Genotyping ofLeptospiraspp. in wild rats leads to first time detection ofL. kirshneriserovar Mozdok in Serbia

1 1 Full title: Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri serovar 2 Mozdok in Serbia 3 Short title: Multilocus sequence typing of Leptospira spp. 4 Vladimir Gajdov 1*¶, Goran Jokic 2¶, Sara Savic 1¶, Marina Zekic 1¶, Tanja Blazic 2¶, Milica Rajkovic 3¶, Tamas 5 Petrovic 1¶ 6 1Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia; 7 2Institute of Pesticides and Environmental Protection, Belgrade, Serbia 8 3Institute for Medical Research, Belgrade, Serbia 9 * Corresponding author: 10 E-mail: [email protected] 11 ¶These authors contributed equally to this work. 12 Abstract 13 This study aimed to investigate the prevalence and molecular characterization of Leptospira species in 14 Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ 15 molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and 16 distribution of Leptospira species within the region. A comprehensive molecular analysis was conducted on 17 kidney samples obtained from Norway rats ( Rattus novegicus) in urban environments. The study utilized 18 molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing 19 sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2 and secY genes. These 20 methodologies were applied to ascertain the presence and characterize different Leptospira species and 21 serotypes, respectively. The findings revealed the presence of two Leptospira species and three separate .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 2 22 serotypes in the Belgrade area. Moreover, this study identified the presence of L. kirschneri serovar 23 Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This 24 pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in 25 Serbia. The study underscores the importance of employing molecular typing methods to gain insights into 26 the epidemiology and characterization of Leptospira species. These findings significantly contribute to both 27 local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of 28 effective control strategies and interventions. 29 Keywords: molecular characterization; multilocus sequence typing, sequencing, epidemiology, rat; 30 zoonosis. 31 Author summary 32 In our recent study, we explored the presence and performed molecular typing of the Leptospira species, 33 the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was 34 conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often 35 transmitted through contact with water contaminated by infected animals. Our focus was on 36 understanding which types of Leptospira were present in these animals. Excitingly, we discovered a 37 particular strain of Leptospira, known as L. kirshneri serovar Mozdok, for the first time in Serbia. This 38 finding is significant because it sheds light on the presence and spread of different Leptospira serovars in 39 Serbia. It also raises awareness about the potential health risks associated with this serovar, which was 40 previously unknown in the area. Our work fits into a broader context of disease surveillance and public 41 health. By identifying the types of Leptospira present in a specific region, we can better understand the 42 risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not 43 just important for scientists studying infectious diseases; it has real implications for public health officials, 44 veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the 45 need for ongoing monitoring of Leptospira in wildlife, to protect both animal and human health. .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 3 46 1. Introduction 47 Leptospirosis, a zoonotic disease caused by pathogenic spirochaetes of the genus Leptospira, is constantly 48 present in some parts of the world and holds significant relevance in both veterinary and public health 49 contexts due to its ability to cross over between humans, domestic animals, wildlife and even environment 50 (water). Reported cases of leptospirosis are global with over one million cases annualy, leading to 51 approximately 60,000 fatalities [1]. To date, a minimum of 64 distinct Leptospira species have been 52 validated worldwide using the