Research Project: Intervention Strategies for Spirochete Diseases

Location: Infectious Bacterial Diseases Research

2022 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
During FY2022, new species and serovars of Leptospira were isolated from dairy cows and bulls. Isolates are being characterized by genome sequencing and serotyping. As vaccine protection is generally limited by serotype, understanding circulating serotypes and strains is critical for disease intervention strategies. In a similar manner, research characterizing the microbiome of bacteria within digital dermatitis (DD) lesions in cattle and wild elk proceeded with the goal of elucidating the pathogenesis of this polymicrobial disease. Specifically, research studies were focused on characterizing the influences of stage of development and duration of persistence on the microbiome of DD lesions. A group of novel, environmentally-safe chemicals, biocides, were evaluated for their ability to act as disinfectants on anaerobic and aerobic bacteria including those associated with DD lesions. The effect of organic materials on the ability of biocides to function as disinfectants was also characterized.

Accomplishments
1. Protein expression by Leptospira. Proteins expressed by Leptospira at body temperatures may be candidates for effective vaccines. Leptospirosis is a zoonotic disease that causes reproductive losses and infertility in cattle with L. borgpetersenii serovar Hardjo being the most prevalent isolate associated with infections in cattle. Leptospira borgpetersenii serovar Hardjo can only be isolated and propagated at 29C. Since pathogenic species of Leptospira are known to modify antigen expression when exposed to different incubation temperatures, ARS scientists in Ames, Iowa, developed and used a new media to characterize protein expression of serovar Hardjo at temperatures encountered during host infection. Leptospira proteins were identified that had increased expression at body temperature that may be upregulated during in vivo infection. These proteins may be more immunogenic in vivo and therefore attractive targets for development of novel vaccines. This work will be of interest to stakeholders, regulatory personnel, and researchers with interest in leptospirosis and leptospirosis vaccines.

2. New Leptospira species and serovars identified in dairy cows. Leptospirosis is a disease that infects multiple species, including humans, that causes reproductive losses and infertility in dairy cattle. Vaccines against leptospirosis are only efficacious for the serotype(s) included in the vaccine. ARS scientists in Ames, Iowa, made the first isolation of L borgpetersenii serovar Tarassovi from a dairy cow in Minnesota, and the first isolation of L. santarosai serogroup Pyrogenes from a dairy cow in Puerto Rico. These isolations suggest a possibility that current commercial cattle vaccines may need modification to include these new serotypes. This work will be of interest to stakeholders, regulatory personnel, and researchers with interest in leptospirosis and leptospirosis vaccines.

3. Foamy macrophages in animal leptospirosis models. In the United States, Leptospira bacterin vaccines must satisfy codified regulations that require their evaluation in a hamster model of leptospirosis. ARS scientists in Ames, Iowa, observed a novel immune cell type that circulates in the blood of hamsters infected with leptospires. The novel immune cell type was consistent with lipid filled foamy macrophages (FM). This unique finding of circulating FM in the hamster model of leptospirosis suggests novel, unexplored, immune activated pathways that are induced by exposure to pathogenic Leptospira. This work will be of interest to stakeholders, regulatory personnel, and researchers with interest in leptospirosis and leptospirosis vaccines.

