The COVID-19 pandemic represents one of the most significant public health events of the last century. As with other coronaviruses (SARS, MERS) the role of animals is of intense interest. Believed to have originated in bats, the role of other animals in the epidemiology of the SARS-CoV-2 pandemic is still unclear, as is the range of susceptible hosts. American bison were intranasally infected with SARS-CoV-2 and monitored for seroconversion and the presence of viral RNA in oronasal secretions and feces. Although clinical signs were not seen, permissiveness of bison to infection with SARS-CoV-2 was manifest by seroconversion, the presence of viral RNA in oronasal secretions, persistence of viral RNA in lymphoid tissue, and viral associated interstitial pneumonia. Retrospective sequencing of the inoculum revealed a common in vitro adaptation in the furin cleavage site of the spike protein that may have reduced in vivo viral fitness. As such, we cannot exclude the possibility that use of an isolate with an intact furin cleavage motif would more efficiently infect bison.

USDA confirmed by whole genome sequence the first detection of HPAI H5N1 clade 2.3.4.4b genotype D1.1 in dairy cattle. While genotype D1.1 has been the dominant strain circulating in migratory birds in North America, the Nevada cases represent the first detection of a genotype other than B3.13 in cattle and the second known spillover event from wild birds into lactating dairy cattle. D1.1 clinical presentation in dairy herds in both Nevada and Arizona was mild compared to HPAI B3.13. However, this is based on a small number of affected herds and may not be the case for the broader population. Here we sought to experimentally reproduce infection of dairy cattle with HPAI H5N1 genotype D1.1. and also sought determine if cattle with serum antibodies following natural infection with HPAI B3.13 were protected against reinfection with HPAI D1.1. Four adult Holstein lactating cows were moved into ABSL-3-Ag containment, two cows free of influenza A virus and two cows free of influenza A virus, but with serum antibodies from a natural H5N1 infection (genotype B3.13 ). All cows were inoculated via the intramammary route with 1 ml of 1 x 105.4 TCID50/ml A/dairy cattle/Nevada/24-002644-003/2025 into two contralateral quarters. The drop in milk production and rumination observed in this study were similar to those reported in experimental intramammary challenge of lactating cows with HPAI B3.13, as well as natural infections, indicating that clinical presentation of HPAI D1.1 was similar in severity to experimental challenge with HPAI B3.13. Unlike the HPAI B3.13 intramammary challenges, HPAI D1.1 migrated and infected a non-inoculated quarter. The two B3.13 convalescent cows were susceptible to reinfection with D1.1, demonstrating clinical signs including a drop in milk production and rumination, pyrexia, and mastitis. However, milk production and rumen motility recovered more quickly in the two convalescent cows than in the two naïve cows and pyrexia was not as severe. Viral RNA was also not detected in the milk of the convalescent cows after 10 DPI while it was detected in the milk of the naïve cows for the durations of the study. Furthermore, while viral RNA was detected in the milk of both convalescent cows, no viable virus was isolated. While convalescent cows with serum but not milk antibodies to B3.13 are susceptible to reinfection with D1.1 and clinical disease antibodies can transudate into the milk and bind virus, likely preventing further spread throughout the herd. The single-nucleotide variant analyses of whole genome sequences virus recovered from the milk of previously naïve cows also uncovered some potentially important patterns. Genes HA and MP were found to have strong evidence for natural selection and analysis indicates a fitness advantage is conferred through some key mutations that could lead to antigenic drift and immune escape.

Bovine tuberculosis (bTB), caused by infection with Mycobacterium bovis, continues to be an animal and zoonotic concern in many parts of the world, including the United States. Long-standing eradication programs have been successful at lowering prevalence of disease in many countries; however, disease eradication has not been achieved. One major obstacle to eradication is the presence of various wildlife reservoirs for M. bovis, such as white-tailed deer (Odocoileus virginianus), which serve as a source of spill-back to cattle herds. A potential method to reduce intra- and inter-species disease transmission of M. bovis between wildlife and domestic livestock includes vaccination of wildlife species. Oral vaccination of white-tailed deer with the human tuberculosis vaccine, M. bovis bacillus Calmette-Guérin (BCG) has been demonstrated to afford some level of protection against experimental challenge. However, vaccinating wildlife presents its own challenges, primarily due to the need of a delivery platform that could be implemented at scale and would not require animal handling. Oral vaccine delivery units or baits are an effective means of delivering vaccine to wildlife populations. Therefore, we explored whether sodium alginate spheres could be used as a delivery platform for BCG for vaccination of white-tailed deer. We assessed the development of peripheral immune responses following BCG vaccination and demonstrated that passive administration of BCG via alginate spheres results in antigen-specific cellular responses, similar to oral administration of BCG. Our data characterize the kinetics of cellular responses elicited by oral vaccination and suggest passive oral administration of BCG as a potential means to vaccinate free-ranging white-tailed deer.

