Research Accomplishment Reports 2010

Novel, Protectively Immunogenic, Surface Exposed, and Secreted Proteins of Streptococcus equi

J.F. Timoney, S. Artiushin
Department of Veterinary Sciences

 

Non-Technical Summary

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.

2010 Project Description

A Streptococcus equi expression DNA library was screened with convalescent serum and mucosal IgA. Twenty surface exposed or secreted proteins were identified, expressed in E. coli, and purified. IgG and IgA responses of horses to the proteins following infection were measured and the more reactive proteins selected for functional and immunization studies. Six proteins (SeM, Se44.7, Se75.3, Se42.0, Se110.0 and Se18.9) expressed by S. equi but not by S. zooepidemicus were incorporated in a subcutaneous vaccine administered to 8 Welsh weanling ponies.

Another group of 8 ponies was immunized with a vaccine composed of 5 proteins (SzPSE, Se73.9, Se51.9, Se44.2 and Se46.8) involved in fimbrial synthesis and adhesion. All ponies and 5 controls were later exposed to S. equi by commingling challenge and observed daily for signs of infection. A second vaccination trial was performed with a modification of Vaccine 3 that contained an additional protein (IdeE) and QuilA instead of Alhydrogel as adjuvant. The bactericidal effect of the secreted IdE on equine neutrophils for S. equi was characterized.

The antiphagocytic activity of the complement control (factor H) protein Se18.9 was also studied. A fusion of Se18.9 to the central region of SeM was used to immunize a group of ponies by the intranasal route. Potency and efficacy of the vaccine was later tested by commingling challenge. The early stages of S. equi infection were studied in yearling ponies/horses using a combination of labeled organisms, histochemistry and culture. Uptake and survival of S. equi by equine monocytes was investigated by a combination of confocal microscopy, FACS analysis and culture.

The effect of allelic variation in the N terminal region of SeM was studied using a panel of monoclonal antibodies, fibrinogen binding, opsonic antibody, and mapping of B and T cell epitopes. A helicase-dependent nucleic acid amplification technology was developed for rapid detection of S. equi DNA in clinical specimens.

2010 Impact

Vaccines composed of combinations of recombinant and secreted proteins of S. equi although immunogenic and safe were generally of low efficacy under conditions of commingling exposure. This conclusion including the trial of Vaccine 3 that incorporated S. equi proteins IdeE and IdeE2 (Se44.2) previously shown in a European study to provide protection against cultured S. equi. These data therefore indicate that vaccine efficacy should be determined by commingling challenge. Proteins of S. equi involved in fimbrial structure/adhesion did not show promise as protective antigens. A fusion peptide of Se18.9 to SeM was effective as a mucosal and systemic immunogen following intranasal administration to weanling ponies. Five of 8 immunized ponies were resistant to commingling challenge. Se18.9 was shown to bind strongly to tonsillar epithelium and elicit strong specific IgA responses. IdE, a partial homologue of CD11b, was shown to reduce the bactericidal activity of equine neutrophils for S. equi. S. equi was shown to enter susceptible ponies via crypts of the nasopharyngeal and lingual tonsils. Entry was rapid (few hours), did not involve a prior colonization event, and very few organisms invaded. Organisms were visible in epithelial cells and in macrophage-like cells in the subepithelial lymphoid follicles. Draining lymph nodes sometimes contained viable S. equi within 3 hours of inoculation of the horse. By 3 days, large numbers of extracellular S. equi were present in tonsillar tissues. S. equi could not be detected in tonsils of horses inoculated a few weeks following recovery from strangles, an indication that acquired immunity blocks entry of S. equi. Participation of tonsillar macrophages in acquired tonsillar immune clearance was cofirmed by results of experiments that showed equine monocytes engest and kill opsonized S. equi with a high degree of efficacy. An effective strangles vaccine must be capable of achieving this effect. Allelic variation in the N terminus of SeM was shown to involve a conformational epitope reactive with mucosal IgA. The significance of this with respect to protective immunity is probably minimal. The helicase dependent rapid assay for S. equi in clinical specimens was sensitive (10 CFU S. equi). Primers from the Se18.9 gene were well suited for use in the assay.

2010 Publications

Timoney, J.F. 2010. Streptococcus. In: Pathogenesis of Bacterial Infections in Animals, 4th Edition, Eds: C.L. Gyler, J.F. Prescott, J.G. Songer and C.O. Thoen. Blackwell Publishing, pp 51-73.

Causey, R.C., Artiushin, S.C., Crowley, I.F., Weber, J.A., Homola, A.D., Kelley, A., Stephenson, C.A., Opitz, H.J., Guilmain, S. and Timoney, J.F. 2010. Immunization of the equine uterus against Streptococcus equi subsp. zooepidemicus using an intranasal attenuated Salmonella vector. Veterinary Journal 184: 156-161.