{"paper_id":"1aea0efe-3827-447f-96fb-1fef7a30cdec","body_text":"1\n1 Full title: Genotyping of Leptospira spp. in wild rats leads to first time detection of L. kirshneri  serovar \n2 Mozdok in Serbia\n3 Short title: Multilocus sequence typing of Leptospira spp. \n4 Vladimir Gajdov 1*¶, Goran Jokic 2¶, Sara Savic 1¶, Marina Zekic 1¶, Tanja Blazic 2¶, Milica Rajkovic 3¶, Tamas \n5 Petrovic 1¶\n6 1Scientific Veterinary Institute “Novi Sad”, Novi Sad, Serbia;\n7 2Institute of Pesticides and Environmental Protection, Belgrade, Serbia\n8 3Institute for Medical Research, Belgrade, Serbia\n9 * Corresponding author:\n10 E-mail: vladimir.g@niv.ns.ac.rs\n11 ¶These authors contributed equally to this work.\n12 Abstract\n13 This study aimed to investigate the prevalence and molecular characterization of Leptospira species in \n14 Belgrade, Serbia, an area where this disease is underexplored. Specifically, the study sought to employ \n15 molecular and multilocus sequence typing analyses to fill the gap in understanding the diversity and \n16 distribution of Leptospira species within the region. A comprehensive molecular analysis was conducted on \n17 kidney samples obtained from Norway rats ( Rattus novegicus) in urban environments. The study utilized \n18 molecular diagnostic techniques including real-time PCR targeting the lipL32 gene and performing \n19 sequence-based typing schemes utilizing adk, icdA, lipL32, lipL41, rrs2 and secY genes. These \n20 methodologies were applied to ascertain the presence and characterize different Leptospira species and \n21 serotypes, respectively. The findings revealed the presence of two Leptospira species and three separate \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n2\n22 serotypes in the Belgrade area. Moreover, this study identified the presence of L. kirschneri serovar \n23 Mozdok in Serbia for the first time, a significant discovery previously undocumented in the region. This \n24 pioneering investigation sheds light on the molecular diversity and prevalence of Leptospira species in \n25 Serbia. The study underscores the importance of employing molecular typing methods to gain insights into \n26 the epidemiology and characterization of Leptospira species. These findings significantly contribute to both \n27 local and global perspectives on leptospirosis epidemiology, providing vital insights for the development of \n28 effective control strategies and interventions.\n29 Keywords: molecular characterization; multilocus sequence typing, sequencing, epidemiology, rat; \n30 zoonosis.\n31 Author summary\n32 In our recent study, we explored the presence and performed molecular typing of the Leptospira species, \n33 the bacteria responsible for leptospirosis, in wild rats in Serbia. This was the first time such a study was \n34 conducted in the region. Leptospirosis is a serious disease that affects both animals and humans, often \n35 transmitted through contact with water contaminated by infected animals. Our focus was on \n36 understanding which types of Leptospira were present in these animals. Excitingly, we discovered a \n37 particular strain of Leptospira, known as L. kirshneri  serovar Mozdok, for the first time in Serbia. This \n38 finding is significant because it sheds light on the presence and spread of different Leptospira serovars in \n39 Serbia. It also raises awareness about the potential health risks associated with this serovar, which was \n40 previously unknown in the area. Our work fits into a broader context of disease surveillance and public \n41 health. By identifying the types of Leptospira present in a specific region, we can better understand the \n42 risks to public health and take steps to prevent and control the spread of leptospirosis. This discovery is not \n43 just important for scientists studying infectious diseases; it has real implications for public health officials, \n44 veterinarians, and anyone concerned with preventing and treating leptospirosis. Our findings highlight the \n45 need for ongoing monitoring of Leptospira in wildlife, to protect both animal and human health.