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Md Kaosar Yazdani-Ibn-Taz,1 Myint Myint Han,1 Sven Jonuscheit,1 Andrew Collier,1,2 Jane E Nally,1 Suzanne Hagan1 1School of Health and Life Sciences, Glasgow Caledonian University (GCU), Glasgow G4 0BA, UK; 2Diabetes Day Centre, University Hospital Ayr, Ayr KA6 6DX, UK Purpose: The purpose of the study was to assess the relationship between patient-reported severity of dry eye disease (DED), quality of life (QoL), presence of diabetic retinopathy (DR) and length of disease duration in people with type 1 diabetes mellitus (DM1) and type 2 diabetes mellitus (DM2).Patients and methods: A survey of 152 people (110 with and 42 without diabetes). All participants completed the Ocular Surface Disease Index (OSDI) and Dry Eye-related Quality of Life Score (DEQS) questionnaires.Results: Forty-four percent of all diabetic subjects reported dry eye symptoms, compared to 29% in the control group. Patients with DM2 reported dry eye symptoms more frequently than those with DM1 (55% and 27% respectively, P=0.001). Dry eye severity was linked to a significant deterioration in QoL in both types of diabetes (DM1, r=0.609 and P=0.036; DM2, r=0.417 and P=0.011). Irrespective of DR, the presence of DED was significantly higher in DM2 compared to DM1 (with DR, P=0.011; without DR, P=0.018).Conclusion: Dry eye symptoms are associated with reduced QoL and are more common in people with DM2 than in DM1, irrespective of DR status. Routine clinical screening for severe DED could potentially allow for a timely and more effective treatment and could contribute to mitigating the dry eye-associated reduction in QoL in those with DM2. Keywords: ocular surface, dry eye, symptoms, retinopathy

Jennifer H Wilson-Welder, David P Alt, Jarlath E Nally Bacterial Diseases of Livestock Research Unit, National Animal Disease Center, Agricultural Research Service, United States Department of Agriculture, Ames, IA, USA Abstract: Digital dermatitis (DD) is a multifactorial polymicrobial infectious disease originally described in dairy cattle, but is increasingly recognized in beef cattle, sheep, and more recently, elk and goats. Clinical bovine lesions typically appear on the plantar surface of the hind foot from the interdigital space and heel bulb to the accessory digits, with a predilection for skin–horn junctions. Lesions present as a painful ulcerative acute or chronic inflammatory process with differing degrees of severity. This variability reflects disease progression and results in a number of different clinical descriptions with overlapping pathologies that ultimately have a related bacterial etiology. The goal of this review article is to provide a concise overview of our current understanding on digital dermatitis disease to facilitate clinical recognition, our current understanding on the causative agents, and recent advances in our understanding of disease transmission. Keywords: Digital dermatitis, treponemes, lameness, ruminant

Leptospira borgpetersenii commonly causes human leptospirosis, including severe disease. The first published analysis of L. borgpetersenii , performed on two strains of serovar Hardjo (L550 and JB197), concluded that the L. borgpetersenii genome is in the process of genome decay with functional consequences leading to a more obligately host-dependent life cycle. Yet whole genome analysis has only been carried out on few strains of L. borgpetersenii , with limited closed genomes and comprehensive analysis. Herein we report the complete, circularized genomes of seven non-Hardjo Leptospira borgpetersenii isolates from human leptospirosis patients in Sri Lanka. These isolates (all ST144) were found to be nearly identical by whole genome analysis; serotyping showed they are a novel serovar. We show that the L. borgpetersenii isolated from humans in Sri Lanka are less genomically decayed than previously reported isolates: fewer pseudogenes (N=141) and Insertion Sequence (IS) elements (N=46) compared to N=248, N=270, and N=400 pseudogenes, and N=121 and N=116 IS elements in published L. borgpetersenii Hardjo genomes (L550, JB197 and TC112). Compared to previously published L. borgpetersenii whole genome analyses showing two to three VM proteins in L. borgpetersenii isolates from cattle, rats and humans, we found that all of the human L. borgpetersenii isolates from Sri Lanka, including previously reported serovar Piyasena, have 4 encoded VM proteins, one ortholog of L. interrogans Copenhageni LIC12339 and 3 orthologs of LIC12844. Our findings of fewer pseudogenes, IS elements and expansion of the LIC12844 homologs of the PF07598 family in these human isolates suggests that this newly identified L. borgpetersenii serovar from Sri Lanka has unique pathogenicity. Comparative genome analysis and experimental studies of these L. borgpetersenii isolates will enable deeper insights into the molecular and cellular mechanisms of leptospirosis pathogenesis., Competing Interests: Declaration of Competing Interest Some of work reported here has been filed in patent applications from Yale University. JMV and spouse have an equity interest in Luna Bioscience, Inc, which may have a future interest in licensing this work. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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 (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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Hamond, Adam, Stone, LeCount, Anderson, Putz, Camp, Hicks, Stuber, van der Linden, Bayles, Sahl, Schlater, Wagner and Nally.)

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.

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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hamond, Dirsmith, LeCount, Soltero, Rivera-Garcia, Camp, Anderson, Hicks, Galloway, Sutherland, Schafer, Goris, van der Linden, Stuber, Bayles, Schlater and Nally.)

Leptospirosis is a global zoonotic bacterial disease which is a threat for humans and most mammals. Bacterin vaccines for leptospirosis are available however they are severely limited in cross protection between serogroups. Leptospira typically colonize the kidneys of reservoir hosts where they are subsequently shed in the urine and persist in the environment and can thus be indirectly or directly transmitted to incidental hosts. Leptospira borgpetersenii serovar Hardjo is the primary cause of leptospirosis in cattle which can result in abortion, unhealthy calves, and rebreed problems. This dataset comprises proteomic profiles of four strains of L. borgpetersenii serovar Hardjo propagated at the routinely utilized culture temperature of 29 °C, and a newly achieved culture temperature of 37 °C, which more closely emulates the temperature of an infected host. The strains analyzed include JB197 (established strain that causes Hardjo atypical acute disease in the hamster model of leptospirosis), HB203 (established strain, causes typical chronic disease in hamsters), as well as TC129 and TC273 (recently isolated strains from the central United States). Differential expression profiles were detected not only between strains but also within strains between culture temperatures. Mass spectrometry data are available via ProteomeXchange with identifier PXD032831., Competing Interests: The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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 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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Hamond, LeCount, Putz, Bayles, Camp, Goris, van der Linden, Stone, Schlater, Sahl, Wagner and Nally.)

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.

Pathogenic species of Leptospira cause leptospirosis, a global zoonotic disease affecting humans and all major livestock species. Cattle act as a reservoir host for L . borgpetersenii serovar Hardjo which colonize the kidneys and reproductive tract from which they are excreted and transmitted to other cattle via urine, semen or uterine discharges. Bovine leptospirosis results in reproductive failure, abortion, stillbirth and loss of milk production, and is an occupational risk for those working with infected animals. A recent study determined that 7.2% of cattle from an abattoir in the central United States were actively shedding pathogenic Leptospira . Here, we report and compare the complete genome sequence of four recent isolates of L . borgpetersenii serovar Hardjo designated strain TC112, TC147, TC129, and TC273., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Fernandes, Putz, Stasko, Lippolis, Nascimento and Nally.)

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).)

Pulmonary hypertension is a serious clinical condition characterised by increased pulmonary arterial pressure. This can lead to right ventricular failure which can be fatal. Connexins are gap junction-forming membrane proteins which serve to exchange small molecules of less than 1 kD between cells. Connexins can also form hemi-channels connecting the intracellular and extracellular environments. Hemi-channels can mediate adenosine triphosphate release and are involved in autocrine and paracrine signalling. Recently, our group and others have identified evidence that connexin-mediated signalling may be involved in the pathogenesis of pulmonary hypertension. In this review, we discuss the evidence that dysregulated connexin-mediated signalling is associated with pulmonary hypertension.

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 is a world-wide zoonotic disease caused by pathogenic Leptospira and can be asymptomatic or can cause clinical signs ranging from influenza-like to multi-organ failure and death in severe cases. While species and strain specificity can play a major role in disease presentation, the hamster is susceptible to most leptospiral infections and is the model of choice for vaccine efficacy testing. During evaluation of blood smears from hamsters challenged with different species and strains of Leptospira, a circulating population of large, mononuclear, lipid-filled cells, most similar to foamy macrophages (FMs), was detected. Circulating FMs were identified by Giemsa staining and verified by scanning and transmission electron microscopy. FMs were found in the circulating blood of all Leptospira-challenged hamsters, indicating that the finding was not species or strain specific, although higher numbers of FMs tended to correlate with severity of disease. The unique finding of circulating FMs in the hamster model of leptospirosis can yield additional insights into the pathogenesis of leptospirosis and other diseases that induce circulating FMs., (Published by Elsevier Ltd.)

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.

Background: The prevalence of diabetes is increasing in low and middle-income countries (LMICs) and over two-thirds of these are not diagnosed. Consequently, diabetes complications usually exist at the time of diagnosis. Foot ulcers is a leading cause of disability and mortality among diabetes patients., Purpose: To assess the knowledge and experiences of adult patients with Diabetes on diabetes complications and self-management practices with emphasis on foot care., Methodology: This applied phenomenological study design. Twenty patients attending Diabetes clinics were purposively sampled from two hospitals in Ghana. Face-to-face semi-structured interviews were conducted to evaluate patient's understanding of diabetes and self-management practices. The interviews were audio-taped, transcribed, and analysed to generate themes using the constant comparison method., Results: Three-quarters of the participants in the study correctly defined diabetes as high blood glucose levels, but few knew the risk factors and complications of diabetes. Stroke and Hypertension were the most popular complications known, whiles diabetes foot complications were the least known. Almost all participants showed awareness of dietary self-management practices, but few had limited knowledge in foot care practices., Conclusion: Diabetes education in LMICs should promote self-management practices, especially foot care and clear dietary guidelines. There is also opportunity to invest in specialist diabetes training for healthcare providers and increase community-based care for people living with diabetes in Ghana., Competing Interests: No authors have competing interests.

Leptospirosis is a global neglected zoonosis, responsible for at least 1 million cases per year and almost 60 thousand deaths. The disease is caused by pathogenic and virulent bacteria of the genus Leptospira, either by direct contact with the bacteria or indirectly by exposure to contaminated water or soil. Domestic and wild animals act as reservoir hosts of infection, shedding leptospires from colonized renal tubules of the kidney, via urine, into the environment. The generation of mutant strains of Leptospira is critical to evaluate and understand pathogenic mechanisms of infection. CRISPR interference (CRISPRi) has proven to be a straightforward, affordable, and specific tool for gene silencing in pathogenic Leptospira. Therefore, the methodological details of obtaining the plasmid constructs containing both dCas9 and guide RNA, delivery of plasmids to Leptospira by conjugation with the E. coli strain β2163, and transconjugant recovery and evaluation, will be described. In addition, the recently described Hornsby-Alt-Nally (HAN) media allows for the relatively rapid isolation and selection of mutant colonies on agar plates.

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.)