Review Publications
Putz, E.J., Sivasankaran, S.K., Fernandes, L.G., Brunelle, B.W., Lippolis, J.D., Alt, D.P., Bayles, D.O., Hornsby, R.L., Nally, J.E. 2021. Distinct transcriptional profiles of Leptospira borgpetersenii serovar Hardjo strains JB197 and HB203 cultured at different temperatures. PLOS Neglected Tropical Diseases. 15(4). Article e0009320. https://doi.org/10.1371/journal.pntd.0009320.
Cranford, H.M., Taylor, M., Browne, A., Alt, D.P., Anderson, T., Hammond, C., Hornsby, R.L., Lecount, K., Schlater, L., Stuber, T., Dewilde, L., Burke-France, V.J., Ellis, E.M., Nally, J.E., Bradford, B. 2021. Prevalence of pathogenic Leptospira in livestock in St. Croix, U.S. Virgin Islands. Tropical Medicine and Infectious Disease. 6(2), Article 85. https://doi.org/10.3390/tropicalmed6020085.
Fernandes, L.G., Hornsby, R.L., Nascimento, A., Nally, J.E. 2021. Application of CRISPR interference (CRISPRi) for gene silencing in pathogenic species of Leptospira. The Journal of Visualized Experiments (JoVE). 174. Article e62631. https://doi.org/10.3791/62631.
Nally, J.E., Hornsby, R.L., Alt, D.P. 2021. Antigen specific urinary immunoglobulin in reservoir hosts of leptospirosis. Veterinary Sciences. 8(9). Article 178. https://doi.org/10.3390/vetsci8090178.
Cranford, H.M., Browne, A.S., Lecount, K., Anderson, T., Hamond, C., Schlater, L., Stuber, T., Burke-France, V.J., Taylor, M., Harrison, C.J., Matias, K.Y., Medley, A., Rossow, J., Wiese, N., Jankelunas, L., De Wilde, L., Mehalick, M., Blanchard, G.L., Garcia, K.R., Mckinley, A.S., Lombard, C.D., Valiulis, J., Bradford, B., Berentsen, A., Salzer, J.S., Galloway, R., Schafer, I.J., Bisgard, K., Roth, J., Ellis, B.R., Ellis, E.M., Nally, J.E. 2021. Mongooses (Urva auropunctata) as reservoir hosts of Leptospira species in the United States Virgin Islands, 2019–2020. PLOS Neglected Tropical Diseases. 15(11). Article e0009859. https://doi.org/10.1371/journal.pntd.0009859.
Putz, E.J., Andreasen, C.B., Stasko, J.A., Fernandes, L.G., Palmer, M.V., Rauh, M.J., Nally, J.E. 2021. Circulating foamy macrophages in the golden syrian hamster (mesocricetus auratus) model of Leptospirosis. Journal of Comparative Pathology. 189(10):98-109. https://doi.org/10.1016/j.jcpa.2021.10.004.
Hamond, C., Browne, A.S., Hornsby, R.L., Lecount, K., Anderson, T., Stuber, T., De Wilde, L.H., Cranford, H.M., Browne, S.K., Blanchard, G., Horner, D., Taylor, M.L., Evans, M., Roth, J., Bisgard, K., Salzer, J., Schafer, I., Ellis, B., Alt, D.P., Schlater, L., Nally, J.E., Ellis, E.M. 2022. Assessing rodents as carriers of pathogenic leptospires in the U.S. Virgin Islands: the risk to animal and public health. Scientific Reports. 12. Article 1132. https://doi.org/10.1038/s41598-022-04846-3.
Fernandes, L.G., Putz, E.J., Stasko, J.A., Lippolis, J.D., Nascimento, A.L., Nally, J.E. 2022. Evaluation of LipL32 and LigA/LigB knockdown mutants in Leptospira interrogans Serovar Copenhageni: impacts to proteome and virulence. Frontiers in Microbiology. 12. Article 799012. https://doi.org/10.3389/fmicb.2021.799012.
Putz, E.J., Bayles, D.O., Alt, D.P., Nally, J.E. 2022. Complete genome sequence of four strains of Leptospira borgpetersenii serovar Hardjo isolated from cattle in the Central United States. Journal of Genomics. 10. Pages 45-48. https://doi.org/10.7150/jgen.69822.
Hamond, C., LeCount, K., Putz, E.J., Bayles, D.O., Camp, P., Goris, M.G., Van Der Linden, H., Stone, N.E., Schlater, L., Sahl, J.W., Wagner, D.M., Nally, J.E. 2022. Bovine leptospirosis due to persistent renal carriage of Leptospira borgpetersenii serovar Tarassovi. Frontiers in Veterinary Science. 9. Article 848664. https://doi.org/10.3389/fvets.2022.848664.
Sykes, J., Reagan, K., Nally, J.E., Galloway, R., Haake, D. 2022. Role of diagnostics in epidemiology, management, surveillance, and control of leptospirosis. Pathogens. 11(4). Article 395. https://doi.org/10.3390/pathogens11040395.
Putz, E.J., Fernandes, L., Sivasankaran, S., Bayles, D.O., Alt, D.P., Lippolis, J.D., Nally, J.E. 2022. Some like it hot, some like it cold; Proteome comparison of Leptospira borgpetersenii serovar Hardjo strains propagated at different temperatures. Journal of Proteomics. 262(2022). Article 104602. https://doi.org/10.1016/j.jprot.2022.104602.
Wilson-Welder, J.H., Olsen, S.C., Alt, D.P., Bayles, D.O., Han, S., Mansfield, K. 2022. Lesion material from Treponema-associated Hoof Disease of wild elk induces disease pathology in the sheep Digital Dermatitis model. Frontiers in Veterinary Science. 8. Article 782149. https://doi.org/10.3389/fvets.2021.782149.
Stone, N.E., Hall, C.M., Ortiz, M., Hutton, S., Santana-Popper, E., Celona, K.R., Williamson, C.H., Bratsch, N., Fernandes, L.G., Busch, J.D., Pearson, T., Rivera-Garcia, S., Soltero, F., Galloway, R., Weiner, Z., Hoffmaster, A.R., Sahl, J.E., Nally, J.E., Wagner, D.M. 2022. Divergent lineages of pathogenic Leptospira species are widespread and persisting in the environment in Puerto Rico, USA. PLOS Neglected Tropical Diseases. 16(5). e0009959. https://doi.org/10.1371/journal.pntd.0009959.