Surveillance of human leptospirosis infections in Ukraine between 2018 and 2023.

Leptospirosis is a bacterial disease that affects both humans and animals worldwide. Currently, a positional war is ongoing in Ukraine, and the military is encountering a significant number of rodents in trenches and dugouts, which are known reservoirs for Leptospira, the causative agent of leptospirosis-a potentially dangerous infectious disease with a high mortality rate. The civilian population is also at potential risk of leptospirosis. The destruction of the Kakhovka Dam on June 6, 2023, has led to widespread devastation and human suffering. In the short term, there is a significant risk of rodent-borne diseases such as leptospirosis. We utilized data from the Ukrainian Centre for Disease Prevention Control and observed a substantial increase in prevalence in 2023. The notification rate in Ukraine in 2023 was 1.06 per 100,000 persons, which is higher than that of other countries in the European Union. Particular attention is being given to Zakarpattia Oblast, located on the western border of Ukraine, which shares boundaries with Romania, Hungary, Poland, and Slovakia, with an extremely high incidence rate of 12.08 per 100,000 persons. Based on these findings, we recommend education and awareness campaigns, vaccination, personal protective measures, and improved surveillance to address the increasing incidence of leptospirosis in Ukraine.

Comprehensive Analysis of Antiphage Defense Mechanisms: Serovar-Specific Patterns.

Leptospirosis is a major zoonotic disease caused by pathogenic spirochetes in the genus Leptospira, affecting over a million people annually and causing approximately 60,000 deaths. Leptospira interrogans, a key causative agent, likely possesses defense systems against bacteriophages (leptophages), yet these systems are not well understood. We analyzed 402 genomes of L. interrogans using the DefenseFinder tool to identify and characterize the antiphage defense systems. We detected 24 unique systems, with CRISPR-Cas (Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated proteins), PrrC, Borvo, and Restriction-Modification (R-M) being the most prevalent. Notably, Cas were identified in all strains, indicating their central role in phage defense. Furthermore, there were variations in the antiphage system distribution across different serovars, suggesting unique evolutionary adaptations. For instance, Retron was found exclusively in the Canicola serovar, while prokaryotic Argonaute proteins (pAgo) were only detected in the Grippotyphosa serovar. These findings significantly enhance our understanding of Leptospira's antiphage defense mechanisms. They reveal the potential for the development of serovar-specific phage-based therapies and underscore the importance of further exploring these defense systems.

Leptospiral LipL45 lipoprotein undergoes processing and shares structural similarities with bacterial sigma regulators.

Leptospirosis is a widespread zoonotic infectious disease of human and veterinary concern caused by pathogenic spirochetes of the genus Leptospira. To date, little progress towards understanding leptospiral pathogenesis and identification of virulence factors has been made, which is the main bottleneck for developing effective measures against the disease. Some leptospiral proteins, including LipL32, Lig proteins, LipL45, and LipL21, are being considered as potential virulence factors or vaccine candidates. However, their function remains to be established. LipL45 is the most expressed membrane lipoprotein in leptospires, upregulated when the bacteria are transferred to temperatures resembling the host, expressed during infection, suppressed after culture attenuation, and known to suffer processing in vivo and in vitro, generating fragments. Based on body of evidence, we hypothesized that the LipL45 processing might occur by an auto-cleavage event, deriving two fragments. The results presented here, based on bioinformatics, structure modeling analysis, and experimental data, corroborate that LipL45 processing probably includes a self-catalyzed non-proteolytic event and suggest the participation of LipL45 in cell-surface signaling pathways, as the protein shares structural similarities with bacterial sigma regulators. Our data indicate that LipL45 might play an important role in response to environmental conditions, with possible function in the adaptation to the host.

Current treatment options for leptospirosis: a mini-review.

Leptospirosis, one of the most common global zoonotic infections, significantly impacts global human health, infecting more than a million people and causing approximately 60,000 deaths annually. This mini-review explores effective treatment strategies for leptospirosis, considering its epidemiology, clinical manifestations, and current therapeutic approaches. Emphasis is placed on antibiotic therapy, including recommendations for mild and severe cases, as well as the role of probiotics in modulating the gut microbiota. Furthermore, novel treatment options, such as bacteriophages and newly synthesized/natural compounds, are discussed, and the findings are expected to provide insights into promising approaches for combating leptospirosis.

