Leptospira interrogans Sensu Lato in Wild Small Mammals in Three Moravian Localities of the Czech Republic

Leptospirosis is a widespread zoonosis, affecting humans, domestic animals and wildlife, with small mammals as a reservoir of this infection. In recent years, this disease has been re-emerging and affects approximately 1 million people all over the world each year. Due to this disease having a significant health impact, it is important to identify the source and method of infection. The risk of Leptospira sp. infection is higher mainly in the cities of developed and industrialised countries. The aim of the study was the detection of antibodies against Leptospira sp. in some wild small mammals captured in the Czech Republic. In total, samples of 855 animals captured in three locations of Moravia during a six-year study (2010–2015) were examined by a microscopic agglutination test, using eight serovars of Leptospira interrogans sensu lato, representing serogroups Grippotyphosa, Icterohaemorrhagiae, Australis, Canicola, Sejroe, Javanica, Pomona and Pyrogenes, as antigens. Antibodies to Leptospira sp. were detected in 6.1% (52/855) of animals, with a prevalence of 6.4% (51/801) and 1.9% (1/54) in rodents and insectivores, respectively. The only statistically significant difference (p ≤ 0.05) was in prevalence between individual species (0–33%), while there were no differences in sex (6.7% in females and 5.1% in males), locality (1.8–8%) and year of trapping (0–8.4%). Only two serovars, L. interrogans serovar Pomona and L. interrogans serovar Grippotyphosa, were detected in 5.5% and 0.5% of animals, respectively. The prevailing serovar of pathogenic L. interrogans s.l. can be identified in a number of infected people in the Czech Republic. The composition of vaccines should be based on the current occurrence of Leptospira serovars in the actual territory. For this reason, the occurrence of Leptospira and its serovars should therefore be regularly monitored.

In the Czech Republic, the annual incidence of human leptospirosis does not change significantly, and a higher number of patient cases occurs with occasional outbreaks during the periodic overpopulation of wild small mammals or after unexpected flood events [9]. For example, a three-times higher incidence of leptospirosis was recorded after the floods in 1997 and 2002 [9]. With respect to the general importance of spirochaetal zoonoses and the increasing number of patients with Lyme borreliosis and leptospirosis in the Czech Republic in recent years [10], the role of reservoirs should be considered. Leptospirosis may cause very serious damage to tissues and organs and thus has a significant health impact, affecting an estimated 1.03 million humans annually worldwide and causing 58,900 deaths [11].
The aim of this study was to determine the prevalence of antibodies to Leptospira in some wild small mammals captured in the Czech Republic, to identify the causative agent of leptospirosis among small mammals and the serovar of Leptospira circulating among them.

