomona (Pomona strain, Pomona sero- i = 1, 2, …, n. group), Canicola (Hond Utrecht IV strain, Canicola The maximum likelihood method was used to estimate serogroup), Hardjo (Hardjoprajitno strain, Sejroe sero- the model’s coefficients. The Wald test was used to evalu - group), Ballum (MUS127 strain, Ballum serogroup), ate the significance of individual variables. Evaluation Australis (Ballico strain, Australis serogroup), Bataviae of model fit to data was performed using the likelihood (Swart strain, Bataviae serogroup), Saxkoebing (MUS ratio (LR) test. 24 strain, Sejroe serogroup) and Poi (Poi strain, Javanica Five predictors (4 qualitative and 1 quantitative) were serogroup) [8, 9]. The selection of the serovars used was included in the modelling: based on their common identification in previous Euro - pean studies [10–13] reporting Leptospira spp. in wild carnivores. • sampling season (spring: March–May, summer: Each serovar was grown in 10  mL of Ellinghausen– June–August, autumn: September–November, or McCullough–Johnson–Harris (EMJH) medium, at winter: December–Feburary); 30 ± 1 °C for at least 4 but no more than 8 days depend- • sex (male, female); ing on the serovar. The concentration of bacteria was • age (young, adult); adjusted to 1–2 × 10   cells/mL using a Helber count- • province (LD: Łódzkie; MP: Lesser Poland; MA: Mas- ing chamber. The sera were initially diluted 1:50 and ovia; OP: Opolskie; PK: Subcarpathia; PM: Pomera- screened for antibodies to the 12 serovars. A volume nia; SL: Silesia; SW: Świętokrzyskie; WM: Warmia- of each antigen equal to the diluted serum volume was Masuria); (Fig. 1) and added to each well with a final serum dilution of 1:100 • fox density in counties in 2015 (No/km ). in the screening test. The final concentration of antigen after mixing with the diluted serum was 1–2 × 10   cells/ The dependent variable was the qualitative result of the mL. The plates were incubated at 30 ± 1 °C for 2–4 h and study. Analysis was performed for results without dis- subsequently examined by dark-field microscopy. The tinguishing between serovars (Leptospira spp.: positive/ Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 3 of 9 Table 1 Total number of red foxes from Poland hunted in 9 Polish provinces between 2014 and 2015 Sex Females Males Unknown Total No % of anti-Leptospira sp. of seropositive antibodies positive (95% Age Adult Young Unknown Adult Young Unknown Unknown CI) Result (Leptospira sp.) − + − + − + − + − + − + − Province/season LD 45 14 35 14 81 27 26 12 254 67 26.4 (21.1–32.3) Spring 12 4 3 11 4 4 1 39 9 Autumn 12 2 9 1 15 3 7 49 6 Winter 21 8 23 13 55 20 15 11 166 52 MP 56 18 60 8 92 23 51 12 320 61 19.1 (14.9–23.8) Unknown 36 10 37 3 75 13 28 5 207 31 Spring 17 6 5 2 12 6 7 2 57 16 Autumn 3 2 18 3 5 4 16 5 56 14 MA 47 30 80 42 199 72 36.2 (29.5–43.3) Unknown 47 30 80 42 199 72 OP 25 13 29 6 42 24 25 9 173 52 30.1 (23.3–37.5) Summer 4 1 11 3 7 5 11 3 45 12 Autumn 21 12 18 3 35 19 14 6 128 40 PK 19 17 1 38 24 2 101 42 41.6 (31.9–51.8) Unknown 19 17 1 38 24 2 101 42 PM 43 17 7 4 36 16 3 5 131 42 32.1 (24.2–40.8) Winter 43 17 7 4 36 16 3 5 131 42 SL 61 11 62 8 106 8 55 9 1 321 36 11.2 (8.0–15.2) Spring 7 2 3 9 2 5 1 1 30 5 Summer 13 2 22 4 18 3 17 5 84 14 Autumn 23 5 28 1 48 2 22 2 131 10 Winter 18 2 9 3 31 1 11 1 76 7 SW 40 8 73 11 73 14 36 5 260 38 14.6 (10.6–19.5) Spring 8 1 5 5 2 21 3 Summer 5 2 13 10 3 9 42 5 Autumn 3 1 27 7 15 3 10 3 69 14 Winter 24 4 28 4 43 6 17 2 128 16 WM 73 36 30 26 87 70 34 19 375 151 40.3 (35.3–45.4) Winter 73 36 30 26 87 70 34 19 375 151 Total 343 117 315 94 47 31 517 182 268 95 82 42 1 2134 561 26.3 (24.4–28.2) LD Łódzkie, MP Leser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SL Silesia, SW Świętokrzyskie, WM Warmia-Masuria Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 4 of 9 Fig. 1 Geographic distribution of red foxes seropositive for pathogenic Leptospira in Poland. LD Łódzkie, MP Lesser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SL Silesia, SW Świętokrzyskie, WM Warmia-Masuria, DS Lower Silesia, KP Kuyavian-Pomerania, LB Lubuskie, LU Lubelskie, PD Podlaskie, WP Greater Poland, ZP West Pomerania negative) and for each serovar separately. The selection of variables for modelling was based on analytical stepping Table 2 Dichotomous coding for  qualitative variables methods (step-wise). For qualitative variables, 0–1 cod- with an example of sampling season ing for k − 1 variables was used (Table 2). Sampling season Spring Autumn Winter The following classes of variables were reference classes in models: ‘summer’ for sampling season, ‘female’ for sex, Spring 1 0 0 ‘young’ for age and ‘SL’ for province. Parameters of sig- Summer 0 0 0 nificant and best fit logistic regression models obtained Autumn 0 1 0 for each analysis are shown in Table  3. The accepted Winter 0 0 1 Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 5 of 9 Table 3 Results of the best fit logistic regression models obtained for each analysis Significance assessment Independent variable Coefficient (β ) Std. error P value (Wald) Odds ratio Confidence Confidence of model (P value of LR OR − 95% OR + 95% test) Models for infection of Leptospira sp. (without distinction of serovars) < 0.001 Absolute term (β ) − 2.92912 0.296482 < 0.001 0.05 0.03 0.10 LD 1.216036 0.233494 < 0.001 3.37 2.13 5.33 MP 0.671037 0.228562 0.003 1.96 1.25 3.06 MA 1.68051 0.237135 < 0.001 5.37 3.37 8.55 OP 1.388372 0.247953 < 0.001 4.01 2.46 6.52 PK 1.769046 0.269941 < 0.001 5.87 3.45 9.96 PM 1.534127 0.265823 < 0.001 4.64 2.75 7.81 SW 0.555254 0.259964 0.03 1.74 1.05 2.90 WM 1.630786 0.20659 < 0.001 5.11 