AMR mechanisms in L. interrogans serovars: a comprehensive study.

Antimicrobial resistance (AMR) is one of the global health challenges of the 21st century. Data regarding AMR mechanisms in Leptospira interrogans, the causative agents of leptospirosis, have been relatively limited. Therefore, our study aimed to identify resistance genes and explore potential resistance mechanisms specific to particular serovars. We conducted a comprehensive analysis of 98 Leptospira strains, representing 10 common serovars, using whole-genome sequencing (WGS) FASTA files. Employing the PATRIC tool from the Bacterial and Viral Bioinformatics Resource Center (BV-BRC), we scrutinized the genomes for AMR genes. Our investigation revealed 32 genes associated with AMR, with 20 key genes consistently prevalent across most strains. Notably, we identified unique efflux pump systems in serovar Pomona, indicating distinctive resistance mechanisms in this serovar. In summary, our findings shed light on the genetic landscape of AMR in Leptospira, uncovering both common and serovar-specific resistance elements. The presence of unique efflux pump systems in serovar Pomona introduces a novel dimension to our understanding of resistance mechanisms. These insights underscore the importance of tailored intervention strategies and collaborative efforts between human and veterinary healthcare professionals, as well as environmental scientists, to address the complex dynamics of leptospirosis and its implications for antibiotic resistance.

Exploring Leptospira interrogans FDAARGOS_203: Insights into AMR and Anti-Phage Defense.

Leptospira, which are known to be important disease-causing agents transmitted between animals and humans, result in significant illness and, in some cases, significant death in human populations. This purpose of this study was to examine the genomic structure of Leptospira interrogans serovar Copenhageni strain FDAARGOS_203 to identify the specific genetic factors that contribute to antimicrobial resistance (AMR) and defense against phages. The genome, consisting of two contigs totaling 4,630,574 base pairs, underwent thorough examination for protein-coding sequences, transfer RNA genes, and ribosomal RNA genes. A total of twenty-two antibiotic resistance genes that specifically target essential cellular processes such as cell wall synthesis, DNA replication, and protein synthesis have been identified. Significant among these were gidB, gdpD, and ggsA, each involved in separate aspects of antibiotic resistance. In addition, the investigation explored the defense mechanisms of bacteriophages, revealing the presence of defense islands that contain a range of anti-phage systems, including RM_Type_IV, PrrC, Borvo, CAS_Class1-Subtype-IC, and CAS_Class1-Subtype-IB. This comprehensive genomic analysis enhances our understanding of the molecular mechanisms that determine Leptospira's ability to adapt to various environments. The identified genetic factors linked to AMR and defense against phages not only enhance our scientific comprehension, but also provide a basis for focused interventions to reduce the impact of leptospirosis.

High Exposure to Livestock Pathogens in Southern Pudu (Pudu puda) from Chile.

A significant gap in exposure data for most livestock and zoonotic pathogens is common for several Latin America deer species. This study examined the seroprevalence against 13 pathogens in 164 wild and captive southern pudu from Chile between 2011 and 2023. Livestock and zoonotic pathogen antibodies were detected in 22 of 109 wild pudus (20.18%; 95% CI: 13.34-29.18) and 17 of 55 captive pudus (30.91%; 95% CI: 19.52-44.96), including five Leptospira interrogans serovars (15.38% and 10.71%), Toxoplasma gondii (8.57% and 37.50%), Chlamydia abortus (3.03% and 12.82%), Neospora caninum (0.00% and 9.52%), and Pestivirus (8.00% and 6.67%). Risk factors were detected for Leptospira spp., showing that fawn pudu have statistically significantly higher risk of positivity than adults. In the case of T. gondii, pudu living in "free-range" have a lower risk of being positive for this parasite. In under-human-care pudu, a Pestivirus outbreak is the most strongly suspected as the cause of abortions in a zoo in the past. This study presents the first evidence of Chlamydia abortus in wildlife in South America and exposure to T. gondii, L. interrogans, and N. caninum in wild ungulate species in Chile. High seroprevalence of livestock pathogens such as Pestivirus and Leptospira Hardjo in wild animals suggests a livestock transmission in Chilean template forest.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms.