average nucleotide identity (ANI) values of their genomes. While rats are 53 traditionally known as the primary reservoirs for pathogenic Leptospira species, there have been numerous 54 reports on various vertebrate and invertebrate hosts as excreting this pathogen through their urine. Wild 55 and domestic mammals [2,3], livestock [4,5], amphibians [6], reptiles [7] and bats [8] also appear to play 56 significant roles in the spread of Leptospira sp. Human infections typically result from exposure to soil or 57 water contaminated with Leptospira, mostly from the urine of reservoir animals [9]. Detecting Leptospira 58 through traditional growth on media can be problematic due to their slow growth, making it impractical for 59 timely diagnoses. To address this, molecular diagnostic methods, such as the real-time PCR of the lipL32 60 gene, have been developed [10, 11]. PCR-based amplification of secY and ompL1 genes using species- 61 specific primers and probes has been used to identify Leptospira species directly from clinical samples. 62 These assays can identify common pathogenic Leptospira species when combined with a lipL32 assay, 63 including L. borgpetersenii, L. interrogans, L. kirschneri, and Leptospira noguchii [12]. Furthermore, 64 sequence-based typing schemes utilizing gene targets like 16S rRNA rrs2, secY, and lfb1, or adk, icdA, 65 lipL32, lipL41, rrs2 and secY have been developed for Leptospira [13,14]. For example, a ∼435-bp fragment 66 of the secY gene shows good phylogenetic discrimination between pathogenic Leptospira species. 67 Sequence-based methods can also be applied directly to clinical samples to determine the infecting species 68 and genotype, as well as investigate links between human and animal Leptospira infection [15]. In Serbia, 69 the presence of pathogenic Leptospira sp. has been documented in various animals including small wild 70 mammals [16], however most of the studies in Serbia have been focused on seroprevalence and .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 4 71 seroepidemiological detection of antibodies in samples from cats [17], dogs [18], cattle and sheep [19] and 72 humans [20]. To the best of our knowledge this is the first study to perform molecular and multilocus 73 sequence typing analysis of Leptospira species in Serbia. Moreover, this study revealed the presence of 74 Leptospira kirshneri serovar Mozdok in Serbia for the first time. 75 2. Results 76 All 344 samples were analyzed for the presence of pathogenic Leptospira species. In kidney tissues, 77 Leptospira spp. was detected in a total of 103 out of 344 individuals (29.94 %, 95% CI: 25.15-35.09) upon 78 amplification by qPCR (Table 4). A total of 27 out of 103 positive samples (with Ct values between 20 and 79 28) were used in this study. Among all samples, the BLASTn analysis indicated that 26 sequences were 80 affiliated with the L. interrogans, and 1 sequence exhibited the closest resemblance to the L. kirschneri 81 (with 100% identity). The calculated sequence similarity of our samples with a cutoff value of 95% 82 performed with Biopython was in concordance with the BLASTn results and for some of the samples it was 83 possible to determine the serovar. For the final and definite characterization of our samples we 84 determined the allele profile using the MLST scheme 3 from the PubMLST (https://pubmlst.org/Leptospira) 85 database [24]. The MLST analysis yielded the following results: 11 of our samples belong to L. interrogans 86 serovar Copenhageni, 12 to L. interrogans serovar Icterohaemorrhagiae and one to L. kirschneri serovar 87 Mozdok. For the rest 3 of our samples, we were only able to determine the taxonomy to the level of 88 species (L. interrogans) due to lower sequence quality. 89 Table 4: The presence of Leptospira spp. in Norway rat kidney tissues, collected in the period 2020.-2022. 90 in Belgrade, Serbias 2020 2021 2022 Sex Number of individuals MS*±SE Number of individuals MS*±SE Number of individuals MS*±SE Negative .