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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., Competing Interests: The authors declare no conflict of interest.

Leptospirosis is a global zoonotic disease affecting humans, domestic, and wild animals. Leptospira are typically shed in the urine of reservoir hosts which persist in suitable environments where incidental host transmission occurs after direct contact with infected urine or contaminated environments. Interestingly, serologically identical L. borgpetersenii serovar Hardjo strains JB197 and HB203 show divergent disease severity in the hamster model; JB197 causes severe acute infection while HB203 causes persistent chronic infection. Historically, serovar Hardjo was limited to culture at 29 °C, but utilization of HAN media allows propagation from host tissues at 37 °C. Here, the proteome of strains JB197 and HB203 were characterized after culture from experimentally challenged hamsters at 29 °C and 37 °C. Comparative analyses of JB197 and HB203 samples cultured at 29 °C yielded 425 significantly differentially expressed (DE) proteins, while strains at 37 °C yielded 613 DE proteins including prominent outer membrane proteins and known virulence factors. In agreement, membrane protein GO terms were identified by STRING network analyses along with numerous metabolic KEGG pathways consistent with condition differences. Within strain, JB197 cultured at 29 °C vs 37 °C identified 529 DE proteins, while HB203 identified 524 DE proteins. Investigating differential protein profiles provide insights into strain specific behaviors with implications for better understanding host-pathogen interactions, disease transmission, and response to environmental conditions which can contribute to vaccine development, diagnostic improvement, and ultimately leptospirosis control. SIGNIFICANCE: Leptospirosis is a devastating zoonotic disease affecting humans, wild and domestic animals around the globe. Different species and serovars of Leptospira can affect various animal host species differently; for instance, a serovar that is asymptomatic in the rat may cause severe disease in a dog or human. These differences in host response are not only found at the species and serovar level for Leptospira, but also at the strain level. A prime example comes from strains JB197 and HB203, both species L. borgpetersenii, both serovar Hardjo. Interestingly, JB197 causes a severe acute infection in the hamster while HB203 causes an asymptomatic chronic infection. Understanding these unique relationships between pathogen and host species is important, especially in the context of prevention technologies such as vaccine design, where the strain of Leptospira used as a bacterin might have different efficiencies in different hosts. In this study, proteomic profiles of strains JB197 and HB203 were analyzed, and results revealed diverse protein expression profiles of outer membrane proteins, as well as proteins functioning in motility and growth., Competing Interests: Declaration of competing interest The authors have no competing interests to report., (Published by Elsevier B.V.)

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., Competing Interests: The authors declare no conflict of interest.

Leptospirosis is an archetypal One Health problem as described in the companion Currents in One Health article in the October 2022 issue of the Journal of the American Veterinary Medical Association by Sykes et al. A thorough understanding of leptospirosis requires a detailed analysis of the elaborate interplay among pathogenic leptospiral strains, host species, and the environment. Such an understanding is required to inform appropriate preventative measures including vaccine design, prophylaxis efforts, educational programs that help to reduce exposure to pathogenic spirochetes, as well as policy development. Because of the complex epidemiology of leptospirosis, a One Health approach as defined by the One Health Initiative Task Force is critical-an approach that calls for "the collaborative efforts of multiple disciplines working locally, nationally, and globally, to attain optimal health for people, animals and our environment." Over the last three decades, progressive advances in cutting-edge molecular typing techniques, as well as our ability to rapidly generate and share large amounts of sequence data through establishment and growth of databases, have been central to accelerating a One Health understanding of the epidemiology of leptospirosis. Nevertheless, our dependence on serotype information because of the serovar-specific nature of current vaccines means that laborious serotyping efforts continue. With the advent of new approaches such as mRNA vaccines that are based on lipopolysaccharide immunogens, sequence- and/or proteomics-based typing methods may replace these methods.

Leptospirosis is a quintessential one health disease of humans and animals caused by pathogenic spirochetes of the genus Leptospira. Intra- and interspecies transmission is dependent on 1) reservoir host animals in which organisms replicate and are shed in urine over long periods of time, 2) the persistence of spirochetes in the environment, and 3) subsequent human-animal-environmental interactions. The combination of increased flooding events due to climate change, changes in human-animal-environmental interactions as a result of the pandemic that favor a rise in the incidence of leptospirosis, and under-recognition of leptospirosis because of nonspecific clinical signs and severe signs that resemble COVID-19 represents a "perfect storm" for resurgence of leptospirosis in people and domestic animals. Although often considered a disease that occurs in warm, humid climates with high annual rainfall, pathogenic Leptospira spp have recently been associated with disease in animals and humans that reside in semiarid regions like the southwestern US and have impacted humans that have a wide spectrum of socioeconomic backgrounds. Therefore, it is critical that physicians, veterinarians, and public health experts maintain a high index of suspicion for the disease regardless of geographic and socioeconomic circumstances and work together to understand outbreaks and implement appropriate control measures. Over the last decade, major strides have been made in our understanding of the disease because of improvements in diagnostic tests, molecular epidemiologic tools, educational efforts on preventive measures, and vaccines. These novel approaches are highlighted in the companion Currents in One Health by Sykes et al, AJVR, September 2022.