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1-130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1. SIGNIFICANCE: The comparative analysis established an array of specific proteins in pathogenic strain that will narrow down the identification of immune protective proteins that will help fight leptospirosis.

Global proteome of the saprophytic strain Leptospira biflexa and comparative analysis with pathogenic strain Leptospira interrogans uncover new pathogenesis mechanisms

Leptospira is a genus of bacteria that includes free-living saprophytic species found in water or soil, and pathogenic species, which are the etiologic agents of leptospirosis. Besides all the efforts, there are only a few proteins described as virulence factors in the pathogenic strain L. interrogans. This work aims to perform L. biflexa serovar Patoc1 strain Paris global proteome and to compare with the proteome database of pathogenic L. interrogans serovar Copenhageni strain Fiocruz L1–130. We identified a total of 2327 expressed proteins of L. biflexa by mass spectrometry. Using the Get Homologues software with the global proteome of L. biflexa and L. interrogans, we found orthologous proteins classified into conserved, low conserved, and specific proteins. Comparative bioinformatic analyses were performed to understand the biological functions of the proteins, subcellular localization, the presence of signal peptide, structural domains, and motifs using public softwares. These results lead to the selection of 182 low conserved within the saprophyte, and 176 specific proteins of L. interrogans. It is anticipated that these findings will indicate further studies to uncover virulence factors in the pathogenic strain. This work presents for the first time the global proteome of saprophytic strain L. biflexa serovar Patoc, strain Patoc1.

Updated ACVIM consensus statement on leptospirosis in dogs.

Since publication of the last consensus statement on leptospirosis in dogs, there has been revision of leptospiral taxonomy and advancements in typing methods, widespread use of new diagnostic tests and vaccines, and improved understanding of the epidemiology and pathophysiology of the disease. Leptospirosis continues to be prevalent in dogs, including in small breed dogs from urban areas, puppies as young as 11 weeks of age, geriatric dogs, dogs in rural areas, and dogs that have been inadequately vaccinated for leptospirosis (including dogs vaccinated with 2-serovar Leptospira vaccines in some regions). In 2021, the American College of Veterinary Internal Medicine (ACVIM) Board of Regents voted to approve the topic for a revised Consensus Statement. After identification of core panelists, a multidisciplinary group of 6 experts from the fields of veterinary medicine, human medicine, and public health was assembled to vote on the recommendations using the Delphi method. A draft was presented at the 2023 ACVIM Forum, and a written draft posted on the ACVIM website for comment by the membership before submission to the editors of the Journal of Veterinary Internal Medicine. This revised document provides guidance for veterinary practitioners on disease in dogs as well as cats. The level of agreement among the 12 voting members (including core panelists) is provided in association with each recommendation. A denominator lower than 12 reflects abstention of ≥1 panelists either because they considered the recommendation to be outside their scope of expertise or because there was a perceived conflict of interest.

Degradation of p0071 and p120-catenin during adherens junction disassembly by Leptospira interrogans.

Leptospira interrogans disseminates hematogenously to reach the target organs by disrupting epithelial adherens junctions (AJs), thus causing leptospirosis, which is a globally neglected zoonotic disease. L. interrogans induces E-cadherin (E-cad) endocytosis and cytoskeletal rearrangement during AJ disassembly, but the detailed mechanism remains unknown. Elucidation of AJ disassembly mechanisms will guide new approaches to developing vaccines and diagnostic methods. In this study, we combine proteomic and imaging analysis with chemical inhibition studies to demonstrate that disrupting the AJs of renal proximal tubule epithelial cells involves the degradation of two armadillo repeat-containing proteins, p0071 and p120-catenin, that stabilize E-cad at the plasma membrane. Combining proteasomal and lysosomal inhibitors substantially prevented p120-catenin degradation, and monolayer integrity destruction without preventing p0071 proteolysis. In contrast, the pan-caspase inhibitor Z-VAD-FMK inhibited p0071 proteolysis and displacement of both armadillo repeat-containing proteins from the cell-cell junctions. Our results show that L. interrogans induces p120-catenin and p0071 degradation, which mutually regulates E-cad stability by co-opting multiple cellular degradation pathways. This strategy may allow L. interrogans to disassemble AJs and disseminate through the body efficiently.

