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Porcine reproductive and respiratory syndrome (PRRS), caused by the highly variable PRRS virus (PRRSV), presents a significant challenge to the swine industry due to its pathogenic and economic burden. The virus evades host immune responses, particularly interferon (IFN) signaling, through various viral mechanisms. Traditional vaccines have shown variable efficacy in the field, prompting the exploration of novel vaccination strategies. This study investigates a reverse genetics approach to develop a modified live virus (MLV) vaccine expressing the potent antiviral cytokine interferon-ω5 (IFN-ω5) to combat PRRSV. The study utilizes an infectious cDNA clone of PRRSV, incorporating genetic modifications for IFN-ω5 expression. A comparative evaluation, including in vitro and particularly in vivo assessments here, was conducted to determine the vaccine's efficacy. Results indicate that pigs vaccinated with the IFN-ω5 MLV exhibited significant differences compared to the mock group in terms of body temperature, weight gain, antibody response, viral load, cytokine profile, and lung lesions following PRRSV challenge. This study underscores the potential of reverse genetics and IFN-ω5 expression as a promising strategy for developing effective PRRSV vaccines. The findings provide valuable insights into the mechanisms of immune response and viral pathogenesis, highlighting the importance of early immune activation in combating PRRSV infection., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2025 The Authors. Published by Elsevier B.V. All rights reserved.)

Identifying cellular markers within archived formalin-fixed, paraffin-embedded (FFPE) tissues is critical for understanding tissue landscapes impacting animal health, but in situ detection methods are limited in veterinary species by a restricted toolbox of species-compatible immunoreagents. We identify antibodies with conserved in situ reactivity to IBA-1 (macrophages/dendritic cells), CD3ε (T cells), Pax5 (B cells), Ki-67 (cycling cells), and cytokeratin type I/II (epithelial cells) in FFPE tissues of pigs, cattle, and white-tailed deer. Multiplexed brightfield detection (IBA-1/CD3ε/Pax5) in lymph nodes of all three species demonstrated species-specific and species-conserved features of cellular architecture. Multiplexed fluorescent staining in pig lymph nodes for IBA-1/CD3ε/Pax5/Ki-67 allowed detection of colocalizing signals and identification of active germinal centers. Antibody compatibility with RNA in situ hybridization was confirmed for all antibodies in all species, allowing co-detection of RNA markers, which is a strategy highly useful in veterinary species where protein-reactive reagents are often lacking. Multiplexed protein and RNA staining was performed in tonsil tissue of a pig infected with Senecavirus A, enabling identification of virally-infected cell types via simultaneous detection of host cell type-specific proteins and virus-specific RNA. Findings have important applications for future in situ identification and comparative study of tissue landscapes and immunopathology in a diverse range of veterinary species., Competing Interests: Declaration of Competing Interest 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., (Published by Elsevier B.V.)

Vaccines that cross-protect across serovars of Salmonella enterica ( Salmonella ) would be a beneficial intervention against emerging and persistent Salmonella isolates of concern for the turkey industry. The 2017-2019 foodborne outbreak of Salmonella enterica serovar Reading ( S . Reading) revealed the need for effective control of this serovar in turkey production. This study evaluated two live-attenuated Salmonella vaccines, an internally developed cross-protective vaccine and a commercially available vaccine, against an outbreak-associated strain of S . Reading in turkeys. At 1 day and 3 weeks of age, male turkey poults were either mock-vaccinated with phosphate buffered saline (PBS) or given one of the vaccines by oral gavage (primary and booster) or aerosol spray (primary) then drinking water (booster). At 7 weeks of age, poults were challenged with 10 9 colony forming units (CFU) of S . Reading; a mock-vaccinated group was mock-challenged with PBS. Colonization of the cecal contents and cecal tonsil was 1.5-3 log 10 CFU/g lower in vaccinated birds than mock-vaccinated birds at 7 and/or 14 days post-inoculation (DPI). Salmonella dissemination to the spleen was significantly reduced by both vaccines. Gene expression of intestinal transporters (such as SCNN1B and SLC10A2 ) and tight junction proteins was significantly decreased in the turkey cecal tonsil transcriptome at 2 DPI with S . Reading. Vaccination with either vaccine mitigated most cecal tonsil gene expression responses to S . Reading challenge. Therefore, both the internally developed vaccine and commercial vaccine were cross-protective against colonization and dissemination, and both were able to limit transcriptional changes from challenge in intestinal health-related genes in the cecal tonsil, thereby providing vaccination efficacy and impact data against S . Reading in turkeys., 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 © 2024 Monson, Gurung, Bearson, Whelan, Trachsel, Looft, Sylte and Bearson.)

Human seasonal H3 clade 3C3a influenza A viruses (IAV) were detected four times in U.S. pigs from commercial swine farms in Michigan, Illinois, and Virginia in 2019. To evaluate the relative risk of this spillover to the pig population, whole genome sequencing and phylogenetic characterization were conducted, and the results revealed that all eight viral gene segments were closely related to 2018-2019 H3N2 human seasonal IAV. Next, a series of in vitro viral kinetics, receptor binding, and antigenic characterization studies were performed using a representative A/swine/Virginia/A02478738/2018(H3N2) (SW/VA/19) isolate. Viral replication kinetic studies of SW/VA/19 demonstrated less efficient replication curves than all 10 swine H3N2 viruses tested but higher than three human H3N2 strains. Serial passaging experiments of SW/VA/19 in swine cells did not increase virus replication, but changes at HA amino acid positions 9 and 159 occurred. In swine transmission studies, wild-type SW/VA/19 was shed in nasal secretions and transmitted to all indirect contact pigs, whereas the human seasonal strain A/Switzerland/9715293/2013(H3N2) from the same 3C3a clade failed to transmit. SW/VA/19 induced minimal macroscopic and microscopic lung lesions. Collectively, these findings demonstrate that these human seasonal H3N2 3C3a-like viruses did not require reassortment with endemic swine IAV gene segments for virus shedding and transmission in pigs. Limited detections in the U.S. pig population in the subsequent period of time suggest a yet-unknown restriction factor likely limiting the spread of these viruses in the U.S. pig population.IMPORTANCEInterspecies human-to-swine IAV transmission occurs globally and contributes to increased IAV diversity in pig populations. We present data that a swine isolate from a 2018-2019 human-to-swine transmission event was shed for multiple days in challenged and contact pigs. By characterizing this introduction through bioinformatic, molecular, and animal experimental approaches, these findings better inform animal health practices and vaccine decision-making. Since wholly human seasonal H3N2 viruses in the United States were not previously identified as being transmissible in pigs (i.e., reverse zoonosis), these findings reveal that the interspecies barriers for transmission to pigs may not require significant changes to all human seasonal H3N2, although additional changes may be required for sustained transmission in swine populations., Competing Interests: The authors declare no conflict of interest.