Leptospirosis is a global zoonotic disease affecting humans and livestock species. Bacterin vaccines lack cross protection between serogroups, and include multiple serovars propagated at 29 °C. Recent work demonstrated substantial variation in the transcriptome of identical species and serovars of Leptospira. Here, substantial differences in protein abundance profiles were identified in Leptospira borgpetersenii serovar Hardjo; strain HB203, which was isolated in the 1980s, compared to newer strains TC129 and TC273 isolated in 2016, and whether they were propagated at the routine temperature of 29 °C, compared to 37 °C which more closely emulates host infection. While 388 and 385 significantly differentially expressed (DE) proteins (FDR of 0.01) were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 29 °C respectively, only 66 and 4 DE proteins were identified in HB203 versus TC129, and HB203 versus TC273 when propagated at 37 °C respectively. Within each strain comparing temperatures, HB203 had 524 significantly DE proteins, TC129 had 347 DE proteins, and TC273 had 569 DE proteins. Data are available via ProteomeXchange with identifier PXD032831. Results highlight significant differential protein expression among identical serovars of L. borgpetersenii suggesting that bacterin vaccine design can benefit from consideration of strains employed and effects of temperature on growth. SIGNIFICANCE: Leptospirosis is a zoonotic disease caused by spirochete bacteria of the genus Leptospira. While leptospirosis affects over one million people per year, symptoms range vastly in severity from completely asymptomatic, to flu-like, to multi-organ failure and death in severe cases. Incidental hosts become infected after encountering pathogens directly from contact with another host, including domestic or wildlife animals, or indirectly from contaminated environments. Though animal vaccines exist, they lack cross protection across serogroups, and instead rely on inclusion of multiple carefully selected serovars from laboratory strains prepared at ~29 °C. Recent interest in gene expression at the Leptospira strain level, along with a newly achieved culture temperature of 37 °C (which more closely resembles host body temperature), led us to investigate the proteomic profiles of an older, established challenge strain HB203 in comparison to TC129 and TC273, two strains isolated in 2016 from abattoir cattle in the central United States. Herein, we identify substantial proteomic differences not only between strains of the same species and serovar, but notably between growth temperatures, collectively suggesting that bacterin vaccine composition may benefit from investigating strain selection and the temperature employed for growth of the bacteria used in bacterin preparation., (Published by Elsevier B.V.)

Background: Leptospirosis, caused by Leptospira bacteria, is a common zoonosis worldwide, especially in the tropics. Reservoir species and risk factors have been identified but surveys for environmental sources are rare. Furthermore, understanding of environmental Leptospira containing virulence associated genes and possibly capable of causing disease is incomplete, which may convolute leptospirosis diagnosis, prevention, and epidemiology., Methodology/principal Findings: We collected environmental samples from 22 sites in Puerto Rico during three sampling periods over 14-months (Dec 2018-Feb 2020); 10 water and 10 soil samples were collected at each site. Samples were screened for DNA from potentially pathogenic Leptospira using the lipL32 PCR assay and positive samples were sequenced to assess genetic diversity. One urban site in San Juan was sampled three times over 14 months to assess persistence in soil; live leptospires were obtained during the last sampling period. Isolates were whole genome sequenced and LipL32 expression was assessed in vitro. We detected pathogenic Leptospira DNA at 15/22 sites; both soil and water were positive at 5/15 sites. We recovered lipL32 sequences from 83/86 positive samples (15/15 positive sites) and secY sequences from 32/86 (10/15 sites); multiple genotypes were identified at 12 sites. These sequences revealed significant diversity across samples, including four novel lipL32 phylogenetic clades within the pathogenic P1 group. Most samples from the serially sampled site were lipL32 positive at each time point. We sequenced the genomes of six saprophytic and two pathogenic Leptospira isolates; the latter represent a novel pathogenic Leptospira species likely belonging to a new serogroup., Conclusions/significance: Diverse and novel pathogenic Leptospira are widespread in the environment in Puerto Rico. The disease potential of these lineages is unknown but several were consistently detected for >1 year in soil, which could contaminate water. This work increases understanding of environmental Leptospira diversity and should improve leptospirosis surveillance and diagnostics., Competing Interests: The authors have declared that no competing interests exist.

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., (© 2022. The Author(s).)

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., Competing Interests: The authors have declared that no competing interests exist.

Leptospirosis causes abortion, premature birth, and stillbirth in cattle, but the mechanisms remain unclear. Infected cattle shed Leptospira intermittently and present a range of clinical symptoms, making diagnosis difficult. The primary route of Leptospira transmission in any animal is the colonization of the renal tubule and excretion by urine; however, Leptospira can also colonize the female reproductive tract of cows and can be transmitted by semen. Vaccination against Leptospira in the US is routine in cattle, but immunity is not guaranteed. The cell wall of Leptospira contains toll-like receptor agonists including peptidoglycan and lipopolysaccharide. The capacity of Leptospira to initiate an innate inflammatory response from uterine endometrial cells is unknown but may be a cause of reproductive failure. Using cell culture, we tested the capacity of bovine endometrial epithelial cells or human monocytes to elicit an inflammatory response to Leptospira borgpetersenii serovar Hardjo strain TC273. Cells were exposed to either heat-killed Leptospira, Leptospira outer membrane, Escherichia coli lipopolysaccharide, Pam3CSK4 or medium alone for 2 to 24 h. Exposure of bovine endometrial epithelial cells or human monocytes to heat-killed Leptospira or Leptospira outer membrane did not induce the expression of IL1A , IL1B , IL6, or CXCL8 , while exposure to E. coli lipopolysaccharide or Pam3CSK4 increased the expression of IL1A , IL1B , IL6, and CXCL8 compared to control cells. This data suggest that Leptospira does not trigger a classical inflammatory response in endometrial cells. Understanding the interaction between Leptospira and the female reproductive tract is important in determining the mechanisms of Leptospirosis associated with reproductive failure., Lay Summary: Cows infected with the Leptospira have abortion and stillbirth. It is not known how Leptospira causes pregnancy failure in the cow. We tested if Leptospira causes inflammation in cells of the uterus which triggers pregnancy failure. We collected cells from the uterus of healthy cows at the abattoir and placed them into culture with Leptospira and measured the expression of genes associated with inflammation. To our surprise, cells of the uterus did not respond to Leptospira ; however, the same cells did respond to other disease-causing bacteria found in the uterus. This suggests that cells of the uterus can recognize bacteria and produce an inflammatory reaction but not in response to Leptospira . This finding suggests the immune system of the uterus cannot detect Leptospira which may go on to cause reproductive failure in cows. Understanding how Leptospira interact with cells of the uterus will help reduce pregnancy failure of cows with leptospirosis., (© The authors.)