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 5 female 23 220.23±13.37 69 243.55±13.76 38 201.89±17.01 male 32 249.37±19.69 49 238.16±12.82 30 233.83±18.29 Positive female 16 258.75±22.71 16 250.44±23.08 24 250.42±16.11 male 20 197.25±22.39 15 277.00±22.58 12 253.83±28.58 91 * - mean average weight (g) 92 3. Discussion 93 There is a growing interest in the surveillance of Leptospira spp. hosts, and investigations into the 94 prevalence of this pathogen in wild mammals across Europe are on the rise and the significance of rodents 95 as reservoirs for various Leptospira serovars has been extensively explored worldwide with various results. 96 It is well-established that wild rats (Rattus spp.) are the principal sources of Leptospira infection, 97 particularly in urban and peri-domestic environments [25]. The brown rat is known as the primary host of 98 L. interrogans related to the serogroup Icterohaemorrhagiae, which is responsible for the most severe 99 forms of the disease in humans [26]. This study aimed to examine the circulating Leptospira strains in wild 100 rats, utilizing qPCR for initial detection of pathogenic Leptospira and MLST analysis for molecular 101 characterization. Our findings confirm that wild rats harbor different serovars of pathogenic Leptospira 102 spp. which pose threat to both animal and public health, highlighting the importance of continuous 103 monitoring the presence and diversity of these bacteria in wild animals. The identification of L. interrogans 104 serovar Icterohaemorrhagiae and L. interrogans serovar Copenhageni aligns with studies from all over 105 Europe: in Sicily the bacteria has been detected in stray dogs and cats [27]; In Sardinia authors have 106 reported pathogenic Leptospira in hedgehogs, mustelids and wild rodents [28]; In Germany, researchers in 107 one study reported that 6% of the tested animals (various small mammals) exhibited positive results for L. 108 kirschneri and L. interrogans [29], while L. interrogans serovar Icterohaemorrhagiae has been reported in 109 wild rats all over the world [25] which is not surprising given that it represents the most common serovar 110 in animals and humans. Additionally, this study relied on the utilization of the adk, icdA, lipL32, lipL41, rrs2 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 6 111 and secY partial genes as a means for molecular typing and differentiating Leptospira serovars. The results 112 obtained using these genes align with those obtained from other MLST analyses. Although Leptospirosis 113 has been the subject of numerous studies across various geographical regions, this present investigation in 114 Serbia marks a significant contribution to the field. Prior research in Serbia had mainly focused on 115 seroprevalence and seroepidemiological studies [16-20]. However, our study distinguishes itself as the first 116 in Serbia to employ molecular and multilocus sequence typing analysis for Leptospira species. This unique 117 approach has yielded in discovering the presence of Leptospira kirshneri serovar Mozdok in Serbia. This 118 marks the first documented occurrence of this serovar in the country. Similar reports have been 119 documented in Croatia (30). The comprehensive and systematic testing conducted in our study, which 120 included various Leptospira genes, facilitated the detailed characterization of positive samples. The 121 sequencing and BLASTn analysis unveiled a predominance of L. interrogans in our samples, reinforcing its 122 role as a common pathogenic Leptospira species. Further analysis, including the calculation of sequence 123 similarity and allele profiling using the PubMLST database, refined our understanding of the Leptospira 124 strains present. Notably, our findings unveiled specific serovars, such as L. interrogans serovar 125 Copenhageni and L. interrogans serovar Icterohaemorrhagiae, underscoring the diversity of Leptospira 126 strains within the Belgrade region. The significance of our discovery of Leptospira kirshneri serovar Mozdok 127 in Serbia extends beyond the confines of our study. This novel serovar presence has far-reaching 128 implications for vaccine strategies and epidemiological studies in both human and veterinary 129 epidemiology. The discovery of Leptospira kirshneri serovar Mozdok in Serbia introduces a new dimension 130 to vaccine development