Leptospirosis is a re-emerging zoonosis of worldwide distribution . The infection is caused by pathogenic spirochaetes of the genus Leptospira and despite affecting most mammals, domestic and wild, rodents are considered the main reservoirs and shedders .
The wild boar (Sus scrofa) is widely distributed in Europe, and its populations have increased in the last decades . Their large movements and dispersion can potentially result in the spread of several infectious diseases, namely emerging and re-emerging zoonosis, such as leptospirosis.
Studies in European wild boar populations seem to indicate that this species might play an important role in the epidemiological cycle of leptospirosis [3, 4], being a potential transmission source of pathogenic leptospires to humans, livestock and other local sylvatic species, that share the same geographical areas [5, 6].
The goal of this study was to determine the prevalence of specific antibodies against Leptospira interrogans sensu lato (s.l.) in wild boar from the Trás-os-Montes region, Northern Portugal, in order to improve the understanding of the role of this species in the epidemiology of leptospirosis in this region. Similar studies have been made in other European countries, but never in Portugal.
Wild boar hunts were selected from a list of hunts planned to occur between October and February of the 2011/2012 and 2012/2013 hunting seasons in the Trás-os-Montes region. A total of 27 hunts were attended during the sampling period, taking into consideration the spatial distribution of the hunts in the districts of Vila Real and Bragança, in order to cover as much area as possible. Convenience sampling was performed taking into account the active collaboration of hunters and hunt managers (i.e. by authorizing biological sample collection), and the number of animals shot.
Blood samples were collected from 101 wild boars slaughtered during the above-mentioned hunting seasons. The blood was centrifuged to extract the serum and then stored at −20 °C until serological analysis. Additional animal information was also collected: district of origin, gender and age. Age estimation was based on tooth eruption patterns and according to three age groups: juveniles (<12 months), subadults (12–24 months) and adults (>24 months) .
To detect the presence of leptospiral antibodies, serum samples were tested by the microscopic agglutination test (MAT) , using the following 17 live serovars (local and reference strains): Altodouro (strain Rim 139-local isolate), Arboreae (Arborea), Autumnalis (Akiyami A), Bratislava (Jêz Bratislava), Bataviae (Van Tienen), Canicola (Hond Utrecht IV), Celledoni (Celledoni), Valbuzzi (Valbuzzi), Hebdomadis (Hebdomadis), Copenhageni (M20), Icterohaemorrhagiae (RGA), Mini (Sari), Panama (CZ 214), Mozdok (5621), Hardjobovis (Lely 607), Wolffi (3705) and Tarassovi (Perepelitsin).
Sera were screened at a 1:50 dilution, and positive samples were titrated in serial twofold dilutions to determine the end-point titre. Samples with titres of 1:50 were considered to provide evidence of exposure to Leptospira interrogans s.l., since higher dilutions may not detect the infection at an early stage or represent a residual level of antibodies years after the exposure to the agent .
Statistical analysis was performed using JMP® v. 9.0.1 (SAS Institute Inc., USA). Leptospiral seroreactivity status (positive or negative) was used as dependent variable, and age (juveniles, subadults, adults), gender (males and females), and district of origin (Vila Real or Bragança) as independent variables for χ
2 tests. A P value <0·05 was considered significant. Risk factors for the presence of antibodies against Leptospira were assessed by nominal logistic regression analysis. The strength of association between seropositivity and other variables were estimated by the calculation of odds ratio (OR). An OR value with a lower limit of 95% confidence interval (CI) >1 was taken to indicate a significant association between variables.
The wild boar population analysed encompassed 101 animals categorized by: gender (33 males, 68 females), district of origin (56 from Bragança, 45 from Vila Real), and by age group (27 juveniles, 35 subadults, 39 adults). After preliminary statistical analysis, the animals were regrouped in just two groups: juveniles and adults (‘adult’ group encompasses both subadult and adult animals). Reorganization of data added robustness to the statistical results, rendering the variable ‘age’ more explanatory and statistically significant in the context of leptospiral seropositivity in the analysed population. The serological results confirmed the presence of antibodies against nine pathogenic serovars of Leptospira (Tarassovi, Altodouro, Autumnalis, Bratislava, Copenhageni, Mozdok, Arboreae, Ballum, Icterohaemorrhagiae) in the wild boar population from the Trás-os-Montes region (Table 1). Seroreactivity or cross-reactions of a serum sample to multiple serovars within a serogroup or different serogroup strains was frequently observed, as stated previously . The serogroup of the strain that had reacted with the highest titre was considered to be the presumptive serogroup of the strain responsible for the infection.
Table 1. Distribution of positive wild boar from the Trás-os-Montes region (Northern Portugal), according to serogroup and serovar reactivities detected by MAT
Serological titres ranged from 1:50 to 1:1600, and the majority (63·6%) of positive samples demonstrated low titres (⩽1:100). This finding is consistent with other serosurvey studies in which low titres were frequently observed [6, 9, 10]. This fact is the reason why, for further statistical analysis, the authors considered sera samples with titres ⩾1:50 as positive, bearing in mind that in epidemiological studies it is much more important to establish the animals' exposure to the aetiological agent independently of determining if there is any evidence of current infection [8, 11]. The highest titres ⩾1:1600 were obtained for just one sample, against serovar Bratislava. This sample also showed reactivity to multiple serovars at lower titres.