This study evaluated influenza A virus (IAV) detection and genetic diversity over time, specifically at the human-swine interface in breeding and nursery farms. Active surveillance was performed monthly in five swine farms in the Midwest United States targeting the employees, the prewean piglets at sow farms, and the same cohort of piglets in downstream nurseries. In addition, information was collected at enrollment for each employee and farm to assess production management practices, IAV vaccination status, diagnostic procedures, and biosecurity. Farm employee and swine samples were screened by IAV reverse transcription real-time polymerase chain reaction (RT-rtPCR), followed by IAV subtyping RT-rtPCR and whole genome sequencing on PCR-positive samples. This study showed higher positivity of IAV RNA detection in nursery pigs compared to prewean pigs, and more whole genome sequences were also obtained in the nursery phase. Surveillance of farm employees revealed two detections of H3N2 representing the 2022-2023 human IAV season, confirming the presence of influenza in farm employees while present at work, and thus highlighting the importance of biosecurity measures at the human-swine interface. This study highlights the importance of routine active surveillance to understand the dynamics of IAV at the farm level in both farm employees and swine.

Pathogenic Leptospira are spirochetes that cause leptospirosis, a worldwide zoonotic disease. Leptospirosis affects humans and animals, with approximately 1 million human infections and 60,000 deaths per year. The diversity of leptospiral strains and serovars allied to the fact that pathogenesis is not yet fully understood, make the development of an effective vaccine against leptospirosis a challenge. Outer membrane and secreted proteins are considered potential antigens since they play a vital role in mediating interactions with host molecules. Several domains or motifs have been reported to participate in the leptospiral infection process. Among them, leucine-rich repeat (LRR) proteins have been highlighted as attractive multipurpose proteins, exhibiting a broad spectrum of ligands and having a putative role in bacterial pathogenesis. Indeed, genome annotation of leptospiral species pointed out that LRR proteins are predominant in pathogenic strains, a feature that corroborates this hypothesis. A few LRR proteins of L. santarosai , L. borgpetersenii and L. interrogans have been studied and their possible role in virulence was proposed. Yet, a mechanistic and broad investigation of LRR proteins was not fully performed. In this review, a comprehensive in silico analysis of 21 LRR proteins of L. interrogans was performed in relation to structure, function, dynamics and virulent potential that will contribute to understanding the key role of these domains in the underlying mechanisms of leptospiral infection., 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 © 2024 Foltran, Gaspar, Silva, Pires, Andrade, Costa, Paixao, Fernandes, Teixeira and Nascimento.)

High mortality in bobwhite quail chicks ( Colinus virginianus ) (35%-85%) was reported from a grower flock in Iowa during July and August of 2022. Two diagnostic submissions of dead, 3-day-old quail chicks were received. Postmortem examination revealed multifocal, pinpoint, pale tan foci in the liver of all birds. Histologic examination revealed moderate to severe, acute, multifocal, random necrotizing hepatitis with multinucleated cells and dystrophic mineralization. Metagenomic sequencing of liver detected orthoreovirus. A high level of avian reovirus (ARV) RNA was identified by real-time reverse transcriptase-PCR. ARV was successfully isolated from liver and lung on the Leghorn male hepatoma cell line. In addition, electron microscopy revealed orthoreovirus viral particles and virus factories in the formalin-fixed livers and viral-infected cell culture. This case highlights ARV as a potential cause of hepatitis in quail chicks and should be considered in the differential diagnosis., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

The genus Leptospira comprises unique atypical spirochete bacteria that includes the etiological agent of leptospirosis, a globally important zoonosis. Biofilms are microecosystems composed of microorganisms embedded in a self-produced matrix that offers protection against hostile factors. Leptospires form biofilms in vitro, in situ in rice fields and unsanitary urban areas, and in vivo while colonizing rodent kidneys. The complex three-dimensional biofilm matrix includes secreted polymeric substances such as proteins, extracellular DNA (eDNA), and saccharides. The genus Leptospira comprises pathogenic and saprophytic species with the saprophytic L. biflexa being commonly used as a model organism for the genus. In this study, the growth and formation of biofilms by L. biflexa was investigated not just at 29 °C, but at 37 °C/5 % CO 2 , a temperature similar to that encountered during host infection. Planktonic free-living L. biflexa grow in HAN media at both 29 °C and 37 °C/5 % CO 2, but cells grown at 37 °C/5 % CO 2 are longer (18.52 μm ± 3.39) compared to those at 29 °C (13.93 μm ± 2.84). Biofilms formed at 37 °C/5 % CO 2 had more biomass compared to 29 °C, as determined by crystal violet staining. Confocal microscopy determined that the protein content within the biofilm matrix was more prominent than double-stranded DNA, and featured a distinct layer attached to the solid substrate. Additionally, the model enabled effective protein extraction for proteomic comparison across different biofilm phenotypes. Results highlight an important role for proteins in biofilm matrix structure by leptospires and the identification of their specific protein components holds promise for strategies to mitigate biofilm formation., 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., (© 2024 The Authors.)