strategies. Serovars play a crucial role in vaccine formulation, as they determine 131 the specific Leptospira strains that the vaccine should target. The presence of a novel serovar implies the 132 need for the inclusion of this serovar in regional or local vaccine formulations. Failure to account for the 133 presence of this serovar could compromise the effectiveness of vaccines in protecting both human and 134 animal populations. Consequently, our findings serve as a critical foundation for the adaptation of vaccine 135 strategies to the unique epidemiological landscape of Serbia. The present vaccine strategies in Serbia .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 7 136 include preparations for different animals which contain L. interrogans serovar Icterohaemorrhagiae, 137 Canicola, Copenhageni and Bratislava, L. kirshneri serovar Grippotyphosa. Regarding leptospiros 138 epidemiology, the identification of L. kirshneri serovar Mozdok opens doors to a more comprehensive 139 understanding of the disease's distribution and dynamics in the region. The serovar's presence highlights 140 the complexity of Leptospira populations in Serbia and warrants further investigation into its reservoir 141 hosts and transmission dynamics. Epidemiological studies must now consider the unique characteristics of 142 this serovar, as it may exhibit distinct patterns of host adaptation and disease transmission. Understanding 143 the prevalence and distribution of this serovar is crucial for developing effective control measures, both in 144 terms of prevention and treatment. Moreover, the discovery emphasizes the importance of continued 145 surveillance and monitoring of Leptospira diversity in the region, as new serovars may continue to emerge 146 over time. In conclusion, our study has provided valuable insights into the presence and diversity of 147 Leptospira species in Serbia. The discovery of L. kirshneri serovar Mozdok serves as a pivotal point for 148 advancing vaccine strategies and epidemiological research in the region. By adapting our approaches to the 149 unique characteristics of this novel serovar, we can better address the challenges of leptospirosis and work 150 towards more effective prevention and control measures for both human and veterinary health. 151 Furthermore, the presence of Leptospira kirshneri serovar Mozdok opens new avenues for epidemiological 152 research in Serbia. This novel serovar's presence highlights the complexity of Leptospira populations in the. 153 Further research is essential to unveil the full implications of this discovery and to refine our understanding 154 of the epidemiological landscape in Serbia. 155 156 4. Materials and methods 157 4.1 Animal Collection 158 The research was conducted in accordance with ethical principles and was approved by the Ministry of 159 Agriculture, Forestry and Water Management (Republic of Serbia) - Veterinary Directorate (No. 323-07- .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 8 160 04943/2020-05/2, 29.05.2020 and 323-07-04155/2023-05/2, 16.05.2023). During 2020, 2021 and 2022, a 161 total of 344 (186 female and 158 male) carcasses of Norway rats ( Rattus norvegicus) were collected in the 162 broad environs of Belgrade City. Carcasses were collected predominantly in their urban and suburban 163 habitats. The largest number of individuals was collected after the implementation of control measures or 164 the implementation of monitoring measures. The collected carcasses were kept in a freezer at -20 ◦C for a 165 short time, until further processing. During autopsy, the kidneys were separated for further analysis and 166 the morphological data, body weight and sex of the animals were recorded. 