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ARS Home » Midwest Area » Ames, Iowa » National Animal Disease Center » Infectious Bacterial Diseases Research » Research » Research Project #441160

Research Project: Intervention Strategies for Spirochete Diseases

Location: Infectious Bacterial Diseases Research

2023 Annual Report

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.

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
To address Objective 1, studies were conducted characterizing circulating Leptospira in livestock populations. This included recovery pf Leptospira circulating in dairy cattle in CA and spirochete bacteria within polymerase chain reaction (PCR)-positive bovine semen. Recovered isolates were characterized by genome sequencing and serotyping to determine the specific Leptospira serovar. As vaccine protection is generally limited by serovar, understanding circulating serotypes and strains is critical for development of successful disease intervention strategies. As L. borgpetersenii is the most prevalent species of Leptospira associated with animal infections, the genomes of multiple serovars within this Leptospira species were sequenced, and their respective proteomes characterized to identify conserved and differentially expressed proteins that could be used in novel vaccines or diagnostics assays. In studies addressing Objective 2, archived digital dermatitis (DD) lesion material was expanded in vivo using a sheep model of infection and bacterial populations characterized. Analysis of samples collected at various timepoints post-infection demonstrated that the most predominant bacterial phyla within lesions were Firmicutes, Bacteroidota and Spirochaetae. In vitro bacterial culture was conducted to obtain isolates within these phyla to determine individual bacterial species, in addition to recovering isolates of Mycoplasma and minor phyla, which are hypothesized to contribute to DD lesion development. This work allows characterization of bacterial populations which induce lesions of DD, thereby enabling development of intervention strategies. Environmentally safe chemical compounds which inhibit or kill harmful bacteria (biocides) were evaluated for their inactivation properties on bacterial isolates. Based on these studies, a topical balm containing a hydrazone-based biocide was developed and tested under in vivo conditions on DD lesions. The topical treatment was similar in efficacy to an antibiotic containing paste in treating DD lesions under experimental conditions. Through an MTRA with a commercial company, further studies are being conducted with the topical biocide treatment under field conditions.

1. Novel assay for Leptospira identification. Novel assay to determine genomes of Leptospira directly from field samples. In collaboration with academic scientists, ARS scientists in Ames, Iowa, validated a culture-independent DNA capture and enrichment system for characterizing Leptospira genomic data from field samples. The assay greatly increases recovery of Leptospira DNA from field samples and facilitates robust species identification and high-resolution genotyping. Sequence data of recovered DNA is similar in quality to data from in vitro cultured isolates. Implementation of this DNA capture and enrichment assay improves identification of Leptospira in unculturable samples and expands understanding of Leptospira populations and genomic diversity. The assay will improve epidemiologic knowledge of Leptospira distribution, facilitate development of improved diagnostics and vaccines, and will be of interest to diagnostic personnel, scientists, and livestock producers. The assay has already been used to demonstrate that U.S. cattle can be infected with more than one species of Leptospira concurrently.

2. Leptospira concurrent infections. Confirmation of Leptospira concurrent infections to inform potential transmission of the disease in animals. It has been hypothesized that domestic and wildlife animal reservoirs of leptospirosis can be co-infected and shed multiple species of Leptospira concurrently. This would impact the development of intervention strategies. However, definitive microbiologic evidence of this phenomena in any species was lacking. ARS scientists in Ames, Iowa, developed a novel culture strategy that was applied to samples from rodents suspected of being co-infected with multiple Leptospira species. Results definitively prove the hypothesis that reservoir hosts can be colonized with more than one species of Leptospira concurrently. Demonstration of co-infection of Leptospira in a reservoir host has implications for understanding the epidemiology and transmission of spirochete disease in human and animal populations, and will be of interest to producers, veterinarians, and public health professionals.

3. Identification of new Leptospira species. Leptospirosis is a zoonotic disease with multiple serovars that can cause chronic disease in human and animal reservoir hosts. Recovery of Leptospira from environmental samples is difficult but may allow identification of new species. ARS scientists in Ames, Iowa, isolated two novel spirochetes (designated strains LGVF01 and LGVF02) from soil samples in San Juan, Puerto Rico using microbiological techniques and confirmed using various tests. Genome sequence analysis indicated that both strains are members of a novel species within the pathogenic (P1) clade of the Leptospira genus. Although highly homologous (99.2% nucleotide identity), the two strains had much greater nucleotide differences when compared to previously described leptospiral species (<93.2% nucleotide identity). Serotyping suggests strain LGVF02 represents a new novel Leptospira serovar. Collectively, these two novel strains represent a new species of pathogenic Leptospira for which the name Leptospira sanjuanensis sp. nov is proposed. This work will be of interest to producers, veterinarians, and public health professionals.

Review Publications
Sykes, J.E., Gamage, C.D., Haake, D.A., Nally, J.E. 2022. Understanding leptospirosis: application of state-of-the-art molecular typing tools with a One Health lens. American Journal of Veterinary Research. 83(10).
Fernandes, L.G., Stone, N.E., Roe, C.C., Goris, M.G., Van Der Linden, H., Sahl, J.W., Wagner, D.E., Nally, J.E. 2022. Leptospira sanjuanensis sp. nov., a pathogenic species of the genus Leptospira isolated from soil in Puerto Rico. International Journal of Systematic and Evolutionary Microbiology. 72(10). eArticle 5560.
Sykes, J.E., Haake, D.A., Gamage, C.D., Mills, W., Nally, J.E. 2022. A Global One Health Perspective on Leptospirosis in Humans and Animals. Journal of the American Veterinary Medical Association. 260(13). p. 1589-1596.