CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira.

Leptospirosis, caused by pathogenic bacteria from the genus Leptospira, is a global zoonosis responsible for more than one million human cases and 60,000 deaths annually. The disease also affects many domestic animal species. Historically, genetic manipulation of Leptospira has been difficult to perform, resulting in limited knowledge on pathogenic mechanisms of disease and the identification of virulence factors. The application of CRISPR/Cas9 and its variations have helped fill these gaps but the generation of knockout mutants remains challenging because double-strand breaks (DSBs) inflicted by Cas9 nuclease are lethal to Leptospira cells. The novel CRISPR prime editing (PE) strategy is the first precise genome-editing technology that allows deletions, insertions, and base substitutions without introducing DSBs. This revolutionary technique utilizes a nickase Cas9 that cleaves a single strand of DNA, coupled with an engineered reverse transcriptase and a modified single-guide RNA (termed prime editing guide RNA) containing an extended 3' end with the desired edits. We demonstrate the application of CRISPR-PE in both saprophytic and pathogenic Leptospira from multiple species and serovars by introducing deletions or insertions into target DNA with a remarkable precision of just one nucleotide. Additionally, we demonstrate the ability to genetically manipulate Leptospira borgpetersenii, a prevalent pathogenic species of humans, domestic cattle, and wildlife animals. Rapid plasmid loss by mutated strains in liquid culture allows for the generation of knockout strains without selective markers, which can be readily used to elucidate virulence factors and develop optimized bacterin and/or live vaccines against leptospirosis.IMPORTANCELeptospirosis is a geographically widespread bacterial zoonosis. Genetic manipulation of pathogenic Leptospira spp. has been laborious and difficult to perform, limiting our ability to understand how leptospires cause disease. The application of the CRISPR/Cas9 system to Leptospira enhanced our ability to generate knockdown and knockout mutants; however, the latter remains challenging. Here, we demonstrate the application of the CRISPR prime editing technique in Leptospira, allowing the generation of knockout mutants in several pathogenic species, with mutations comprising just a single nucleotide resolution. Notably, we generated a mutant in the Leptospira borgpetersenii background, a prevalent pathogenic species of humans and cattle. Our application of this method opens new avenues for studying pathogenic mechanisms of Leptospira and the identification of virulence factors across multiple species. These methods can also be used to facilitate the generation of marker-less knockout strains for updated and improved bacterin and/or live vaccines.

Host population structure and rare dispersal events drive leptospirosis transmission patterns among Rattus norvegicus in Boston, Massachusetts, US.

Leptospirosis (caused by pathogenic bacteria in the genus Leptospira ) is prevalent worldwide but more common in tropical and subtropical regions. Transmission can occur following direct exposure to infected urine from reservoir hosts, such as rats, or a urine-contaminated environment, which then can serve as an infection source for additional rats and other mammals, including humans. The brown rat, Rattus norvegicus , is an important reservoir of leptospirosis in urban settings. We investigated leptospirosis among brown rats in Boston, Massachusetts and hypothesized that rat dispersal in this urban setting influences the movement, persistence, and diversity of Leptospira . We analyzed DNA from 328 rat kidney samples collected from 17 sites in Boston over a seven-year period (2016-2022); 59 rats representing 12 of 17 sites were positive for Leptospira . We used 21 neutral microsatellite loci to genotype 311 rats and utilized the resulting data to investigate genetic connectivity among sampling sites. We generated whole genome sequences for 28 Leptospira isolates obtained from frozen and fresh tissue from some of the 59 Leptospira -positive rat kidneys. When isolates were not obtained, we attempted Leptospira genomic DNA capture and enrichment, which yielded 14 additional Leptospira genomes from rats. We also generated an enriched Leptospira genome from a 2018 human case in Boston. We found evidence of high genetic structure and limited dispersal among rat populations that is likely influenced by major roads and/or other unknown dispersal barriers, resulting in distinct rat population groups within the city; at certain sites these groups persisted for multiple years. We identified multiple distinct phylogenetic clades of L. interrogans among rats, with specific clades tightly linked to distinct rat populations. This pattern suggests L. interrogans persists in local rat populations and movement of leptospirosis in this urban rat community is driven by rat dispersal. Finally, our genomic analyses of the 2018 human leptospirosis case in Boston suggests a link to rats as the source. These findings will be useful for guiding rat control and human leptospirosis mitigation efforts in this and other urban settings.

