Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri serovar Mozdok in Serbia

Introduction This study aimed to investigate the prevalence and molecular characterization of Leptospira species in Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and distribution of Leptospira species within the region. Methods A comprehensive molecular analysis was conducted on kidney samples obtained from Norway rats (Rattus norvegicus) in the urban environment. The study utilized molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2, and secY genes. These methodologies were applied to ascertain the presence and characterize different Leptospira species and serovars, respectively. Results The findings revealed the presence of two Leptospira species and three separate serovars in the Belgrade area. This study identified the presence of L. kirschneri serovar Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in Serbia. Discussion The study underscores the importance of employing molecular typing methods to gain insights into the epidemiology and characterization of Leptospira species. These findings significantly contribute to both local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of effective control strategies and interventions. Summary In our recent study, we explored the presence and performed molecular typing of the Leptospira species, the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often transmitted through contact with water contaminated by infected animals. Our focus was on understanding which types of Leptospira were present in these animals. Excitingly, we discovered a particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This finding is significant because it sheds light on the presence and spread of different Leptospira serovars in Serbia. It also raises awareness about the potential health risks associated with this serovar, which was previously unknown in the area. Our work fits into a broader context of disease surveillance and public health. By identifying the types of Leptospira present in a specific region, we can better understand the risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not just important for scientists studying infectious diseases; it has real implications for public health officials, veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the need for ongoing monitoring of Leptospira in wildlife and synanthropic fauna, to protect both animal and human health.


Introduction
Leptospirosis, a zoonotic disease caused by pathogenic spirochaetes of the genus Leptospira, is constantly present in some parts of the world and holds significant relevance in both veterinary and public health contexts due to its ability to cross over between humans, domestic animals, wildlife and even environment (water).Reported cases of leptospirosis are global with over one million cases annualy, leading to approximately 60,000 fatalities (Costa et al., 2015).To date, a minimum of 64 distinct Leptospira species have been validated worldwide using the average nucleotide identity (ANI) values of their genomes (Vincent et al., 2019).While rats are traditionally known as the primary reservoirs for pathogenic Leptospira species, there have been numerous reports on various vertebrate and invertebrate hosts as excreting this pathogen through their urine.Wild and domestic mammals (Arent et al., 2017;Vieira et al., 2017), livestock (Shiokawa et al., 2019;Zhang et al., 2019), amphibians (Dezzutto et al., 2017), reptiles (Rodamilans et al., 2020) and bats (Mateus et al., 2019) also appear to play significant roles in the spread of Leptospira sp.Human infections typically result from exposure to soil or water contaminated with Leptospira, mostly from the urine of reservoir animals (Adler and de la Peña Moctezuma, 2010).Detecting Leptospira through traditional growth on media can be problematic due to their slow growth, making it impractical for timely diagnoses.To address this, molecular diagnostic methods, such as the real-time PCR of the lipL32 gene, have been developed (Ferreira et al., 2014;Wu et al., 2014).PCR-based amplification of secY and ompL1 genes using species-specific primers and probes has been used to identify Leptospira species directly from clinical samples.These assays can identify common pathogenic Leptospira species when combined with a lipL32 assay, including L. borgpetersenii, L. interrogans, L. kirschneri, and Leptospira noguchii (Victoria et al., 2008).Furthermore, sequence-based typing schemes utilizing gene targets like 16S rRNA rrs2, secY, and lfb1, or adk, icdA, lipL32, lipL41, rrs2 and secY have been developed for Leptospira (Ahmed et al., 2006;Morey et al., 2006).For example, a ∼ 435-bp fragment of the secY gene shows good phylogenetic discrimination between pathogenic Leptospira species.Sequence-based methods can also be applied directly to clinical samples to determine the infecting species and genotype, as well as investigate links between human and animal Leptospira infection (Hamond et al., 2015).In Serbia, the presence of pathogenic Leptospira sp. has been documented in various animals including small wild mammals (Blagojević et al., 2019), however most of the studies in Serbia have been focused on seroprevalence and seroepidemiological detection of antibodies in samples from cats (Obrenović et al., 2014), dogs (Vojinović et al., 2022) and humans (Svirčev et al., 2009).To the best of our knowledge this is the first study to perform molecular and multilocus sequence typing analysis of Leptospira species in Serbia.Moreover, this study revealed the presence of Leptospira kirshneri serovar Mozdok in Serbia for the first time.