Background: Leptospirosis is a zoonotic, bacterial disease, posing significant health risks to humans, livestock, and companion animals around the world. Symptoms range from asymptomatic to multi-organ failure in severe cases. Complex species-specific interactions exist between animal hosts and the infecting species, serovar, and strain of pathogen. Leptospira borgpetersenii serovar Hardjo strains HB203 and JB197 have a high level of genetic homology but cause different clinical presentation in the hamster model of infection; HB203 colonizes the kidney and presents with chronic shedding while JB197 causes severe organ failure and mortality. This study examines the transcriptome of L. borgpetersenii and characterizes differential gene expression profiles of strains HB203 and JB197 cultured at temperatures during routine laboratory conditions (29°C) and encountered during host infection (37°C)., Methodology/principal Findings: L. borgpetersenii serovar Hardjo strains JB197 and HB203 were isolated from the kidneys of experimentally infected hamsters and maintained at 29°C and 37°C. RNAseq revealed distinct gene expression profiles; 440 genes were differentially expressed (DE) between JB197 and HB203 at 29°C, and 179 genes were DE between strains at 37°C. Comparison of JB197 cultured at 29°C and 37°C identified 135 DE genes while 41 genes were DE in HB203 with those same culture conditions. The consistent differential expression of ligB, which encodes the outer membrane virulence factor LigB, was validated by immunoblotting and 2D-DIGE. Differential expression of lipopolysaccharide was also observed between JB197 and HB203., Conclusions/significance: Investigation of the L. borgpetersenii JB197 and HB203 transcriptome provides unique insight into the mechanistic differences between acute and chronic disease. Characterizing the nuances of strain to strain differences and investigating the environmental sensitivity of Leptospira to temperature is critical to the development and progress of leptospirosis prevention and treatment technologies, and is an important consideration when serovars are selected and propagated for use as bacterin vaccines as well as for the identification of novel therapeutic targets., Competing Interests: The authors have declared that no competing interests exist.

This study examined the humoral and cellular response of cattle vaccinated with two commercial leptospiral vaccines, Leptavoid and Spirovac, and a novel bacterin vaccine using Seppic Montanide oil emulsion adjuvant. Vaccination was followed by experimental challenge. All vaccinated cattle were protected from colonization of the kidney and shedding of Leptospira in urine, as detected by culture and immunofluorescence assay. Agglutinating antibody titers were detected in vaccinated cattle at 4 weeks following vaccination, with small anamnestic response detected following experimental challenge. Only animals vaccinated with the oil emulsion-adjuvanted bacterin produced significant IgG2 titers following vaccination, and nonvaccinated animals produced serum IgA titers after experimental challenge. CD4 + and γδ T cells from vaccinated cattle proliferated when cultured with antigen ex vivo Cellular responses included a marked proliferation of γδ T cells immediately following experimental challenge in vaccinated cattle and release of gamma interferon (IFN-γ), interleukin 17a (IL-17a), and IL-12p40 from stimulated cells. Proliferative and cytokine responses were found not just in peripheral mononuclear cells but also in lymphocytes isolated from renal lymph nodes at 10 weeks following experimental challenge. Overall, effects of leptospirosis vaccination and infection were subtle, resulting in only modest activation of CD4 + and γδ T cells. The use of Seppic Montanide oil emulsion adjuvants may shorten the initiation of response to vaccination, which could be useful during outbreaks or in areas where leptospirosis is endemic. IMPORTANCE Leptospirosis is an underdiagnosed, underreported zoonotic disease of which domestic livestock can be carriers. As a reservoir host for Leptospira borgpetersenii serovar Hardjo, cattle may present with reproductive issues, including abortion, birth of weak or infected calves, or failure to breed. Despite years of study and the availability of commercial vaccines, detailed analysis of the bovine immune response to vaccination and Leptospira challenge is lacking. This study evaluated immunologic responses to two efficacious commercial vaccines and a novel bacterin vaccine using an adjuvant chosen for enhanced cellular immune responses. Antigen-specific responsive CD4 and γδ T cells were detected following vaccination and were associated with release of inflammatory cytokines IFN-γ and IL-17a after stimulation. CD4 and γδ cells increased in the first week after infection and, combined with serum antibody, may play a role in clearance of bacteria from the blood and resident tissues. Additionally, these antigen-reactive T cells were found in the regional lymph nodes following infection, indicating that memory responses may not be circulating but are still present in regional lymph nodes. The information gained in this study expands knowledge of bovine immune response to leptospirosis vaccines and infection. The use of oil emulsion adjuvants may enhance early immune responses to leptospiral bacterins, which could be useful in outbreaks or situations where leptospirosis is endemic.

Leptospirosis is a global zoonosis causing significant economic losses for cattle production. Current cattle vaccines against leptospirosis need improvement to provide efficacy against multiple serovars, reduce shedding in urine, and to induce earlier and more robust immune responses. In this study, Leptospira borgpetersenii serovar Hardjo strain 203 antigen was combined with novel adjuvants (a biodegradable polyanhydride compressed rod implant (VPEAR), poly(diaminosulfide) microparticles, a water-oil-water emulsion adjuvant, and aluminum hydroxide) to develop novel vaccines. Cattle were immunized twice, at a 4 week interval, with inoculums containing adjuvants alone or leptospira antigens and immune responses were compared to responses of cattle receiving a commercial monovalent leptospirosis vaccine (Spirovac). All animals were inoculated with a single dose of Spirovac at 20 weeks to assess antigen recall responses. Serum antibody responses were increased (P > 0.05) at 8 and 20 weeks after vaccination in cattle receiving inoculums containing leptospira antigens combined with water-oil-emulsion, poly(diaminosulfide) microparticles (PNSN-MP), or aluminum hydroxide and in cattle vaccinated with Spirovac. Humoral responses were predominantly IgG1 isotypes. Antigen-specific proliferative responses were detected after initial vaccination in cattle vaccinated with Spirovac, PNSN-MP and water-oil-water treatments. Most proliferative responses occurring within CD4+ and gamma delta T cell populations expressing CD45RO and CD25 markers, a response consistent with an effector memory phenotype. Antigen-specific immune responses were not detected in cattle vaccinated with VPEAR after initial inoculation, but were detected in the antigen recall responses. PBMCs from cattle vaccinated with Spirovac, oil-water-oil, or PNSN-MP treatments had increased (P < 0.05) IL-17A release after in vitro stimulation with leptospirosis antigens, whereas all groups produced IFN-γ and IL-17A after in vitro stimulation during the antigen recall response. Our data demonstrates that combining leptospirosis antigens with these adjuvants enhances immunogenicity in cattle. Interpretative Summary: Vaccination of livestock is a key mechanism for minimizing transmission of leptospirosis, a zoonotic disease. Leptospirosis vaccines for cattle need to be improved to provide greater levels of protection from kidney colonization, better immune responses, and protection against multiple serovars. This could be accomplished using new vaccine adjuvants. In this study, several novel adjuvants were evaluated for their ability to induce effective immune responses in cattle to leptospira antigens as compared to currently available vaccines. Data suggested that vaccines containing biodegradable polymer microparticles and oil-emulsion adjuvants induced similar or greater immune responses as compared to a commercial vaccine. Our data suggest these new vaccine formulations warrant further investigation as new vaccine formulations for cattle and other livestock., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: [Jones is listed as inventor on Patent No.: 14/814148 “VACCINE DELIVERY DEVICES”. Wilson-Welder, Wafa, Bowden and Salem are listed as inventor Patent Application Serial No.: 62/946,474 “POLY(DIAMINOSULFIDE) PARTICLE-BASED VACCINE” Filed December 11, 2019]., (Published by Elsevier Ltd.)

Leptospirosis is a debilitating infectious disease that detrimentally affects both animals and humans; therefore, disease prevention has become a high priority to avoid high incidence rates of disease in the herd and break the transmission cycle to humans. Thus, there remains an important unmet need for a prophylactic vaccine that can provide long-term immunity against leptospirosis in cattle. Herein, a novel vaccine formulation was developed where poly(diaminosulfide) polymer was employed to fabricate microparticles encapsulating the antigen of Leptospira borgpetersenii serovar Hardjo strain HB15B203 (L203-PNSN). A prime-boost vaccination with a L203-PNSN microparticle formulation increased the population of L203-specific CD3 + T cells and CD21 + B cells to levels that were significantly higher than those of cattle vaccinated with L203-AlOH or the vehicle control (empty PNSN microparticles and blank AlOH). In addition, L203-PNSN was demonstrated to stimulate durable humoral immune responses as evidenced by the increases in the antibody serum titers following the vaccination. It was also found that cattle vaccinated with L203-PNSN produced higher macroscopic agglutinating titers than cattle in other groups. Thus, it can be concluded that L203-PNSN is a novel first-in-class microparticle-based Leptospira vaccine that represents a powerful platform with the potential to serve as a prophylactic vaccine against leptospiral infection in cattle.

Pathogenic leptospires colonize the renal tubules of reservoir hosts of infection, including cattle, and are excreted via urine. In order to identify circulating serovars of pathogenic leptospires in beef cattle, and their associated rates of urinary excretion, a cross sectional study was performed. Fifty urine samples were collected one day each month over 12 consecutive months (N = 600), directly from the bladder of beef cattle at a single slaughter facility and assessed for the presence of leptospires by culture and the fluorescent antibody test (FAT). Where possible, a matched serum sample was also collected for the microscopic agglutination test (MAT). Forty-three urine samples were either culture positive or FAT positive, indicating that 7.2% of sampled beef cattle were actively excreting leptospires in urine. Twenty-three urine samples were culture positive. Sequence analysis of 16S ribosomal DNA and secY indicated that all isolates were Leptospira borgpetersenii. Typing by serology indicated that all isolates were serogroup Sejroe. An overall seroprevalence of 20% (MAT ≥ 1:25) was determined; positive bovine sera was most reactive to serogroup Sejroe (serovar Hardjo) (8.1%), and serogroup Australis (serovar Bratislava) (6.7%). There was poor correlation between seroprevalence and excretion of leptospires since 18/43 (41.9%) cattle, which were positive by culture or FAT, were seronegative. The virulence of two selected isolates of L. borgpetersenii was confirmed by experimental infection in small animal models of infection. Results confirm that L. borgpetesenii continues to circulate in beef cattle and that multiple diagnostic assays are required to detect active shedding. These findings also highlight beef cattle as a reservoir host for the potential zoonotic transmission of leptospires., (Copyright © 2018. Published by Elsevier B.V.)

