Location: Infectious Bacterial Diseases Research
2025 Annual Report
Objectives
Objective 1: Characterize leptospires circulating in dairy cows to advance the development of efficacious intervention strategies.
Sub-objective 1.1: Detect and classify leptospires circulating in dairy cows.
Sub-objective 1.2: Characterization of recent isolates of pathogenic leptospires.
Sub-objective 1.3: Enhanced bacterin intervention strategies.
Objective 2: Characterize the pathogenesis of DD for the development of more effective intervention strategies.
Sub-objective 2.1: Characterization of Treponema and other pathogens in DD.
Sub-objective 2.2: Development of host immune response to antigens from DD lesions.
Sub-objective 2.3: Development of effective intervention strategies for DD.
Approach
Leptospirosis and digital dermatitis (DD) are two different diseases caused by two separate groups of bacteria in the Phylum Spirochaetes that have substantial impact on livestock production. Multiple serovars of Leptospira interrogans are the leading cause of acute lethal leptospirosis in humans and domestic animals while serovar Hardjo of L. borgpetersenii is the leading cause of bovine disease, causing reproductive failure and persistent shedding via urine to maintain disease transmission. Current bovine bacterin vaccines are limited in efficacy. To improve on this, Objective 1 will characterize leptospires circulating in dairy cows to advance the development of efficacious intervention strategies. It is first necessary to identify those species and serovars of Leptospira currently circulating in animal populations, as described in subobjective 1.1. Given the importance of L. borgpetersenii to animal disease, subobjective 1.2. will include comparative genomics and proteomics of multiple serovars within Leptospira borgpetersenii to identify conserved pathogenic mechanisms of infection and candidate vaccinogens as potential recombinant subunit vaccines. We hypothesize that efficacy of bacterins can be further improved to provide heterologous protection using growth media that more closely emulates that encountered during host infection, as proposed in subobjective 1.3. Unlike leptospirosis, DD is an infectious polymicrobial skin infection in which Treponema species are found at the invading edge of the lesions. Causing painful ulcerative proliferative and necrotizing lesions on the skin at or near the hooves, DD is a significant cause of lameness in both dairy and feedlot cattle. Aside from lameness being a significant animal welfare issues, DD leads to decreased production, higher treatment costs and premature culling. Objective 2 will characterize the pathogenesis of DD for the development of more effective intervention strategies. Since the etiology of DD is not fully characterized, subobjective 2.1 will use bacterial 16S rRNA gene sequencing to characterize bacterial community of early DD lesions as induced in a sheep model, compare the sheep model to naturally infected bovine DD lesions in order to determine a core consortium of Treponema and other pathogens present. After obtaining isolates representing this core consortium, a defined mixture of Treponema species and other bacterial pathogens will be used to induce lesions in the sheep model. Subobjective 2.2. will then characterize the bovine innate immune responses to Treponema, and how that may affect lesion healing. Since current mitigation strategies use heavy metal, formalin-containing footbaths or topical antibiotics, subobjective 2.3. will evaluate novel alternative antimicrobial compounds for topical treatment of DD lesions. Understanding pathogenic mechanisms used by Spirochetes, specific species causing disease and host-pathogen interactions, are critical for the development of efficacious diagnostics, vaccines, and therapeutics for control of infection in domestic livestock.
Progress Report
Objective 1, studies continue to identify those species and serovars of Leptospira that circulate in bovines, and other domestic, wildlife and environmental sources that facilitate disease transmission to bovines. Isolates are comprehensively characterized by high resolution genome sequencing and serotyping and are being used by diagnostic laboratories to enhance surveillance. Genotyping (analyzing genetic differences) of more isolates is providing new epidemiological outputs that are critical to elucidate sources of infection. A pipeline of genetic tools continues to be optimized to genetically manipulate pathogenic species of Leptospira to identify virulence factors and develop efficacious bacterin vaccines that can be used to generate DIVA (Differentiating Infected from Vaccinated Animals) vaccines that do not interfere with diagnostics. Comparative protein analyses of different serovars of L. borgpetersenii is being performed. New bacterin strategies continue to be developed and evaluated.
Objective 2, populations of bacteria within digital dermatitis lesions were characterized from field samples collected during the 2024 National Animal Health Monitoring System (NAHMS) survey (a natioinal sampling to evaluate disease prevalence) to identify casuses of lameness. In addition, two species of Mycoplasma (M. farmentans and M. adleri) were recovered from digital dermatitis lesions suggesting a role in disease pathogenesis. New isolates of Treponema spp., the bacteria hypothesized to be the most important factor in lesion development, were recovered.
Accomplishments
1. CRISPR DNA editing as a new genetic tool for Leptospira. Leptospirosis is a significant disease in livestock that causes reproductive issues and production losses. Improving the ability to genetically modify Leptospira spp. is a crucial step toward advancing understanding of their biology leading to improved vaccines and diagnostics. ARS scientists in Ames, Iowa, developed a novel CRISPR gene editing procedure as the first precise technology to create DNA deletions, insertions, and substitutions within Leptospira genome. The technique was used to make genetic changes with remarkable precision within pathogenic Leptospira including Leptospira borgpetersenii, the leading cause of bovine leptospirosis. The technique allows the creation of clean knockout strains without selective markers (i.e. antibiotic resistance). Recombinant strains were created and used to characterize virulence factors in an effort to develop more protective vaccines for cattle. More protective vaccines and identification of new diagnostic targets will reduce the economic losses caused by leptospirosis in domestic livestock. This work will be of interest to producers, clinical veterinarians, and scientists with an interest in leptospirosis.
2. Control of gene expression in Leptospirosis to identify virulence genes. Leptospirosis is an endemic disease in livestock causing reproductive losses. ARS scientists in Ames, Iowa, developed an inducible heterologous protein expression system in Leptospira spp using a Cas9 system. After expression of targeted genes, genes essential for pathogenesis and non-coding RNAs were identified. This technique advances knowledge on the basic biology and virulence of Leptospira to facilitate the development of strategies to limit disease transmission and provides novel targets to test as subunit recombinant protein vaccines. More protective vaccines and identification of new diagnostic targets will reduce the economic losses caused by leptospirosis in domestic livestock. This work will be of interest to producers, clinical veterinarians, and scientists with an interest in leptospirosis.
3. Identification of new Leptospira species to improve diagnosis and intervention strategies. Leptospirosis is an endemic disease in livestock causing reproductive losses. ARS scientists in Ames, Iowa, identified and characterized five new species of Leptospira from water sources in central Iowa. Genome sequence analyses distinguished them from other Leptospira species and included pathogenic and saprophylic species. Knowledge of new pathogenic strain enhances the ability to diagnose leptospirosis in domestic livestock thereby enhancing intervention strategies to limit disease transmission and prevent economic losses to livestock producers. This work will be of interest to diagnostic laboratories, regulatory and public health personnel, and researchers with an interest in leptospirosis.
4. Development of effective intervention strategies for digital dermatitis in livestock. Digital dermatitis is a contagious bacterial skin infection affecting the feet of livestock causing lameness and associated production losses. Alternative, non-antibiotic, antimicrobial treatments are urgently needed due to widespread economic losses this disease causes. ARS scientists in Ames, Iowa, evaluated zinc sulfate, cuminaldehyde guanylhdrazone, and sodium bisulfate as treatments for digital dermatitis lesions. As a foot bath, cuminaldehyde guanylhdrazone reduced lesions by approximately 60% in infected cattle and zinc sulfate reduced lesions in sheep by greater than 80%. Cuminaldehyde guanylhdrazone was effective in lowering the pH in bedding and reduced the presence of the bacterial pathogen that causes foot rot (Fusobacterium). Treatment with sodium bisfulfate was also associated with better rates of weight gain and reduced incidence of lameness in cattle under field conditions. This work demonstrates intervention strategies that can reduce lameness and prevent associated production costs in domestic livestock. This work will be of interest to livestock owners, clinical veterinarians, and researchers with an interest in lameness.