Abstract Background Mycobacterium bovis BCG is the human tuberculosis vaccine and is the oldest vaccine still in use today with over 4 billion people vaccinated since 1921. The BCG vaccine has also been investigated experimentally in cattle and wildlife by various routes including oral and parenteral. Thus far, oral vaccination studies of cattle have involved liquid BCG or liquid BCG incorporated into a lipid matrix. Lyophilization is an established technique used for stabilizing bioproducts such as vaccines. Methods In the current study, cattle were vaccinated in two phases. In each phase, cattle were divided into three groups. Group 1 received BCG injected SQ, Group 2 received liquid BCG delivered to the posterior oral cavity, Group 3 orally consumed lyophilized BCG contained within a gelatin capsule placed within a small amount of a commercial alfalfa product. Results No vaccinated cattle were positive by an interferon gamma release assay. All but 4 animals were negative by tuberculin skin testing prior to vaccination: the 4 non-negative animals being categorized as suspects. Sixteen weeks post-vaccination all but 1 animal was negative, it being categorized as a suspect. An in vitro antigen stimulation assay and flow cytometry were used to detect antigen-specific CD4, CD8 and γδ T cell responses following vaccination. Oral vaccination of animals with lyophilized BCG did not result in any increases in the frequency of CD4, CD8 or γδ T cell proliferative or IFN-γ responses at any of the time points analyzed in either phase 1 or 2. In contrast, vaccination with BCG SQ and liquid BCG delivered to the posterior pharynx, resulted in an increase in the frequency of proliferating and IFN-γ-producing CD4 T cells with peak responses at 9–12 weeks post-vaccination. Similar to oral lyophilized BCG vaccinated animals, we did not observe any significant increases in the frequency of CD8 and γδ T cell proliferative and IFN-γ responses following SQ or oral liquid vaccinated animals. Conclusions These data would suggest that vaccination with oral lyophilized BCG does not induce a measurable, antigen-specific cell mediated responses in the periphery, when compared to BCG administered SQ or liquid BCG administered via the oral route. However, vaccination with either SQ or liquid BCG delivered to the posterior pharynx does induce measurable CD4 T cell responses in the periphery.

Mycoplasma bovis (M. bovis) is an emerging pathogen in American bison (Bison bison) responsible for high mortality epizootics of severe pneumonia and systemic disease. Though M. bovis poses a significant threat to bison conservation and ranching, there are no commercial vaccines licensed for use in this species. To this end, novel modified-live Mannheimia haemolytica (M. haemolytica) vaccine strains, serotypes 1 and 6, secreting inactive leukotoxin fused to truncated M. bovis Elongation Factor Thermal unstable (EFTu) and Heat shock protein (Hsp) 70 (EFTu-Hsp70-ΔlktCAV4) were evaluated for efficacy in bison. Modified-live M. haemolytica were administered intranasally and both serotypes were sporadically recovered from bison nasal swabs. Bison inoculated with the two M. haemolytica EFTu-Hsp70- ΔlktCAV4 strains produced systemic and mucosal antibodies to the M. bovis antigens and M. haemolytica surface antigens prior to intranasal challenge with M. bovis. Following intranasal M. bovis challenge no differences were observed in lung bacterial counts nor in lung lesion formation between bison that received the M. haemolytica EFTu-Hsp70-ΔlktCAV4 strains or the ΔlktCAV4, lacking the M. bovis antigens. Despite this, fewer M. bovis positive tissue swabs were collected from animals administered the M. haemolytica EFTu-Hsp70-ΔlktCAV4. These results demonstrate that modified-live M. haemolytica vaccine strains expressing M. bovis antigens failed to provide full protection against M. bovis infection in bison.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) remains a health risk for humans and other domestic and wildlife species. Recently, North American elk have been identified as seropositive for SARS-CoV-2, thus posing a potential threat to humans and other mammals. In this work, we characterized the peripheral transcriptomic response to experimental SARS-CoV-2 infection in calves and adult elk at different time points. Significantly differentially expressed genes were identified at 2-, 5-, and 14-days post inoculation (pi) for both age groups. Adult elk presented the greatest number of differentially expressed (DE) genes at all time points, including many genes associated with viral response, immune activation, antibody production, as well as genes associated with coronavirus disease (COVID-19), and coronavirus GO terms and KEGG pathways. Calves presented DE genes associated with viral responses at 5 days pi as well as neurodegenerative-associated genes at 14 days pi. Both adults and calves showed predicted activation of the ISGF3 and IFN type I pathways at day 2 pi and, globally, increased activity related to the coronavirus pathway disease at 5 and 14 days pi. Collectively, this work provides valuable data characterizing the cervid immune response of elk to viral diseases as well as the response of wildlife to SARS-CoV-2 infection.