Identification of equine mares as reservoir hosts for pathogenic species of Leptospira.

Equine leptospirosis can result in abortion, stillbirth, neonatal death, placentitis, and uveitis. Horses can also act as subclinical reservoir hosts of infection, which are characterized as asymptomatic carriers that persistently excrete leptospires and transmit disease. In this study, PCR and culture were used to assess urinary shedding of pathogenic Leptospira from 37 asymptomatic mares. Three asymptomatic mares, designated as H2, H8, and H9, were PCR-positive for lipL32, a gene specific for pathogenic species of Leptospira. One asymptomatic mare, H9, was culture-positive, and the recovered isolate was classified as L. kirschneri serogroup Australis serovar Rushan. DNA capture and enrichment of Leptospira genomic DNA from PCR-positive, culture-negative samples determined that asymptomatic mare H8 was also shedding L. kirschneri serogroup Australis, whereas asymptomatic mare H2 was shedding L. interrogans serogroup Icterohaemorrhagiae. Sera from all asymptomatic mares were tested by the microscopic agglutination test (MAT) and 35 of 37 (94.6%) were seropositive with titers ranging from 1:100 to 1:3200. In contrast to asymptomatic mares, mare H44 presented with acute spontaneous abortion and a serum MAT titer of 1:102,400 to L. interrogans serogroup Pomona serovar Pomona. Comparison of L. kirschneri serogroup Australis strain H9 with that of L. interrogans serogroup Pomona strain H44 in the hamster model of leptospirosis corroborated differences in virulence of strains. Since lipopolysaccharide (LPS) is a protective antigen in bacterin vaccines, the LPS of strain H9 (associated with subclinical carriage) was compared with strain H44 (associated with spontaneous abortion). This revealed different LPS profiles and immunoreactivity with reference antisera. It is essential to know what species and serovars of Leptospira are circulating in equine populations to design efficacious vaccines and diagnostic tests. Our results demonstrate that horses in the US can act as reservoir hosts of leptospirosis and shed diverse pathogenic Leptospira species via urine. This report also details the detection of L. kirschneri serogroup Australis serovar Rushan, a species and serotype of Leptospira, not previously reported in the US.

Concurrent colonization of rodent kidneys with multiple species and serogroups of pathogenic Leptospira.

Rodents are important reservoir hosts of pathogenic leptospires in the US Virgin Islands. Our previous work determined that trapped rodents were colonized with Leptospira borgpetersenii serogroup Ballum (n = 48) and/or Leptospira kirschneri serogroup Icterohaemorrhagiae (n = 3). In addition, nine rodents appeared to be colonized with a mixed population comprising more than one species/serogroup. The aim of this study was to validate this finding by characterizing clonal isolates derived from cultures of mixed species. Cultures of presumptive mixed species (designated LR1, LR5, LR37, LR57, LR60, LR61, LR68, LR70, and LR72) were propagated in different media including Hornsby-Alt-Nally (HAN) media, incubated at both 29℃ and 37℃, and T80/40/LH incubated at 29℃. Polyclonal reference antisera specific for serogroup Ballum and Icterohaemorrhagiae were used to enrich for different serogroups followed by subculture on agar plates. Individual colonies were then selected for genotyping and serotyping. Of the nine cultures of mixed species/serogroups, a single clonal isolate was separated in five of them: L. borgpetersenii serogroup Ballum in LR1, LR5, and LR37, and L. kirschneri serogroup Icterohaemorrhagiae in LR60 and LR72. In four of the cultures with mixed species (LR57, LR61, LR68, and LR70), clonal isolates of both L. borgpetersenii serogroup Ballum and L. kirschneri serogroup Icterohaemorrhagiae were recovered. Our results definitively establish that rodents can be colonized with more than one species/serogroup of Leptospira concurrently. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.IMPORTANCEPathogenic Leptospira, the causative agent of human and animal leptospirosis, comprise a diverse genus of species/serogroups which are inherently difficult to isolate from mammalian hosts due to fastidious growth requirements. Molecular evidence has indicated that reservoir hosts of Leptospira may shed multiple species concurrently. However, evidence of this phenomena by culture has been lacking. Culture is definitive and is essential for comprehensive characterization of recovered isolates by high-resolution genome sequencing and serotyping. In this work, a protocol using recently developed novel media formulations, in conjunction with reference antisera, was developed and validated to demonstrate the recovery of multiple species/serogroups of pathogenic Leptospira from the same host. The identification and characterization of multiple species/serogroups of Leptospira from individual reservoir hosts of infection are essential to understand the epidemiology and transmission of disease to both human and domestic animal populations.

