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Polymerase chain reaction-restriction fragment length polymorphism analysis of the SzP gene of Streptococcus zooepidemicus isolated from the respiratory tract of horses

Toru Anzai DVM, PhD1, John F. Timoney MVB, DSc2, Yasushi Kuwamoto DVM3, Ryuichi Wada DVM, PhD4, Msaaki Oikawa DVM, PhD5, and Tohru Higuchi DVM, PhD6
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  • 1 Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Kokubunji-machi, Shimotsuga-gun, Tochigi 329-0412, Japan.
  • | 2 Gluck Equine Research Center, Department of Veterinary Science, University of Kentucky, Lexington, KY 40546-0099.
  • | 3 Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Kokubunji-machi, Shimotsuga-gun, Tochigi 329-0412, Japan.
  • | 4 Epizootic Research Station, Equine Research Institute, Japan Racing Association, 1400-4 Shiba, Kokubunji-machi, Shimotsuga-gun, Tochigi 329-0412, Japan.
  • | 5 Equine Research Institute, Japan Racing Association, 321- 4, Togami-cho, Utsunomiya-shi, Tochigi 320-0856, Japan.
  • | 6 Mitsuishi Animal Clinic Center, Agriculture Mutual Aid Association of Hidaka District, 200 Higashihohrai, Mitsuishi-cho, Mitsuishi-gun, Hokkaido 059-3105, Japan.

Abstract

Objective—To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds.

Sample Population—10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia.

Procedure—Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, genotypes were obtained with primer sets from the SzP gene, followed by restriction endonuclease digestion of the amplicons.

Results—Unique genotypic patterns were obtained with a primer set designed from both ends of the structural gene and the restriction endonuclease Dde I. Forty-five isolates from the lymphoid tissue within the pharyngeal recess (ie, pharyngeal tonsil) of yearlings included 10 SzP genotypes and SzP phenotypes. Isolates from the trachea of each yearling were of a single genotype that was also present among isolates from the pharyngeal tonsil of the same horses. Isolates from tracheal aspirates of foals belonged to 14 genotypes.

Conclusion and Clinical Relevance—Analysis of the SzP gene by use of PCR-RFLP was effective for molecular typing of strains of S zooepidemicus in the study of respiratory tract disease in horses. Results of PCR-RFLP analysis indicate that a single strain of S zooepidemicus can migrate from the pharyngeal tonsil to the trachea at a high rate in horses undergoing long distance transportation. (Am J Vet Res 2002;63:1298–1301)

Abstract

Objective—To develop polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis for molecular typing of strains of Streptococcus zooepidemicus and to use the new typing method to analyze a collection of isolates from the respiratory tract of Thoroughbreds.

Sample Population—10 strains of S zooepidemicus, 65 isolates from the respiratory tract of 9 yearlings following long distance transportation, and 89 isolates from tracheal aspirates of 20 foals with pneumonia.

Procedure—Phenotypic variations in the SzP protein were detected by western immunoblot analysis. Using PCR-RFLP analysis, genotypes were obtained with primer sets from the SzP gene, followed by restriction endonuclease digestion of the amplicons.

Results—Unique genotypic patterns were obtained with a primer set designed from both ends of the structural gene and the restriction endonuclease Dde I. Forty-five isolates from the lymphoid tissue within the pharyngeal recess (ie, pharyngeal tonsil) of yearlings included 10 SzP genotypes and SzP phenotypes. Isolates from the trachea of each yearling were of a single genotype that was also present among isolates from the pharyngeal tonsil of the same horses. Isolates from tracheal aspirates of foals belonged to 14 genotypes.

Conclusion and Clinical Relevance—Analysis of the SzP gene by use of PCR-RFLP was effective for molecular typing of strains of S zooepidemicus in the study of respiratory tract disease in horses. Results of PCR-RFLP analysis indicate that a single strain of S zooepidemicus can migrate from the pharyngeal tonsil to the trachea at a high rate in horses undergoing long distance transportation. (Am J Vet Res 2002;63:1298–1301)