Pathogenic species of Leptospira cause leptospirosis, a bacterial zoonotic disease with a global distribution affecting over one million people annually. Rats are regarded as one of the most significant reservoir hosts of infection for human disease, and in the absence of clinical signs of infection, excrete large numbers of organisms in their urine. A unique biological equilibrium exists between pathogenic leptospires and reservoir hosts of infection, but surprisingly, little is known concerning the host's cellular immune response that facilitates persistent renal colonization. To address this deficiency, we established and applied an immunocompetent inbred rat model of persistent renal colonization; leptospires were detected in urine of experimentally infected rats by 3 weeks post-infection and remained positive until 8 weeks post-infection. However, there was little, if any, evidence of inflammation in colonized renal tubules. At 8 weeks post-infection, a robust antibody response was detected against lipopolysaccharide and protein outer membrane (OM) components. Purified B and T cells derived from the spleen of infected and non-infected rats proliferated in response to stimulation with 0.5 μg of OM fractions of Leptospira , including CD4+ T cells, which comprised 40% of proliferating cells, compared to 25% in non-infected controls. However, analysis of gene expression did not determine which immunoregulatory pathways were activated. Lymphocytes purified from the lymph node draining the site of colonization, the renal lymph node, also showed an increase in percentage of proliferating B and T cells. However, in contrast to a phenotype of 40% CD4+ T cells in the spleen, the phenotype of proliferating T cells in the renal lymph node comprised 65% CD4+ T cells. These results confirm that the renal lymph node, the local lymphoid organ, is a dominant site containing Leptospira reactive CD4+ T cells and highlight the need to consider the local, vs. systemic, immune responses during renal colonization infection. The use of inbred immunocompetent rats provides a novel tool to further elucidate those pathophysiological pathways that facilitate the unique biological equilibrium observed in reservoir hosts of leptospirosis.

The greater white-toothed shrew (Crocidura russula) is an invasive mammalian species that was first recorded in Ireland in 2007. It currently occupies an area of approximately 7,600 km2 on the island. C. russula is normally distributed in Northern Africa and Western Europe, and was previously absent from the British Isles. Whilst invasive species can have dramatic and rapid impacts on faunal and floral communities, they may also be carriers of pathogens facilitating disease transmission in potentially naive populations. Pathogenic leptospires are endemic in Ireland and a significant cause of human and animal disease. From 18 trapped C. russula, 3 isolates of Leptospira were cultured. However, typing of these isolates by standard serological reference methods was negative, and suggested an, as yet, unidentified serovar. Sequence analysis of 16S ribosomal RNA and secY indicated that these novel isolates belong to Leptospira alstonii, a unique pathogenic species of which only 7 isolates have been described to date. Earlier isolations were limited geographically to China, Japan and Malaysia, and this leptospiral species had not previously been cultured from mammals. Restriction enzyme analysis (REA) further confirms the novelty of these strains since no similar patterns were observed with a reference database of leptospires. As with other pathogenic Leptospira species, these isolates contain lipL32 and do not grow in the presence of 8-azagunaine; however no evidence of disease was apparent after experimental infection of hamsters. These isolates are genetically related to L. alstonii but have a novel REA pattern; they represent a new serovar which we designate as serovar Room22. This study demonstrates that invasive mammalian species act as bridge vectors of novel zoonotic pathogens such as Leptospira., Competing Interests: The authors have declared that no competing interests exist.

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a particularly severe form of leptospirosis. LPHS is increasingly recognized in both humans and animals and is characterized by rapidly progressive intra-alveolar haemorrhage leading to high mortality. The pathogenic mechanisms of LPHS are poorly understood which hampers the application of effective treatment regimes. In this study a 2-D guinea pig proteome lung map was created and used to investigate the pathogenic mechanisms of LPHS. Comparison of lung proteomes from infected and non-infected guinea pigs via differential in-gel electrophoresis revealed highly significant differences in abundance of proteins contained in 130 spots. Acute phase proteins were the largest functional group amongst proteins with increased abundance in LPHS lung tissue, and likely reflect a local and/or systemic host response to infection. The observed decrease in abundance of proteins involved in cytoskeletal and cellular organization in LPHS lung tissue further suggests that infection with pathogenic Leptospira induces changes in the abundance of host proteins involved in cellular architecture and adhesion contributing to the dramatically increased alveolar septal wall permeability seen in LPHS., Biological Significance: The recent completion of the complete genome sequence of the guinea pig (Cavia porcellus) provides innovative opportunities to apply proteomic technologies to an important animal model of disease. In this study, the comparative proteomic analysis of lung tissue from experimentally infected guinea pigs with leptospiral pulmonary haemorrhage syndrome (LPHS) revealed a decrease in abundance of proteins involved in cellular architecture and adhesion, suggesting that loss or down-regulation of cytoskeletal and adhesion molecules plays an important role in the pathogenesis of LPHS. A publically available guinea pig lung proteome map was constructed to facilitate future pulmonary proteomics in this species., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Leptospiral pulmonary haemorrhage syndrome (LPHS) is a severe form of leptospirosis. Pathogenic mechanisms are poorly understood. Lung tissues from 26 dogs with LPHS, 5 dogs with pulmonary haemorrhage due to other causes and 6 healthy lungs were labelled for IgG (n=26), IgM (n=25) and leptospiral antigens (n=26). Three general staining patterns for IgG/IgM were observed in lungs of dogs with LPHS with most tissues showing more than one staining pattern: (1) alveolar septal wall staining, (2) staining favouring alveolar surfaces and (3) staining of intra-alveolar fluid. Healthy control lung showed no staining, whereas haemorrhagic lung from dogs not infected with Leptospira showed staining of intra-alveolar fluid and occasionally alveolar septa. Leptospiral antigens were not detected. We conclude that deposition of IgG/IgM is demonstrable in the majority of canine lungs with naturally occurring LPHS, similar to what has been described in other species. Our findings suggest involvement of the host humoral immunity in the pathogenesis of LPHS and provide further evidence to support the dog as a natural disease model for human LPHS., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Background: Leptospirosis is caused by pathogenic spirochetes of the genus Leptospira. The bacteria enter the human body via abraded skin or mucous membranes and may disseminate throughout. In general the clinical picture is mild but some patients develop rapidly progressive, severe disease with a high case fatality rate. Not much is known about the innate immune response to leptospires during haematogenous dissemination. Previous work showed that a human THP-1 cell line recognized heat-killed leptospires and leptospiral LPS through TLR2 instead of TLR4. The LPS of virulent leptospires displayed a lower potency to trigger TNF production by THP-1 cells compared to LPS of non-virulent leptospires., Methodology/principal Findings: We investigated the host response and killing of virulent and non-virulent Leptospira of different serovars by human THP-1 cells, human PBMC's and human whole blood. Virulence of each leptospiral strain was tested in a well accepted standard guinea pig model. Virulent leptospires displayed complement resistance in human serum and whole blood while in-vitro attenuated non-virulent leptospires were rapidly killed in a complement dependent manner. In vitro stimulation of THP-1 and PBMC's with heat-killed and living leptospires showed differential serovar and cell type dependence of cytokine induction. However, at low, physiological, leptospiral dose, living virulent complement resistant strains were consistently more potent in whole blood stimulations than the corresponding non-virulent complement sensitive strains. At higher dose living virulent and non-virulent leptospires were equipotent in whole blood. Inhibition of different TLRs indicated that both TLR2 and TLR4 as well as TLR5 play a role in the whole blood cytokine response to living leptospires., Conclusions/significance: Thus, in a minimally altered system as human whole blood, highly virulent Leptospira are potent inducers of the cytokine response.