A total of 66 (65·4%) wild boar showed antibodies at a titre of ⩾1:50 and 34·7% at a titre of ⩾1:100, the cut-off titre most currently used by other authors in identical studies [6, 9, 10, 12]. The seropositivity rate obtained in this study is greater than those observed comparatively in other European countries such as Sweden (3·1%) , Italy (6%) , Spain (14·4%) , Germany (18%)  and the Czech Republic (16·9%) , but lower than the rates reported in Slovenia (45·8%)  and Croatia (35·0%) .
Titres to Tarassovi and Pomona serogroups were the most common (23·8% and 19·8%, respectively) with titres ranging from 1:50 to 1:400 (Table 1). Seroreactivity to Pomona group was mainly due to serovar Altodouro, which was responsible for 16·8% of the observed reactivity. Serovar Altodouro, a new serovar from the Leptospira kirschneri genospecies, was recently isolated from a Mus musculus rodent species in the Trás-os-Montes region [1, 16]. Later studies in dogs from Greece  have shown that this serovar seems highly reactive, which, in addition to our findings, may indicate the need to include serovar Altodouro as antigen in coming leptospirosis serological studies across Europe, concerning both domestic and wild populations. Several other strains from the Pomona group have been previously isolated in Portugal: serovar Mozdok strains from pigs  and small mammals , and serovar Tsaratsovo from horses . Pigs are commonly considered reservoirs of strains from serogroup Tarassovi  and Pomona , and serovars Bratislava and Muenchen (Australis group) were previously isolated in pigs from Northern Ireland . The role of the wild boar is questionable as a potential host for Tarassovi because strains from this serogroup were never isolated from this species, but strains from Pomona, Australis and Icterohaemorrhagiae groups have been isolated from wild boar kidneys in Croatia , as well as strains from Pomona serovar in Italy .
Several authors have reported seropositivity to different serovars in wild boar populations worldwide [6, 10, 12–15]. Seroreactivity to Tarassovi has been frequently reported [6, 15, 23] and is, similarly to our study, the most reactive serovar observed in wild boar from Slovenia . Antibodies against Pomona is the most frequently reported leptospiral seroreactivity in wild boar populations in Spain [13, 24], and Germany , and the second most common in Croatia .
In our study, titres to Autumnalis (7·9%), Australis (6·9%), Icterohaemorrhagiae (4%) and Ballum (3%) groups were less frequently observed. Antibodies to serovar Bratislava were less frequently detected than expected, since reactivity to strains of the Australis serogroup is much more often observed, and were the most prevalent in similar studies conducted in Italy  and Sweden .
Adult wild boars showed a higher seropositivity rate (71·6%) than juveniles (48·2%), and the differences were statistically significant (P < 0·05) (Table 2). Age seems to be an important risk factor for the presence of leptospiral antibodies in wild boar from the Trás-os-Montes region (OR 2·72, 95% CI 1·096-6·742), as shown in similar studies from the Czech Republic , Germany  and Croatia . These results could obviously be anticipated since the possibility of direct or indirect contact with leptospirosis increases over the lifespan.
Table 2. Wild boar distribution data and results of statistical analysis of serological reactivity to Leptospira strains (Trás-os-Montes region, Northern Portugal)
Gender-wise, males showed a higher seropositivity rate (72·7%) than females (61·8%). Nevertheless, significant statistical differences (P > 0·05) were not found (Table 2). These findings corroborate the data presented in studies from Slovenia , Germany  and the Czech Republic .
Regarding district, the seropositivity rate was higher in Bragança (76·8%) than in Vila Real (51·1%) with significant statistical differences (P < 0·05) (Table 2). District of origin seems to be an important risk factor for the presence of leptospiral antibodies in wild boar from the Trás-os-Montes region (OR 3·16, 95% CI 1·349-7·412). Other studies in the Trás-os-Montes region corroborate this tendency. Similar results were obtained for indigenous Maronesa cattle from the Trás-os Montes region . Climate and land use distribution seem to explain differences within each of these areas. Bragança is much more rural than the district of Vila Real, with a higher percentage of forested land, especially broad-leaved forest. This type of land cover benefits high humidity in the soil, thus contributing to the maintenance of leptospires in the environment for longer periods of time .
In the highly forested region of Trás-os-Montes, hunting activities have great economic impact, putting humans in closer contact with wild animals (reservoirs for these spirochaetes), especially occupational risk groups (hunters, gamekeepers, other forestry-related professionals).
From a One Health perspective, these study results revealed that wild boar should be considered as a potential source of dissemination of pathogenic leptospires for humans, domestic animals and other wild species in shared geographical regions, particularly in the Trás-os-Montes region.
We thank Teresa Carreira of the Group of Leptospirosis and Lyme Borreliosis, Unit of Medical Microbiology, Institute of Hygiene and Tropical Medicine (IHMT), University Nova of Lisboa (UNL), who kindly provided several Leptospira strains. The authors also thank Professor Madalena Vieira-Pinto of the Department of Veterinary Sciences, University of Trás-os-Montes and Alto Douro (UTAD), Catarina Coelho of the Agrarian School of Viseu (ESAV), Polytechnic Institute of Viseu, Eduardo Sousa, Bruno Vinhas and the hunting associations and hunt organizers for their collaboration and support during field work carried out during the wild boar hunting seasons of 2011/12 and 2012/13 in the Trás-os-Montes region.
This study was supported by funding from several ecological monitoring projects of the Laboratory of Applied Ecology (University of Trás-os-Montes and Alto Douro, UTAD) and by the Portuguese Foundation for Science and Technology (FCT) through project PEst-OE/AGR/UI4033/2014.
DECLARATION OF INTEREST
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