167 4.2 DNA extraction, molecular detection, sequencing and MLST analysis 168 DNA was extracted from the kidney using the Quick-DNA MiniPrep kit (Zymo Research, Australia, Cat. no. 169 D3024), according to manufacturers’ instructions. Due to validate the extraction processes and all 170 downstream steps, nuclease-free water and DNA extracted from Leptospira positive samples were used as 171 positive and negative controls, respectively. DNA extracted from each sample was stored at −20 °C until 172 downstream use. To distinguish between pathogenic and non-pathogenic Leptospira, we performed qPCR 173 targeting the lipL32 partial target genes. Specifically, we used primers LipL32F (5’-GGA TCC GTG TAG AAA 174 GAA TGT CGG-3’) and LipL32R (5’-GTC ACC ATC ATC ATC ATC GTC C-3’) to amplify a 101 bp fragment of the 175 lipL32 gene, which was detected by the probe LipL32P (6-carboxyfluorescein [FAM]-5’-ATG CCT GAC CAA 176 ATC GCC AAA GCT GCG AAA-3’-Black Hole Quencher 1 [BHQ1]) [10]. An internal control, represented by 177 exogenous DNA added before the extraction phase, representing simultaneously the extraction and PCR 178 amplification control (qPCR Extraction Control RED, Meridian Bioscience, UK) was also included. The qPCR 179 was carried out in a 12 μL reaction mixture containing 3 μL of Leptospira spp. genomic DNA, 0.5 μL 180 (concentration of 20 pmol/μL) of forward and reverse primer and probe and 5 μL (concentration of 181 10pmol/μL) of FastGene 2x PROBE Universal (Nippon Genetics, Germany) and 2.5 μL of PCR water. All 182 reactions were conducted in duplicates using a 7500 Fast Real-Time PCR System (Applied Biosystems, 183 ThermoFisher, USA) with the following conditions: initial denaturation at 95°C for 2 min, followed by 45 184 cycles of denaturation at 95°C for 20 s, and annealing/elongation at 65°C for 50 s. Each PCR test included a .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 9 185 negative control (DNA extracted from water) and a positive control (DNA extracted Leptospira spp. positive 186 samples). Among the positive samples obtained through qPCR, only those with threshold cycle (Ct) values 187 lower than or equal to 30 underwent further analysis. Specifically, 27 kidney samples and 27 Leptospira 188 isolates were subjected to PCR using a set of primers amplifying adk, icdA, lipL32, lipL41, rrs2 and secY 189 partial genes (Table 1) [14]. PCR reagents and their volumes, as well as PCR cycling conditions are shown in 190 Table 2 and Table 3, respectively. The PCR products were visualized by electrophoresis on a 1.5% agarose 191 gel and examined under UV transillumination. 192 Table 1: Details of gene loci and the corresponding primer sequences used for MLST Analysis Gene Locus Gene size (bp) Genome position PCR product (bp) Size of polymorphic sequence (bp) Primer sequences 5’-3’ adk LIC12852 564 3458298– 3458861 531 430 F-GGGCTGGAAAAGGTACACAA R-ACGCAAGCTCCTTTTGAATC icdA LIC13244 1197 3979829– 3981025 674 557 F-GGGACGAGATGACCAGGAT R-TTTTTTGAGATCCGCAGCTTT lipL32 LIC11352 819 1666299– 1667117 474 474 F-ATCTCCGTTGCACTCTTTGC R-ACCATCATCATCATCGTCCA lipL4l LIC12966 1068 3603575– 3604642 520 518 F-TAGGAAATTGCGCAGCTACA R-GCATCGAGAGGAATTAACATCA rrs2 LIC11508 1512 1862433– 1863944 541 452 F-CATGCAAGTCAAGCGGAGTA R-AGTTGAGCCCGCAGTTTTC secY LIC12853 1383 3458869– 3460251 549 549 F-ATGCCGATCATTTTTGCTTC R-CCGTCCCTTAATTTTAGACTTCTTC 193 194 Table 2: Reagents and Volumes .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 10 Reagent Volume per Reaction - µL DNA Template (Leptospira DNA) 3 Forward Primer 1 (concentration of 20 pmol/μL Reverse Primer 1 (concentration of 20 pmol/μL HotStarTaq Master Mix 12.5 Sterile Water 7.5 Total Reaction Volume 25 195 196 Table 3: PCR Cycling Conditions Step Temperature (°C) Time Number of Cycles Initial Denaturation 95 15 minutes 1 Denaturation 95 30 seconds Annealing 58 30 seconds Extension 72 1 minute 35 Final Extension 72 10 1 Hold 4 ∞ 1 197 198 We purified (GeneJET PCR Purification Kit, ThermoFisher Scientific, USA, cat. no. K0702) and sent all 199 positive amplicons for genes listed in Table 2 to Macrogen Europe for Sanger sequencing. Sequences were .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 11 200 analyzed and edited using the Staden package [21]. Consensus sequence validation was performed against 201 a custom Leptospira database using nucleotide blast (BLASTn) [22] Each allele and the allelic profiles (adk- 202 icdA-lipL32-lipL41-rrs2-secY) were submitted to the Leptospira database [23] 203 (http://pubmlst.org/Leptospira, accessed in October 2023) for ST assignment. Sequence similarity of our 204 samples was performed with a custom reference database using Biopython [24]. All sequences were 205 submitted to NCBI’s GenBank under the following accession numbers: OR920389 - OR920523 for adk, icdA, 206 lipL32, LipL41 and secY, while for rrs2 OR912477-OR912503. 