3.41 7.66 Fox density (No/km ) 1.142803 0.307487 < 0.001 3.14 1.72 5.73 < 0.001 Absolute term (β ) − 1.50766 0.198255 < 0.001 0.22 0.15 0.33 Spring 0.267965 0.280004 0.34 1.31 0.75 2.26 Autumn 0.0834 0.232275 0.72 1.09 0.69 1.71 Winter 0.688467 0.211402 0.001 1.99 1.31 3.01 Model for Icterohaemorrhagiae 0.003 Absolute term (β ) − 6.41457 0.839692 < 0.001 0.002 0.0003 0.008 Fox density (No/km ) 2.913659 0.989553 0.003 18.42 2.65 128.30 Adult − 1.18961 0.553268 0.03 0.30 0.10 0.90 Model for Grippotyphosa 0.001 Absolute term (β ) − 5.71115 0.543301 < 0.001 0.003 0.001 0.01 Fox density (No/km ) 2.364823 0.677533 < 0.001 10.64 2.82 40.19 Model for Sejroe 0.015 Absolute term (β ) − 3.43721 0.318798 < 0.001 0.03 0.02 0.06 LD 1.130284 0.386552 0.003 3.10 1.45 6.61 MP 0.35268 0.419714 0.40 1.42 0.62 3.24 MA 0.85591 0.422493 0.04 2.35 1.03 5.39 OP 0.110974 0.514159 0.83 1.12 0.41 3.06 PK 1.228934 0.461089 0.008 3.42 1.38 8.44 PM 1.047612 0.44818 0.02 2.85 1.18 6.87 SW 0.408686 0.434724 0.35 1.50 0.64 3.53 WM 0.880843 0.376223 0.02 2.41 1.15 5.05 Model for Australis < 0.001 Absolute term (β ) − 6.36907 0.610643 < 0.001 0.002 0.0005 0.01 Fox density (No/km ) 2.843724 0.730836 < 0.001 17.18 4.10 72.02 Models for Saxkoebing 0.024 Absolute term (β ) − 2.77882 0.325736 < 0.001 0.06 0.03 0.12 Spring 0.445929 0.436438 0.31 1.56 0.66 3.68 Autumn 0.408323 0.368228 0.27 1.50 0.73 3.10 Winter 0.806557 0.34165 0.02 2.24 1.15 4.38 Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 6 of 9 Table 3 (continued) Significance assessment Independent variable Coefficient (β ) Std. error P value (Wald) Odds ratio Confidence Confidence of model (P value of LR OR − 95% OR + 95% test) < 0.001 Absolute term (β ) − 2.94772 0.25661 < 0.001 0.05 0.03 0.09 LD 1.010782 0.318737 0.002 2.75 1.47 5.13 MP 0.715284 0.318663 0.03 2.04 1.09 3.81 MA 1.257746 0.322546 < 0.001 3.52 1.87 6.62 OP 1.021486 0.343397 0.003 2.78 1.42 5.45 PK 1.939495 0.341159 < 0.001 6.96 3.56 13.58 PM 1.452948 0.341859 < 0.001 4.28 2.19 8.36 SW − 0.63976 0.461137 0.17 0.53 0.21 1.30 WM 1.121314 0.296979 < 0.001 3.07 1.71 5.49 Models for Poi < 0.001 Absolute term (β ) − 5.45234 0.689411 < 0.001 0.004 0.001 0.02 LD 2.814176 0.617742 < 0.001 16.68 4.97 56.01 MP 1.032509 0.682165 0.13 2.81 0.74 10.70 MA 3.445862 0.612244 < 0.001 31.37 9.44 104.22 OP 3.293047 0.618209 < 0.001 26.92 8.01 90.51 PK 2.239957 0.687842 0.001 9.39 2.44 36.19 PM 2.960175 0.643965 < 0.001 19.30 5.46 68.24 SW 2.610502 0.629299 < 0.001 13.61 3.96 46.74 WM 3.666819 0.591781 < 0.001 39.13 12.26 124.88 Fox density (No/km ) 1.043369 0.476866 0.03 2.84 1.11 7.23 < 0.001 Absolute term (β ) − 2.89037 0.342638 < 0.001 0.06 0.03 0.11 Spring 0.375612 0.464447 0.42 1.46 0.59 3.62 Autumn 0.519876 0.383324 0.18 1.68 0.79 3.57 Winter 1.368756 0.353764 < 0.001 3.93 1.96 7.87 0.003 Absolute term (β ) − 2.30544 0.125369 0 0.10 0.08 0.13 Adult 0.437116 0.152208 0.004 1.55 1.15 2.09 LD Łódzkie, MP Leser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SW Świętokrzyskie, WM Warmia-Masuria that all provinces had significantly greater odds for hav - significance level was alpha = 0.05. STATISTICA data ing seropositive foxes than the reference SL province, in analysis software in version 10 (StatSoft, Inc.) and Arc- which the lowest percentage of seropositive foxes was GIS 10.4.1 for Desktop Standard (ESRI, Inc.) were used observed. The highest odds ratio (OR = 5.87) with the for statistical and spatial data analysis. Red fox demo- highest seroprevalence was shown for the PK province. graphics were derived from the Polish Hunting Associa- In addition, with an increase of fox density by one animal tion-PZL [6]. per km , the probability of detecting seropositive animals increased more than threefold and it almost doubled in Results winter when compared to summer. However due to data Antibodies against a Leptospira serovar was found in 561 deficiencies e.g. sampling date, seasonal influence on the serum samples (26.3%). The highest seroprevalence was obtained serological results was analysed using a separate observed in foxes hunted in the Subcarpathia (41.6%) logistic regression model. and Warmia-Masuria provinces (40.3%) (Table  1, Fig.  1). Based on analyses for individual serovars, an increase Specific antibodies were mainly directed against Poi of fox density by one animal per k m increased the risk (12.4%), Saxkoebing (11.3%), and Sejroe (6.0%) serovars of being seropositive by 2.8, 10.6, 17.2 and 18.4 times for with serum antibody titres up to 1:25,600 in individual the serovars Poi, Grippotyphosa, Australis and Ictero- animals (Table 4). When analysing the logistic regression haemorrhagiae, respectively. The models also show a sig - model of positive and negative serostatus (excluding data nificant influence of the province on the proportion of related to individual Leptospira serovars), a significant seropositive samples. A significantly higher risk of being influence of the area (province) and associated density seropositive to Sejroe serovar was observed in the LD of foxes on the serostatus was found. The model showed Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 7 of 9 Table 4 Distribution of pathogenic Leptospira antibody titers for 561 positive red foxes hunted during season 2014–2015 in Poland Serovar No of antibody-positive samples (%) Prevalence of serovar (95% 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:25600 Total CI) (%) Icterohaemorrhagiae 8 (0.4) 3 (0.1) 3 (0.1) 1 (0.05) 2 (0.1) 1 (0.05) 0 0 0 18 0.8 (0.5–1.3) Grippotyphosa 6 (0.3) 16 (0.75) 8 (0.4) 4 (0.2) 1 (0.05) 2 (0.1) 0 0 0 37 1.7 (1.2–2.4) Sejroe 39 (1.8) 37 (1.7) 