Animal collection
The research was conducted in accordance with ethical principles and was approved by the Ministry of Agriculture, Forestry and Water Management (Republic of Serbia) -Veterinary Directorate (No. 323-07-04943/2020-05/2, 29.05.2020 and 323-07-04155/2023-05/2, 16.05.2023).During 2020, 2021 and 2022, a total of 344 (186 female and 158 male) carcasses of Norway rats (Rattus norvegicus) were collected in the broad environs of Belgrade City, predominantly in their urban and suburban 163 habitats.With the aim of collecting material that would be of good quality for further analysis of the presence of bacteria in the tissues, animals were collected by trapping (snap traps with fish mixture with peanut butter and oat flakes as a bait) and carcasses were collected daily and kept at 4°C during transportation to the laboratory where they were measured, followed by necropsy, during which the kidneys were removed and kept at −20°C until further processing and analysis.The average body mass of all used animals (± SD) was 236.97 ± 99.31 g (range 30-498 g).The average body length of the individuals was 201.79 ± 40.09 cm (range 91-374 cm), while the average tail length was 168.14 ± 30.48 cm (range 80-230 cm).Carcasses were collected predominantly in their urban and suburban habitats.The largest number of individuals was collected after the implementation of control measures or the implementation of monitoring measures.The collected carcasses were kept in a freezer at −20°C for a short time, until further processing.During autopsy, the kidneys were separated for further analysis and the morphological data, body weight and sex of the animals were recorded.

DNA extraction, molecular detection, sequencing, and MLST analysis
DNA was extracted from the kidney using the Quick-DNA MiniPrep kit (Zymo Research, USA, Cat.no.D3024) according to manufacturers' instructions.To validate the extraction processes and all downstream steps, nuclease-free water and DNA extracted from Leptospira positive samples were used as negative and positive controls, respectively.DNA extracted from each sample was stored at −20°C until downstream use.To distinguish between pathogenic and non-pathogenic Leptospira, we performed qPCR targeting the lipL32 partial target genes.Specifically, we used primers LipL32F (5′-GGA TCC GTG TAG AAA GAA TGT CGG-3′) and LipL32R (5'-GTC ACC ATC ATC ATC ATC GTC C-3′) to amplify a 101 bp fragment of the lipL32 gene, which was detected by the probe LipL32P (6-carboxyfluorescein [FAM]-5′-ATG CCT GAC CAA ATC GCC AAA GCT GCG AAA-3'-Black Hole Quencher 1 [BHQ1]) (Wu et al., 2014).An internal control, represented by exogenous DNA added before the extraction phase, representing simultaneously the extraction and PCR amplification control (qPCR Extraction Control RED, Meridian Bioscience, UK) was also included.The qPCR was carried out in a 12 μL reaction mixture containing 3 μL of Leptospira spp.genomic DNA, 0.5 μL (concentration of 20 pmol/μL) of forward and reverse primer and probe and 5 μL (concentration of 10 pmol/μL) of FastGene 2x PROBE Universal (Nippon Genetics, Germany) and 2.5 μL of PCR water.All reactions were conducted in duplicates using a 7,500 Fast Real-Time PCR System (Applied Biosystems, ThermoFisher, USA) with the following conditions: initial denaturation at 95°C for 2 min, followed by 45 cycles of denaturation at 95°C for 20 s, and annealing/elongation at 65°C for 50 s.Each PCR test included a negative control (DNA extracted from water) and a positive control (DNA extracted Leptospira spp.positive samples).Among the positive samples obtained through qPCR, only those with threshold cycle (Ct) values lower than or equal to 30 underwent further analysis.Specifically, 27 kidney samples and 27 Leptospira DNAs were subjected to PCR using a set of primers amplifying adk, icdA, lipL32, lipL41, rrs2 and secY partial genes (Table 1) (Ahmed et al., 2006).PCR reagents and their volumes, as well as PCR cycling conditions are shown in Tables 2, 3, respectively.The PCR products were visualized by electrophoresis on a 1.5% agarose gel and examined under UV transillumination.We purified the amplicons using the GeneJET PCR Purification Kit (ThermoFisher Scientific, USA, cat.no.K0702) and sent them to Macrogen Europe (Amsterdam, Netherlands) for Sanger sequencing.Sequences were analyzed and edited using the Staden package (Staden et al., 2003).Consensus sequence validation was performed against a custom Leptospira database using nucleotide blast (BLASTn) (Altschul et al., 1990).Each allele and the allelic profiles (adk-icdA-lipL32-lipL41-rrs2-secY) were submitted to the Leptospira database (Jolley et al., 2018) for ST assignment. 1 Sequence similarity of our samples was performed with a custom reference database using Biopython (Cock et al., 2009).All sequences were submitted to NCBI's GenBank under the following accession numbers: OR920389 -OR920523 for adk, icdA, lipL32, LipL41 and secY, while for rrs2 OR912477-OR912503.

