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Novel, Protectively Immunogenic, Surface Exposed, and Secreted Proteins of Streptococcus equi
J.F. Timoney, S. Artiushin
Department of Veterinary Sciences
The purpose of the research is to identify proteins of Streptococcus equi, the cause of equine strangles that might be included in new, more effective vaccines. These proteins will be identified by probing a genomic DNA library of S. equi and its recently determined genomic sequence. The gene for each interesting protein will be inactivated to determine whether and how it contributes to virulence. Finally, ponies will be vaccinated with pools of newly identified interesting proteins and challenged with virulent S. equi to evaluate immune status.
2009 Project Description
Groups of ponies were immunized with 2 different sets of recombinant surface exposed and secreted proteins of Streptococcus equi. Group 1, consisting of 8 four-year-old ponies, was immunized subcutaneously with 200 ug of each of the following proteins expressed in E. coli as his-tag fusions: SeM, Se18.9, IdeE, Se110.0, Se44.2 (IdeE2), Se42.0. Se75.3. These proteins were suspended in 2 ml phosphate buffered saline (PBS) containing 10 mg/ml Quil A and 1:10,000 merthiolate. Group 1 ponies were immunized twice at an interval of 21 days and bled on Days 0, 16, and 36. Group 2 pones were immunized intranasally with recombinant Se18.9 fused to the central region of SeM (3 ponies) or to Se46.8 (4 ponies). Fusion proteins were suspended in PBS at a concentration of 60 ug/ml and 4 mls were administered intranasally via a Devilbiss nasal atomizer to each pony on Days 0 and 18. Nasal washes and bloods were collected on days 2, 18 and 40. All vaccinated ponies were commingled with a seeder pony inoculated intranasally with 8x108 CFU of S. equi CF32 7 days earlier. Ponies were then monitored daily for pyrexia, cough, lymphadenopathy and nasal discharge. Serum samples from Group 1 ponies were assayed separately for IgG specific for each protein. Sera of Group 2 animals were assayed for IgG specific for Se18.9, SeM and Se46.9. Their nasal washes were assayed for IgA specific for these proteins.
Both the subcutaneous and intranasal vaccines were well tolerated. Quil A caused a temporary subcutaneous firm, non-painful swelling that persisted for 3 days. Ponies of Group 1 made strong serum IgG responses to all 7 recombinant proteins indicating that Quil A was an effective adjuvant in the horse. Intranasal vaccination with fusions of Se18.9 to SeM and Se44.2 also stimulated strong serum IgG responses to these proteins. Mucosal IgA responses varied between horses. Data as yet incomplete from commingling challenge of both groups indicate that neither vaccine was effective in preventing infection in the majority of ponies. However, disease was mild in most vaccinates that become infected.
Timoney, J.F., De Negri, R., Sheoran, A., and Forster, N. (2009) Affects of N-terminal variation in the SeM protein of Streptococcus equi on antibody and fibrinogen binding. Vaccine doi:10.1016/j.vaccine.2009.11.064
Causey, R.C., Artiushin, S.C., Crowley, I.F., Weber, J.A., Honiola, A.D., Kelley, A., Stephenson, L.A., Opitz, H.M., Guilmain, S. and Timoney, J.F. (2009) Immunization of the equine uterus against Streptococcus equi subspecies zooepidemicus using an intranasal attenuated Salmonella vector. Vet. J. doi:10.1016/j.tvjl.2009.05.001