30 (1.4) 16 (0.75) 2 (0.1) 2 (0.1) 0 0 1 (0.05) 127 6.0 (5.0–7.0) Tarassovi 0 1 (0.05) 0 0 0 0 0 0 0 1 0.1 (0.0–0.3) Pomona 7 (1.5) 7 (1.5) 8 (0.4) 8 (0.4) 2 (0.1) 2 (0.1) 0 0 0 34 1.6 (1.1–2.2) Canicola 0 2 (0.1) 1 (0.05) 0 0 0 0 0 0 3 0.1 (0.0–0.4) Australis 7 (1.5) 11 (0.5) 1 (0.05) 7 (1.5) 2 (0.1) 0 0 0 0 28 1.3 (0.9–1.9) Saxkoebing 63 (3.0) 55 (2.6) 66 (3.1) 44 (2.1) 8 (0.4) 3 (0.1) 2 (0.1) 0 1 (0.05) 242 11.3 (10.0–12.8) Ballum 0 1 (0.05) 1 (0.05) 1 (0.05) 0 0 0 0 0 3 0.1 (0.0–0.4) Poi 64 (3.0) 68 (3.2) 63 (3.0) 34 (1.6) 11 (0.5) 19 (0.9) 4 (0.2) 1 (0.05) 1 (0.05) 265 12.4 (11.1–13.9) Bataviae 1 (0.05) 1 (0.05) 3 (0.1) 3 (0.1) 0 1 (0.05) 0 0 0 9 0.4 (0.2–0.8) Hardjo 0 2 (0.1) 0 1 (0.05) 0 0 1 (0.05) 0 0 4 0.2 (0.1–0.5) Antibodies against serovar Poi were the most com- (OR = 3.1), MA (OR = 2.4), PK (OR = 3.4), PM (OR = 2.9) monly detected. Exposure of foxes to this serovar is not and WM (OR = 2.4) provinces compared to the SL surprising given the results of previous Polish studies province. where serogroup Javanica (to which serovar Poi belongs) When compared to the reference SL province, antibod- was also reported in horses, goats, and sheep [15–17]. ies to the Saxkoebing and Poi serovars were more preva- Besides serovar Poi, antibodies against serovar Sejroe lent in foxes from all provinces except SW (OR from 2.0 were also prevalent in foxes. This is consistent with other to 7.0), and MP province (OR from 9.4 to 39.1) respec- studies as serovars Hardjo, Sejroe and Saxkoebing (all tively. An impact of the season on the seroprevalence belonging to the Sejroe serogroup) are widely prevalent to particular serovars was observed. Antibodies against in animals in Europe [18–21]. MAT reactions to serovar serovars Saxkoebing and Poi were ~ 2 and 4 times more Hardjo commonly detected in sheep and cattle [18–20, frequent, respectively, during the winter period than dur- 22, 23] were not common in foxes. The presence of sero - ing summer. The age of the foxes influenced the serosta - positive animals to this serogroup could be mainly attrib- tus for some serovars such as Icterohaemorrhagiae that uted to Sejroe or Saxkoebing serovars (Table  4). It may was detected more frequently in young foxes (OR = 3.3) be associated with fox diet as the main source of food and Poi found more often in adults (OR = 1.5) (Table  3). for red foxes are wild small mammals, which are known Using a one-factor model the association between influ - reservoirs of Saxkoebing and Sejroe serovars [24]. Anti- ence of sex on serostatus was not significant (LR-test bodies to Sejroe serogroup were previously detected in P = 0.0525, OR = 1.44, 95% CI 0.99–2.09). pigs, dogs, horses and cattle in Poland confirming a wide - spread exposure of different animal species to leptospires Discussion from this serogroup [15, 25–28]. In addition, this indi- Other serological surveys have shown that red foxes are cates an endemic occurrence of this serovar and a pos- frequently exposed to Leptospira spp. of different sero - sible role of the environment in pathogen transmission. vars [10, 11, 13]. However this is the first prevalence The observed regional differences in exposure to different study on the occurrence of antibodies to a broad range Leptospira serovars may be related to active circulation of of Leptospira serovars in a red fox population in eastern Leptospira spp. in the environment [12]. Europe. The high seroprevalence (26.3%) in red foxes in Studies conducted in other European countries pro- Poland is comparable to that found in Spain (47.1%) [10] vide scientific evidences that the most common serovar and Croatia (31.3%) [13] but higher than in other Euro- among red foxes is serovar Icterohaemorrhagiae [10, 11, pean countries such as Germany (1.9%) [14] and Norway 13], which however seems to be rare in the Polish red fox (9.9%) [11]. Hypothetically any pathogenic Leptospira population (Table 4). As leptospires are sensitive to desic- may infect domestic and wild animals, but in practice cation, the regional differences in climate conditions may only a small number of serovars are endemic in any par- have a significant influence on seroprevalence in general ticular region. Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 8 of 9 to warmly thank Artur Rzeżutka for useful comments and editing of the or for some serovars in particular. In that aspect, Poland manuscript. differs from other countries such as Spain and Croatia where the seroprevalence of Leptospira spp. in foxes has Competing interests The authors declare that they have no competing interests. been investigated [10, 13]. Although the studies were conducted on a reasonable Availability of data and materials number of hunted animals originating from different All data generated or analysed during this study are included in this published article. locations across the country, the number of tested serum samples of red foxes did not fully reflect the size of the Consent for publication animal population present in the studied provinces. It Not applicable. could be taken as a major limitation to interpretation of Ethics approval and consent to participate the occurrence and prevalence of tested Leptospira sero- All procedures were carried out according to the ethical standards for the use vars in the Polish population of red foxes. Nevertheless, of animal samples. The study was approved by the Ethical Committee of the University of Life Sciences in Lublin under Grant of Approval No. 30/2016. the findings still provide useful data on the seroepidemi - ology of red foxes exposed to different Leptospira sero - Funding