A Rare Complication of Leptospirosis: Weil's Disease Diagnosed in the United States.

Leptospirosis is mostly found in tropical regions such as Latin America and Southeast Asia. Here we present a case of leptospirosis in the United States in a 43-year-old African American male who had complications such as sepsis, acute renal failure, hyperbilirubinemia, and transaminitis. Through this case report, we want to highlight the rare occurrence of this infection in the United States. It should be considered high in the list of differential diagnoses for patients traveling to underdeveloped countries and participating in adventure sports. Early recognition and treatment of leptospirosis are essential in decreasing life-threatening complications.

Protective role of stratum corneum in percutaneous Leptospira infection in a hamster model.

Leptospira enters humans and animals through injured skin or mucous membranes by direct or indirect contact with urine excreted from infected reservoirs. Individuals with cut or scratched skin are at high risk of infection and are recommended to be protected from contact with Leptospira, but the risk of infection via skin without apparent wounds is unknown. We hypothesized that the stratum corneum of the epidermis might prevent percutaneous invasion of leptospires. We established a stratum corneum deficient model of hamsters using the tape stripping method. The mortality rate of hamsters lacking stratum corneum that were exposed to Leptospira was higher than that of controls with shaved skin, and was not significantly different from an epidermal wound group. These results indicated that the stratum corneum plays a critical role in protecting the host against leptospiral entry. We also examined the migration of leptospires through the monolayer of HaCaT cells (human keratinocyte cell line) using Transwell. The number of pathogenic leptospires penetrating the HaCaT cell monolayers was higher than that of non-pathogenic leptospires. Furthermore, scanning and transmission electron microscopic observations revealed that the bacteria penetrated the cell monolayers through both intracellular and intercellular routes. This suggested that pathogenic Leptospira can migrate easily through keratinocyte layers and is associated with virulence. Our study further highlights the importance of the stratum corneum as a critical barrier against the invasion of Leptospira found in contaminated soil and water. Hence, preventative measures against contact infection should be taken, even without visible skin wounds.

Complete genome sequences of twelve strains of Leptospira interrogans isolated from humans in Sri Lanka.

Leptospirosis, a major zoonotic disease caused by pathogenic Leptospira spp. is recognized globally as an emerging zoonotic disease. Whole-genome sequencing reveals hidden messages about Leptospira's pathogenesis. We used Single Molecule Real-Time (SMRT) sequencing to obtain complete genome sequences of twelve L. interrogans isolates from febrile patients from Sri Lanka for a comparative whole genome sequencing study. The sequence data generated 12 genomes with a coverage greater than X600 with sizes ranging from 4.62 Mb to 5.16 Mb, and a G + C content ranging from 35.00% to 35.42%. The total number of coding sequences predicted by the NCBI (National Center for Biotechnology Information) genome assembly platform ranged from 3845 to 4621 for the twelve strains. Leptospira serogroup with similar-sized LPS biosynthetic loci that belonged to the same clade had a close relationship in the phylogenetic analysis. Nonetheless, variations in the genes encoding sugar biosynthesis were found in the serovar determinant region (rfb locus). Type I and Type III CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems were found in all of the strains. Genome BLAST Distance Phylogeny of these sequences allowed for detailed genomic strain typing. These findings may help us better understand the pathogenesis, develop a tools for early diagnosis, comparative genomic analysis and evolution of Leptospira.

Outbreak of leptospirosis amidst COVID'19 in Tanzania: a new threat? Short communication.