Influenza A viruses (IAV) of subtypes H1N1, H1N2, and H3N2 are endemic in US domestic swine populations and contribute to significant economic losses annually and pose a persistent pandemic threat. Adjuvanted, whole-inactivated virus (WIV) vaccines are the primary countermeasure to control IAV in swine. The compositions of these vaccines are matched for hemagglutinin (HA) strain and content, often ignoring the other IAV glycoprotein, the neuraminidase (NA). The IAV NA is immunogenic and antibodies targeting epitopes adjacent to the active site have been shown to inhibit the sialidase activity of NA thereby reducing virus replication and shedding. To assess the ability of neuraminidase inhibiting (NAI) antibodies induced from WIV administration to protect swine from challenge with IAV containing homologous and heterologous NA, we produced WIV composed of viruses with an irrelevant mismatched H9 HA but expressing NA proteins from two predominant clades (N2-2002A.2 and N22002B.2) currently circulating in US domestic swine populations. Pigs that received two doses of H9N2 WIV developed vaccine-specific neuraminidase inhibition antibodies and when challenged with a wild-type H3N2 virus containing homologous NA, displayed reduced virus shedding in the upper respiratory tract and decreased virus titers in the lung compared to unvaccinated controls. Pigs challenged with H3N2 containing a heterologous NA also had reduced virus titers in the nasal swab and BALF samples. Together these results show that NAI antibodies cross-protected across phylogenetic clades and reduced virus replication and shedding in swine., Competing Interests: Declaration of competing interest 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., (Copyright © 2024. Published by Elsevier Ltd.)

Respiratory syncytial virus (RSV) is a leading cause of respiratory infection, hospitalization and death in infants worldwide. No fully effective RSV therapy using direct antivirals is marketed. Since clinical efficacy data from naturally infected patients for such antivirals are not available yet, animal studies are indispensable to predict therapeutic intervention. Here, we report the impact of an RSV fusion inhibitor, JNJ-49214698, on severe RSV-associated acute lower respiratory tract infection (ALRTI) in neonatal lambs. Randomized animals were treated once daily with 25 mg/kg JNJ-49214698, starting either before RSV infection, 1 day post-infection or as late as peak lung viral load on Day 3 post-infection. Treatment efficacy was assessed by scoring clinical signs of illness, development of RSV-induced gross and microscopic lung lesions and measuring virus titres in the lungs. Treatment with JNJ-49214698 was very effective in all treatment groups. Even in animals for which treatment was delayed until peak viral load was reached, a reduced amount and severity of gross and microscopic lesions, as well as RSV titres and RNA levels, were found. These results strongly suggest that treatment with small-molecule fusion inhibitors is an effective strategy to treat patients who are diagnosed with an RSV-induced ALRTI.

Chicken ceca are a rich source of bacteria, including zoonotic pathogens such as Salmonella enterica. The microbiota includes strains/species carrying antimicrobial resistance genes and horizontal transfer of resistance determinants between species may increase the risk to public health and farming systems. Possible sources of these antimicrobial resistance donors - the eggshell carrying bacteria from the hen vertically transmitted to the offspring, or the barn environment where chicks are hatched and raised - has been little explored. In this study, we used Salmonella enterica serovar Heidelberg to evaluate if layer chicks raised in different environments (using combinations of sterilized or non-sterile eggs placed in sterilized isolation chambers or non-sterile rooms) acquired transferable tetracycline resistance genes from surrounding bacteria, especially Escherichia coli. Two-day old chicks were challenged with an antibiotic-susceptible S. Heidelberg strain SH2813 nal R and Salmonella recovered from the cecum of birds at different timepoints to test the in vivo acquisition of tetracycline resistance. Tetracycline-resistant E. coli isolates recovered from birds from the in vivo experiment were used to test the in vitro transfer of tetracycline resistance genes from E. coli to Salmonella. Even though Salmonella SH2813 nal R colonized the 2-day old chicks after oral challenge, tetracycline-resistant Salmonella transconjugants were not recovered, as previously observed. In vitro experiments provided similar results. We discuss several hypotheses that might explain the absence of transconjugants in vitro and in vivo, despite the presence of diverse plasmids in the recovered E. coli. The factors that can inhibit/promote antimicrobial resistance transfers to Salmonella for different plasmid types need further exploration., Competing Interests: Declaration of Competing Interest 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., (Published by Elsevier B.V.)

Peyer's patches (PPs) are B cell-rich sites of intestinal immune induction, yet PP-associated B cell signaling, activation, and differentiation are poorly defined. Single-cell and spatial transcriptomics were completed to study B cells from porcine jejunum and ileum containing PPs. Intestinal locations had distinct immune landscapes, including more follicular B cells in ileum and increased MHC-II-encoding gene expression in jejunal B cells. Despite distinct landscapes, conserved B cell dynamics were detected across intestinal locations, including B cell signaling to CD4 + macrophages that are putative phagocytic, cytotoxic, effector cells and deduced routes of B cell activation/differentiation, including resting B cells migrating into follicles to replicate/divide or differentiate into antibody-secreting cells residing in intestinal crypts. A six-biomarker panel recapitulated transcriptomics findings of B cell phenotypes, frequencies, and spatial locations via ex vivo and in situ staining. Findings convey conserved B cell dynamics across intestinal locations containing PPs, despite location-specific immune environments. Results establish a benchmark of B cell dynamics for understanding intestinal immune induction important to promoting gut/overall health., Competing Interests: Declaration of Competing Interest 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., (Published by Elsevier Inc.)