207 4.3 Statistical analysis 208 Mean prevalence and confidence intervals (95% CI) for Leptospira spp. were determined using the Clopper 209 and Pearson method. An alpha value <0.05 was considered the threshold for statistical significance. 210 Conflict of interest: The authors declare that they have no known competing financial interests or personal 211 relationships that could have appeared to influence the work reported in this paper. 212 Funding Source: This work was funded by Ministry of Science, Technological Development and Innovation 213 of Republic of Serbia by the Contract of implementation and funding of research work of NIV-NS in 2023, 214 Contract No: 451-03-47/2023-01/200031. 215 Ethical Approval statement: Ethics review and approval for this study were obtained from the Ministry of 216 Agriculture, Forestry and Water Management (Republic of Serbia) - Veterinary Directorate (No. 323-07- 217 04943/2020-05/2, 29.05.2020 and 323-07-04155/2023-05/2, 16.05.2023). 218 219 6. References 220 [1] Costa F, Hagan JE, Calcagno JI, et al. Global Morbidity and Mortality of Leptospirosis: a Systematic 221 review. PLOS Neglected Tropical Diseases. 2015;9(9):e0003898. doi:10.1371/journal.pntd.0003898 222 [2] Arent Z, Gilmore C, Ayanz JMSM, Neyra L, García-Peña FJ. Molecular epidemiology of Leptospira 223 serogroup pomona infections among wild and domestic animals in Spain. Ecohealth. 2017;14(1):48-57. 224 doi:10.1007/s10393-017-1210-8 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 12 225 [3] Vieira AS, Pinto PS, Lilenbaum W. A systematic review of leptospirosis on wild animals in Latin America. 226 Tropical Animal Health and Production. 2017;50(2):229-238. doi:10.1007/s11250-017-1429-y 227 [4] Shiokawa K, Welcome S, Kenig M, Lim B, Rajeev S. Epidemiology of Leptospira infection in livestock 228 species in Saint Kitts. Tropical Animal Health and Production. 2019;51(6):1645-1650. doi:10.1007/s11250- 229 019-01859-5 230 [5] Zhang C, Xu J, Zhang T, et al. Genetic characteristics of pathogenic Leptospira in wild small animals and 231 livestock in Jiangxi Province, China, 2002–2015. PLOS Neglected Tropical Diseases. 2019;13(6):e0007513. 232 doi:10.1371/journal.pntd.0007513 233 [6] Dezzutto D, Barbero R, Canale G, et al. Detection of Leptospira spp. in Water Turtle (Trachemys scripta) 234 Living in Ponds of Urban Parks. Veterinary Sciences. 2017;4(4):51. doi:10.3390/vetsci4040051 235 [7] Rodamilans GM, Fonseca MS, Paz LN, et al. Leptospira interrogans in wild Boa constrictor snakes from 236 Northeast Brazil peri‑urban rainforest fragments. Acta Tropica. 2020;209:105572. 237 doi:10.1016/j.actatropica.2020.105572 238 [8] Mateus JE, Gómez N, Herrera-Sepúlveda MT, Hidalgo M, Pérez-Torres J, Cuervo C. Bats are a potential 239 reservoir of pathogenic Leptospira species in Colombia. Journal of Infection in Developing Countries. 240 2019;13(04):278-283. doi:10.3855/jidc.10642 241 [9] Adler B, De La Peña Moctezuma A. Leptospira and leptospirosis. Veterinary Microbiology. 2010;140(3- 242 4):287-296. doi:10.1016/j.vetmic.2009.03.012 243 [10] Wu Q, Prager KC, Goldstein T, et al. Development of a real-time PCR for the detection of pathogenic 244 Leptospira spp. in California sea lions. Diseases of Aquatic Organisms. 2014;110(3):165-172. 245 doi:10.3354/dao02752 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 13 246 [11] Ferreira A, Costa P, Rocha T, et al. Direct detection and differentiation of pathogenic Leptospira 247 species using a Multi-Gene targeted real time PCR approach. PLOS ONE. 2014;9(11):e112312. 248 doi:10.1371/journal.pone.0112312 249 [12] Victoria B, Ahmed A, Zuerner RL, et al. Conservation of the S10-spc-α Locus within Otherwise Highly 250 Plastic Genomes Provides Phylogenetic Insight into the Genus Leptospira. PLOS ONE. 2008;3(7):e2752. 251 doi:10.1371/journal.pone.0002752 252 [13] Morey RE, Galloway RL, Bragg SL, Steigerwalt AG, Mayer LW, Levett PN. Species-Specific identification 253 of Leptospiraceae by 16S RRNA gene sequencing. Journal of Clinical Microbiology. 