\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n3\n46 1. Introduction\n47 Leptospirosis, a zoonotic disease caused by pathogenic spirochaetes of the genus Leptospira, is constantly \n48 present in some parts of the world and holds significant relevance in both veterinary and public health \n49 contexts due to its ability to cross over between humans, domestic animals, wildlife and even environment \n50 (water). Reported cases of leptospirosis are global with over one million cases annualy, leading to \n51 approximately 60,000 fatalities [1]. To date, a minimum of 64 distinct Leptospira species have been \n52 validated worldwide using the average nucleotide identity (ANI) values of their genomes. While rats are \n53 traditionally known as the primary reservoirs for pathogenic Leptospira species, there have been numerous \n54 reports on various vertebrate and invertebrate hosts as excreting this pathogen through their urine. Wild \n55 and domestic mammals [2,3], livestock [4,5], amphibians [6], reptiles [7] and bats [8] also appear to play \n56 significant roles in the spread of Leptospira sp. Human infections typically result from exposure to soil or \n57 water contaminated with Leptospira, mostly from the urine of  reservoir animals [9]. Detecting Leptospira \n58 through traditional growth on media can be problematic due to their slow growth, making it impractical for \n59 timely diagnoses. To address this, molecular diagnostic methods, such as the real-time PCR of the lipL32 \n60 gene, have been developed [10, 11]. PCR-based amplification of secY and ompL1 genes using species-\n61 specific primers and probes has been used to identify Leptospira species directly from clinical samples. \n62 These assays can identify common pathogenic Leptospira species when combined with a lipL32 assay, \n63 including L. borgpetersenii, L. interrogans, L. kirschneri, and Leptospira noguchii [12]. Furthermore, \n64 sequence-based typing schemes utilizing gene targets like 16S rRNA rrs2, secY, and lfb1, or adk, icdA, \n65 lipL32, lipL41, rrs2 and secY have been developed for Leptospira [13,14]. For example, a ∼435-bp fragment \n66 of the secY gene shows good phylogenetic discrimination between pathogenic Leptospira species. \n67 Sequence-based methods can also be applied directly to clinical samples to determine the infecting species \n68 and genotype, as well as investigate links between human and animal Leptospira infection [15].  In Serbia, \n69 the presence of pathogenic Leptospira sp. has been documented in various animals including small wild \n70 mammals [16], however most of the studies in Serbia have been focused on seroprevalence and \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n4\n71 seroepidemiological detection of antibodies in samples from cats [17], dogs [18], cattle and sheep [19] and \n72 humans [20]. To the best of our knowledge this is the first study to perform molecular and multilocus \n73 sequence typing analysis of Leptospira species in Serbia. Moreover, this study revealed the presence of \n74 Leptospira kirshneri serovar Mozdok in Serbia for the first time.\n75 2. Results \n76 All 344 samples were analyzed for the presence of pathogenic Leptospira species. In kidney tissues, \n77 Leptospira spp. was detected in a total of 103 out of 344 individuals (29.94 %, 95% CI: 25.15-35.09) upon \n78 amplification by qPCR (Table 4). A total of 27 out of 103 positive samples (with Ct values between 20 and \n79 28) were used in this study. Among all samples, the BLASTn analysis indicated that 26 sequences were \n80 affiliated with the L. interrogans, and 1 sequence exhibited the closest resemblance to the L. kirschneri \n81 (with 100% identity). The calculated sequence similarity of our samples with a cutoff value of 95% \n82 performed with Biopython was in concordance with the BLASTn results and for some of the samples it was \n83 possible to determine the serovar. For the final and definite characterization of our samples we \n84 determined the allele profile using the MLST scheme 3 from the PubMLST (https://pubmlst.org/Leptospira) \n85 database [24]. The MLST analysis yielded the following results: 11 of our samples belong to L. interrogans \n86 serovar Copenhageni, 12 to L. interrogans serovar Icterohaemorrhagiae and one to L.  kirschneri serovar \n87 Mozdok. For the rest 3 of our samples, we were only able to determine the taxonomy to the level of \n88 species (L.  interrogans) due to lower sequence quality. \n89 Table 4: The presence of Leptospira spp. in Norway rat kidney tissues, collected in the period 2020.-2022. \n90 in Belgrade, Serbias\n2020 2021 2022\nSex Number of \nindividuals\nMS*±SE\nNumber of \nindividuals\nMS*±SE\nNumber of \nindividuals\nMS*±SE\nNegative\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n5\nfemale 23 220.23±13.37 69 243.55±13.76 38 201.89±17.01\nmale 32 249.37±19.69 49 238.16±12.82 30 233.83±18.29\nPositive\nfemale 16 258.75±22.71 16 250.44±23.08 24 250.42±16.11\nmale 20 197.25±22.39 15 277.00±22.58 12 253.83±28.58\n91 * - mean average weight (g)\n92 3. Discussion\n93 There is a growing interest in the surveillance of Leptospira spp. hosts, and investigations into the \n94 prevalence of this pathogen in wild mammals across Europe are on the rise and the significance of rodents \n95 as reservoirs