The ability to reliably induce bovine digital dermatitis (DD) in naive calves provides unique opportunities to evaluate immune responses of the calves to infection after disease induction, during healing, and after subsequent re-infection. Dairy calves infected in a previous induction trial were held until lesions resolved and were then re-infected in parallel with naïve calves. Humoral and cell-mediated responses were assessed via serum antibody titer and lymphocyte proliferation analysis with responses of previously infected calves compared with responses of the newly infected calves and naïve calves. In addition, feet of calves in both treatment groups were photographed and scored by a single blinded observer using a previously described induced lesion scoring system. All naïve calves developed lesions after initial infection whereas only 5 of 8 calves developed lesions consistent with DD after a second experimental infection. In the naïve group, lesions commensurate with DD occurred in 15 of 26 experimentally infected feet with 6 feet not included in the analysis due to bandage failure. In comparison, calves in the second infection group developed lesions in 10 of 25 infected feet. Humoral responses or cellular proliferative responses did not differ between the two treatment groups or between calves which developed or did not develop lesions after experimental infection. Our results indicate that resolution of lesions after DD infection, immunity only provides partial protection against reinfection. Further studies are needed to determine immune mechanisms that provide the observed partial protection against reinfection with DD., 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 © 2024 Coatney, Krull, Gorden, Shearer, Humphrey, Olsen, Plummer and Wilson-Welder.)

Introduction: Bordetella bronchiseptica is bacterial pathogen that is pervasive in swine populations and serves multiple roles in respiratory disease., Methods: This study utilized whole-genome sequencing (WGS) analysis to assess the sequence type (ST), identify the genetic diversity of genes predicted to encode regulatory and virulence factors, and evaluated any potential antimicrobial resistance harbored by B. bronchiseptica isolates obtained from swine within the U.S., Results: While a generally high degree of genomic conservation was observed among the swine B. bronchiseptica isolates, genetic diversity was identified within the fimNX locus and among the sequence type six (ST6) isolates. The majority of B. bronchiseptica isolates exhibited phenotypic resistance to four antibiotic classes, however, only three antimicrobial resistance genes were identified., Discussion: Combined the data suggests that B. bronchiseptica isolates are not serving as a source of antimicrobial resistance gene transference in the swine production environment., 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 © 2024 Nicholson and Shore.)

Mycobacterium bovis , the causative agent of bovine tuberculosis (bTB), is a zoonotic pathogen that contributes to economic losses in the cattle industry and poses a public health risk worldwide. Bacillus Calmette-Guerin, or BCG, is a live attenuated strain of M. bovis that is used for human vaccination against tuberculosis and is considered a potential vaccine candidate against bTB. However, BCG affords widely variable levels of protection against challenge and interferes with current diagnostic methods, and as such, it is not currently approved for use as a livestock or wildlife vaccine in the United States. Many efforts have been made to develop bTB vaccines that are reliable and do not interfere with diagnostic testing, but BCG continues to be the most effective option. Previous work has shown that a T helper 1 immune response is essential for protection against virulent M. bovis infection, characterized by CD4 + central and effector memory T cells. In an effort to identify an efficacious bTB intervention strategy, the study presented here used an in vitro recall response assay and concurrent evaluation of CD4 + T cell proliferation and cytokine production to characterize the surface and functional phenotypes of memory responses to BCG vaccination in cattle. Our findings enhance understanding of the bovine immune response to BCG and provide insights into the development of improved vaccines for the control of bTB., 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 © 2024 Sterle, Putz, Olsen and Boggiatto.)

Gonadotropins and progestins are the primary regulators of follicle maturation and ovulation in fish, and they require complex communication among the oocyte and somatic cells of the follicle. The major progestin and the maturation-inducing hormone in salmonids is 17α,20β-dihdroxy-4-pregnen-3-one (17,20βP), and traditional nuclear receptors and membrane steroid receptors for the progestin have been identified within the follicle. Herein, RNA-seq was used to conduct a comprehensive survey of changes in gene expression throughout the intact follicle in response to in vitro treatment with these hormones to provide a foundation for understanding the coordination of their actions in regulating follicle maturation and preparation for ovulation. A total of 5292 differentially expressed genes were identified from our transcriptome sequencing datasets comparing four treatments: fresh tissue; untreated control; 17,20βP-treated; and salmon pituitary homogenate-treated follicles. Extensive overlap in affected genes suggests many gonadotropin actions leading to the acquisition of maturational and ovulatory competence are mediated in part by gonadotropin induction of 17,20βP synthesis. KEGG analysis identified signaling pathways, including MAPK, TGFβ, FoxO, and Wnt signaling pathways, among the most significantly enriched pathways altered by 17,20βP treatment, suggesting pervasive influences of 17,20βP on actions of other endocrine and paracrine factors in the follicle complex.

Pathogenic spirochaetes of the genus Leptospira are the etiological agents of leptospirosis, a zoonotic infection worldwide. The disease is considered an emerging and re-emerging threat due to global warming, followed by heavy rainfall and flooding when outbreaks of leptospirosis occur. Adhesion to host tissues is mediated by surface/extracellular proteins expressed by pathogens during infection. Leucine-rich repeat (LRR) domain-containing proteins seem to be important for the virulence of pathogenic Leptospira and their role has been recently examined. Here, we report the characterization of two LRR-proteins encoded by LIC11051 and LIC11505. They present 7 and 17 LRR motifs, respectively. LIC11051 was found mainly in the P1 subclade, whereas LIC11505 was identified with higher identity in subclade P1, but was also found in subclades P2, S1, and S2. The recombinant proteins were recognized by antibodies in leptospirosis serum samples, suggesting their expression during infection. rLIC11505 contains a broad spectrum of ligands, including GAG and integrin receptors, whereas rLIC11051 showed limited binding activity. The attachment of proteins to ligands was specific, dose-dependent, and saturable. Compared to their role in adhesion, both proteins were shown to be secreted, with the ability to reassociate with the bacteria. Taken together, our data suggested that LIC11051 and LIC11505 participate in leptospiral pathogenesis. To the best of our knowledge, this is the first report showing leptospiral LRR-proteins exhibiting GAG and integrin receptor-binding properties., 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 © 2024 Foltran, Teixeira, Romero, Fernandes and Nascimento.)