2006;44(10):3510-3516. 254 doi:10.1128/jcm.00670-06 255 [14] Ahmed N, Devi SM, De Los Angeles Valverde M, et al. Multilocus sequence typing method for 256 identification and genotypic classification of pathogenic Leptospira species. Annals of Clinical Microbiology 257 and Antimicrobials. 2006;5(1):28. doi:10.1186/1476-0711-5-28 258 [15] Hamond C, Pestana CP, Medeiros MA, Lilenbaum W. Genotyping of Leptospira directly in urine 259 samples of cattle demonstrates a diversity of species and strains in Brazil. Epidemiology and Infection. 260 2015;144(1):72-75. doi:10.1017/s0950268815001363 261 [16] Blagojević J, Šekler M, Rajičić M, et al. The prevalence of pathogenic forms of Leptospira in natural 262 populations of small wild mammals in Serbia. Acta Veterinaria Hungarica. 2019;67(3):338-346. 263 doi:10.1556/004.2019.035 264 [17] Obrenović S, Radojičić S, Stević N, Bogunović D, Vakanjac S, Valčić M. Seroprevalence of Cat 265 leptospirosis in Belgrade (Serbia). Acta Veterinaria-beograd. 2014;64(4):510-518. doi:10.2478/acve-2014- 266 0047 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 14 267 [18] Vojinović D, Žutić J, Vasić A, Stanojević S, Spalević L, Sapundžić ZZ. A serological survey of canine 268 leptospirosis in the city of Belgrade, Serbia. Veterinarski Glasnik. 2022;76(1):47-55. 269 doi:10.2298/vetgl210708001v 270 [19] Ramin AG, Azizzadeh F. Seroepidemiological detection of antibodies against Leptospira spp using 271 microscopic agglutination test in Urmia cows and sheep. Acta Veterinaria-beograd. 2013;63(1):53-61. 272 doi:10.2298/avb1301053r 273 [20] Svirčev Z, Marković SB, Vukadinov J, et al. Leptospirosis distribution related to freshwater habitats in 274 the Vojvodina region (Republic of Serbia). Science in China Series C-Life Sciences. 2009;52(10):965-971. 275 doi:10.1007/s11427-009-0124-2 276 [21] Staden R, Beal KF, Bonfield JK. The Staden Package, 1998. In: Humana Press eBooks. ; 2003:115-130. 277 doi:10.1385/1-59259-192-2:115 278 [22] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. Journal of 279 Molecular Biology. 1990;215(3):403-410. doi:10.1016/s0022-2836(05)80360-2 280 [23] Jolley KA, Bray JE, Maiden MCJ. Open-access bacterial population genomics: BIGSdb software, the 281 PubMLST.org website and their applications. Wellcome Open Research. 2018;3:124. 282 doi:10.12688/wellcomeopenres.14826.1 283 [24] Cock PJA, Antão T, Chang JT, et al. Biopython: freely available Python tools for computational 284 molecular biology and bioinformatics. Bioinformatics. 2009;25(11):1422-1423. 285 doi:10.1093/bioinformatics/btp163 286 [25] Boey K, Shiokawa K, Rajeev S. Leptospira infection in rats: A literature review of global prevalence and 287 distribution. PLOS Neglected Tropical Diseases. 2019;13(8):e0007499. doi:10.1371/journal.pntd.0007499 288 [26] Haake DA, Levett PN. Leptospirosis in humans. In: Current Topics in Microbiology and Immunology. ; 289 2014:65-97. doi:10.1007/978-3-662-45059-8_5 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint 15 290 [27] Grippi F, Cannella V, Macaluso G, et al. Serological and Molecular Evidence of Pathogenic Leptospira 291 spp. in Stray Dogs and Cats of Sicily (South Italy), 2017–2021. Microorganisms. 2023;11(2):385. 292 doi:10.3390/microorganisms11020385 293 [28] Piredda I, Ponti MN, Palmas B, et al. Molecular Typing of Pathogenic Leptospira Species Isolated from 294 Wild Mammal Reservoirs in Sardinia. Animals. 2021;11(4):1109. doi:10.3390/ani11041109 295 [29] Obiegala A, Woll D, Karnath C, et al. Prevalence and Genotype Allocation of Pathogenic Leptospira 296 Species in Small Mammals from Various Habitat Types in Germany. PLOS Neglected Tropical Diseases. 297 2016;10(3):e0004501. doi:10.1371/journal.pntd.0004501 298 [30] Majetić Š, Galloway L, Sabljić R, et al. Epizootiological survey of small mammals as Leptospira spp. 299 reservoirs in Eastern Croatia. Acta Tropica. 2014;131:111-116. doi:10.1016/j.actatropica.2013.12.009 .CC-BY 4.0 International licenseperpetuity. It is made available under a preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in The copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint