Research Article: Prevalence and Genotype Allocation of Pathogenic Leptospira Species in Small Mammals from Various Habitat Types in Germany

Date Published: March 25, 2016

Publisher: Public Library of Science

Author(s): Anna Obiegala, Dietlinde Woll, Carolin Karnath, Cornelia Silaghi, Susanne Schex, Sandra Eßbauer, Martin Pfeffer, Mathieu Picardeau.

Abstract: Small mammals serve as most important reservoirs for Leptospira spp., the causative agents of Leptospirosis, which is one of the most neglected and widespread zoonotic diseases worldwide. The knowledge about Leptospira spp. occurring in small mammals from Germany is scarce. Thus, this study’s objectives were to investigate the occurrence of Leptospira spp. and the inherent sequence types in small mammals from three different study sites: a forest in southern Germany (site B1); a National Park in south-eastern Germany (site B2) and a renaturalised area, in eastern Germany (site S) where small mammals were captured. DNA was extracted from kidneys of small mammals and tested for Leptospira spp. by real-time PCR. Positive samples were further analysed by duplex and conventional PCRs. For 14 positive samples, multi locus sequence typing (MLST) was performed. Altogether, 1213 small mammals were captured: 216 at site B1, 456 at site B2 and 541 at site S belonging to following species: Sorex (S.) araneus, S. coronatus, Apodemus (A.) flavicollis, Myodes glareolus, Microtus (Mi.) arvalis, Crocidura russula, Arvicola terrestris, A. agrarius, Mustela nivalis, Talpa europaea, and Mi. agrestis. DNA of Leptospira spp. was detected in 6% of all small mammals. At site B1, 25 small mammals (11.6%), at site B2, 15 small mammals (3.3%) and at site S, 33 small mammals (6.1%) were positive for Leptospira spp. Overall, 54 of the positive samples were further determined as L. kirschneri, nine as L. interrogans and four as L. borgpetersenii while five real-time PCR-positive samples could not be further determined by conventional PCR. MLST results revealed focal occurrence of L. interrogans and L. kirschneri sequence type (ST) 117 while L. kirschneri ST 110 was present in small mammals at all three sites. Further, this study provides evidence for a particular host association of L. borgpetersenii to mice of the genus Apodemus.

Partial Text: Leptospira spp. are helical-shaped bacteria and form a particular group of causative agents for the zoonotic disease Leptospirosis. Leptospira spp. are transmitted through infected urine of small mammals or contaminated water via the direct contact to skin lesions or conjunctivae [1]. Small mammals are described as the most important maintenance reservoirs in nature and thus as an essential vector for several pathogenic Leptospira spp. [2, 3, 4, 5]. Leptospirosis is considered the most widespread zoonotic disease worldwide, which is of emerging concern [6]. In the past, Leptospirosis was described to be a disease of occupational risk for harvesters, miners, veterinarians and rodent control workers in Europe [2, 7]. Nowadays, it is increasingly linked to recreational outdoor activities, such as water sports and adventure travels [5, 8]. However, partially due to the broad variety of clinical symptoms, which are nonspecific, the awareness for this disease is not yet present especially in temperate regions [5, 9]. The estimated incidence of clinical cases per year is 0.2 / 100,000 in Germany [10]. Severe cases associated with rats have also been reported [8, 11]. Recently, human cases, which were linked to contaminated water or soil, occurred in Austria [12, 13, 14]. Furthermore leptospirosis outbreaks were reported among triathletes and strawberry harvesters in Germany [15, 16]. The clinical severity of Leptospira spp. infection depends on the virulence of the infecting Leptospira serovar as well as on the health status of the patient [3]. The taxonomy of Leptospira spp. is complex. To date, ten different pathogenic Leptospira species with more than 300 serovars, grouped in 20 serogroups are known [17]. The term serogroup is of taxonomic importance and defines groups with antigenetically related serovars. However identical serovars may belong to different Leptospira species [2]. Duplex PCR [18] and detailed sequence typing are used for the characterisation of Leptospira spp. strains and genotypes while the microscopic agglutination test (MAT) which is important for the categorisation of serovars, is still the gold standard in routine diagnostics [19]. Most commonly, human clinical cases in Europe are caused by L. interrogans and/or Leptospira spp. serovar Grippotyphosa [12, 13, 16, 20]. A recent study from Poland reported also antibody titres in humans against the serovars Australis, Autumnalis, Hebdomadis, Hardjo, Sejroe, Zanoni, Bataviae, Bratislava, Canicola and Grippotyphosa, belonging to 3 species, L. interrogans, L. borgpetersenii and L. kirschneri[21]. Studies from Germany and France reported high prevalences for Leptospira spp. in small mammals which are likely responsible for simultaneous human leptospirosis cases [16, 20]. So far there are only a few studies reporting moderate to high prevalences in small mammals, beavers (Castor fiber) and wild boars (Sus scrofa) from Germany [5, 8, 22]. Little is known about the prevalence and the geographic distribution of pathogenic Leptospira spp. in rodent maintenance hosts in Germany. Possible host-pathogen associations were not further determined thus far.

This study focussed on pathogenic Leptospira species in small mammals from selected habitats in Germany. Studies on prevalences for Leptospira spp. in mammals in Europe are rare and focussed mainly on larger rodent species such as rats (Rattus norvegicus) which are considered to be the major source of Leptospira infection for humans [34, 35]. High prevalences (20–88%) in Rattus norvegicus have been reported from different European countries such as Turkey, France and Denmark [36, 37, 38]. Studies from Germany, Switzerland, the Netherlands, Croatia and Austria showed the occurrence of Leptospira spp. in a wide range of different small mammal species including M. glareolus, Apodemus spp., Mi. arvalis, Mus musculus, Castor fiber and Sorex spp. (2.9–71.4%)[5, 22, 39, 40, 41, 42, 43]. This study’s prevalences show a similar wide range in prevalence regarding the investigated rodent species (5.3–42.9%). The highest prevalence in small mammals was detected at site B1, a forest in southern Bavaria in comparison to the other two study sites. A recent German study showed high prevalences of leptospiral DNA in Mi. arvalis and A. agrarius (12–14%) which are supposed to be the most common carrier hosts for L. kirschneri [5]. In this study the highest prevalence was also found in both of these rodent species for L. kirschneri. Leptospira kirschneri was detected in almost all investigated rodent species (M. glareolus, Mi. arvalis, A. flavicollis, A. agrarius) with the exception of Mi. agrestis suggesting that this Leptospira species has a broad host range and is well adapted to a number of different small mammal species. Additionally, L. kirschneri was found in Mustela nivalis but not in Sorex spp. which therefore may play a subordinate role as maintenance host for L. kirschneri.