Chronic wasting disease (CWD) is a prion disease afflicting wild and farmed elk. CWD prions (PrP Sc ) are infectious protein conformations that replicate by inducing a natively expressed prion protein (PrP C ) to refold into the prion conformation. Mass spectrometry was used to study the prions resulting from a previously described experimental inoculation of MM132, ML132, and LL132 elk with a common CWD inoculum. Chymotryptic digestion times and instrument parameters were optimized to yield a set of six peptides, TNMK, MLGSAMSRPL, LLGSAMSRPL, ENMYR, MMER, and VVEQMCITQYQR. These peptides were used to quantify the amount, the M132 and L132 polymorphic composition, and the extent of methionine oxidation of elk PrP Sc . The amount (ng/g brain tissue) of PrP Sc present in each sample was determined to be: MM132 (5.4 × 10 2 ± 7 × 10 1 ), ML132 (3.3 × 10 2 ± 6 × 10 1 and 3.6 × 10 2 ± 3 × 10 1 ) and LL132 (0.7 × 10 2 ± 1 × 10 1 , 0.2 × 10 2 ± 0.2 × 10 1 , and 0.2 × 10 2 ± 0.5 × 10 1 ). The proportion of L132 polymorphism in ML132 (heterozygous) PrP Sc from CWD-infected elk was determined to be 43% ± 2% or 36% ± 3%. Methionine oxidation was detected and quantified for the M132 and L132 polymorphisms in the samples. In this way, mass spectrometry can be used to characterize prion strains at a molecular level.

Mammalian prion diseases are infectious neurodegenerative diseases caused by the self-templating form of the prion protein PrP Sc . Much evidence supports the hypothesis that prions exist as a mixture of a dominant strain and minor prion strains. While it is known that prions can infect new species, the relative contribution of the dominant prion strain and minor strains in crossing the species barrier is unknown. We previously identified minor prion strains from a biologically cloned drowsy (DY) strain of hamster-adapted transmissible mink encephalopathy (TME). Here we show that these minor prion strains have increased infection efficiency to rabbit kidney epithelial cells that express hamster PrP C compared to the dominant strain DY TME. Using protein misfolding cyclic amplification (PMCA), we found that the dominant strain DY TME failed to convert mouse PrP C to PrP Sc , even after several serial passages. In contrast, the minor prion strains isolated from biologically cloned DY TME robustly converted mouse PrP C to PrP Sc in the first round of PMCA. This observation indicates that minor prion strains from the mutant spectra contribute to crossing the species barrier. Additionally, we found that the PMCA conversion efficiency for the minor prion strains tested was significantly different from each other and from the short-incubation period prion strain HY TME. This suggests that minor strain diversity may be greater than previously anticipated. These observations further expand our understanding of the mechanisms underlying the species barrier effect and has implications for assessing the zoonotic potential of prions., Importance: Prions from cattle with bovine spongiform encephalopathy have transmitted to humans, whereas scrapie from sheep and goats likely has not, suggesting that some prions can cross species barriers more easily than others. Prions are composed of a dominant strain and minor strains, and the contribution of each population to adapt to new replicative environments is unknown. Recently, minor prion strains were isolated from the biologically cloned prion strain DY TME, and these minor prion strains differed in properties from the dominant prion strain, DY TME. Here we found that these minor prion strains also differed in conversion efficiency and host range compared to the dominant strain DY TME. These novel findings provide evidence that minor prion strains contribute to interspecies transmission, underscoring the significance of minor strain components in important biological processes., Competing Interests: The authors declare no conflict of interest.

Streptococcus suis negatively impacts swine health, posing diagnostic and preventative challenges. S. suis can induce disease and also quietly reside on mucosal surfaces. The limited use of diagnostic tools to identify disease-associated strains and rule out differential diagnoses, alongside the complex ecology of S. suis , poses significant challenges in comprehending this important pathogen and defining pathotypes. This study evaluated 2,379 S . suis central nervous system (CNS) isolates from diagnostic submissions between 2015 and 2019. Isolates originating from submissions with histologic evidence of CNS infection ( n = 1,032) were further characterized by standard and advanced diagnostic techniques. We identified 29 S . suis serotypes and 4 reclassified serotypes as putative causes of CNS disease. Among these, serotypes 1 and 7 emerged as the predominant putative causes of CNS infection (32% of submissions). Furthermore, 51 sequence types (STs), of which 15 were novel, were detected with ST1 predominating. Through whole-genome sequencing of 145 isolates, we observed that five commonly used virulence-associated genes (VAGs; epf , mrp , sly , ofs , and srtF ) were not present in most disease-associated isolates, and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) yielded false-positive results in 7% of isolates. These data indicate that (i) clinical signs and site of isolation alone are insufficient for defining a pathotype, (ii) S. suis serotypes and STs associated with CNS infection are more diverse than previously reported, (iii) MALDI-TOF MS may need to be supplemented with additional diagnostic tools for precise S. suis identification, and (iv) VAGs remain an unreliable means for identifying isolates associated with CNS disease.IMPORTANCE Streptococcus suis is an important and complex systemic bacterial pathogen of swine. Characterization of S. suis strains originating from pigs with histologic confirmation of neurologic disease is limited. Review of swine diagnostic submissions revealed that fewer than half of cases from which S. suis was isolated from the brain had histologic evidence of neurologic disease. This finding demonstrates that clinical signs and site of isolation alone are not sufficient for identifying a neurologic disease-associated strain. Characterization of strains originating from cases with evidence of disease using classic and advanced diagnostic techniques revealed that neurologic disease-associated strains are diverse and commonly lack genes previously associated with virulence., Competing Interests: The authors declare no conflict of interest.