Total thyroxine (T4) concentrations are lower in healthy greyhounds compared to most other non-sighthound breeds. In humans, variations in the structure or concentration of the major thyroid hormone binding proteins are responsible for most reported differences between total T4 concentrations in healthy individuals from different ethnic groups or other subpopulations. The aim of this study was to determine if such variations are also responsible for the lower total T4 concentrations in greyhounds. The predicted protein sequences of thyroxine-binding globulin (TBG), transthyretin and albumin were determined in liver tissue from a euthyroid greyhound with decreased T4 concentration and a Jack Russell terrier using reverse-transcriptase PCR. Sequences were compared to each other and online reference sequences. Serum proteins from 21 greyhounds and 21 non-sighthound dogs were separated by denaturing electrophoresis and immunoblots probed with polyclonal antibodies to human TBG and transthyretin. Reactive bands were quantified by densitrometry, expressed relative to the mean of reference samples included in each gel. Serum albumin concentrations were measured using a commercially-available assay. Several SNPs were identified but none was thought likely to explain the lower total T4 concentrations in greyhounds. There was no significant difference between the quantity of any of the binding proteins in serum from greyhounds and non-sighthound dogs. However, total T4 and transthyretin concentrations were highly correlated in the greyhound group (r = 0.73, P = 0.0002). Variation in the sequence of thyroid hormone binding proteins is not responsible for low greyhound total T4 concentrations. Further evaluation of the role of transthyretin is warranted., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Animal production and health are of significant economic importance, particularly regarding the world food supply. Animal and veterinary sciences have evolved immensely in the past six decades, particularly in genetics, nutrition, housing, management and health. To address major challenges such as those posed by climate change or metabolic disorders, it is of utmost importance to use state-of-the-art research tools. Proteomics and the other post-genomic tools (transcriptomics or metabolomics) are among them. Proteomics has experienced a considerable development over the last decades. This brought developments to different scientific fields. The use and adoption of proteomics tools in animal and veterinary sciences has some limitations (database availability or access to proteomics platforms and funding). As a result, proteomics' use by animal science researchers varies across the globe. In this viewpoint article, we focus on the developments of domestic animal proteomics over the last decade in different regions of the globe and how the researchers have coped with such challenges. In the second part of the article, we provide examples of funding, educational and laboratory establishment initiatives designed to foster the development of (animal-based) proteomics. International scientific collaboration is a definitive and key feature in the development and advancement of domestic animal proteomics. SIGNIFICANCE: Animal production and health are very important for food supply worldwide particularly as a source of proteinaceous foods. Animal and veterinary sciences have evolved immensely in the last decades. In order to address the major contemporary challenges facing animal and veterinary sciences, it is of utmost importance to use state-of-the-art research tools such as Proteomics and other Omics. Herein, we focus on the major developments in domestic animal proteomics worldwide during the last decade and how different regions of the world have used the technology in this specific research field. We address also major international efforts aiming to increase the research output in this area and highlight the importance of international cooperation to address specific problems inherent to domestic animal proteomics., (Copyright © 2021 Elsevier B.V. All rights reserved.)

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.

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 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., Competing Interests: EW, HA, CH, KO, LL, CR, VB, JN, DA, MR, PD, PF, AK No competing interests declared

Leptospirosis is a neglected, widespread zoonosis caused by pathogenic species of the genus Leptospira, and is responsible for 60,000 deaths per year. Pathogenic mechanisms of leptospirosis remain poorly understood mainly because targeted mutations or gene silencing in pathogenic Leptospira continues to be inherently inefficient, laborious, costly and difficult to implement. In addition, pathogenic leptospires are highly fastidious and the selection of mutants on solid agar media can take up to 6 weeks. The catalytically inactive Cas9 (dCas9) is an RNA-guided DNA-binding protein from the Streptococcus pyogenes CRISPR/Cas system and can be used for gene silencing, in a strategy termed CRISPR interference (CRISPRi). Here, this technique was employed to silence genes encoding major outer membrane proteins of pathogenic L. interrogans. Conjugation protocols were optimized using the newly described HAN media modified for rapid mutant recovery at 37 °C in 3% CO 2 within 8 days. Complete silencing of LipL32 and concomitant and complete silencing of both LigA and LigB outer membrane proteins were achieved, revealing for the first time that Lig proteins are involved in pathogenic Leptospira serum resistance. Gene silencing in pathogenic leptospires and rapid mutant recovery will facilitate novel studies to further evaluate and understand pathogenic mechanisms of leptospirosis.

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 ., (Copyright © 2020 Putz and Nally.)

