Progress 09/01/02 to 08/31/05
Outputs
Projective objectives that were met: 1. To characterize uterine, vaginal and nasal antibody responses against S typhimurium and SzP protein of S zooepidemicus MB9, following intranasal vaccination of S typhimurium expressing MB9 SzP protein. Significant findings: Vaccinated horses remained normal on physical examination, with no evidence of pyrexia, inappetance or diarrhea. Significant (P < 0.05) anti-LPS responses were seen post vaccination in serum, nasal, and uterine washings of vaccinated but not control horses. Significant anti-SzP-MB9 responses were seen in nasal washings of vaccinated animals, but not in serum or uterine washings. Pre-existing levels of antibodies to SzP-MB9 were high in serum of all vaccinated horses. Increased post-vaccination uterine response to SzP-MB9 appeared in 3 horses with low pre-vaccination anti-SzP-MB9 IgA in uterine washings. In the other 2 horses, which had high pre-vaccination anti-SzP-MB9 IgA, post-vaccination uterine responses
were slightly negative. By absorbing out cross-reacting antibodies to other streptococcal strains in sera, we have now been able to detect significant immune respsonses to SzP-MB9 that were previously undetected. This improved ability to detect SzP-MB9 antibodies has allowed us to demonstrate improved immunogenicity of the vaccine, meeting objective 1. Project objectives not met and explanation 2. To determine the effect of vaccination on uterine clearance of an intrauterine challenge of S zooepidemicus MB9. This is the last phase of the project to be completed. The planned trial for summer 2005 was postponed while we developed improved antibody detection for SzP-MB9. With the improved antibody detection we can now use a larger group of horses and are not required to pre-select horses with low SzP-MB9 titers. This will improve the chances of successfully demonstrating protective effect of the vaccine.
Impacts
This poject has provided $30,000 for the Witter center over two years, much of it in student labor, purchase of feed for research horses, and installation of ligts. Several undergraduate students used this project for their senior thesis research. This project involved close collaboration with the Gluck Center for Equine Research at the University of Kentucky. Safety and potential efficacy of this vaccine has been demonstrated. Students learned how to restrain, vaccinate, draw blood, and collect nasal and uterine washings. Findings to date were presented at the Society of Theriogenology annual conference in Lexington Kentucky in August 2004, a meeting of veterinary researchers and practitioners. A list of graduate students who completed degrees under the terminating project: Ian Crowley (In progress)
Publications
- Causey R, Artiushin SC, Weber JA, *Crowley IF, Homola AD, Opitz HM, Stephenson LA, Guilmain S, Timoney JF (2005) Immunization of the equine uterus using an intranasal attenuated Salmonella vector. (The Veterinary Journal - under review).
- *Causey R, Artiushin S, Weber J, Opitz 0, Crowley P, Homola A, Stephenson L, Guilmain S, Timoney J. The Uterine Mucosal Immune Response in Mares Following Intranasal Vaccination. (Society for Theriogenology Annual Conference August 2004).
- Causey R, Artiushin S, Weber J, Opitz M, Crowley I, Homola A, Stephenson L, Guilmain S, Timoney J. Intranasal Vaccination of Mares to Protect Against Streptococcal Uterine Infections. (Eighth Annual Conference of Vaccine Research, National Foundation of Infectious Diseases, May 4-6 2005).
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Progress 10/01/03 to 09/30/04
Outputs
Introduction: Intranasal Salmonella vectors show promise in stimulating immunity to various pathogens at mucosal sites throughout the body. An attenuated strain of Salmonella enteritica serotype typhimurium (S typhimurium MGN 707) expressing the protective protein of Streptococcus equi subspecies zooepidemicus Moore and Bryans serovar 9 (SzP-MB9) was used to explore the feasibility of using intranasal Salmonella vectors to boost equine uterine immunity. Materials and Methods: Ten reproductively sound Standardbred mares were randomly assigned into vaccinated (n=5) and non-vaccinated (n=5) groups. Mares in the vaccinated group received 2 doses, 2 weeks apart, of 2.5 X 109 CFU of live vaccine intranasally, delivered in 2 ml of normal saline via a hand-pumped nebulizer. Serum, nasal and uterine washings were collected from both groups before and approximately 6 and 12 weeks after vaccination. Salmonella cultures were performed on samples from pastures and drinking water
of both groups before and after vaccination. During the week after each vaccination, mares in the vaccinated group received daily physical examinations and fresh fecal samples were collected rectally for Salmonella culture. Immune responses were assessed by Enzyme-Linked-Immunosorbent-Assay (ELISA). Plates were coated with lipopolysaccharide (LPS) of S typhimurium and purified recombinant SzP-MB9 separately. Serum responses were detected using protein-G conjugated to horseradish peroxidase (HRP); nasal and uterine responses were detected by anti-equine-IgA HRP conjugate. Ortho-phenylene-diamine (OPD) was used as substrate for color development. Optical density was measured by spectrophotometer, and results analyzed by one-way, analysis of variance. Results: Salmonella was not recovered from any of the samples submitted for culture. Vaccinated horses remained normal on physical examination, with no evidence of pyrexia, inappetance or diarrhea. Significant (P < 0.05) anti-LPS responses
were seen post vaccination in serum, nasal, and uterine washings of vaccinated but not control horses. Significant anti-SzP-MB9 responses were seen in nasal washings of vaccinated animals, but not in serum or uterine washings. Pre-existing levels of antibodies to SzP-MB9 were high in serum of all vaccinated horses. Increased post-vaccination uterine response to SzP-MB9 appeared in 3 horses with low pre-vaccination anti-SzP-MB9 IgA in uterine washings. In the other 2 horses, which had high pre-vaccination anti-SzP-MB9 IgA, post-vaccination uterine responses were slightly negative. Discussion and Conclusion: These data confirm that intranasal vaccination with attenuated S typhimurium may safely stimulate nasal and uterine immune responses in horses. However, pre-existing antibodies in uterine fluid may have affected responses to SzP-MB9.
Impacts
This project has supported 3 undergraduate senior research projects for the academic year 2003/2004 and continues to support a Master's Student. Demonstration of a uterine IgA response to intranasal vaccination provides important basic information about the common equine mucosal immune system.
Publications
- R Causey, S Artiushin, J Weber, M Opitz, I Crowley, A Homola, L Stephenson, S Guilmain, J Timoney. The Uterine Mucosal Immune Response in Mares Following Intranasal Vaccination. Society for Theriogenology Annual Conference Proceedings, August 2004.
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Progress 10/01/02 to 09/30/03
Outputs
Consistent with the first objective 7 mares were vaccinated twice in August 2003 with Salmonella typhimurium expressing the MB9 SzP protein of Streptococcus zooepidemicus. Seven mares were also vaccinated with Salmonella typhimurium not expressing the MB9 SzP protein. Prior to vaccination, serum, nasal and uterine lavage samples were taken on two estrous periods. Following vaccination samples were taken in September, in estrus, and in November, without determining stage of cycle. Samples are scheduled for January, March and May. Following re-vaccination of the horses in the same groups in Summer 2004, all horses will be challenged intrauterinely with Streptococcus zooepidemicus.
Impacts
This project has supported 3 undergraduate senior research projects for the academic year 2003/2004. Demonstration of a uterine IgA response to intranasal vaccination will provide important basic information about the common equine mucosal immune system.
Publications
- No publications reported this period
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