H5 influenza is considered a potential pandemic threat. Recently, H5 viruses belonging to clade 2.3.4.4b have caused large outbreaks in avian and multiple nonhuman mammalian species. Previous studies have identified molecular phenotypes of the viral hemagglutinin (HA) protein that contribute to pandemic potential in humans, including cell entry, receptor preference, HA stability, and reduced neutralization by polyclonal sera. However, prior experimental work has only measured how these phenotypes are affected by a handful of the >10,000 different possible amino-acid mutations to HA. Here, we use pseudovirus deep mutational scanning to measure how all mutations to a 2.3.4.4b H5 HA affect each phenotype. We identify mutations that allow HA to better bind α2-6-linked sialic acids and show that some viruses already carry mutations that stabilize HA. We also measure how all HA mutations affect neutralization by sera from mice and ferrets vaccinated against or infected with 2.3.4.4b H5 viruses. These antigenic maps enable rapid assessment of when new viral strains have acquired mutations that may create mismatches with candidate vaccine virus, and we show that a mutation present in some recent H5 HAs causes a large antigenic change. Overall, the systematic nature of deep mutational scanning combined with the safety of pseudoviruses enables comprehensive measurements of the phenotypic effects of mutations that can inform real-time interpretation of viral variation observed during surveillance of H5 influenza., Competing Interests: J.D.B. and B.D. are inventors on Fred Hutch licensed patents related to the pseudovirus deep mutational scanning technique used in this paper and a provisional patent describing stabilizing mutations to HA.. J.D.B. consults for Apriori Bio, Invivyd, the Vaccine Company, Moderna, and GSK. B.D. consults for Moderna. N.P.K. is a cofounder, shareholder, paid consultant, and chair of the scientific advisory board of Icosavax, Inc. The King lab has received unrelated sponsored research agreements from Pfizer and GlaxoSmithKline., (Copyright: This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.)

Mannheimia haemolytica is a major bacterial pathogen associated with broncho- and fibrinous pneumonia in ruminants. Here, we report the complete genome sequence of an isolate of serotype A2 M. haemolytica (D95) recovered from a pneumonic ovine lung. The D95 genome has a size of 2.7 Mb and contains 2,720 genes., Competing Interests: The authors declare no conflict of interest.

Understanding the molecular mechanisms underlying immune response can allow informed decisions in drug or vaccine development, and aid in the identification of biomarkers to predict exposure or evaluate treatment efficacy. The objective of this study was to identify differentially expressed transfer RNA-derived fragments (tRFs) in calves challenged with Mycoplasma bovis ( M. bovis ) or co-infected with M. bovis and bovine viral diarrhea virus (BVDV). Serum, white blood cells (WBC), liver, mesenteric lymph node (MLN), tracheal-bronchial lymph node (TBLN), spleen, and thymus were collected from Control ( n  = 2), M. bovis (MB; n  = 3), and co-infected (Dual; n = 3) animals, and small RNAs extracted for sequencing. An average of 94% of reads were derived from 5` halves and/or 5` tRFs in serum, liver, WBC, TBLN, spleen, MLN, and thymus. The expression of tRFs in lymphatic tissues (MLN, TBLN, Thymus, Spleen) were highly correlated with each other (r ≥ 0.82), but not with serum and WBC. A total of 25 and 65 differentially expressed tRFs were observed in liver and thymus, respectively. There were no differentially expressed tRFs found in other tissues analyzed. Nineteen thymus tRFs were differentially expressed in Dual compared to Control and MB, and the predicted targets of these tRFs were associated with MAPK signaling pathways and ERK1 and ERK2 cascades. The differentially expressed tRFs found in thymus and liver may underlie mechanisms of thymic depletion or liver inflammation previously observed in BVDV. Additional studies should be pursued to investigate differential expression of the predicted tRF targets., 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 Goldkamp, Atchison, Falkenberg, Dassanayake, Neill and Casas.)

Shiga toxin - producing Escherichia coli (STEC) are major foodborne pathogens that result in thousands of hospitalizations each year in the United States. Cattle, the natural reservoir, harbor STEC asymptomatically at the recto-anal junction (RAJ). The molecular mechanisms that allow STEC and non-STEC E. coli to adhere to the RAJ are not fully understood, in part because most adherence studies utilize human cell culture models. To identify a set of bovine-specific E. coli adherence factors, we used the primary RAJ squamous epithelial (RSE) cell-adherence assay to coculture RSE cells from healthy Holstein cattle with diverse E. coli strains from bovine and nonbovine sources. We hypothesized that a comparative genomic analysis of the strains would reveal factors associated with RSE adherence. After performing adherence assays with historical strains from the E. coli Reference Center ( n = 62) and strains newly isolated from the RAJ ( n = 15), we used the bioinformatic tool Roary to create a pangenome of this collection. We classified strains as either low or high adherence and using the Scoary program compiled a list of accessory genes correlated with the "high adherence" strains. While none of the correlations were statistically significant, several gene clusters were associated with the high-adherence phenotype, including two that encode uncharacterized proteins. We also demonstrated that non-STEC E. coli strains from the RAJ are more adherent than other isolates and can outcompete STEC in coculture with RSEs. Further analysis of adherence-associated gene clusters may lead to an improved understanding of the molecular mechanisms of RSE adherence and may help develop probiotics targeting STEC in cattle., Importance: E. coli strains that produce Shiga toxin cause foodborne illness in humans but colonize cattle asymptomatically. The molecular mechanisms that E. coli uses to adhere to cattle cells are largely unknown. Various strategies are used to control E. coli in livestock and limit the risk of outbreaks. These include vaccinating animals against common E. coli strains and supplementing their feed with probiotics to reduce the carriage of pathogens. No strategy is completely effective, and probiotics often fail to colonize the animals. We sought to clarify the genes required for E. coli adherence in cattle by quantifying the attachment to bovine cells in a diverse set of bacteria. We also isolated nonpathogenic E. coli from healthy cows and showed that a representative isolate could outcompete pathogenic strains in cocultures. We propose that the focused study of these strains and their adherence factors will better inform the design of probiotics and vaccines for livestock., Competing Interests: The authors declare no conflict of interest.