Discussion
Leptospirosis is an infection of global importance. Antibodies to Leptospira sp. were detected by MAT, e.g., in 92% of various species of rats from Philippines [12], in 68% of R. norvegicus from Brazil [13], and in 52% of R. norvegicus captured near human dwellings in Argentina [14]. Molecular methods were used in studies from the Canary Islands, where L. interrogans serovar Copenhageni and L. borgpetersenii were found in 14.8% of small mammals [15], and L. interrogans s.l. (L. borgpetersenii, L. interrogans, L. kirschneri, and L. weilli) was found in 7% of small mammals from Southeast Asia [16]. High prevalence of 22% was noted also in humans from Brazil, when 812 suspected cases of leptospirosis were examined from the national reference laboratory by MAT, with the most prevalent serogroup being Icterohaemorrhagie, followed by Pomona, Ballum, and Canicola [17].
In Slovakia, antibodies to Leptospira spp. were detected by MAT in 5% of 11 species of wild mammals [18]. In Croatia, the prevalence of Leptospira spp. in small rodents by MAT was 64% with serogroups L. australis and L. grippotyphosa [19] and 12.7% with serovars Sejroe, Pomona, and Australis [20]. In France, the prevalence of Leptospira spp. in small mammals by MAT was 30.8% with the main serovar L. icterohaemorrhagiae [21], 53% by MAT and PCR with the predominant serovar Icterohaemorrhagiae, followed by Sejroe, Grippotyphosa, and L. interrogans serogroup Australis [22], and 19.2% by RT-PCR with serotype L. interrogans [23]. The prevalence of Leptospira spp. in rats caught in six localities in Denmark in 2006-2007 was 48-89% by PCR [24]; some of the samples were examined by MAT, showing the most common serogroup to be Pomona, Sejroe, and Icterohaemorrhagiae. In rats, the prevalence of Leptospira spp. was recorded as 14% in England, using different diagnostic tests [25], or 45.5% in Italy, using PCR [26]. In Germany, leptospiral DNA was detected by duplex PCR in 10% of wild small mammals, with 13% in Microtus spp., 11% in Apodemus spp., and 6% in Clethrionomys spp. [27]. In Germany, Romanian and Slovakian harvesters working outdoors in nature had a 49% prevalence of Leptospira spp., as tested by MAT and confirmed by ELISA, with main serotypes being Grippotyphosa, Pomona, Bratislava, and other serogroups (Copenhageni and Pomona) [28]. New, unexpected cases of zoonoses, specifically leptospirosis, can still appear. An example is the unusual increase in icteric bovine aborted foetuses in Belgium in 2014 [29]. Most foetuses presented jaundice and splenomegaly, and cows undergoing icteric abortions had antibodies against Leptospira serogroups Australis or Grippotyphosa.
The notification rate of leptospirosis in the European Union in 2014 was 0.23 cases per 100,000 inhabitants, which represents a two-fold increase in comparison with the average number of confirmed cases in previous years. However, on the other hand, this average of the European number is equal to the average number of patients in the Czech Republic [30]. The seroprevalence of Leptospira in wild small mammals caught in the Czech Republic can be compared with the incidence of human leptospirosis in the observed years 2010-2015 ( Figure 1, [10]), with an average of 20 human patients per year that became ill. In the following years, it was, e.g., 25 patients that suffered from disease in 2019 (approximately 0.25 cases per 100,000), 29 patients in 2020 (approximately 0.29 cases per 100,000) and 31 patients in 2021 (which corresponds to approximately 0.31 cases per 100,000 inhabitants). These data demonstrate that human leptospirosis in the Czech Republic appears annually in similar numbers [31], but occasional outbreaks are observed during the periodic overpopulation of wild small mammals or after floods, e.g., in the years 1997 (92 patients) and 2002 (94 patients). Four cases of Weil disease (leptospirosis) reported in 1997 were fatal.
However, Ref. [4] warned that the incidence of leptospirosis may be significantly underestimated, because many cases are asymptomatic or with a slight clinical manifestation and, moreover, some cases may be inaccurately diagnosed, diagnosed late, or misdiagnosed. In the Czech Republic, a decreasing trend with occasional fluctuations in the incidence of human leptospirosis was observed in 2008 compared to recent decades, but future trends are hardly predictable. Thus, the monitoring of Leptospira circulating in the environment is essential for a description of the actual epidemiological situation, management of the disease, and its future prevention.

Value of Prevalence/ Incidence
Year

Incidence of leptospirosis in Czech Republic
Prevalence of Leptospira sp. antibodies in small mammals