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection.

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch's molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen's ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

DNA Capture and Enrichment: A Culture-Independent Approach for Characterizing the Genomic Diversity of Pathogenic Leptospira Species.

Because they are difficult to culture, obtaining genomic information from Leptospira spp. is challenging, hindering the overall understanding of leptospirosis. We designed and validated a culture-independent DNA capture and enrichment system for obtaining Leptospira genomic information from complex human and animal samples. It can be utilized with a variety of complex sample types and diverse species as it was designed using the pan-genome of all known pathogenic Leptospira spp. This system significantly increases the proportion of Leptospira DNA contained within DNA extracts obtained from complex samples, oftentimes reaching >95% even when some estimated starting proportions were <1%. Sequencing enriched extracts results in genomic coverage similar to sequenced isolates, thereby enabling enriched complex extracts to be analyzed together with whole genome sequences from isolates, which facilitates robust species identification and high-resolution genotyping. The system is flexible and can be readily updated when new genomic information becomes available. Implementation of this DNA capture and enrichment system will improve efforts to obtain genomic data from unculturable Leptospira-positive human and animal samples. This, in turn, will lead to a better understanding of the overall genomic diversity and gene content of Leptospira spp. that cause leptospirosis, aiding epidemiology and the development of improved diagnostics and vaccines.

Animals Exposed to Leptospira Serogroups Not Included in Bacterins in the United States and Puerto Rico.

Leptospirosis is a worldwide zoonotic disease. Pathogenic leptospires colonize the renal tubules and genital tract of animals and are excreted via urine. Transmission occurs via direct contact or through contaminated water or soil. The microscopic agglutination test (MAT) is the gold standard for the serodiagnosis of leptospirosis. The present study aims to evaluate animal exposure to Leptospira in the U.S. and Puerto Rico during the period 2018-2020. The presence of antibodies against pathogenic Leptospira spp. was assessed with the MAT according to the standards of the World Organisation for Animal Health. A total of 568 sera were submitted for diagnostic, surveillance, or import/export testing from the U.S. and Puerto Rico. Seropositivity (≥1:100) was 51.8% (294/568) with agglutinating antibodies found in 115 (39.1%) cattle, 84 (28.6%) exotic animals, 38 (12.9%) horses, 22 (7.5%) goats, 15 (5.1%) dogs, 11 (3.7%) swine, and 9 (3.1%) sheep. The most detected serogroups were Australis, Grippotyphosa, and Ballum. The results showed that animals were exposed to serogroups/serovars not included in commercial bacterins such as Ballum, Bratislava (only in swine vaccine), and Tarassovi. Our findings suggest that more studies should include culture and concomitant genotyping to reduce animal disease and zoonotic risk through efficacious vaccine and diagnostic strategies.

MPL36, a major plasminogen (PLG) receptor in pathogenic Leptospira, has an essential role during infection