Highly pathogenic avian influenza (HPAI) H5N1 haemagglutinin clade 2.3.4.4b was detected in the USA in 2021. These HPAI viruses caused mortality events in poultry, wild birds and wild mammals. On 25 March 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multistate investigation into milk production losses 1 . More than 200 positive herds were identified in 14 US states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production and thick yellow milk 2,3 . The diagnostic investigation revealed viral RNA in milk and alveolar epithelial degeneration and necrosis and positive immunoreactivity of glandular epithelium in mammary tissue. A single transmission event, probably from birds, was followed by limited local transmission and onward horizontal transmission of H5N1 clade 2.3.4.4b genotype B3.13 (ref.  4 ). Here we sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. Heifers were inoculated by an aerosol respiratory route and cows by an intramammary route. Clinical disease was mild in heifers, but infection was confirmed by virus detection, lesions and seroconversion. Clinical disease in lactating cows included decreased rumen motility, changes to milk appearance and production losses. Infection was confirmed by high levels of viral RNA detected in milk, virus isolation, lesions in mammary tissue and seroconversion. This study provides the foundation to investigate additional routes of infection, pathogenesis, transmission and intervention strategies., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Histophilus somni is an important causative agent of bovine respiratory disease complex. Here, we report the complete genome sequence of a Histophilus somni strain 91, which was isolated from a pneumonic lung tissue sample collected from a beef calf., Competing Interests: The authors declare no conflict of interest.

Johne's disease (JD; paratuberculosis) control programs have been regionally implemented across the globe, but few have successfully eradicated the pathogen (Mycobacterium avium ssp. paratuberculosis (MAP)) causing this disease. The limited success may partly be attributed to excluding young stock (calves and replacement heifers or bulls) from testing strategies aimed at identifying MAP-infected cattle. Young stock can shed MAP in feces and can have detectable MAP-specific antibodies in blood, as confirmed in experimentally and naturally infected cattle. Furthermore, MAP transmission causes new infections in young stock. Calves and heifers are often included in JD management strategies on dairy farms but excluded from conventional diagnostic tests due to a presumed lag between infection and detection of MAP shedding and/or MAP-specific serum antibodies. We summarize evidence of MAP shedding early in the course of infection and discuss promising diagnostics, testing and management strategies to support inclusion of young stock in JD control programs. Improvements in fecal Polymerase Chain Reaction, interferon-gamma release assay (IGRA), and enzyme-linked immunosorbent assay (ELISA) enable earlier detection of MAP and specific early immune responses. Studies on IGRA and ELISA have focused on evaluation of new antigens and optimal age of testing. There are new diagnostics, including phage-based tests to detect viable MAP, and gene expression patterns and metabolomics to detect MAP-infected young stock. In addition, refinements in testing and management of calves and heifers may enable reductions in MAP prevalence. We provide recommendations for dairy farmers, researchers, veterinarians, and other stakeholders that may improve JD control programs with an objective to control and potentially eradicate JD. Additionally, we have identified the most pressing gaps in knowledge that currently hamper inclusion of young stock in JD prevention and control programs. In summary, transmission among young stock may cause new MAP infections, and appropriate use of new diagnostic tests, testing and management strategies for young stock may improve the efficacy of JD control programs., (© 2025, The Authors. Published by Elsevier Inc. on behalf of the American Dairy Science Association®. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).)

Shiga toxin-producing Escherichia coli (STEC) is a significant public health threat due to its ability to cause severe gastrointestinal diseases in humans, ranging from diarrhea to life-threatening conditions such as hemorrhagic colitis and hemolytic uremic syndrome (HUS). As the primary reservoir of STEC, cattle play a crucial role in its transmission through contaminated food and water, posing a considerable risk to human health. This comprehensive review explores host-pathogen interactions during STEC colonization of the bovine gut, focusing on the role of gut microbiota in modulating these interactions and influencing disease outcomes. We integrated findings from published transcriptomics, proteomics, and genomics studies to provide a thorough understanding of how STEC adheres to and colonizes the bovine gastrointestinal tract. The insights from this review offer potential avenues for the development of novel preventative and therapeutic strategies aimed at controlling STEC colonization in cattle, thereby reducing the risk of zoonotic transmission.

Escherichia coli O157:H7-adulterated food products are associated with disease outbreaks in humans. Although cattle feces are a source for E. coli O157:H7 contamination, it is unclear if human-associated outbreak isolates differentially colonize and shed in the feces of cattle from that of non-outbreak isolates. It is also unclear if phenotypes, such as biofilm formation, cell attachment, or toxin production, differentiate environmental E. coli O157:H7 isolates from those associated with human illness. The objective of this study was to compare the genotypes and phenotypes of a diverse set of E. coli O157:H7 isolates, with the intent of identifying differences that could inform cattle colonization and fecal shedding, along with virulence potential in humans. Isolates differed in attachment phenotypes on human Caco-2 cells and bovine-derived recto-anal junction squamous epithelial cells, with curli having a strong impact on attachment to the human-derived cell line. The prototypical E. coli O157 isolate EDL933 had the greatest expression of the adhesin gene iha , yet it had decreased expression of the virulence genes stx2 , eae , and ehxA compared the lineage I/II isolates RM6067W and/or FRIK1989. Strong or weak biofilm production was not associated with significant differences in cattle colonization or shedding, suggesting biofilms may not play a major role in cattle colonization. No significant differences in cattle colonization and fecal shedding were detected, despite genomic and in vitro phenotypic differences. The outbreak isolate associated with the greatest incidence of hemolytic uremic syndrome, RM6067W, induced the greatest Vero cell cytotoxicity and had the greatest stx2 gene expression., Importance: Foodborne illness has major impacts on global health and imposes financial hardships on food industries. Escherichia coli serotype O157:H7 is associated with foodborne illness. Cattle feces are a source of E. coli O157:H7, and routine surveillance has led to an abundance of E. coli O157:H7 genomic data. The relationship between E. coli O157:H7 genome and phenotype is not clearly discerned for cattle colonization/shedding and improved understanding could lead to additional strategies to limit E. coli O157:H7 in the food chain. The goal of the research was to evaluate genomic and phenotypic attributes of E. coli O157:H7 associated with cattle colonization and shedding, environmental persistence, and human illness. Our results indicate variations in biofilm formation and in vitro cellular adherence was not associated with differences in cattle colonization or shedding. Overall, processes involved in cattle colonization and various phenotypes in relation to genotype are complex and remain not well understood., Competing Interests: The authors declare no conflict of interest.