Reservoir hosts of pathogenic Leptospira species, including wild and domestic animal species, persistently excrete leptospires from infected renal tubules via urine into the environment. Reservoir hosts are typically asymptomatic. In contrast to the hamster model of leptospirosis (described in UNIT 12E.2), which emulates the severe form of human leptospirosis, the rat model of chronic leptospirosis emulates the asymptomatic infection observed in reservoir hosts. Rats are the primary reservoir host for pathogenic Leptospira species associated with the severe forms of human leptospirosis., (© 2011 by John Wiley & Sons, Inc.)

In the past decade, leptospirosis has emerged as a globally important infectious disease. It occurs in urban environments of industrialised and developing countries, as well as in rural regions worldwide. Mortality remains significant, related both to delays in diagnosis due to lack of infrastructure and adequate clinical suspicion, and to other poorly understood reasons that may include inherent pathogenicity of some leptospiral strains or genetically determined host immunopathological responses. Pulmonary haemorrhage is recognised increasingly as a major, often lethal, manifestation of leptospirosis, the pathogenesis of which remains unclear. The completion of the genome sequence of Leptospira interrogans serovar lai, and other continuing leptospiral genome sequencing projects, promise to guide future work on the disease. Mainstays of treatment are still tetracyclines and beta-lactam/cephalosporins. No vaccine is available. Prevention is largely dependent on sanitation measures that may be difficult to implement, especially in developing countries.

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 PCRpositive 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, culturenegative 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A One Health approach to the epidemiology, management, surveillance, and control of leptospirosis relies on accessible and accurate diagnostics that can be applied to humans and companion animals and livestock. Diagnosis should be multifaceted and take into account exposure risk, clinical presentation, and multiple direct and/or indirect diagnostic approaches. Methods of direct detection of Leptospira spp. include culture, histopathology and immunostaining of tissues or clinical specimens, and nucleic acid amplification tests (NAATs). Indirect serologic methods to detect leptospiral antibodies include the microscopic agglutination test (MAT), the enzyme-linked immunosorbent assay (ELISA), and lateral flow methods. Rapid diagnostics that can be applied at the point-of-care; NAAT and lateral flow serologic tests are essential for management of acute infection and control of outbreaks. Culture is essential to an understanding of regional knowledge of circulating strains, and we discuss recent improvements in methods for cultivation, genomic sequencing, and serotyping. We review the limitations of NAATs, MAT, and other diagnostic approaches in the context of our expanding understanding of the diversity of pathogenic Leptospira spp. Novel approaches are needed, such as loop mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR)-based approaches to leptospiral nucleic acid detection. [ABSTRACT FROM AUTHOR]

Leptospirosis is a worldwide zoonosis caused by pathogenic species of the genus Leptospira. The recent application of CRISPR interference (CRISPRi) to Leptospira facilitates targeted gene silencing and provides a new tool to investigate pathogenic mechanisms of leptospirosis. CRISPRi relies on the expression of a catalytically "dead" Cas9 (dCas9) and a single-guide RNA (sgRNA). Previously, our group generated a LipL32 and a double LigA/LigB (LigAB) mutant, which, in the current study, are characterized by whole-cell proteomics in comparison with control leptospires harboring plasmid expressing dCas9 alone. Comparison of control and LigAB mutant leptospires identified 46 significantly differentially expressed (DE) proteins, including 27 proteins that were less abundant and 19 proteins that were more abundant in the LigAB mutant compared with the control. Comparison of the control and LipL32 mutant leptospires identified 243 DE proteins, of which 84 proteins were more abundant and 159 were less abundant in the LipL32 mutant strain. Significantly increased amounts of known virulence impactors and surface membrane receptors, including LipL45, LipL31, LigB, and LipL41, were identified. The virulence of LipL32 and LigAB mutants were evaluated in the hamster model of leptospirosis; the LigAB mutant was unable to cause acute disease although mutant leptospires could still be recovered from target organs, albeit at a significantly lower bacterial burden (<850 and <16-fold in liver and kidney, respectively, in comparison with control), indicating attenuation of virulence and a shift to chronic bacterial persistence. Notably, the LipL32 mutant displayed augmented virulence as evidenced by early onset of clinical symptoms and increased numbers of circulating foamy macrophages. Validation of LipL32 and LigAB mutants recovered from liver and kidney in the presence or absence of antibiotic selection revealed high plasmid stability and, by extension, gene silencing in vivo. Collectively, this work emphasizes the advantages and feasibility of using CRISPRi technology to evaluate and characterize virulence factors of leptospires and their respective host–pathogen interactions in animal models of leptospirosis. Importantly, it also provides insight into the requirements of LigA and LigB for acute disease and explores the impact of silencing expression of lipL32 , which resulted in substantial changes in amounts of outer membrane proteins. [ABSTRACT FROM AUTHOR]