for various Leptospira serovars has been extensively explored worldwide with various results. \n96 It is well-established that wild rats (Rattus  spp.) are the principal sources of Leptospira infection, \n97 particularly in urban and peri-domestic environments [25]. The brown rat is known as the primary host of \n98 L. interrogans related to the serogroup Icterohaemorrhagiae, which is responsible for the most severe \n99 forms of the disease in humans [26]. This study aimed to examine the circulating Leptospira strains in wild \n100 rats, utilizing qPCR for initial detection of pathogenic Leptospira and MLST analysis for molecular \n101 characterization. Our findings confirm that wild rats harbor different serovars of pathogenic Leptospira \n102 spp. which pose threat to both animal and public health, highlighting the importance of continuous \n103 monitoring the presence and diversity of these bacteria in wild animals. The identification of L. interrogans \n104 serovar Icterohaemorrhagiae  and L. interrogans serovar Copenhageni aligns with studies from all over \n105 Europe: in Sicily the bacteria has been detected in stray dogs and cats [27]; In Sardinia authors have \n106 reported pathogenic Leptospira in hedgehogs, mustelids and wild rodents [28]; In Germany, researchers in \n107 one study reported that 6% of the tested animals (various small mammals) exhibited positive results for L. \n108 kirschneri and L. interrogans [29], while L. interrogans serovar Icterohaemorrhagiae has been reported in \n109 wild rats all over the world [25] which is not surprising given that it represents the most common serovar \n110 in animals and humans. Additionally, this study relied on the utilization of the adk, icdA, lipL32, lipL41, rrs2 \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n6\n111 and secY partial genes as a means for molecular typing and differentiating Leptospira serovars. The results \n112 obtained using these genes align with those obtained from other MLST analyses. Although Leptospirosis \n113 has been the subject of numerous studies across various geographical regions, this present investigation in \n114 Serbia marks a significant contribution to the field. Prior research in Serbia had mainly focused on \n115 seroprevalence and seroepidemiological studies [16-20]. However, our study distinguishes itself as the first \n116 in Serbia to employ molecular and multilocus sequence typing analysis for Leptospira species. This unique \n117 approach has yielded in discovering the presence of Leptospira kirshneri serovar Mozdok in Serbia. This \n118 marks the first documented occurrence of this serovar in the country. Similar reports have been \n119 documented in Croatia (30). The comprehensive and systematic testing conducted in our study, which \n120 included various Leptospira genes, facilitated the detailed characterization of positive samples. The \n121 sequencing and BLASTn analysis unveiled a predominance of L. interrogans in our samples, reinforcing its \n122 role as a common pathogenic Leptospira species. Further analysis, including the calculation of sequence \n123 similarity and allele profiling using the PubMLST database, refined our understanding of the Leptospira \n124 strains present. Notably, our findings unveiled specific serovars, such as L. interrogans serovar \n125 Copenhageni and L. interrogans serovar Icterohaemorrhagiae, underscoring the diversity of Leptospira \n126 strains within the Belgrade region. The significance of our discovery of Leptospira kirshneri serovar Mozdok \n127 in Serbia extends beyond the confines of our study. This novel serovar presence has far-reaching \n128 implications for vaccine strategies and epidemiological studies in both human and veterinary \n129 epidemiology. The discovery of Leptospira kirshneri serovar Mozdok in Serbia introduces a new dimension \n130 to vaccine development strategies. Serovars play a crucial role in vaccine formulation, as they determine \n131 the specific Leptospira strains that the vaccine should target. The presence of a novel serovar implies the \n132 need for the inclusion of this serovar in regional or local vaccine formulations. Failure to account for the \n133 presence of this serovar could compromise the effectiveness of vaccines in protecting both human and \n134 animal populations. Consequently, our findings serve as a critical foundation for the adaptation of vaccine \n135 strategies to the unique epidemiological landscape of Serbia. The present vaccine strategies in Serbia \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n7\n136 include preparations for different animals which contain L. interrogans serovar Icterohaemorrhagiae, \n137 Canicola, Copenhageni