Frequent interspecies transmission of human influenza A viruses (FLUAV) to pigs contrasts with the limited subset that establishes in swine. While hemagglutinin mutations are recognized for their role in cross-species transmission, the contribution of neuraminidase remains understudied. Here, the NA's role in FLUAV adaptation was investigated using a swine-adapted H3N2 reassortant virus with human-derived HA and NA segments. Adaptation in pigs resulted in mutations in both HA (A138S) and NA (D113A). The D113A mutation abolished calcium (Ca 2+ ) binding in the low-affinity Ca 2+ -binding pocket of NA, enhancing enzymatic activity and thermostability under Ca 2+ -depleted conditions, mirroring swine-origin FLUAV NA behavior. Structural analysis predicts that swine-adapted H3N2 viruses lack Ca 2+ binding in this pocket. Further, residue 93 in NA (G93 in human, N93 in swine) also influences Ca 2+ binding and impacts NA activity and thermostability, even when D113 is present. These findings demonstrate that mutations in influenza A virus surface proteins alter evolutionary trajectories following interspecies transmission and reveal distinct mechanisms modulating NA activity during FLUAV adaptation, highlighting the importance of Ca 2+ binding in the low-affinity calcium-binding pocket., (© 2024. The Author(s).)

Mycoplasma hyopneumoniae (M. hyopneumoniae) is a significant porcine respiratory disease complex pathogen, prompting many swine farms and production systems to pursue M. hyopneumoniae elimination strategies. Antibody testing is cost-effective in demonstrating sustained freedom from M. hyopneumoniae, often replacing PCR testing on deep tracheal swabs. The process typically involves testing a subpopulation of the herd using an M. hyopneumoniae screening antibody ELISA, with non-negative results further assessed through confirmatory testing, such as PCR. Recently, a commercial (Biovet) fluorescent microsphere immunoassay (FMIA) for detecting M. hyopneumoniae antibodies has been introduced as an alternative to ELISA. Its performance was compared to three commercial ELISAs (Idexx, Hipra, and Biochek) using experimental serum samples from pigs inoculated with M. hyopneumoniae, M. hyorhinis, M. hyosynoviae, M. flocculare, or mock-inoculated with Friis medium. FMIA consistently detected M. hyopneumoniae at earlier time points than the ELISAs, although two false-positive results were encountered using the manufacturer's recommended cutoff. ROC analysis allowed for the evaluation of various cutoffs depending on testing objectives. Poisson regression of misclassification error counts detected no difference in the Biovet FMIA and Hipra ELISA but significantly fewer misclassification errors than Idexx and Biocheck ELISAs. This study showed FMIA as a suitable alternative to traditional ELISAs for screening purposes due to its superior antibody detection rate at early stages. Alternatively, adopting a more stringent cutoff to improve diagnostic specificity could position the FMIA as a viable confirmatory test option. Overall, FMIA is an optimal choice for M. hyopneumoniae antibody surveillance testing, offering versatility in testing strategies (e.g., triplex FMIA M. hyopneumoniae/PRRSV types 1 and 2) and contributing to improved diagnostic capabilities in porcine health management., Competing Interests: Declaration of Competing Interest 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., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Poultry-associated salmonellosis results in significant costs to poultry producers and consumers. Given the vertically integrated nature of the United States poultry industry, a better understanding of Salmonella ecology throughout all levels of poultry production is essential. One nexus point is the hatchery, where eggs from multiple broiler breeder farms are incubated and hatched, with the chicks being sent to numerous farms; therefore, the hatchery represents an ideal area to understand preharvest Salmonella ecology and flow. To achieve this, a commercial broiler hatchery was biomapped, focusing on Salmonella prevalence and serotype diversity among four major sample type categories (Air, Egg, Water, Facility) across five different places in the prehatch, hatch, and posthatch areas. Following two sets of eggs from broiler breeder farms over two production days, the overall Salmonella prevalence was 26% (48/184). Of the positive samples, the highest prevalence was observed in swabs taken from the floor drains in the facility and transport truck (56%), as well as in the hatch and posthatch hatchery areas (50%). Kentucky (n = 17), Gaminara (n = 12), and Alachua (n = 11) were the dominant Salmonella serotypes, with serotypes of greatest outbreak concern from chickens (Enteritidis) representing only 6.25% (3/48) of all recovered Salmonella isolates. The posthatch transport area, including the underfloor reservoirs of the transport trucks, not only harbored Enteritidis but also the enrichment broths from these Salmonella-positive samples also possessed sequences matching the commercial live-attenuated vaccine Typhimurium strain according to CRISPR SeroSeq analyses. These findings highlight the complex diversity of commercial hatchery Salmonella populations, including identifying facility floor drains and transport trucks as potentially important critical control points for hatchery managers to focus their Salmonella mitigation efforts to reduce loads and serotypes entering live production farms., Competing Interests: Declaration of competing interest 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., (Published by Elsevier Inc.)

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.