Leptospirosis, a zoonosis with worldwide distribution, is caused by pathogenic spirochetes belonging to the genus Leptospira. Bacterial outer membrane proteins (OMPs), particularly those with surface-exposed regions, play crucial roles in pathogen dissemination and virulence mechanisms. Here we characterized the leptospiral Membrane Protein L36 (MPL36), a rare lipoprotein A (RlpA) homolog with a C-terminal Sporulation related (SPOR) domain, as an important virulence factor in pathogenic Leptospira. Our results confirmed that MPL36 is surface exposed and expressed during infection. Using recombinant MPL36 (rMPL36) we also confirmed previous findings of its high plasminogen (PLG)-binding ability determined by lysine residues of the C-terminal region of the protein, with ability to convert bound-PLG to active plasmin. Using Koch’s molecular postulates, we determined that a mutant of mpl36 has a reduced PLG-binding ability, leading to a decreased capacity to adhere and translocate MDCK cell monolayers. Using recombinant protein and mutant strains, we determined that the MPL36-bound plasmin (PLA) can degrade fibrinogen. Finally, our mpl36 mutant had a significant attenuated phenotype in the hamster model for acute leptospirosis. Our data indicates that MPL36 is the major PLG binding protein in pathogenic Leptospira, and crucial to the pathogen’s ability to attach and interact with host tissues during infection. The MPL36 characterization contributes to the expanding field of bacterial pathogens that explore PLG for their virulence, advancing the goal to close the knowledge gap regarding leptospiral pathogenesis while offering a novel potential candidate to improve diagnostic and prevention of this important zoonotic neglected disease.

Leptospira borgpetersenii serovar Hardjo and Leptospira santarosai serogroup Pyrogenes isolated from bovine dairy herds in Puerto Rico.

Leptospirosis is one of the most common zoonotic diseases in the world and endemic in the Caribbean Islands. Bovine leptospirosis is an important reproductive disease. Globally, cattle are recognized as a reservoir host for L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges, and can result in abortion and poor reproductive performance. The dairy industry in Puerto Rico comprises up to 25% of agriculture-related income and is historically the most financially important agricultural commodity on the island. In this study, we report the isolation of two different pathogenic Leptospira species, from two different serogroups, from urine samples collected from dairy cows in Puerto Rico: L. borgpetersenii serogroup Sejroe serovar Hardjo and L. santarosai serogroup Pyrogenes. Recovered isolates were classified using whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, and immunoblotting. These results demonstrate that dairy herds in Puerto Rico can be concurrently infected with more than one species and serovar of Leptospira, and that bacterin vaccines and serologic diagnostics should account for this when applying intervention and diagnostic strategies.

Bovine Leptospirosis Due to Persistent Renal Carriage of Leptospira borgpetersenii Serovar Tarassovi.

Leptospirosis is a global zoonotic disease that causes significant morbidity and mortality in human and animal populations. Leptospira interrogans is a leading cause of human disease, and L. borgpetersenii is a leading cause of animal disease. Cattle are reservoir hosts of L. borgpetersenii serovar Hardjo, which is transmitted via urine, semen, and uterine discharges resulting in abortion and poor reproductive performance. Bovine bacterin vaccines can only protect against those serovars included in vaccine formulations and typically include serovar Hardjo among others. Genotyping and serotyping represent two different and unique methods for classifying leptospires that do not always correlate well; comprehensive characterization using either method requires recovery of isolates from infected animals. In this study, we report for the first time, isolation of L. borgpetersenii serovar Tarassovi from the urine of a dairy cow in the U.S. The classification of the isolate, designated strain MN900, was confirmed by whole-genome sequencing, serotyping with reference antisera and monoclonal antibodies, Matrix Assisted Laser Desorption/Ionization (MALDI), and immunoblotting with reference antisera. Strain MN900 was excreted in urine samples for 18 weeks even as the cow was seronegative for serovar Tarassovi. Strain MN900 has an unusual morphology since it is not as motile as other leptospires and lacks hooked ends. Serovar Tarassovi is not included in U.S. bacterin vaccines. These results demonstrate the importance of culture and concomitant genotyping and serotyping to accurately classify leptospires, and as required to design efficacious vaccine and diagnostic strategies to not only limit animal disease but reduce zoonotic risk.

Detection of Leptospira interrogans in Wild Sambar Deer (Rusa unicolor), Brazil.