IntroductionBrucella abortus and Mycobacterium bovis, the causative agents of bovine brucellosis and tuberculosis respectively, are zoonotic bacterial pathogens that both contribute to major economic losses in the cattle industry and pose a human health risk worldwide. Co-infections of cattle with B. abortus and M. bovis have been identified in various developing countries, necessitating the development of an efficacious strategy for controlling both important zoonotic diseases even in the event of co-infection. Brucella abortus strain RB51, a live attenuated vaccine for bovine brucellosis that is currently used in the US, is highly effective at preventing reproductive failure due to brucellosis in cattle. Bacillus Calmette-Guérin (BCG) is a live attenuated vaccine strain of M. bovis that provides protection against bovine tuberculosis in cattle but is not currently licensed for use in the US.MethodsThe study presented here compares functional Th1 responses of RB51 + BCG vaccinated cattle to responses of RB51-only and BCG-only vaccinated cattle to evaluate the feasibility of a combined vaccination strategy for controlling both bovine brucellosis and tuberculosis.ResultsThis work identified that peripheral blood mononuclear cells (PBMC) from RB51 vaccinates proliferate not only in response to stimulation with killed RB51 but also in response to mycobacterial antigen PPDb. Combination vaccinates show significantly more CD4+ T cell proliferation than single BCG vaccinates when stimulated with PPDb, while no differences were observed between RB51 and combination vaccinates stimulated with RB51.Discussion/conclusionSignificantly enhanced BCG-specific Th1 responses in combination vaccinates compared to BCG-only vaccinates suggest that combining vaccinations for B. abortus and M. bovis may alter the host CD4+ T cell response.

Mycobacterium bovis is the primary cause of tuberculosis in animals, most notably cattle. In cattle and other susceptible hosts, the hallmark lesion of tuberculosis is the granuloma. Granulomas represent the host–pathogen interface where disease outcome is determined; therefore, it is critical to understand host–pathogen interactions at the granuloma level. Granulomas are highly structured lesions with distinct cellular compartments for T cells, macrophages, multinucleated giant cells, and B cells. A recognized but poorly understood morphologic feature of many granulomas is the presence of structures resembling follicular or germinal center-like arrangements of B cells known as tertiary lymphoid organs, or tertiary lymphoid structures. Pulmonary granulomas from cattle experimentally infected with M. bovis were collected at 15-, 30-, 90-, 180- and 270-days post-infection and examined for the presence of tertiary lymphoid-like structures. Follicle-like structures associated with granulomas were first seen 90 days after infection and persisted in later time points. Compartmentalization of T cells and B cells similar to follicles in germinal centers of lymph nodes was demonstrated using in situ hybridization. Additionally, the presence and arrangement of myeloid cells, endothelial cells, T follicular helper cells, and chemokines critical to tertiary lymphoid structure formation was shown to be similar to lymph node follicles and that described for tertiary lymphoid structures in other species. This represents the first demonstration by in situ hybridization of the similarities of follicle-like structures associated with pulmonary bovine tuberculous granulomas to tertiary lymphoid structures in other species and follicles within secondary lymphoid organs such as lymph nodes.