vars in this part of Europe and their role as an important This study was supported by the Polish National Science Centre (Grant No. 2013/09/B/NZ7/02563). The open access publication fee was funded by the source of zoonotic Leptospira spp. for humans. KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal—Safe Food”, Ministry of Science and Higher Education resolution no. Conclusions 05-1/KNOW2/2015”. Red foxes of central and eastern Poland, particularly in the Subcarpathia and Warmia-Masuria regions, are Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- highly exposed to Leptospira spp. Due to the high preva- lished maps and institutional affiliations. lence of foxes, their predatory behaviour and their varied diet mainly composed of small mammals, they could be Received: 7 September 2017 Accepted: 24 May 2018 considered as sentinel animals of environmental contam- ination with leptospires. Interactions between animals require further epidemiological investigations to eluci- References date the role of wild carnivores as a reservoir of rarely 1. Jancloes M, Bertherat E, Scheider C, Belmain S, Munoz-Zanzi C, Hartskeerl occurring Leptospira serovars pathogenic for other ani- R, et al. Towards a “one health” strategy against leptospirosis. Planet@Risk. mals and humans. 2014;2:204–6 (Special Issue on One Health (Part I/II): GRF Davos). 2. 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Washington, D.C. 2017. https ://www.natio nalge ograp hic.com/anima ls/mamma ls/r/red-fox/. Accessed 04 Mar 2018. Authors’ contributions 6. Polish Hunting Association-PZL. Warsaw. 2015. http://www.czemp JZ designed and coordinated the study. SZ and AN were responsible for the in.pzlow .pl/palio /html.run?_Insta nce=pzl_www&_PageI D=21&_ laboratory work and preliminary data analysis under the supervision of JZ, AJ CAT=CZEMP IN.MATER IALY. Accessed 04 Mar 2018. and ZP. AS was involved in the analyses of epidemiological data. LB performed 7. Stritof Majetic Z, Galloway R, Ruzic Sabljic E, Milas Z, Mojcec Perko V, the statistical analyses. ZA, AJ and AB had the main responsibility of check- Habus J, et al. Epizootiological survey of small mammals as Leptospira ing and authoring the manuscript. All authors read and approved the final spp. reservoirs in Eastern Croatia. Acta Trop. 2014;131:111–6. manuscript. 8. OIE Terrestrial Manual 2014. Chapter 2.1.12. 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Serosurvey for canine distemper virus, canine adenovirus, Leptospira inter- Acknowledgements rogans, and Toxoplasma gondii in free-ranging canids in Scandinavia and We are grateful to the hunters of all nine provinces. Many thanks to Jolanta Svalbard. J Wildl Dis. 2010;46:474–80. Sajna from the Warsaw Veterinary Hygiene Research Station (Ostrołęka 12. Moinet M, Fournier-Chambrillon C, André-Fontaine G, Aulagnier S, field division) and to Zofia Klimczak from the Bydgoszcz Veterinary Hygiene Mesplède A, Blanchard B. Leptospirosis in free-ranging endangered Research Station for data and serum samples from foxes; without their help this investigation would not have been possible. The authors would like Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 9 of 9 European mink (Mustela lutreola) and other small carnivores (Mustelidae, 21. Lange S. Seroepidemiological studies of the detection of leptospires Viverridae) from southwestern France. 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Seroprevalence of 12 serovars of pathogenic Leptospira in red foxes (Vulpes vulpes) in Poland

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Abstract

Background: Leptospira spp. infect humans and a wide range of domestic and wild animals, but certain species such as small rodents and red foxes (Vulpes vulpes) play a particular role as reservoirs and transmission of leptospirosis as they easily adapt to many habitats including human environments. To investigate the significance of red foxes in the epidemiology of leptospirosis in Poland, a seroprevalence survey was conducted. During the 2014–2015 hunt- ing season, blood samples of 2134 red foxes originating from the central-eastern part of Poland were collected. Serum samples were tested by a microscopic agglutination test for the presence of specific antibodies to Leptospira serovars Icterohaemorrhagiae, Grippotyphosa, Sejroe, Tarassovi, Pomona, Canicola, Hardjo, Ballum, Australis, Bataviae, Saxkoebing and Poi. Results: Antibodies to at least one serovar were detected in 561 sera (26.3%). The highest seroprevalence was found in the Subcarpathia (41.6%) and Warmia-Masuria (40.3%) provinces. Antibodies were mainly directed against serovars Poi (12.4%), Saxkoebing (11.3%), and Sejroe (6.0%). Conclusions: Exposure of red foxes to certain Leptospira serovars seems to be common in central and eastern Poland. In addition, the high prevalence of antibodies against Leptospira spp. in foxes may indicate a potential risk of infection for humans and other species coming into contact with these animals. Keywords: Leptospirosis, Prevalence, Red fox, Serology, Vulpes vulpes, Zoonosis Background rodents. In addition to rodents, other wild animal species Leptospirosis caused by pathogenic spirochetes of such as the red fox (Vulpes vulpes) may act as a reservoir the genus Leptospira is an important but sometimes [2]. The bacteria are occasionally transmitted through direct contact with mammal hosts, but the majority are neglected infection that affects people and animals usually transmitted via contact with contaminated soil worldwide. Leptospirosis is a re-emerging major public and water [3], where leptospires’ survival outside