Leptospirosis is an emerging zoonotic disease caused by bacteria of the genus Leptospira. Wild animals may present acute disease or become chronic hosts. The present study aimed to identify Leptospira spp. infection and determine circulating serogroups in free-ranging sambar deer (Rusa unicolor) in a fragment of peri-urban tropical forest in northeastern Brazil. Blood samples were collected and subjected to microscopic agglutination testing (MAT) and PCR analysis (genes lipL32 and secY). Anti-Leptospira antibodies were detected in 60% of the animals tested, with serogroups Cynopteri (33.4%), Hebdomadis (22.2%) and Grippotyphosa (22.2%) identified as most prevalent. DNA corresponding to the pathogenic species Leptospira interrogans was detected in 2/15 (13.3%) of the samples tested. Sambar deer experience a high level of exposure to Leptospira spp. in our epidemiological setting. It is important to emphasize the implementation of effective measures (i.e., maintaining habitats and reducing human contact) for the conservation of endangered species.

Assessing rodents as carriers of pathogenic Leptospira species in the U.S. Virgin Islands and their risk to animal and public health.

Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the genus Leptospira. We sought to determine if rodents in U.S. Virgin Islands (USVI) are carriers of Leptospira. In total, 140 rodents were sampled, including 112 Mus musculus and 28 Rattus rattus. A positive carrier status was identified for 64/140 (45.7%); 49 (35.0%) were positive by dark-field microscopy, 60 (42.9%) by culture, 63 (45.0%) by fluorescent antibody testing, and 61 (43.6%) by real-time polymerase chain reaction (rtPCR). Molecular typing indicated that 48 isolates were L. borgpetersenii and 3 were L. kirschneri; the remaining nine comprised mixed species. In the single culture-negative sample that was rtPCR positive, genotyping directly from the kidney identified L. interrogans. Serotyping of L. borgpetersenii isolates identified serogroup Ballum and L. kirschneri isolates as serogroup Icterohaemorrhagiae. These results demonstrate that rodents are significant Leptospira carriers and adds to understanding the ecoepidemiology of leptospirosis in USVI.

Sero-prevalence of anti-Leptospira antibodies and associated risk factors in rural Rwanda: A cross-sectional study.

BACKGROUND: Leptospirosis is a zoonotic disease transmitted through the urine of wild and domestic animals, and is responsible for over 50,000 deaths each year. In East Africa, prevalence varies greatly, from as low as 7% in Kenya to 37% in Somalia. Transmission epidemiology also varies around the world, with research in Nicaragua showing that rodents are the most clinically important, while studies in Egypt and Chile suggest that dogs may play a more important role. There are no published studies of leptospirosis in Rwanda. METHODS & FINDINGS: We performed a cross-sectional survey of asymptomatic adults recruited from five occupational categories. Serum samples were tested using ELISA and Microscopic Agglutination Test (MAT). We found that 40.1% (151/377) of asymptomatic adults had been exposed to Leptospira spp. Almost 36.3% of positive subjects reported contact with rats (137/377) which represent 90.7% among positive leptospira serology compared with 48.2% of negative subjects (182/377) which represent 80.5% among negative leptospira serology (OR 2.37, CI 1.25-4.49) and 1.7 fold on prevalence ratio and 2.37 of odd ratio. Furthermore, being a crop farmer was significantly associated with leptospirosis (OR 2.06, CI 1.29-3.28). We identified 6 asymptomatic subjects (1.6%) who met criteria for acute infection. CONCLUSIONS: This study demonstrates a high prevalence of leptospiral antibodies infection among asymptomatic adults in rural Rwanda, particularly relative to neighboring countries. Although positive subjects were more likely to report rat contact, we found no independent association between rats and leptospirosis infection. Nonetheless, exposure was high among crop farmers, which is supportive of the hypothesis that rats together with domestic livestock might contribute to the transmission. Further studies are needed to understand infecting Leptospira servers and elucidate the transmission epidemiology in Rwanda and identify means of host transmitters.

Mongooses (Urva auropunctata) as reservoir hosts of Leptospira species in the United States Virgin Islands, 2019-2020.