As the world is still fighting to combat the coronavirus disease 2019 (COVID-19) virus, the United Republic of Tanzania has been confronting yet another bacterial infection called leptospirosis (LS). It is caused by the spirochete bacteria of genus Leptospira, and has been known to infect several people, already claiming a number of lives. It infects 1 million people annually with ~60 000 deaths having a fatality rate of 6.85% worldwide. COVID has profusely burdened the healthcare system worldwide within the past 2 years; it has sabotaged medical management and brought down resources, which has now made it difficult for any country to withstand another pandemic. LS has overburdened the medical care system of Tanzania abjectly; it is now imperative not to overlook environmental factors, like a flood, the presence of rodents, unsatisfactory socioeconomic conditions in areas where dogs reside, substandard wastewater and garbage disposal facilities, or any other factor which might lead to further spread of LS and put Tanzania in jeopardy.

Lipopolysaccharide and Glycolipoprotein Coordinately Triggered Necroptosis Contributes to the Pathogenesis of Leptospira Infection in Mice.

Leptospirosis is a recurring but neglected zoonotic disease caused by pathogenic Leptospira. The explicit underlying mechanism of necroptosis and its role in Leptospira infection have not yet been elucidated. Here we reported that leptospiral pathogen-associated molecular patterns, lipopolysaccharide, and glycolipoprotein activate the necroptotic RIPK1-RIPK3-MLKL cascade through the TLR4 signaling pathway in mouse macrophages. Using the murine acute leptospirosis model, we reveal that abolition of necroptosis exhibited significantly improved outcomes in acute phases, with enhanced eradication of Leptospira from liver, mild clinical symptoms, and decreased cytokine production. RIPK3 was also found to exert a necroptosis-independent function in CXCL1 production and neutrophil recruitment, with the consequence of improved Leptospira control. These findings improve our understanding of the mechanism of Leptospira-macrophage interactions, indicating potential therapeutic values by targeting necroptosis signaling pathways.

Biocomputational Identification of sRNAs in Leptospira interrogans Serovar Lai.

Regulatory small RNAs (sRNA) are RNA transcripts that are not translated into proteins but act as functional RNAs. Pathogenic Leptospira cause an epidemic spirochaetal zoonosis, Leptospirosis. It is speculated that Leptospiral sRNAs are involved in orchestrating their pathogenicity. In this study, biocomputational approach was adopted to identify Leptospiral sRNAs. In this study, two sRNA prediction programs, i.e., RNAz and nocoRNAc, were employed to screen the reference genome of Leptospira interrogans serovar Lai. Out of 126 predicted sRNAs, there are 96 cis-antisense sRNAs, 28 trans-encoded sRNAs and 2 sRNAs that partially overlap with protein-coding genes in a sense orientation. To determine whether these candidates are expressed in the pathogen, they were compared with the coverage files generated from our RNA-seq datasets. It was found out that 7 predicted sRNAs are expressed in mid-log phase, stationary phase, serum stress, temperature stress and iron stress while 2 sRNAs are expressed in mid-log phase, stationary phase, serum stress, and temperature stress. Besides, their expressions were also confirmed experimentally via RT-PCR. These experimentally validated candidates were also subjected to mRNA target prediction using TargetRNA2. Taken together, our study demonstrated that biocomputational strategy can serve as an alternative or as a complementary strategy to the laborious and expensive deep sequencing methods not only to uncover putative sRNAs but also to predict their targets in bacteria. In fact, this is the first study that integrates computational approach to predict putative sRNAs in L. interrogans serovar Lai. Supplementary Information: The online version contains supplementary material available at 10.1007/s12088-022-01050-9.

In silico identification and characterization of potential druggable targets among hypothetical proteins of Leptospira interrogans serovar Copenhageni: a comprehensive bioinformatics approach.