Highly pathogenic avian influenza (HPAI) clade 2.3.4.4b H5N1 is circulating widely in lactating cows in the United States. Due to the critical need for intervention strategies for this outbreak, we evaluated antibody and cellular immune responses of a clade 2.3.4.4b H5 mRNA-LNP vaccine in calves. We found that the H5 mRNA-LNP vaccine induced a robust antibody and CD8+ T cellular-mediated immune response and conferred protection against clade 2.3.4.4b H5N1 infection. ### Competing Interest Statement S.E.H. and D.W. are co-inventors on patents that describe the use of nucleoside-modified mRNA as a platform to deliver therapeutic proteins and as a vaccine platform. S.E.H reports receiving consulting fees from Sanofi, Pfizer, Lumen, Novavax, and Merck. National Institutes of Health, 75N93021C00015 United States Department of Agriculture, https://ror.org/01na82s61, 5030-32000-231-000-D United States Department of Energy, https://ror.org/01bj3aw27, AC05-06OR23100

IntroductionFree-ranging white-tailed deer (Odocoileus virginianus) are a self-sustaining reservoir for bovine tuberculosis (bTB) in northeastern lower Michigan, (United States) continually putting the area’s cattle industry at risk. Liberal recreational deer harvest, baiting bans, and mitigation measures on farms have reduced but not eliminated bTB in deer nor have they eliminated transmission to cattle. With apparent prevalence in deer being low (1–2%) but constant, vaccination could be an additional tool to aid in addressing the problem and merits investigation. Mycobacterium tuberculosis Bacillus Calmette-Guérin (BCG) vaccine is a widely used human vaccine for tuberculosis that has also been well studied in domestic livestock and wildlife. It is the primary vaccine candidate, and oral delivery is the logical means for delivering it to free-ranging deer, although this has never previously been attempted.Materials and methodsBuilding off methods and strategies developed for vaccinating deer, we incorporated BCG vaccine into vaccine delivery units (DUs), consisting of a food-based matrix. We deployed DUs at sites in Michigan with a historically high prevalence of bTB. At each site, 100 DUs were placed systematically 2.5-m apart on 50-m x 10-m grids and monitored with still and video cameras. Consumption, still images, and video data were analyzed to assess uptake of vaccine DUs by deer.Results and discussionVaccine DUs were deployed in 2024 at 11 agricultural sites on private land which had previously demonstrated moderate to high deer activity and at all but two sites >50% of distributed vaccine DU’s were consumed, with 100% consumed at two sites. Deer learned to seek out and consume vaccine DU’s in just 1 to 3 days, with individuals often eating more than the 1 or 2 needed to vaccinate themselves. This high level of consumption was in spite of an exceptionally warm and dry winter, where deer were less food stressed than usual.

Leptospirosis is a global zoonotic disease affecting humans, wildlife, companion, and domestic animals. Incidental hosts can contract the disease directly or indirectly from asymptomatic reservoir hosts, most commonly small rodents. The Golden Syrian hamster is recognized as the dominant rodent model for acute leptospirosis because the animals are susceptible to many serovars and are used to maintain laboratory strains and test bacterin vaccine efficacy. However, hamsters are primarily used in survival-based studies, and investigations into host immune response and disease pathogenesis are limited. We found that Peromyscus leucopus white-footed deer mice are susceptible to acute leptospirosis, and thus might be an alternative rodent model. Furthermore, similar to hamsters, deer mice produce circulating foamy macrophages in response to Leptospira challenge. Deer mice exhibit differences in response to different serovars, clinical disease severity, kidney and liver lesions, and an overall sex effect, with male mice demonstrating more severe clinical signs and higher bacterial burden.

Background/Objectives: White-tailed deer (Odocoileus virginianus) (WTD) play a central role at the human–livestock–wildlife interface, given their contribution to the spread of diseases that can affect livestock. These include a variety of bacterial, viral, and prion diseases with significant economic impact. Given the implications for WTD as potential reservoirs for a variety of diseases, methods for prevention and disease control in WTD are an important consideration. Methods: Using commercial livestock vaccines against bovine viral diarrhea virus (BVDV) in killed and modified live formulations, we test the ability of WTD to develop humoral and cellular immune responses following vaccination. Results: We demonstrate that, similar to cattle, WTD develop humoral immune responses to both killed and modified live formulations. Conclusions: As the farmed deer industry and the use of livestock vaccines in non-approved species grow, this type of information will help inform and develop improved husbandry and veterinary care practices. Additionally, while we were unable to detect cell-mediated immune responses to the vaccine, we established PrimeFlow as a method to detect IFN-γ responses in specific T cell populations, adding another level of resolution to our ability to understand WTD immune responses.