During 2019-2020, the Virgin Islands Department of Health investigated potential animal reservoirs of Leptospira spp., the bacteria that cause leptospirosis. In this cross-sectional study, we investigated Leptospira spp. exposure and carriage in the small Indian mongoose (Urva auropunctata, syn: Herpestes auropunctatus), an invasive animal species. This study was conducted across the three main islands of the U.S. Virgin Islands (USVI), which are St. Croix, St. Thomas, and St. John. We used the microscopic agglutination test (MAT), fluorescent antibody test (FAT), real-time polymerase chain reaction (lipl32 rt-PCR), and bacterial culture to evaluate serum and kidney specimens and compared the sensitivity, specificity, positive predictive value, and negative predictive value of these laboratory methods. Mongooses (n = 274) were live-trapped at 31 field sites in ten regions across USVI and humanely euthanized for Leptospira spp. testing. Bacterial isolates were sequenced and evaluated for species and phylogenetic analysis using the ppk gene. Anti-Leptospira spp. antibodies were detected in 34% (87/256) of mongooses. Reactions were observed with the following serogroups: Sejroe, Icterohaemorrhagiae, Pyrogenes, Mini, Cynopteri, Australis, Hebdomadis, Autumnalis, Mankarso, Pomona, and Ballum. Of the kidney specimens examined, 5.8% (16/270) were FAT-positive, 10% (27/274) were culture-positive, and 12.4% (34/274) were positive by rt-PCR. Of the Leptospira spp. isolated from mongooses, 25 were L. borgpetersenii, one was L. interrogans, and one was L. kirschneri. Positive predictive values of FAT and rt-PCR testing for predicting successful isolation of Leptospira by culture were 88% and 65%, respectively. The isolation and identification of Leptospira spp. in mongooses highlights the potential role of mongooses as a wildlife reservoir of leptospirosis; mongooses could be a source of Leptospira spp. infections for other wildlife, domestic animals, and humans.

Multidisciplinary approach in the diagnosis of acute leptospirosis in dogs naturally infected by Leptospira interrogans serogroup Icterohaemorrhagiae: A prospective study.

Leptospirosis, a zoonotic disease with worldwide distribution, is caused by spirochetes of the genus Leptospira. In dogs, this disease is frequently misdiagnosed. Few studies have attempted to associate the detection of Leptospira spp. infection with clinicopathological and renal histopathological findings using a multidisciplinary approach. The present study isolated and characterized Leptospira spp. obtained from naturally infected dogs and described relevant clinical and histopathological findings. Blood and urine were collected from 57 dogs with clinical symptomatology suggestive of leptospirosis; 38 cases were confirmed by PCR in urine or by culture or microscopic agglutination testing (titers ≥800). A total of 12 strains of pathogenic Leptospira were isolated from the studied dogs (seven in blood, four in urine and one in both blood and urine samples). All isolates were characterized as Leptospira interrogans serogroup Icterohaemorrhagiae. Of the confirmed cases, almost one-third of the animals had been vaccinated. Our analysis of laboratory testing revealed that azotemia and proteinuria were statistically significant predictors of infection. The main histopathological findings seen in kidney tissues were necrosis, degeneration, tubular regeneration, mononuclear inflammatory infiltrate and congestion. A multidisciplinary approach involving clinicopathological and histopathological characterization of renal involvement can aid in the identification of acute leptospirosis infection.

Exposure and Carriage of Pathogenic Leptospira in Livestock in St. Croix, U.S. Virgin Islands.

From 2019-2020, the Virgin Islands Department of Health (VIDOH) investigated potential animal reservoirs of Leptospira spp., the pathogenic bacteria that cause leptospirosis. We examined Leptospira exposure and carriage in livestock on the island of St. Croix, United States Virgin Islands (USVI). We utilized the microscopic agglutination test (MAT) to evaluate the sera, and the fluorescent antibody test (FAT), real time polymerase chain reaction (rt-PCR), and bacterial culture to evaluate urine specimens from livestock (n = 126): 28 cattle, 19 goats, 46 pigs, and 33 sheep. Seropositivity was 37.6% (47/125) with agglutinating antibodies to the following serogroups identified: Australis, Djasiman, Icterohaemorrhagiae, Ballum, Sejroe, Cynopteri, Autumnalis, Hebdomadis, Pomona, Canicola, Grippotyphosa, and Pyrogenes. Urine from 4 animals (4.0%, 4/101) was positive by rt-PCR for lipL32: 2 sheep, 1 goat, and 1 bull. Sequencing of secY amplicons identified L. interrogans in 1 sheep and 1 bull. Livestock in USVI harbor pathogenic Leptospira bacteria and could play a role in the zoonotic cycle of leptospirosis.