Review Publications
Hamond, C., Tibbs-Cortes, B.W., Fernandes, L.G., Lecount, K., Putz, E.J., Anderson, T., Camp, P., Stuber, T., Hicks, J., Van Der Linden, H., Dos Santos-Ribeiro, P., Bayles, D.O., Schlater, L., Nally, J.E. 2025. Leptospira gorisiae sp. nov, L. cinconiae sp. nov, L. mgodei sp. nov, L. milleri sp. nov and L. iowaensis sp. nov: five new species isolated from water sources in the Midwestern United States. International Journal of Systematic and Evolutionary Microbiology. https://doi.org/10.1099/ijsem.0.006595.
Dos Santos Ribeiro, P., Stasko, J.A., Shircliff, A.L., Fernandes, L.G., Putz, E.J., Andreasen, C., Azevedo, V., Ristow, P., Nally, J.E. 2025. Investigations into the growth and formation of biofilm by Leptospira biflexa at temperatures encountered during infection. Biofilm. https://doi.org/10.1016/j.bioflm.2024.100243.
Fernandes, L.G., Nascimento, A.L., Nally, J.E. 2025. Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation. Scientific Reports. https://doi.org/10.1038/s41598-025-88633-w.
Fernandes, L., Hamond, C., Tibbs-Cortes, B.W., Putz, E.J., Olsen, S.C., Palmer, M.V., Nally, J.E. 2024. CRISPR-prime editing, a versatile genetic tool to create specific mutations with a single nucleotide resolution in Leptospira. mBio. https://doi.org/10.1128/mbio.01516-24.
Imlau, M., Browne, J.A., Browett, S.S., Mcdevitt, A.A., Mcmahon, B.J., Nally, J.E., Jahns, H. 2025. Surveillance for Leptospira sp. in native and invasive shrews in Ireland compared with cohabitating shrews in France. Journal of Wildlife Diseases. 61(2):477-482. https://doi.org/10.7589/JWD-D-24-00084.
Putz, E.J., Andreasen, C., Stasko, J.A., Hamond, C., Olsen, S.C., Nally, J.E., Palmer, M.V. 2025. Circulating foamy macrophages and other features of bacillus Calmette-Guérin challenge in Golden Syrian hamsters. Vaccine. 55. Article 127037. https://doi.org/10.1016/j.vaccine.2025.127037.
Stone, N.E., Hamond, C., Clegg, J.R., Mcdonough, R.F., Bourgeois, R.M., Ballard, R., Thornton, N.B., Nuttall, M., Hertzel, H., Anderson, T., Whealy, R.N., Timm, S., Roberts, A.K., Barragan, V., Phipatanakul, W., Leibler, J.H., Benson, H., Specht, A., White, R., Lecount, K., Furstenau, T.N., Galloway, R.L., Hill, N.J., Madison, J.D., Fofanov, V.Y., Pearson, T., Sahl, J.W., Busch, J.D., Weiner, Z., Nally, J.E., Wagner, D.M., Rosenbaum, M.H. 2025. Host population dynamics influence Leptospira spp. transmission patterns among Rattus norvegicus in Boston, Massachusetts, US. PLOS Neglected Tropical Diseases. 19(4). https://doi.org/10.1371/journal.pntd.0012966.
Hau, S.J., Eberle, K.C., Nally, J.E., Nielsen, D.W., Lippolis, J.D., Brockmeier, S. 2025. Identification of candidate vaccine antigens using 2-D gel electrophoresis and immunoproteomics for cross protection against Glaesserella parasuis. Veterinary Microbiology. 307. Article 110594. https://doi.org/10.1016/j.vetmic.2025.110594.
Putz, E.J., Andreasen, C.B., Boggiatto, P.M., Palmer, M.V., Fernandes, L.G., Tibbs-Cortes, B.W., Stasko, J.A., Hamond, C., Olsen, S.C., Nally, J.E. 2025. Peromyscus spp. Deer Mice as rodent model of acute Leptospirosis. Emerging Infectious Diseases. 31(7). https://doi.org/10.3201/eid3107.241579.