the health problem in many countries and is one of the most host is favoured by warm moist conditions [4]. The red widespread zoonoses. It is an excellent example validat- fox lives throughout Europe, mainly inhabiting forests, ing the “One Health” approach, where the relationship meadows, coastal dunes and urbanized areas [5]. The between humans, animals and ecosystems needs to be Polish hunting statistics for 2015 indicate that the pop considered in order to better understand and manage a - disease [1]. Some serovars of Leptospira can chronically ulation of red foxes in Poland is 190,000–200,000 indi- infect domestic and wild animals and in particular small viduals, with a tendency to remain stable [6]. Red foxes prey upon small rodents, among other animals and the red fox may transmit leptospirosis to humans. A recent *Correspondence: jaca@piwet.pulawy.pl study indicate that small mammals might be an impor- Swine Diseases Department, National Veterinary Research Institute, Partyzantow 57, 24-100 Pulawy, Poland tant source of human leptospirosis as both rodents and Full list of author information is available at the end of the article © The Author(s) 2018. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. 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. Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 2 of 9 humans share infections caused by Leptospira spp. from titre was defined as the highest dilution where ≥ 50% of the same serogroups [7]. The aim of the present study the antigen suspension added to the tested serum was was to determine the seroprevalence for Leptospira spp. agglutinated. When agglutination was observed, the rel- in red foxes from central and eastern Poland. evant sera were end-point tested using twofold dilutions ranging from 1:100 to 1:25,600. The quality control of the MAT was performed by using Methods certified reference Leptospira strains and anti-Leptospira Sample collection and study area rabbit antisera (Veterinary Sciences Division, AFBI, Blood samples from red foxes (n = 2134) were collected OIE Leptospira Reference Laboratories, Belfast, and the during the 2014–2015 hunting seasons in Poland. Blood WHO/FAO and National Collaborating Centre for Refer- was taken from the thoracic cavity or heart of animals ence and Research on Leptospirosis, Royal Tropical Insti- culled primarily through the rabies monitoring pro- tute (KIT), Amsterdam, the Netherlands). Testing of the gram. Sex and geographic location were recorded and age samples was conducted at the National Reference Labo- was determined by the degree of dentine surface wear ratory of Leptospirosis, National Veterinary Research and tooth eruption (juveniles: < 1  year; mature > 1  year) Institute in Pulawy, Poland using an accredited method (Table  1). The samples originated from 134 counties according to PN/EN ISO/IEC 17025-2005. of nine provinces of Poland and were mainly collected from the central and eastern (49–55°N, 17–23°E) parts of the country (Fig.  1). Blood samples were centrifuged Statistical analysis at 4500  g for 30  min and serum stored at − 20  °C until Statistical analysis was used to study the impact of the analysis. season, sex, age, region and population density of foxes on Leptospira seroprevalence. It was based on logistic regression models to describe the influence of several Microscopic agglutination test variables X , X , …, X on the dichotomous variable Y: Serum samples were tested by a microscopic agglutina- 1 2 n tion test (MAT) using a range of 12 Leptospira serovars (β + β x ) 0 i i i=1 representative of 10 serogroups found in Europe: Ictero- e P(Y = 1|x , x , . . . , x ) = 1 2 n n haemorrhagiae (RGA strain, representing the Ictero- β + β x ( 0 i i) i=1 1 + e haemorrhagiae serogroup), Grippotyphosa (Moskva V strain, Grippotyphosa serogroup), Sejroe (M84 strain, where β is the regression coefficient for i = 0, …, n, χ are Sejroe serogroup), Tarassovi (Perepelicyn strain, Tarass- i i independent variables (measurable or qualitative) for ovi serogroup), Pomona (Pomona strain, Pomona sero- i = 1, 2, …, n. group), Canicola (Hond Utrecht IV strain, Canicola The maximum likelihood method was used to estimate serogroup), Hardjo (Hardjoprajitno strain, Sejroe sero- the model’s coefficients. The Wald test was used to evalu - group), Ballum (MUS127 strain, Ballum serogroup), ate the significance of individual variables. Evaluation Australis (Ballico strain, Australis serogroup), Bataviae of model fit to data was performed using the likelihood (Swart strain, Bataviae serogroup), Saxkoebing (MUS ratio (LR) test. 24 strain, Sejroe serogroup) and Poi (Poi strain, Javanica Five predictors (4 qualitative and 1 quantitative) were serogroup) [8, 9]. The selection of the serovars used was included in the modelling: based on their common identification in previous Euro - pean studies [10–13] reporting Leptospira spp. in wild carnivores. • sampling season (spring: March–May, summer: Each serovar was grown in 10  mL of Ellinghausen– June–August, autumn: September–November, or McCullough–Johnson–Harris (EMJH) medium, at winter: December–Feburary); 30 ± 1 °C for at least 4 but no more than 8 days depend- • sex (male, female); ing on the serovar. The concentration of bacteria was • age (young, adult); adjusted to 1–2 × 10   cells/mL using a Helber count- • province (LD: Łódzkie; MP: Lesser Poland; MA: Mas- ing chamber. The sera were initially diluted 1:50 and ovia; OP: Opolskie; PK: Subcarpathia; PM: Pomera- screened for antibodies to the 12 serovars. A volume nia; SL: Silesia; SW: Świętokrzyskie; WM: Warmia- of each antigen equal