and Bratislava, L. kirshneri serovar Grippotyphosa. Regarding leptospiros \n138 epidemiology, the identification of L. kirshneri serovar Mozdok opens doors to a more comprehensive \n139 understanding of the disease's distribution and dynamics in the region. The serovar's presence highlights \n140 the complexity of Leptospira populations in Serbia and warrants further investigation into its reservoir \n141 hosts and transmission dynamics. Epidemiological studies must now consider the unique characteristics of \n142 this serovar, as it may exhibit distinct patterns of host adaptation and disease transmission. Understanding \n143 the prevalence and distribution of this serovar is crucial for developing effective control measures, both in \n144 terms of prevention and treatment. Moreover, the discovery emphasizes the importance of continued \n145 surveillance and monitoring of Leptospira diversity in the region, as new serovars may continue to emerge \n146 over time. In conclusion, our study has provided valuable insights into the presence and diversity of \n147 Leptospira species in Serbia. The discovery of L. kirshneri serovar Mozdok serves as a pivotal point for \n148 advancing vaccine strategies and epidemiological research in the region. By adapting our approaches to the \n149 unique characteristics of this novel serovar, we can better address the challenges of leptospirosis and work \n150 towards more effective prevention and control measures for both human and veterinary health. \n151 Furthermore, the presence of Leptospira kirshneri serovar Mozdok opens new avenues for epidemiological \n152 research in Serbia. This novel serovar's presence highlights the complexity of Leptospira populations in the. \n153 Further research is essential to unveil the full implications of this discovery and to refine our understanding \n154 of the epidemiological landscape in Serbia.\n155\n156 4. Materials and methods\n157 4.1 Animal Collection\n158 The research was conducted in accordance with ethical principles and was approved by the Ministry of \n159 Agriculture, Forestry and Water Management (Republic of Serbia) - Veterinary Directorate (No. 323-07-\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n8\n160 04943/2020-05/2, 29.05.2020 and 323-07-04155/2023-05/2, 16.05.2023). During 2020, 2021 and 2022, a \n161 total of 344 (186 female and 158 male) carcasses of Norway rats ( Rattus norvegicus) were collected in the \n162 broad environs of Belgrade City. Carcasses were collected predominantly in their urban and suburban \n163 habitats. The largest number of individuals was collected after the implementation of control measures or \n164 the implementation of monitoring measures. The collected carcasses were kept in a freezer at -20 ◦C for a \n165 short time, until further processing. During autopsy, the kidneys were separated for further analysis and \n166 the morphological data, body weight and sex of the animals were recorded.\n167 4.2 DNA extraction, molecular detection, sequencing and MLST analysis\n168 DNA was extracted from the kidney using the Quick-DNA MiniPrep kit (Zymo Research, Australia, Cat. no. \n169 D3024), according to manufacturers’ instructions. Due to validate the extraction processes and all \n170 downstream steps, nuclease-free water and DNA extracted from Leptospira positive samples were used as \n171 positive and negative controls, respectively. DNA extracted from each sample was stored at −20 °C until \n172 downstream use. To distinguish between pathogenic and non-pathogenic Leptospira, we performed qPCR \n173 targeting the lipL32 partial target genes. Specifically, we used primers LipL32F (5’-GGA TCC GTG TAG AAA \n174 GAA TGT CGG-3’) and LipL32R (5’-GTC ACC ATC ATC ATC ATC GTC C-3’) to amplify a 101 bp fragment of the \n175 lipL32 gene, which was detected by the probe LipL32P (6-carboxyfluorescein [FAM]-5’-ATG CCT GAC CAA \n176 ATC GCC AAA GCT GCG AAA-3’-Black Hole Quencher 1 [BHQ1]) [10]. An internal control, represented by \n177 exogenous DNA added before the extraction phase, representing simultaneously the extraction and PCR \n178 amplification control (qPCR Extraction Control RED, Meridian Bioscience, UK) was also included. The qPCR \n179 was carried out in a 12 μL reaction mixture containing 3 μL of Leptospira spp. genomic DNA, 0.5 μL \n180 (concentration of 20 pmol/μL) of forward and reverse primer and probe and 5 μL (concentration of \n181 10pmol/μL) of FastGene 2x PROBE Universal (Nippon Genetics, Germany) and 2.5 μL of PCR water. All \n182 reactions were conducted in duplicates using a 7500 Fast Real-Time PCR System (Applied Biosystems, \n183 ThermoFisher, USA) with the following conditions: initial denaturation at 