Progress 07/01/24 to 06/30/25

Outputs
Target Audience:The target audiences for our study are scientists interested in sheep infectious disease and immunology, veterinarians, sheep farmers and individuals involved in wildlife conservation. In addition, our team is organizing a monthly seminar series at MSU, termed the Sheep Health Research Updates, that is focused on domestic and wild sheep health (https://nios.montana.edu/mbi/sheephealthresearchuntitled.html). This seminar series is attended by MSU faculty and research trainees from the Departments of Microbiology and Cell Biology, the Animal and Range Sciences Department, the Chemistry and Biochemistry Department, MSU Extension and FWP and serves as a local forum to exchange new data and ideas with researchers interested in sheep health and production. Through a virtual option, our seminars also have gained an increasing audience of university and government researchers from other institutions in the Northwest. Presenters include MSU graduate students and faculty as well as collaborators from other institutions. We have discussed our ongoing work on M. ovi vaccine development at these meetings. Initial data on the development of the M. ovipneumoniae vaccine was presented in a public seminar at Montana State University this April by a graduate student in the Bimczok lab, Dr. B. Tegner Jacobson, as part of his dissertation defense. In addition, PI Dr. Bimczok gave an interview discussing our ongoing research with a representative from the Wild SheepFoundation in November 2024, which will be distributed to an audience of wild sheep conversationists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project has provided research training opportunities for two Ph.D. students in the Bimczok laboratory, Mr. B.T. Jacobson and Ms. Sobha Sonar, and for a postdoctoral fellow, Dr. Hannah Marahrens, a small ruminant veterinarian. Ms. Sonar gained experience in the isolation of M. ovipneumoniae from lung samples in the Bimczok lab and isolated multiple vaccine candidate strains. She also received training in ELISA development from co-investigator Dr. Rynda-Apple and was able to validate an in-house ELISA protocol to measure antibody responses to the vaccine and to natural M. ovipneumoniae infection. Mr. Jacobson has been leading the studies on Mycoplasma biofilms in the Bimczok laboratory. He analyzed the genotype of the candidate vaccine strains, performed strain selection for the vaccine, and confirmed biofilm growth for the selected genotyping. He also received internship training in an industry lab, working with our collaborators at SAN group to manufacture the biofilm-based vaccine at their production facility in Germany. Postdoctoral fellow Dr. Marahrens joined our team in February. She has received training in M. ovipneumoniae culture, has established culture conditions for monocyte-derived dendritic cells, and has gained experience with managing large animal experiments. Dr. Marahrens also has gained experience in performing respiratory tract endoscopies in sheep, so that we can administer challenge infections and collect bronchoalveolar lavage samples. The project also provided research training opportunities for three undergraduate students in the Bimczok lab. Two Montana State University students who contributed to the project obtained research credit and gained co-authorships on publications. One of the students enrolled in veterinary school after graduation and is continuing to work on the project through a Summer Veterinary Research Fellowship sponsored by Washington State University. A third undergraduate student is working on the project over the summer with NSF funding through the Extreme Biofilms Research Experience for Undergraduates. Approximately fifteen additional undergraduate students, mostly pre-vet students from Montana State University, were able to gain experience helping with lambing and newborn care forour specific pathogen free sheep flock, but were not supported by this award. How have the results been disseminated to communities of interest?- Public presentation by graduate student at the Montana State University Microbiology and Cell Biology Seminar Series (dissertation defense) - Interview of Dr. Bimczok by representative of the Wild Sheep Foundation (not yet published) - What do you plan to do during the next reporting period to accomplish the goals?During the next reporting period, we will continue our ongoing immunization experiment in the SPF lambs. If antibodies to M. ovipneumoniae are successfully induced by the vaccines, we will move on to the challenge phase of our study. All immunized lambs and placebo-treated control animals will be inoculated intratracheally with the bacterial strains that were included in the two experimental vaccines. During the course of this experiment, we will continue to collect health scoring data including adverse vaccine reactions, will monitor weight gains, and will collect blood, serum, nasal swabs. At the experimental endpoint, lambs will be euthanized and necropsied, and tissue samples and bronchoalveolar lavages will be collected. We will use PCR to assess M. ovipneumoniae infection levels post-challenge, so that we can measure vaccine-mediated protection from infection. We also will measure adaptive immune responses to M. ovipneumoniae by ELISA and T cell stimulation assay. Upon completion of the vaccine and challenge experiments, we will analyze all data from this project and will prepare at least one manuscript describing our study. We also will present our findings at scientific meetings and through local seminars. If the study with the SPF lambs shows that either the regular or the biofilm-based vaccine confer significant protection from M. ovipneumoniae infection, we will test the vaccine in a field setting in a flock of domestic sheep at MSU's Red Bluff research ranch that has a high level of natural infection withM. ovipneumoniae.

Impacts
What was accomplished under these goals? Over the past year, we have designed two multi-strain vaccines that are based on planktonic or biofilm-grown Mycoplasma ovipneumoniaeand that we are currently testing in an immunization and challenge experiment in specific-pathogen-free domestic lambs. Vaccine development: For the vaccine, we isolated field strains of M. ovipneumoniaefrom lungs of lambs that were naturally or experimentally infected withM. ovipneumoniae.Strains were identified asM. ovipneumoniae by microbiology techniques and PCR, and the ability of the strains for form biofilms was confirmed. Illumina and Nanopore sequencing then were used to obtain high resolution genomes of the strains. Three strains that were genetically distinct based on their virulence gene profiles were selected for the vaccines. To produce safe and effective vaccines for administration to lambs, we collaborated with SAN Vet, an autogenic vaccine manufacturer that has experience in the production ofM. ovipneumoniaevaccines for the European market. Two different vaccine formulations were prepared, each containing 1 x 10^8 formalin-killed bacteria mixed with 10% Montanide gel 02 adjuvant: a standard vaccine that was based on planktonicM. ovipneumoniae, and a biofilm vaccine composed of bacteria grown as an adherent biofilm on glass plates, which includes additional antigens found in the biofilm extracellular polymeric matrix. Identity, sterility, and successful inactivation of the bacteria was confirmed. Analysis of immunogenicity: In an ongoing study, we are currently testing the safety and immunogenicity of the two M. ovipneumoniae vaccine formulations. Twenty-one 2-3-month-old male and female specific-pathogen-free lambs were randomly assigned to three groups (n=7 each) and received a subcutaneous injection of (1) saline placebo; (2) the M. ovipneumoniaevaccine generated from bacteria grown under standard culture conditions; or (3) theM. ovipneumoniaevaccine that was generated from bacteria grown as a biofilm.No serious adverse effects beyond occasional fevers and local responses were detected following vaccine administration. A booster injection is scheduled for three weeks after the first dose. Our team is currently collecting health and weight data, data on local and systemic vaccine reactions, nasal swabs, and blood and serum samples to measure vaccine safety and efficacy.To analyze adaptive immune responses to the two vaccines, we developed and validated an enzyme-linked immunosorbent assay (ELISA) to quantify serum antibody responses to M. ovipneumoniae. We also have optimized culture conditions for ovine blood monocyte-derived dendritic cells, so that we can perform T cell stimulation assays with M. ovipneumoniae antigens to measure antigen-specific T cell responses in vaccinated and infected sheep.

Publications