to the diluted serum volume was Masuria); (Fig. 1) and added to each well with a final serum dilution of 1:100 • fox density in counties in 2015 (No/km ). in the screening test. The final concentration of antigen after mixing with the diluted serum was 1–2 × 10   cells/ The dependent variable was the qualitative result of the mL. The plates were incubated at 30 ± 1 °C for 2–4 h and study. Analysis was performed for results without dis- subsequently examined by dark-field microscopy. The tinguishing between serovars (Leptospira spp.: positive/ Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 3 of 9 Table 1 Total number of red foxes from Poland hunted in 9 Polish provinces between 2014 and 2015 Sex Females Males Unknown Total No % of anti-Leptospira sp. of seropositive antibodies positive (95% Age Adult Young Unknown Adult Young Unknown Unknown CI) Result (Leptospira sp.) − + − + − + − + − + − + − Province/season LD 45 14 35 14 81 27 26 12 254 67 26.4 (21.1–32.3) Spring 12 4 3 11 4 4 1 39 9 Autumn 12 2 9 1 15 3 7 49 6 Winter 21 8 23 13 55 20 15 11 166 52 MP 56 18 60 8 92 23 51 12 320 61 19.1 (14.9–23.8) Unknown 36 10 37 3 75 13 28 5 207 31 Spring 17 6 5 2 12 6 7 2 57 16 Autumn 3 2 18 3 5 4 16 5 56 14 MA 47 30 80 42 199 72 36.2 (29.5–43.3) Unknown 47 30 80 42 199 72 OP 25 13 29 6 42 24 25 9 173 52 30.1 (23.3–37.5) Summer 4 1 11 3 7 5 11 3 45 12 Autumn 21 12 18 3 35 19 14 6 128 40 PK 19 17 1 38 24 2 101 42 41.6 (31.9–51.8) Unknown 19 17 1 38 24 2 101 42 PM 43 17 7 4 36 16 3 5 131 42 32.1 (24.2–40.8) Winter 43 17 7 4 36 16 3 5 131 42 SL 61 11 62 8 106 8 55 9 1 321 36 11.2 (8.0–15.2) Spring 7 2 3 9 2 5 1 1 30 5 Summer 13 2 22 4 18 3 17 5 84 14 Autumn 23 5 28 1 48 2 22 2 131 10 Winter 18 2 9 3 31 1 11 1 76 7 SW 40 8 73 11 73 14 36 5 260 38 14.6 (10.6–19.5) Spring 8 1 5 5 2 21 3 Summer 5 2 13 10 3 9 42 5 Autumn 3 1 27 7 15 3 10 3 69 14 Winter 24 4 28 4 43 6 17 2 128 16 WM 73 36 30 26 87 70 34 19 375 151 40.3 (35.3–45.4) Winter 73 36 30 26 87 70 34 19 375 151 Total 343 117 315 94 47 31 517 182 268 95 82 42 1 2134 561 26.3 (24.4–28.2) LD Łódzkie, MP Leser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SL Silesia, SW Świętokrzyskie, WM Warmia-Masuria Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 4 of 9 Fig. 1 Geographic distribution of red foxes seropositive for pathogenic Leptospira in Poland. LD Łódzkie, MP Lesser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SL Silesia, SW Świętokrzyskie, WM Warmia-Masuria, DS Lower Silesia, KP Kuyavian-Pomerania, LB Lubuskie, LU Lubelskie, PD Podlaskie, WP Greater Poland, ZP West Pomerania negative) and for each serovar separately. The selection of variables for modelling was based on analytical stepping Table 2 Dichotomous coding for  qualitative variables methods (step-wise). For qualitative variables, 0–1 cod- with an example of sampling season ing for k − 1 variables was used (Table 2). Sampling season Spring Autumn Winter The following classes of variables were reference classes in models: ‘summer’ for sampling season, ‘female’ for sex, Spring 1 0 0 ‘young’ for age and ‘SL’ for province. Parameters of sig- Summer 0 0 0 nificant and best fit logistic regression models obtained Autumn 0 1 0 for each analysis are shown in Table  3. The accepted Winter 0 0 1 Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 5 of 9 Table 3 Results of the best fit logistic regression models obtained for each analysis Significance assessment Independent variable Coefficient (β ) Std. error P value (Wald) Odds ratio Confidence Confidence of model (P value of LR OR − 95% OR + 95% test) Models for infection of Leptospira sp. (without distinction of serovars) < 0.001 Absolute term (β ) − 2.92912 0.296482 < 0.001 0.05 0.03 0.10 LD 1.216036 0.233494 < 0.001 3.37 2.13 5.33 MP 0.671037 0.228562 0.003 1.96 1.25 3.06 MA 1.68051 0.237135 < 0.001 5.37 3.37 8.55 OP 1.388372 0.247953 < 0.001 4.01 2.46 6.52 PK 1.769046 0.269941 < 0.001 5.87 3.45 9.96 PM 1.534127 0.265823 < 0.001 4.64 2.75 7.81 SW 0.555254 0.259964 0.03 1.74 1.05 2.90 WM 1.630786 0.20659 < 0.001 5.11 3.41 7.66 Fox density (No/km ) 1.142803 0.307487 < 0.001 3.14 1.72 5.73 < 0.001 Absolute term (β ) − 1.50766 0.198255 < 0.001 0.22 0.15 0.33 Spring 0.267965 0.280004 0.34 1.31 0.75 2.26 Autumn 0.0834 0.232275 0.72 1.09 0.69 1.71 Winter 0.688467 0.211402 0.001 1.99 1.31 3.01 Model for Icterohaemorrhagiae 0.003 Absolute term (β ) − 6.41457 0.839692 < 0.001 0.002 0.0003 0.008 Fox density (No/km ) 2.913659 0.989553 0.003 18.42 2.65 128.30 Adult − 1.18961 0.553268 0.03 0.30 0.10 0.90 Model for Grippotyphosa 0.001 Absolute term (β ) − 5.71115 0.543301 < 0.001 0.003 0.001 0.01 Fox density (No/km ) 2.364823 0.677533 < 0.001 10.64 2.82 40.19 Model for Sejroe 0.015 Absolute term (β ) − 3.43721 0.318798 < 0.001 0.03 0.02 0.06 LD 1.130284 0.386552 0.003 3.10 1.45 6.61 MP 0.35268 0.419714 0.40 1.42 0.62 3.24 MA 0.85591 0.422493 0.04 2.35 1.03 5.39 OP 0.110974 0.514159 0.83 1.12 0.41 3.06 PK 1.228934 0.461089 0.008 3.42 1.38 8.44 PM 1.047612 0.44818 0.02 2.85 1.18 6.87 SW 0.408686 0.434724 0.35 1.50 0.64 3.53 WM 0.880843 0.376223 0.02 2.41 1.15 5.05 Model for Australis < 0.001 Absolute term (β ) − 6.36907 0.610643 < 0.001 0.002 0.0005 0.01 Fox density (No/km ) 2.843724 0.730836 < 0.001 17.18 4.10 72.02 Models for Saxkoebing 0.024 Absolute term (β ) − 2.77882 0.325736 < 0.001 0.06 0.03 0.12 Spring 0.445929 0.436438 0.31 1.56 0.66 3.68 Autumn 0.408323 0.368228 0.27 1.50 0.73 3.10 Winter 0.806557 0.34165 0.02 2.24 1.15 4.38 Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 6 of 9 Table 3 (continued) Significance assessment Independent variable Coefficient (β ) Std. error