Domestic and wildlife animal species act as reservoir hosts of leptospirosis, a global zoonotic disease affecting more than 1 million people annually and causing significant morbidity and mortality in domestic animals. In contrast to incidental hosts which present with an array of clinical manifestations, reservoir hosts are typically asymptomatic and can shed leptospires from chronically infected kidneys via urine for extended periods of time. Renal excretion of leptospires occurs despite evidence of a humoral and cellular immune response and is reflective of the unique biological equilibrium that exists between certain animal species and specific serovars of Leptospira. Here, we demonstrate that urinary excretion of leptospires is accompanied by the presence of antigen-specific urinary immunoglobulin. In rats experimentally infected with L. interrogans serovar Copenhageni using the intraperitoneal or conjunctival route of inoculation, urinary immunoglobulin (Ig) G specific for protein antigens was detectable within 1 week. Rat urinary IgG was not bound to urinary-derived leptospires. In cattle that were naturally exposed to, and infected with, L. borgpetersenii serovar Hardjo, urinary IgA specific for protein antigens was detected. Collectively, these results demonstrate that urinary excretion of immunoglobulin specific for leptospires is a hallmark of reservoir hosts of infection. [ABSTRACT FROM AUTHOR]

Leptospirosis is a devastating zoonotic disease affecting people and animals across the globe. Pathogenic leptospires are excreted in urine of reservoir hosts which directly or indirectly leads to continued disease transmission, via contact with mucous membranes or a breach of the skin barrier of another host. Human fatalities approach 60,000 deaths per annum; though most vertebrates are susceptible to leptospirosis, complex interactions between host species and serovars of Leptospira can yield disease phenotypes that vary from asymptomatic shedding in reservoir hosts, to multi-organ failure in incidental hosts. Clinical symptoms of acute leptospirosis reflect the diverse range of pathogenic species and serovars that cause infection, the level of exposure, and the relationship of the pathogen with the given host. However, in all cases, pathogenic Leptospira are excreted into the environment via urine from reservoir hosts which are uniformly recognized as asymptomatic carriers. Therefore, the reservoir host serves as the cornerstone of persistent disease transmission. Although bacterin vaccines can be used to abate renal carriage and excretion in domestic animal species, there is an urgent need to advance our understanding of immune-mediated host–pathogen interactions that facilitate persistent asymptomatic carriage. This review summarizes the current understanding of host–pathogen interactions in the reservoir host and prioritizes research to unravel mechanisms that allow for colonization but not destruction of the host. This information is required to understand, and ultimately control, the transmission of pathogenic Leptospira. [ABSTRACT FROM AUTHOR]

Cattle are susceptible to infection with multiple serovars of pathogenic leptospires, resulting in abortion, stillbirth, premature birth, reproductive failure and milk drop syndrome. Cattle also act as a reservoir host for L. borgpetersenii serovar Hardjo which is excreted from renal tubules via urine into the environment where it persists in suitable moist conditions. Our previous work demonstrated that 7% of urine samples from beef cattle were positive for L. borgpetersenii serovar Hardjo by culture and/or the fluorescent antibody test (FAT). In this study, a real-time PCR (rtPCR) assay was applied to determine the relative performance of rtPCR based detection of L. borgpetersenii serovar Hardjo compared to previously reported culture and FAT techniques. Of 42 bovine urine samples positive for leptospires by culture and/or FAT, 60% (25/42) were positive by rtPCR. Of 22 culture-positive samples, 91% (20/22) were rtPCR-positive. Of 32 FAT-positive samples, 50% (16/32) were rtPCR-positive. For 10 samples that were culture-positive but FAT-negative, 90%(9/10)were rtPCR-positive. For 20 samples that were FAT-positive but culture-negative, 25%(5/20) were rtPCR-positive. Collectively, these results indicate that no single assay is optimal, and the use of more than one assay to detect leptospires in urine from naturally infected cattle is recommended. [ABSTRACT FROM AUTHOR]

Previous studies have suggested that macrophages may contribute to acute Leptospira dissemination, as well as having a major role in kidney fibrosis. Our aim was to characterize the role of macrophages and galectin 3 (Gal-3) on the survival, clinical course, bacterial burden, interstitial nephritis, and chronic kidney fibrosis in Leptospira interrogans serovar Copenhageni (LIC)-induced experimental murine leptospirosis. C57BL/6J mice depleted of macrophages by liposome-encapsulated clodronate treatment and infected with LIC presented a higher bacterial burden, had reduced subacute nephritis and enhanced chronic kidney fibrosis relative to untreated, infected mice. Moreover, LIC infection in mice whose Gal-3 was disrupted (Lgals3 −/–) had a higher bacterial burden and enhanced subacute nephritis and chronic kidney fibrosis when compared to C57BL/6J wild-type mice. Chronic fibrosis did not correlate with higher transcription levels of TGF-β1 or IL-13 in the kidneys. Kidney fibrosis was found in chronically infected rats as well as in wild infected rats. On the other hand, human fibroblast cultures exhibited enhanced differentiation to myofibroblasts after treatment with LIC. Our results demonstrate that macrophages and Gal-3 play a critical role in controlling the LIC burden but has a minor role in subsequent fibrosis. Instead, kidney fibrosis was better correlated with bacterial burden. Taken together, our results do not support a role for macrophages to disseminate leptospires during acute infection, nor in chronic kidney fibrosis. [ABSTRACT FROM AUTHOR]