The causative agent of leptospirosis includes multiple serovars and species of pathogenic leptospires that are excreted via urine from reservoir hosts of infection. Primary isolation takes weeks to months, and is limited to semi-solid media at 28-30 °C. Here we present an alternative media formulation, HAN, compared to commercially available EMJH and the more specialized T80/40/LH media formulations, in semi-solid and liquid compositions, for the primary isolation of two diverse species and serovars of pathogenic leptospires directly from host kidney tissue. All three media types supported the isolation and propagation of L. interrogans serovar Copenhageni strain IC:20:001 in semi-solid media at 29 °C. However, only HAN and T80/40/LH supported the growth of L. borgpetersenii serovar Hardjo strain HB15B203 at 29 °C. In addition, HAN supported primary isolation at 37 °C. Both T80/40/LH and HAN supported primary isolation of strain IC:20:001 in liquid media at 29 °C but only HAN supported growth of strain HB15B203 in liquid media, at both 29 and 37 °C. HAN media supports the primary isolation of fastidious pathogenic leptospires directly from infected host tissue at either 29 or 37 °C: this formulation represents a more defined media for the continued optimization of growth factors required to support the primary isolation of the large and diverse range of species and serovars within the genus Leptospira circulating within domestic and wild animal populations.

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.

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.

MIP and CPAF from Chlamydia have been shown to be effective in inducing immune responses important in clearing chlamydial infections. This study evaluates the protection conferred by MIP and CPAF as novel vaccines in pregnant C. abortus challenged ewes. Fifty C. abortus sero-negative sheep were randomly allocated into 5 groups of 10 according to the treatment they were to receive (1) 100 µg of MBP-MIP (2) 100 µg CPAF (3) 50 µg MBP-MIP and 50 µg CPAF (4) Tris-buffer (negative control) (5) Enzovax (positive control). Booster inoculations were administered 3 weeks after primary inoculations. Blood samples were taken pre-vaccination and weekly for 5 weeks. Five months after vaccination the ewes were mated. Pregnant ewes were then challenged on day 90 of gestation. Blood samples taken at four time-points post challenge were analysed for IFNγ levels, TNFα and IL-10 expression and anti-chlamydial antibody levels. Vaginal swabs, placental and foetal tissue and bacterial shedding were analysed using qPCR to quantify levels of C. abortus. Enzovax was 100% effective with no abortions occurring. The MIP/CPAF combined vaccine offered the greatest protection of the novel vaccines with 67% of ewes giving birth to one or more live lambs equating to a 50% vaccine efficacy rate. MIP and CPAF administered singly did not confer protection. Enzovax and MIP/CPAF vaccinated ewes had longer gestations and lambs with higher birth weights than negative control ewes. Aborting ewes shed higher numbers of C. abortus than ewes that had live lambs, all vaccinated ewes demonstrated lower levels of bacterial shedding than negative control ewes with Enzovax ewes shedding significantly fewer bacteria. Ewes that went on to abort had significantly higher levels of IFNγ and IL-10 at day 35 post challenge and significantly higher levels of anti-chlamydial antibodies at 24 h post lambing compared to ewes that had live lambs., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Bovine digital dermatitis (BDD) is a multifactorial polymicrobial infectious disease associated with multiple species and phylotypes of treponemes. However, despite the abundance of molecular signatures for treponemes that are identified in bovine lesions, relatively few isolates are cultured, and even fewer have been characterized at the level of protein expression. Here we report the successful isolation and characterization of novel strains of T. brennaborense and T. phagedenis from cases of BDD in Iowa dairy cows, and compare them to a well characterized strain of T. phagedenis, and the type strain of the more recently recognized T. pedis. Propagation of T. brennaborense was only possible at room temperature in Cooked Meat Medium, and not in oral treponeme enrichment medium at 37 °C as used for T. phagedenis and T. pedis. A prominent and rapid motility is observed by T. brennaborense under dark-field microscopy. The highly motile T. brennaborense strain 11-3 has an identical enzymatic profile to that of the only other isolate of T. brennaborense to be cultured from a lesion of BDD. Outer membrane protein profiles of each strain were compared by 2-D gel electrophoresis, and the five most abundant proteins in each strain were identified by mass spectrometry. All identified proteins are predicted to have signal peptides. Results identified outer membrane proteins specific to each strain including predicted membrane lipoproteins, ABC transporters and, as yet, uncharacterized proteins. Collectively, our results provide for the identification and characterization of outer membrane components of multiple phylotypes of treponemes associated with BDD which can facilitate development of vaccines and diagnostics in our efforts to eradicate the disease., (Published by Elsevier B.V.)