Statistical analysis
Mean prevalence and confidence intervals (95% CI) for Leptospira spp.were determined using the Clopper and Pearson method.

Results
All 344 samples were analyzed for the presence of pathogenic Leptospira species.In kidney tissues, Leptospira spp. was detected in a total of 103 out of 344 individuals (29.94, 95% CI: 25.15-35.09)upon amplification by qPCR (Table 4).A total of 27 out of 103 positive samples (with Ct values between 20 and 28) were used in this study.Among all samples, the BLASTn analysis indicated that 26 sequences were affiliated with the L. interrogans, and 1 sequence exhibited the closest resemblance to the L. kirschneri (with 100% identity).The calculated sequence similarity of our samples with a cutoff value of 95% performed with Biopython was in concordance with the BLASTn results and for some of the samples it was possible to determine the serovar.For the final and definite characterization of our samples we determined the allele profile using the MLST scheme 3 from the PubMLST database. 2The MLST analysis yielded the following results: 11 of our samples belong to L. interrogans serovar Copenhageni, 12 to L. interrogans serovar Icterohaemorrhagiae (all belong to the serogroup Icterohaemorrhagiae) and one to L. kirschneri serovar Mozdok (serogroup Pomona).For the rest 3 of our samples, we were only able to determine the taxonomy to the level of species (L.interrogans) due to lower sequence quality.