Background: Leptospirosis, a re-emerging disease of global importance caused by pathogenic Leptospira spp., is considered the world's most widespread zoonotic disease. Rats serve as asymptomatic carriers of pathogenic Leptospira and are critical for disease spread. In such reservoir hosts, leptospires colonize the kidney, are shed in the urine, persist in fresh water and gain access to a new mammalian host through breaches in the skin. Methodology/Principal Findings: Previous studies have provided evidence for post-translational modification (PTM) of leptospiral proteins. In the current study, we used proteomic analyses to determine the presence of PTMs on the highly abundant leptospiral protein, LipL32, from rat urine-isolated L. interrogans serovar Copenhageni compared to in vitro-grown organisms. We observed either acetylation or tri-methylation of lysine residues within multiple LipL32 peptides, including peptides corresponding to regions of LipL32 previously identified as epitopes. Intriguingly, the PTMs were unique to the LipL32 peptides originating from in vivo relative to in vitro grown leptospires. The identity of each modified lysine residue was confirmed by fragmentation pattern analysis of the peptide mass spectra. A synthetic peptide containing an identified tri-methylated lysine, which corresponds to a previously identified LipL32 epitope, demonstrated significantly reduced immunoreactivity with serum collected from leptospirosis patients compared to the peptide version lacking the tri-methylation. Further, a subset of the identified PTMs are in close proximity to the established calcium-binding and putative collagen-binding sites that have been identified within LipL32. Conclusions/Significance: The exclusive detection of PTMs on lysine residues within LipL32 from in vivo-isolated L. interrogans implies that infection-generated modification of leptospiral proteins may have a biologically relevant function during the course of infection. Although definitive determination of the role of these PTMs must await further investigations, the reduced immune recognition of a modified LipL32 epitope suggests the intriguing possibility that LipL32 modification represents a novel mechanism of immune evasion within Leptospira. [ABSTRACT FROM AUTHOR]

Leptospirosis, an emerging zoonotic disease with worldwide distribution, is caused by spirochetes belonging to the genus Leptospira. More than 500,000 cases of severe leptospirosis are reported annually, with >10% of these being fatal. Leptospires can survive for weeks in suitably moist conditions before encountering a new host. Reservoir hosts, typically rodents, exhibit little to no signs of disease but shed large numbers of organisms in their urine. Transmission occurs when mucosal surfaces or abraded skin come into contact with infected urine or urine-contaminated water or soil. In humans, leptospires can cause a variety of clinical manifestations, ranging from asymptomatic or mild fever to severe icteric (Weil's) disease and pulmonary haemorrhage. Currently, little is known about how Leptospira persist within a reservoir host. Prior in vitro studies have suggested that leptospires alter their transcriptomic and proteomic profiles in response to environmental signals encountered during mammalian infection. However, no study has examined gene expression by leptospires within a mammalian host-adapted state. To obtain a more faithful representation of how leptospires respond to host-derived signals, we used RNA-Seq to compare the transcriptome of L. interrogans cultivated within dialysis membrane chambers (DMCs) implanted into the peritoneal cavities of rats with that of organisms grown in vitro. In addition to determining the relative expression levels of “core” housekeeping genes under both growth conditions, we identified 166 genes that are differentially-expressed by L. interrogans in vivo. Our analyses highlight physiological aspects of host adaptation by leptospires relating to heme uptake and utilization. We also identified 11 novel non-coding transcripts that are candidate small regulatory RNAs. The DMC model provides a facile system for studying the transcriptional and antigenic changes associated with mammalian host-adaption, selection of targets for mutagenesis, and the identification of previously unrecognized virulence determinants. [ABSTRACT FROM AUTHOR]

Rattus norvegicus is a natural reservoir host for pathogenic species of Leptospira. Experimentally infected rats remain clinically normal, yet persistently excrete large numbers of leptospires from colonized renal tubules via urine, despite a specific host immune response. Whilst persistent renal colonization and shedding is facilitated in part by differential antigen expression by leptospires to evade host immune responses, there is limited understanding of kidney and urinary proteins expressed by the host that facilitates such biological equilibrium. Urine pellets were collected from experimentally infected rats shedding leptospires and compared to urine from non-infected controls spiked with in vitro cultivated leptospires for analysis by 2-D DIGE. Differentially expressed host proteins include membrane metallo endopeptidase, napsin A aspartic peptidase, vacuolar H+ATPase, kidney aminopeptidase and immunoglobulin G and A. Loa22, a virulence factor of Leptospira, as well as the GroEL, were increased in leptospires excreted in urine compared to in vitro cultivated leptospires. Urinary IgG from infected rats was specific for leptospires. Results confirm differential protein expression by both host and pathogen during chronic disease and include markers of kidney function and immunoglobulin which are potential biomarkers of infection. [ABSTRACT FROM AUTHOR]

Abstract: Leptospira interrogans serovar Copenhageni causes pulmonary hemorrhages with respiratory failure, a major cause of death in leptospirosis patients. Protective immunity to Leptospira is known to correlate with the production of leptospiral lipopolysaccharide (L-LPS)-specific agglutinating antibodies. We generated L-LPS-specific mouse monoclonal antibodies (MAbs) and investigated if these MAbs can protect guinea pigs against fatal pulmonary hemorrhages caused by serovar Copenhageni. The MAbs L8H4 and L9B11 against 22kDa L-LPS agglutinated leptospires and completely protected guinea pigs from the development of fatal pulmonary hemorrhages by serovar Copenhageni, whereas the MAb L4C1 against 8kDa L-LPS neither agglutinated the bacteria nor protected the animals against the fatal pulmonary hemorrhages. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]