Incidence of human leptospirosis in the Czech Republic
Prevalence of Leptospira sp. antibodies in wild small mammals Out of a total of 46 captures of small mammals from May to November, the most frequent species among 855 individuals was A. flavicollis (n = 497), followed by M. glareolus (n = 191), A. sylvaticus (n = 57), S. araneus (n = 53), A. agrarius (n = 50), M. arvalis (n = 9), and T. europea (n = 1). A similar representation of animal species was recorded in a previous study from the Czech Republic [32,33], as well as in other European countries, e.g., in Switzerland [34], Croatia [35], and Lithuania [36].
The prevalence of antibodies to eight serovars of Leptospira spp. was tested by MAT, which is considered to be the gold standard for the diagnosis of leptospirosis [37]. In MAT, a panel of live leptospires, belonging to recent isolates and representing the serovars circulating in the Czech Republic, were used. Individual leptospiral serovars have their typical animal reservoirs, which can be both in wild and domestic animals. The most common reservoirs are Rattus norvegicus, Microtus arvalis, and Microtus agrestis [38]. However, a large number of other vertebrates can serve as reservoirs of infection [38]. Previous studies from different regions of the Czech Republic showed a 12% [8] and 9% [39] prevalence of Leptospira spp. in wild small mammals, with M. arvalis being the most infected of five animal species. In our study, M. arvalis also showed the highest prevalence (33%). Among species of small mammals examined in our study, there was high variability (from 1.9% to 33.3%) in the production of antibodies against leptospira spp. The prevalence of Leptospira spp. did not differ according to the sex of animals, localities, or year of sampling.
In the Czech Republic, serovars Grippotyphosa, Icterohaemorrhagiae, Copenhageni, Sejroe, Bratislava, Pomona, Canicola, and Sorex-jalna have so far been isolated from their main hosts, such as M. arvalis, R. norvegicus, R. rattus, Mus musculus, Apodemus sp., S. araneus, Erinaceus sp., Sus sp., and Canis lupus f. familiaris [3,31]. In our study, antibodies to two serovars of L. interrogans (Pomona and Grippotyphosa) were the only ones observed in a six-year study, with the range of 800-3200 and 200-3200 titres, respectively, indicating the gradual development of infection in the host organism. Serovar Grippotyphosa was also the most prevalent serovar in horses [40] and humans [41] in the Czech Republic. In contrast, serovars Copenhageni and Icterohaemorrhagiae, transmitted by rats, are usually responsible for infections in Europe [42]. This is why the geographic location, and the ecology of reservoirs, affect the prevalence of specific serovars involved in infection. Anti-bodies to serovar Pomona were detected only in A. agrarius (8.0%), which belongs to the most important host of this serovar in the Poodří locality, while antibodies against serovar Grippotyphosa were detected in six species in all three localities, which is in accordance with the results from other studies [38,43].
Based on the incidence of leptospirosis in recent years, preventive measures appear to be relatively effective. The increase in the number of patients after the floods in 1997 and 2002 is an exception. However, anti-spread measures were not effective enough during this outbreak [5]. At present, the development of a human vaccine is not an issue in the Czech Republic. However, in the field of veterinary medicine, the vaccination of domestic animals should not be neglected. The composition of vaccines should be based on the current occurrence of Leptospira serovars in the actual territory. For this reason, the occurrence of Leptospira and its serovars should be regularly monitored. Moreover, with respect to potential epidemiological risks, occasional serological surveys of reservoirs and other hosts, as well as of the local human population, should be performed.
This study presents leptospirosis as a disease that is not so epidemiologically significant in our natural conditions as it is in the tropics, but it should not be underestimated in any case. In our 6-year study, two serovars (L. interrogans serovar Pomona and L. interrogans serovar Grippotyphosa) were detected in wild small mammals. With respect to the potential risk of infection, regular monitoring should be conducted not only in humans but also in animals, especially in small mammals, as one of the main sources of Leptospira spp. A total of 855 wild small mammals belonging to 5 species of rodents and 2 species of insectivores were trapped (Table 1). Animals caught by both types of traps were dissected and hearts were removed, cut, and printed on a piece of filter paper, which was placed in the fridge at a temperature of 4 • C. Blood drawn from the carotid artery of anaesthetised living individuals was used to obtain serum, which was stored at −18 • C until assays.

Detection of Antibodies to Leptospira by Microscopic Agglutination Test
Samples on filter papers were examined by a microscopic agglutination test (MAT) [44]. Eight serovars of Leptospira interrogans sensu lato (Lisl) (Grippotyphosa, Icterohaemorrhagiae, Bratislava, Canicola, Sejroe, Sorex jalna, Pomona, and Pyrogenes), belonging to serogroups Grippotyphosa, Icterohaemorrhagiae, Australis, Canicola, Sejroe, Javanica, Pomona, and Pyrogenes, respectively, represent the most prevalent Leptospira serovars in Europe [6]. These serovars were stored long-term in liquid nitrogen at a temperature of −196 • C. Before use, ampoules with Leptospira cultures were thawed and cultured at 28 • C in commercial media (Ellinghausen-McCullough-Johnson-Harris or HIMEDIA Leptospira HiVeg Medium Base, Korthof Modified, REF MV457Z, Test Line, Brno, Czech Republic), with the addition of 10% rabbit serum (Sigma Aldrich, Prague, Czech Republic), to the con-centration of approximately 2 × 10 8 leptospires per ml. The density of Leptospira cultures was determined using a Petroff-Hausser counting chamber after 5-7 days of cultivation. Cultures in concentrations of approximately 2 × 10 8 leptospires per ml were used for MAT. The degree of agglutination (reaction of antigen with antibodies) was evaluated by dark-field microscopy. Samples were marked positive if more than 50% of Leptospira appeared to be agglutinated. Samples with titres ≥100 were considered positive.

Statistical Analysis
The results were statistically analysed, taking into consideration species composition, sex, locality, and year of trapping. Data analysis was performed with Pearson's chi-square test for independence, using STATISTICA Cz 12 [45]. We tested the null hypothesis that Leptospira seroprevalence would not differ in species, sex, locality, and year of trapping. The differences were considered statistically significant if the p-value was <0.05. In the case of a statistically significant difference in seroprevalence in some of the variables, the Scheffé multiple comparison method [45] was subsequently applied. For a detailed analysis of the relationships between species pairs, the odds ratio (OR) was also calculated together with 95% confidence intervals [45].

Data Availability Statement:
The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.