Discussion
There is a growing interest in the surveillance of Leptospira spp.hosts, and investigations into the prevalence of this pathogen in wildlife and synanthropic faunaacross Europe are on the rise and the significance of rodents as reservoirs for various Leptospira serovars has been extensively explored worldwide with various results.It is wellestablished that wild rats (Rattus spp.) are the principal sources of Leptospira infection, particularly in urban and peri-domestic environments (Boey et al., 2019).Rattus norvegicus is known as the primary host of L. interrogans related to the serogroup Icterohaemorrhagiae, which is responsible for the most severe forms of the disease in humans (Haake and Levett, 2014).Our study aimed to examine the circulating Leptospira strains in wild rats, utilizing qPCR for initial detection of pathogenic Leptospira and MLST analysis for molecular characterization.Our findings confirm that wild rats harbor different serovars of pathogenic Leptospira spp.which pose threat to both animal and public health, highlighting the importance of continuous monitoring the presence and diversity of these bacteria in wild animals.The identification of L. interrogans serovar Icterohaemorrhagiae and L. interrogans serovar Copenhageni aligns with studies from all over Europe: in Sicily the bacteria has been detected in stray dogs and cats (Grippi et al., 2023).In Sardinia authors have reported pathogenic Leptospira in hedgehogs, mustelids and wild rodents (Piredda et al., 2021).In Germany, researchers in one study reported that 6% of the tested animals (various small mammals) exhibited positive results for L. kirschneri and L. interrogans (Obiegala et al., 2016), while L. interrogans serovar Icterohaemorrhagiae has been reported in wild rats all over the world (Boey et al., 2019) which is not surprising given that it represents the most common serovar in 2 https://pubmlst.org/Leptospiraanimals and humans.Additionally, this study relied on the utilization of the adk, icdA, lipL32, lipL41, rrs2 and secY partial genes as a means for molecular typing and differentiating Leptospira serovars.The results obtained using these genes align with those obtained from other MLST analyses.Although Leptospirosis has been the subject of numerous studies across various geographical regions, this present investigation in Serbia marks a significant contribution to the field.Prior research in Serbia had mainly focused on seroprevalence and seroepidemiological studies (Svirčev et al., 2009;Obrenović et al., 2014;Vojinović et al., 2022).However, our study distinguishes itself as the first in Serbia to employ molecular and multilocus sequence typing analysis for Leptospira species.This unique approach has yielded in discovering the presence of L. kirshneri serovar Mozdok in Serbia.This marks the first documented occurrence of this serovar in the country.Similar reports have been documented in Croatia (Majetić et al., 2014).The comprehensive and systematic testing conducted in our study, which included various Leptospira genes, facilitated the detailed characterization of positive samples.The sequencing and BLASTn analysis unveiled a predominance of L. interrogans in our samples, reinforcing its role as a common pathogenic Leptospira species.Further analysis, including the calculation of sequence similarity and allele profiling using the PubMLST database, refined our understanding of the Leptospira strains present.Notably, our findings unveiled specific serovars, such as L. interrogans serovar Copenhageni and L. interrogans serovar Icterohaemorrhagiae, underscoring the diversity of Leptospira strains within the Belgrade region.The significance of our discovery of L. kirshneri serovar Mozdok in Serbia extends beyond the confines of our study.This novel serovar presence has potential implications for vaccine strategies and epidemiological studies in both human and veterinary epidemiology.Serovars play a crucial role in vaccine formulation, as they determine the specific Leptospira strains that the vaccine should target.Consequently, our findings serve as a starting point for a more comprehensive and continuous Leptospira sp.surveillance in order to detect new serovars and accordingly adapt management strategies (presently the present vaccine strategies in Serbia include preparations for different animals which contain L. interrogans serovar Icterohaemorrhagiae, Canicola, Copenhageni and Bratislava, L. kirshneri serovar Grippotyphosa) However, it should be noted that this finding does not imply immediate change in vaccine strategy.To date, in Serbia, this serovar has not been confirmed as the causative agent of leptospirosis, most probably due to the lack of testing in patients infected with this bacterium.On the other hand, multiple research has proved that this serovar is clinically relevant since it has been implicated in human and animal leptospirosis (Cunha et al., 2016;Bertasio et al., 2020a,b).The presence of a novel serovar implies the need further explore and determine the presence of this serovar in other animals and/or humans and perform serological tests to screen for seropositive individuals that may have come into contact with this specific bacterium.Regarding leptospiros epidemiology, the identification of L. kirshneri serovar Mozdok opens doors to a more comprehensive understanding of the disease's distribution and dynamics in the

TABLE 2
Reagents and volumes.

TABLE 1
(Majetić et al., 2014;Obiegala et al., 2016)rimer sequences used for MLST analysis.The serovar's presence highlights the complexity of Leptospira populations in Serbia and warrants further investigation into its reservoir hosts and transmission dynamics.While the detection of a single serovar in one rat does not definitively establish that species as a reservoir it should be noted that while our findings do not conclusively determine the rat as a carrier, they are supported by other research [that has identified the same strain in additional rats, suggesting a potential role as carriers(Majetić et al., 2014;Obiegala et al., 2016)].Epidemiological studies must now consider the unique characteristics of this serovar, as it may exhibit distinct patterns of host adaptation and disease transmission.Understanding the prevalence and distribution of this serovar is crucial for developing effective control measures, both in terms of prevention and treatment.Moreover, the discovery emphasizes the importance of continued surveillance and monitoring of Leptospira diversity in the region, as new serovars may continue to emerge over time.In conclusion, our study has provided valuable insights into the presence and diversity of Leptospira species in Serbia.The discovery of L. kirshneri serovar Mozdok serves as a pivotal point for advancing management strategies and epidemiological research in the region.By adapting our approaches to the unique characteristics of this novel serovar, we can better address the challenges of leptospirosis and work toward more effective prevention and control measures for both human and veterinary health.Furthermore, the presence of L. kirshneri serovar Mozdok opens new avenues for epidemiological research in Serbia.Further research is essential to unveil the full implications of this discovery and to refine our understanding of the epidemiological landscape in Serbia.