P value (Wald) Odds ratio Confidence Confidence of model (P value of LR OR − 95% OR + 95% test) < 0.001 Absolute term (β ) − 2.94772 0.25661 < 0.001 0.05 0.03 0.09 LD 1.010782 0.318737 0.002 2.75 1.47 5.13 MP 0.715284 0.318663 0.03 2.04 1.09 3.81 MA 1.257746 0.322546 < 0.001 3.52 1.87 6.62 OP 1.021486 0.343397 0.003 2.78 1.42 5.45 PK 1.939495 0.341159 < 0.001 6.96 3.56 13.58 PM 1.452948 0.341859 < 0.001 4.28 2.19 8.36 SW − 0.63976 0.461137 0.17 0.53 0.21 1.30 WM 1.121314 0.296979 < 0.001 3.07 1.71 5.49 Models for Poi < 0.001 Absolute term (β ) − 5.45234 0.689411 < 0.001 0.004 0.001 0.02 LD 2.814176 0.617742 < 0.001 16.68 4.97 56.01 MP 1.032509 0.682165 0.13 2.81 0.74 10.70 MA 3.445862 0.612244 < 0.001 31.37 9.44 104.22 OP 3.293047 0.618209 < 0.001 26.92 8.01 90.51 PK 2.239957 0.687842 0.001 9.39 2.44 36.19 PM 2.960175 0.643965 < 0.001 19.30 5.46 68.24 SW 2.610502 0.629299 < 0.001 13.61 3.96 46.74 WM 3.666819 0.591781 < 0.001 39.13 12.26 124.88 Fox density (No/km ) 1.043369 0.476866 0.03 2.84 1.11 7.23 < 0.001 Absolute term (β ) − 2.89037 0.342638 < 0.001 0.06 0.03 0.11 Spring 0.375612 0.464447 0.42 1.46 0.59 3.62 Autumn 0.519876 0.383324 0.18 1.68 0.79 3.57 Winter 1.368756 0.353764 < 0.001 3.93 1.96 7.87 0.003 Absolute term (β ) − 2.30544 0.125369 0 0.10 0.08 0.13 Adult 0.437116 0.152208 0.004 1.55 1.15 2.09 LD Łódzkie, MP Leser Poland, MA Masovia, OP Opolskie, PK Subcarpathia, PM Pomerania, SW Świętokrzyskie, WM Warmia-Masuria that all provinces had significantly greater odds for hav - significance level was alpha = 0.05. STATISTICA data ing seropositive foxes than the reference SL province, in analysis software in version 10 (StatSoft, Inc.) and Arc- which the lowest percentage of seropositive foxes was GIS 10.4.1 for Desktop Standard (ESRI, Inc.) were used observed. The highest odds ratio (OR = 5.87) with the for statistical and spatial data analysis. Red fox demo- highest seroprevalence was shown for the PK province. graphics were derived from the Polish Hunting Associa- In addition, with an increase of fox density by one animal tion-PZL [6]. per km , the probability of detecting seropositive animals increased more than threefold and it almost doubled in Results winter when compared to summer. However due to data Antibodies against a Leptospira serovar was found in 561 deficiencies e.g. sampling date, seasonal influence on the serum samples (26.3%). The highest seroprevalence was obtained serological results was analysed using a separate observed in foxes hunted in the Subcarpathia (41.6%) logistic regression model. and Warmia-Masuria provinces (40.3%) (Table  1, Fig.  1). Based on analyses for individual serovars, an increase Specific antibodies were mainly directed against Poi of fox density by one animal per k m increased the risk (12.4%), Saxkoebing (11.3%), and Sejroe (6.0%) serovars of being seropositive by 2.8, 10.6, 17.2 and 18.4 times for with serum antibody titres up to 1:25,600 in individual the serovars Poi, Grippotyphosa, Australis and Ictero- animals (Table 4). When analysing the logistic regression haemorrhagiae, respectively. The models also show a sig - model of positive and negative serostatus (excluding data nificant influence of the province on the proportion of related to individual Leptospira serovars), a significant seropositive samples. A significantly higher risk of being influence of the area (province) and associated density seropositive to Sejroe serovar was observed in the LD of foxes on the serostatus was found. The model showed Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 7 of 9 Table 4 Distribution of pathogenic Leptospira antibody titers for 561 positive red foxes hunted during season 2014–2015 in Poland Serovar No of antibody-positive samples (%) Prevalence of serovar (95% 1:100 1:200 1:400 1:800 1:1600 1:3200 1:6400 1:12800 1:25600 Total CI) (%) Icterohaemorrhagiae 8 (0.4) 3 (0.1) 3 (0.1) 1 (0.05) 2 (0.1) 1 (0.05) 0 0 0 18 0.8 (0.5–1.3) Grippotyphosa 6 (0.3) 16 (0.75) 8 (0.4) 4 (0.2) 1 (0.05) 2 (0.1) 0 0 0 37 1.7 (1.2–2.4) Sejroe 39 (1.8) 37 (1.7) 30 (1.4) 16 (0.75) 2 (0.1) 2 (0.1) 0 0 1 (0.05) 127 6.0 (5.0–7.0) Tarassovi 0 1 (0.05) 0 0 0 0 0 0 0 1 0.1 (0.0–0.3) Pomona 7 (1.5) 7 (1.5) 8 (0.4) 8 (0.4) 2 (0.1) 2 (0.1) 0 0 0 34 1.6 (1.1–2.2) Canicola 0 2 (0.1) 1 (0.05) 0 0 0 0 0 0 3 0.1 (0.0–0.4) Australis 7 (1.5) 11 (0.5) 1 (0.05) 7 (1.5) 2 (0.1) 0 0 0 0 28 1.3 (0.9–1.9) Saxkoebing 63 (3.0) 55 (2.6) 66 (3.1) 44 (2.1) 8 (0.4) 3 (0.1) 2 (0.1) 0 1 (0.05) 242 11.3 (10.0–12.8) Ballum 0 1 (0.05) 1 (0.05) 1 (0.05) 0 0 0 0 0 3 0.1 (0.0–0.4) Poi 64 (3.0) 68 (3.2) 63 (3.0) 34 (1.6) 11 (0.5) 19 (0.9) 4 (0.2) 1 (0.05) 1 (0.05) 265 12.4 (11.1–13.9) Bataviae 1 (0.05) 1 (0.05) 3 (0.1) 3 (0.1) 0 1 (0.05) 0 0 0 9 0.4 (0.2–0.8) Hardjo 0 2 (0.1) 0 1 (0.05) 0 0 1 (0.05) 0 0 4 0.2 (0.1–0.5) Antibodies against serovar Poi were the most com- (OR = 3.1), MA (OR = 2.4), PK (OR = 3.4), PM (OR = 2.9) monly detected. Exposure of foxes to this serovar is not and WM (OR = 2.4) provinces compared to the SL surprising given the results of previous Polish studies province. where serogroup Javanica (to which serovar Poi belongs) When compared to the reference SL province, antibod- was also reported in horses, goats, and sheep [15–17]. ies to the Saxkoebing and Poi serovars were more preva- Besides serovar Poi, antibodies against serovar Sejroe lent in foxes from all provinces except SW (OR from 2.0 were also prevalent in foxes. This is consistent with other to 7.0), and MP province (OR from 9.4 to 39.1) respec- studies as serovars Hardjo, Sejroe and Saxkoebing (all tively. An impact of the season on the seroprevalence belonging