95°C for 2 min, followed by 45 \n184 cycles of denaturation at 95°C for 20 s, and annealing/elongation at 65°C for 50 s. Each PCR test included a \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n9\n185 negative control (DNA extracted from water) and a positive control (DNA extracted Leptospira spp. positive \n186 samples). Among the positive samples obtained through qPCR, only those with threshold cycle (Ct) values \n187 lower than or equal to 30 underwent further analysis. Specifically, 27 kidney samples and 27 Leptospira \n188 isolates were subjected to PCR using a set of primers amplifying adk, icdA, lipL32, lipL41, rrs2 and secY \n189 partial genes (Table 1) [14]. PCR reagents and their volumes, as well as PCR cycling conditions are shown in \n190 Table 2 and Table 3, respectively. The PCR products were visualized by electrophoresis on a 1.5% agarose \n191 gel and examined under UV transillumination.\n192 Table 1: Details of gene loci and the corresponding primer sequences used for MLST Analysis\nGene Locus\nGene \nsize \n(bp)\nGenome \nposition\nPCR \nproduct \n(bp)\nSize of \npolymorphic \nsequence (bp)\nPrimer sequences 5’-3’\nadk LIC12852 564\n3458298–\n3458861\n531 430\nF-GGGCTGGAAAAGGTACACAA\nR-ACGCAAGCTCCTTTTGAATC\nicdA LIC13244 1197\n3979829–\n3981025\n674 557\nF-GGGACGAGATGACCAGGAT\nR-TTTTTTGAGATCCGCAGCTTT\nlipL32 LIC11352 819\n1666299–\n1667117\n474 474\nF-ATCTCCGTTGCACTCTTTGC\nR-ACCATCATCATCATCGTCCA\nlipL4l LIC12966 1068\n3603575–\n3604642\n520 518\nF-TAGGAAATTGCGCAGCTACA\nR-GCATCGAGAGGAATTAACATCA\nrrs2 LIC11508 1512\n1862433–\n1863944\n541 452\nF-CATGCAAGTCAAGCGGAGTA\nR-AGTTGAGCCCGCAGTTTTC\nsecY LIC12853 1383\n3458869–\n3460251\n549 549\nF-ATGCCGATCATTTTTGCTTC\nR-CCGTCCCTTAATTTTAGACTTCTTC\n193\n194 Table 2: Reagents and Volumes\n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n10\nReagent\nVolume per \nReaction - µL\nDNA Template (Leptospira \nDNA)\n3\nForward Primer\n1 (concentration of \n20 pmol/μL\nReverse Primer\n1 (concentration of \n20 pmol/μL\nHotStarTaq Master Mix 12.5\nSterile Water 7.5\nTotal Reaction Volume 25\n195\n196 Table 3: PCR Cycling Conditions \nStep\nTemperature \n(°C)\nTime Number of Cycles\nInitial \nDenaturation\n95 15 minutes 1\nDenaturation 95 30 seconds\nAnnealing 58 30 seconds\nExtension 72 1 minute\n35\nFinal Extension 72 10 1\nHold 4 ∞ 1\n197  \n198 We purified (GeneJET PCR Purification Kit, ThermoFisher Scientific, USA, cat. no. K0702) and sent all \n199 positive amplicons for genes listed in Table 2 to Macrogen Europe for Sanger sequencing. Sequences were \n.CC-BY 4.0 International licenseperpetuity. It is made available under a \npreprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in \nThe copyright holder for thisthis version posted January 11, 2024. ; https://doi.org/10.1101/2024.01.11.575145doi: bioRxiv preprint \n\n11\n200 analyzed and edited using the Staden package [21]. Consensus sequence validation was performed against \n201 a custom Leptospira database using nucleotide blast (BLASTn) [22]  Each allele and the allelic profiles (adk-\n202 icdA-lipL32-lipL41-rrs2-secY) were submitted to the Leptospira database [23] \n203 (http://pubmlst.org/Leptospira, accessed in October 2023) for ST assignment. Sequence similarity of our \n204 samples was performed with a custom reference database using Biopython [24]. All sequences were \n205 submitted to NCBI’s GenBank under the following accession numbers: OR920389 - OR920523 for adk, icdA, \n206 lipL32, LipL41 and secY, while for rrs2 OR912477-OR912503. \n207 4.3 Statistical analysis\n208 Mean prevalence and confidence intervals (95% CI) for Leptospira spp. were determined using the Clopper \n209 and Pearson method. An alpha value <0.05 was considered the threshold for statistical significance.\n210 Conflict of interest: The authors declare that they have no known competing financial interests or personal \n211 relationships that could have appeared to influence the work reported in this paper.\n212 Funding Source: This work was funded by Ministry of Science, Technological Development and Innovation \n213 of Republic of Serbia by the Contract of implementation and funding of research work of NIV-NS in 2023, \n214 Contract No: 451-03-47/2023-01/200031.\n215 Ethical Approval statement: Ethics review and approval for this study were obtained from the Ministry of \n216 Agriculture, Forestry and Water Management (Republic of Serbia) - Veterinary Directorate (No. 323-07-\n217 04943/2020-05/2, 29.05.2020 and 323-07-04155/2023-05/2, 16.05.2023).\n218\n219 6. References \n220 [1] Costa F, Hagan JE, Calcagno JI, et al. Global Morbidity and Mortality of Leptospirosis: a Systematic \n221 review. 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