Leptospirosis is one of the neglected zoonosis, affecting human and animal populations worldwide. Reliable effective therapeutics and concerns to look for more research into the molecular analysis of its genome is therefore needed. In the genomic pool of the Leptospira interrogans many hypothetical proteins are still uncharacterized. In the current research, we performed extensive in silico analysis to prioritize the potential hypothetical proteins of L. interrogans serovar Copenhageni via stepwise reducing the available hypothetical proteins (Total 3606) of the assembly to only 15, based on non-homologous to homosapien, essential, functional, virulent, cellular localization. Out of them, only two proteins WP_000898918.1 (Hypothetical Protein 1) & WP_001014594.1 (Hypothetical Protein 2) were found druggable and involved in protein-protein interaction network. The 3 D structures of these two target proteins were predicted via ab initio homology modeling followed by structures refinement and validation, as no structures were available till date. The analysis also revealed that the functional domains, families and protein-protein interacting partners identified in both proteins are crucial for the survival of the bacteria. The binding cavities were predicted for both the proteins through blind and specific protein-ligand docking with their respective ligands and inhibitors and were found to be in accordance with the druggable sites predicted by DoGSiteScorer. The docking interactions were found within the active functional domains for both the proteins while for Hypothetical Protein 2, the same residues were involved in interactions with Cytidine-5'-triphosphate in blind and specific docking. Furthermore, the simulations of molecular dynamics and free binding energy revealed the stable substrate binding and efficient binding energies, and were in accordance to our docking results. The work predicted two unique hypothetical proteins of L. interrogans as a potential druggable targets for designing of inhibitors for them.Communicated by Ramaswamy H. Sarma.

Multiepitope-based vaccine design by exploring antigenic potential among leptospiral lipoproteins using comprehensive immunoinformatics and structure-based approaches.

Leptospirosis is a tropical and globally neglected zoonotic disease caused by pathogenic spirochetes, Leptospira. Although the disease has been studied for decades, a potent or effective vaccine is not available so far. Efforts are being made to design an efficient vaccine candidate using different approaches. Immunoinformatics approaches have been proven to be promising in terms of time and cost. Here, we used immunoinformatics and structure-based approaches to evaluate antigenic B- and T-cell epitopes present on the leptospiral lipoproteins (LipL). The promiscuous overlapping epitopes (B-cell, T-cell, interferon (IFN)-γ positive, and non-allergens), which can induce humoral, cell-mediated, and innate immunity, were selected to generate a multiepitope chimeric vaccine. To enhance the vaccine immunogenicity, a Toll-like receptor (TLR) agonist was fused to the vaccine with a suitable linker. The chimeric vaccine structure was predicted for molecular docking studies with immune receptors. Moreover, the stability of the vaccine-immune receptor complexes was analyzed by normal mode analysis (NMA). The potency of the vaccine construct was predicted by the immune simulation tool. The study provides additional information toward constructing peptide-based chimeric vaccines  against Leptospira.

Leptospirosis-Induced Septic Shock and Multi-Organ Dysfunction Syndrome: A Complex Case of Zoonotic Infection in a Young Female Patient.

Leptospirosis, a zoonotic infection caused by the Leptospira bacteria, can manifest with varying clinical severities, ranging from subclinical disease to severe multiorgan failure. This progression to severe multiorgan failure, also known as multi-organ dysfunction syndrome (MODS), is a life-threatening condition characterized by the dysfunction of two or more organ systems. Often, MODS is a consequence of events triggered by underlying pathologies, such as severe infections, including those caused by Leptospira. Here, we present a case report of a 29-year-old female patient who initially sought care for increased temperature, fatigue, diarrhea, and vomiting. The patient exhibited signs of septic shock (SS). Her medical history raised suspicion of multiple potential sources of infection. She experienced cat scratch several days before admission, accompanied by an enlarged inguinal lymph node and a history of frequent interaction with animals, as well as freshwater exposure, which prompted investigations into various zoonotic infections. Empiric treatment was started, and, subsequently, after several days, Leptospira titer came back positive. Over the hospitalization course, the patient developed multi-organ failure, which was attributed to the underlying Leptospira infection. The complexity and severity of the patient's condition underscore the far-reaching impact of leptospirosis in precipitating a spectrum of systemic complications culminating in multiorgan failure. The treatment interventions yielded successful outcome, and the patient recovered in one month. This case report highlights the difficulties in diagnosing and treating patients with multiple possible sources of infection. it emphasizes the need for a careful history-taking and high level of suspicion for zoonotic infections in patients with a history of animal exposure and clinical symptoms suggestive of infectious diseases.