to the Sejroe serogroup) are widely prevalent to particular serovars was observed. Antibodies against in animals in Europe [18–21]. MAT reactions to serovar serovars Saxkoebing and Poi were ~ 2 and 4 times more Hardjo commonly detected in sheep and cattle [18–20, frequent, respectively, during the winter period than dur- 22, 23] were not common in foxes. The presence of sero - ing summer. The age of the foxes influenced the serosta - positive animals to this serogroup could be mainly attrib- tus for some serovars such as Icterohaemorrhagiae that uted to Sejroe or Saxkoebing serovars (Table  4). It may was detected more frequently in young foxes (OR = 3.3) be associated with fox diet as the main source of food and Poi found more often in adults (OR = 1.5) (Table  3). for red foxes are wild small mammals, which are known Using a one-factor model the association between influ - reservoirs of Saxkoebing and Sejroe serovars [24]. Anti- ence of sex on serostatus was not significant (LR-test bodies to Sejroe serogroup were previously detected in P = 0.0525, OR = 1.44, 95% CI 0.99–2.09). pigs, dogs, horses and cattle in Poland confirming a wide - spread exposure of different animal species to leptospires Discussion from this serogroup [15, 25–28]. In addition, this indi- Other serological surveys have shown that red foxes are cates an endemic occurrence of this serovar and a pos- frequently exposed to Leptospira spp. of different sero - sible role of the environment in pathogen transmission. vars [10, 11, 13]. However this is the first prevalence The observed regional differences in exposure to different study on the occurrence of antibodies to a broad range Leptospira serovars may be related to active circulation of of Leptospira serovars in a red fox population in eastern Leptospira spp. in the environment [12]. Europe. The high seroprevalence (26.3%) in red foxes in Studies conducted in other European countries pro- Poland is comparable to that found in Spain (47.1%) [10] vide scientific evidences that the most common serovar and Croatia (31.3%) [13] but higher than in other Euro- among red foxes is serovar Icterohaemorrhagiae [10, 11, pean countries such as Germany (1.9%) [14] and Norway 13], which however seems to be rare in the Polish red fox (9.9%) [11]. Hypothetically any pathogenic Leptospira population (Table 4). As leptospires are sensitive to desic- may infect domestic and wild animals, but in practice cation, the regional differences in climate conditions may only a small number of serovars are endemic in any par- have a significant influence on seroprevalence in general ticular region. Żmudzki et al. Acta Vet Scand (2018) 60:34 Page 8 of 9 to warmly thank Artur Rzeżutka for useful comments and editing of the or for some serovars in particular. In that aspect, Poland manuscript. differs from other countries such as Spain and Croatia where the seroprevalence of Leptospira spp. in foxes has Competing interests The authors declare that they have no competing interests. been investigated [10, 13]. Although the studies were conducted on a reasonable Availability of data and materials number of hunted animals originating from different All data generated or analysed during this study are included in this published article. locations across the country, the number of tested serum samples of red foxes did not fully reflect the size of the Consent for publication animal population present in the studied provinces. It Not applicable. could be taken as a major limitation to interpretation of Ethics approval and consent to participate the occurrence and prevalence of tested Leptospira sero- All procedures were carried out according to the ethical standards for the use vars in the Polish population of red foxes. Nevertheless, of animal samples. The study was approved by the Ethical Committee of the University of Life Sciences in Lublin under Grant of Approval No. 30/2016. the findings still provide useful data on the seroepidemi - ology of red foxes exposed to different Leptospira sero - Funding vars in this part of Europe and their role as an important This study was supported by the Polish National Science Centre (Grant No. 2013/09/B/NZ7/02563). The open access publication fee was funded by the source of zoonotic Leptospira spp. for humans. KNOW (Leading National Research Centre) Scientific Consortium “Healthy Animal—Safe Food”, Ministry of Science and Higher Education resolution no. Conclusions 05-1/KNOW2/2015”. Red foxes of central and eastern Poland, particularly in the Subcarpathia and Warmia-Masuria regions, are Publisher’s Note Springer Nature remains neutral with regard to jurisdictional claims in pub- highly exposed to Leptospira spp. Due to the high preva- lished maps and institutional affiliations. lence of foxes, their predatory behaviour and their varied diet mainly composed of small mammals, they could be Received: 7 September 2017 Accepted: 24 May 2018 considered as sentinel animals of environmental contam- ination with leptospires. Interactions between animals require further epidemiological investigations to eluci- References date the role of wild carnivores as a reservoir of rarely 1. Jancloes M, Bertherat E, Scheider C, Belmain S, Munoz-Zanzi C, Hartskeerl occurring Leptospira serovars pathogenic for other ani- R, et al. Towards a “one health” strategy against leptospirosis. Planet@Risk. mals and humans. 2014;2:204–6 (Special Issue on One Health (Part I/II): GRF Davos). 2. 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Acta Veterinaria ScandinavicaSpringer Journals

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