A live attenuated-vaccine model confers cross-protective immunity against different species of the Leptospira genus.

Leptospirosis is the leading zoonotic disease in terms of morbidity and mortality worldwide. Effective prevention is urgently needed as the drivers of disease transmission continue to intensify. The key challenge has been developing a widely applicable vaccine that protects against the >300 serovars that can cause leptospirosis. Live attenuated mutants are enticing vaccine candidates and poorly explored in the field. We evaluated a recently characterized motility-deficient mutant lacking the expression of a flagellar protein, FcpA. Although the fcpA- mutant has lost its ability to cause disease, transient bacteremia was observed. In two animal models, immunization with a single dose of the fcpA- mutant was sufficient to induce a robust anti-protein antibodies response that promoted protection against infection with different pathogenic Leptospira species. Furthermore, characterization of the immune response identified a small repertoire of biologically relevant proteins that are highly conserved among pathogenic Leptospira species and potential correlates of cross-protective immunity.

Leptospirosis is a life-threatening disease with flu-like symptoms that is caused by bacteria known as Leptospira. It is more common in warmer regions with high rainfall, especially in impoverished areas. The disease is spread in the urine of animals such as rodents, farm animals or dogs. Humans and other animals can get leptospirosis when they come in contact with urine-contaminated water and soil. Current measures to control leptospirosis are largely ineffective. Although a vaccine is available for animals, it only protects against a few types of the 300 disease-causing Leptospira bacteria. It also fails to stop the bacteria from colonizing the kidneys of the infected animals, which means that vaccinated animals can still spread disease. Previous research has shown that inactivating a protein called FcpA, which is necessary for Leptospira bacteria to move, can stop them from infecting hamsters. Moreover, when these animals were exposed to the mutant bacteria, they did not get sick nor developed the disease. Here, Wunder et al. tested whether bacteria lacking the FcpA protein could be used as an attenuated vaccine. This form of vaccine contains live bacteria that have been modified to become harmless but are able to train the immune system to produce a long-lasting immune response against the invaders. The results showed that a single dose of the vaccine was enough to prevent hamsters and mice from dying of leptospirosis. It also worked against several types of Leptospira and could stop them from colonizing mice kidneys. Moreover, Wunder et al. found that the immune system targeted specific proteins that were common to various types of Leptospira, which may explain the broad spectrum of protection the vaccine offered. Rapid urbanization and climate change are among the main drivers of leptospirosis. An effective vaccine for this disease would reduce the public health burden by providing protection against leptospirosis and by reducing the spread of the disease. A next step will be to ensure the mutant Leptospira are safe to use in animals and potentially humans.

Detection of Leptospira interrogans DNA in Urine of a Captive Ocelot (Leopardus pardalis).

Dear editor, [...].

Leptospira yasudae sp. nov. and Leptospira stimsonii sp. nov., two new species of the pathogenic group isolated from environmental sources.

Four spirochetes (F1T, B21, YaleT and AMB6-RJ) were isolated from environmental sources: F1T and B21 from soils of an urban slum community in Salvador (Brazil), YaleT from river water in New Haven, Connecticut (USA) and AMB6-RJ from a pond in a horse farm in Rio de Janeiro (Brazil). Isolates were helix-shaped, aerobic, highly motile and non-virulent in a hamster model of infection. Draft genomes of the strains were obtained and analysed to determine the relatedness to other species of the genus Leptospira. The analysis of 498 core genes showed that strains F1T/B21 and YaleT/AMB6-RJ formed two distinct phylogenetic clades within the 'Pathogens' group (group I). The average nucleotide identity (ANI) values of strains F1T/B21 and YaleT/AMB6-RJ to other previously described Leptospira species were below <84 % and <82 %, respectively, which confirmed that these isolates should be classified as representatives of two novel species. Therefore, we propose Leptospirayasudae sp. nov. and Leptospirastimsonii sp. nov. as new species in the genus Leptospira. The type strains are F1T (=ATCC-TSD-163=KIT0259=CLEP00287) and YaleT (=ATCC-TDS-162=KIT0258=CLEP00288), respectively.