Highly pathogenic avian influenza (HPAI) H5N1 of the hemagglutinin clade 2.3.4.4b was detected in the United States in late 2021 and continues to circulate in all four North American flyways to date. In addition to impacting poultry, these HPAI viruses caused mortality events in wild bird species and wild mammals. Transmission in multiple host species raises the concern for mammalian adaptation. On March 25, 2024, HPAI H5N1 clade 2.3.4.4b was confirmed in a dairy cow in Texas in response to a multi-state investigation into milk production losses. Over one hundred positive herds were rapidly identified in Texas and eleven other U.S. states. The case description included reduced feed intake and rumen motility in lactating cows, decreased milk production, and thick yellow milk. The diagnostic investigation revealed detections of viral RNA in milk and mammary tissue with alveolar epithelial degeneration and necrosis, and positive immunoreactivity of glandular epithelium by immunohistochemistry. A single transmission event, likely from avian species to dairy cattle, followed by limited local transmission preceded the onward lateral transmission of H5N1 clade 2.3.4.4b genotype B3.13. We sought to experimentally reproduce infection with genotype B3.13 in Holstein yearling heifers and lactating cows. The heifers were inoculated by an aerosol respiratory route and the cows by an intramammary route. Clinical disease was mild in the 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 consistent with field reports of viral mastitis. 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, transmission, and intervention strategies. ### Competing Interest Statement The authors have declared no competing interest.

Detection of CD3e (T cells), IBA-1 (macrophages/dendritic cells), Pax5 (B cells), Ki-67 (proliferating cells), or pan-cytokeratin type I/II (epithelial cells) in conjunction with an RNA marker (housekeeping gene, UBC, used herein) in formalin-fixed, paraffin-embedded (FFPE) palatine tonsil tissues from pigs, cattle, and white-tailed deer.

White-tailed deer (Odocoileus virginianus) have emerged as a reservoir host for SARS-CoV-2 given their susceptibility to infection and demonstrated high rates of seroprevalence and infection across the United States. As SARS-CoV-2 circulates within free-ranging white-tailed deer populations, there is the risk of transmission to other wildlife species and even back to the human population. The goal of this study was to determine the susceptibility, shedding, and immune response of North American elk (Cervus elaphus canadensis) to experimental infection with SARS-CoV-2, to determine if another wide-ranging cervid species could potentially serve as a reservoir host for the virus. Here we demonstrate that while North American elk do not develop clinical signs of disease, they do develop a neutralizing antibody response to infection, suggesting the virus is capable of replicating in this mammalian host. Additionally, we demonstrate SARS-CoV-2 RNA presence in the medial retropharyngeal lymph nodes of infected elk three weeks after experimental infection. Consistent with previous observations in humans, these data may highlight a mechanism of viral persistence for SARS-CoV-2 in elk.

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.

Detection of CD3e (T cells), IBA-1 (macrophages/dendritic cells), Pax5 (B cells), Ki-67 (proliferating cells), or pan-cytokeratin type I/II (epithelial cells) in formalin-fixed, paraffin-embedded (FFPE) palatine tonsil tissues from pigs, cattle, and white-tailed deer.

Bovine tuberculosis is caused by Mycobacterium bovis, a member of the M. tuberculosis complex of mycobacterial species that cause tuberculosis in humans and animals. Diagnosis of bovine tuberculosis has relied on examinations of cell-mediated immune responses to M. bovis proteins using tuberculin skin testing and/or interferon gamma release assays. Even when using these methods, disease detection during the earliest phases of infection has been difficult, allowing a window for cattle-to-cattle transmission to occur within a herd. Alternative means of diagnosis could include methods to detect M. bovis or M. bovis DNA in bodily fluids such as nasal secretions, saliva, or blood. During the first 8 weeks after experimental aerosol infection of 18 calves, M. bovis DNA was detected in nasal swabs from a small number of calves 5, 6, and 8 weeks after infection and in samples of saliva at 1, 7, and 8 weeks after infection. However, at no time could culturable M. bovis be recovered from nasal swabs or saliva. M. bovis DNA was not found in blood samples collected weekly and examined by real-time PCR. Interferon gamma release assays demonstrated successful infection of all calves, while examination of humoral responses using a commercial ELISA identified a low number of infected animals at weeks 4–8 after infection. Examination of disease severity through gross lesion scoring did not correlate with shedding in nasal secretions or saliva, and calves with positive antibody ELISA results did not have more severe disease than other calves.

ABSTRACT 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. IMPORTANCE Leptospirosis 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.

Simultaneous detection of CD3e (T cells; stained in brown), IBA-1 (macrophages/dendritic cells; stained in blue), and Pax5 (B cells; stained in red) in formalin-fixed, paraffin-embedded (FFPE) tracheobronchial lymph node tissues from pigs, cattle, and white-tailed deer.