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in Journal of the American Veterinary Medical Association

Abstract

Objective—To determine whether streptococcal pneumonia is caused by strains of Streptococcus zooepidemicus similar to those obtained from the tonsils of healthy horses.

Sample Population—5 tonsils from healthy horses, 8 tracheal washes and 6 lung specimens from foals with pneumonia, and 5 nasopharyngeal swab specimens from donkeys with acute bronchopneumonia.

Procedure—Variable M-like protectively immunogenic SzP proteins of 5 isolates of S zooepidemicus from each tonsil and clinical specimen were compared, using immunoblots. The SzP gene of 13 isolates representative of various SzP immunoblot phenotypes from 1 healthy horse and 9 horses and donkeys with pneumonia were sequenced and compared. Cell-associated hyaluronic acid concentration and resistance to phagocytosis of some isolates were measured.

Results—Tonsils of each healthy horse were colonized by several SzP phenotypes similar to those of foals or donkeys with pneumonia. In contrast, multiple isolates from animals with pneumonia had the same SzP phenotype, indicating infection by a single strain or clone. Analysis of the SzP sequence confirmed that differences in immunoblot phenotype were associated with sequence differences and that several SzP genotypes were in healthy horses and animals with pneumonia. Isolates with high concentrations of cell-associated hyaluronic acid were more resistant to phagocytosis.

Conclusions and Clinical Relevance—An SzP-specific immunoblot is a useful, sensitive measure of diversity among strains of S zooepidemicus. Single strains with SzP phenotypes similar to those found in tonsils of healthy horses cause pneumonia. Because of the diversity of SzP phenotype and genotype among isolates from animals with pneumonia, SzP phenotype is not an important determinant of invasiveness or epizootic capabilities. (Am J Vet Res 2000;61:162–166)

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in American Journal of Veterinary Research

Abstract

Objective—To determine whether repetitive sequence-based polymerase chain reaction (rep-PCR) could be used to differentiate Streptococcus equi isolates, to examine S equi isolates from throughout the world, and to determine whether a horse had > 1 subtype of S equi during an outbreak of disease.

Sample Population—An initial group of 32 S equi isolates, 63 S equi isolates from various geographic areas, and 17 S equi isolates obtained during outbreaks of disease.

Procedure—An aliquot of S equi genomic DNA was amplified, using enterobacterial repetitive intergenic consensus primers. Gel electrophoresis was performed on 1.5% agarose gels, and a computed-assisted program was used to compare rep-PCR results.

Results—Use of these primers to analyze 100 ng of S equi genomic DNA resulted in patterns of 6 to 14 bands. The 32 initial isolates were separated into 7 rep- PCR subtypes. There were 30 rep-PCR subtypes found among 29 S equi isolates obtained from Minnesota, Michigan, Canada, and Australia and 34 S equi isolates obtained from Kentucky and other sources. Furthermore, the same clone was identified in several horses during an outbreak of disease. Infected horses on the same farm all had a single clone of S equi.

Conclusion and Clinical Relevance—Analysis of these results suggests that rep-PCR is useful for delineating S equi into rep-PCR subtypes. Results revealed that isolates with the same geographic source or similar date of collection did not always have the same rep-PCR subtype. A single clone of S equi usually predominated during an outbreak of disease. (Am J Vet Res 2000;61:699–705)

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in American Journal of Veterinary Research

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)

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in American Journal of Veterinary Research

Abstract

Objective—To determine concentrations of IgA and IgG subclasses in serum, colostrum, milk, and nasal wash samples of adult horses and foals.

Animals—Seven 2-year-old Welsh ponies, 27 adult mixed-breed horses, and 5 Quarter Horse mares and their foals.

Procedure—Serum was obtained from ponies and adult horses. Colostrum and milk were obtained from mares and serum and nasal wash samples from their foals immediately after parturition and on days 1, 7, 14, 28, 42, and 63. Nasal wash samples were also obtained from 23 adult horses. Concentrations of immunoglobulins were determined by use of inhibition ELISA. To determine transfer of maternal isotypes to foals, concentrations in colostrum and milk were compared with those in foal serum. Serum half-lives of isotypes in foals were also determined.

Results—IgGb was the most abundant isotype in serum and colostrum from adult horses, whereas IgA was the predominant isotype in milk. The major isotype in nasal secretions of adult horses and foals ≥ 28 days old was IgA, but IgGa and IgGb were the major isotypes in nasal secretions of foals ≤ 14 days old. Serum half lives of IgGa, IgGb, IgG(T), and IgA in foals were 17.6, 32, 21, and 3.4 days, respectively.

Conclusions and Clinical Relevance—The early immunoglobulin repertoire of neonatal foals comprised IgGa, IgG(T), and IgA; endogenous synthesis of IgGb could not be detected until 63 days after birth. The restricted repertoire of immunoglobulins in foals may influence humoral immune responses to vaccination. (Am J Vet Res 2000;61:1099–1105)

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in American Journal of Veterinary Research

Abstract

Objective—To develop a method for typing Streptococcus equi on the basis of the DNA sequence of the genes that produce an M-like protein and to compare isolates among the United States, Japan, and other countries.

Sample PopulationS equi strains CF32, Hidaka/95/2, and NCTC9682 as well as 82 other isolates from the United States, Japan, and other countries obtained during 1975 to 2001.

Procedure—DNA sequences of the structural genes ( SeM and SzPSe) that produce M-like proteins were determined for 3 representative strains to find a variable region. Variability in this region of SeM was then determined for the other isolates. Amino acid sequences were deduced and analyzed phylogenetically by use of the neighbor-joining method.

Results—Sequence diversity was detected in the N-terminal region of SeM but not in SzPSe of the 3 representative strains. Base substitutions in the variable region of SeM varied in a nonsynonymous manner, resulting in variation in the amino acid sequence. Eighty-five isolates were categorized as 32 types of SeM on the basis of differences in the deduced amino acid sequences.

Conclusions and Clinical Relevance—This study documented a region in the N-terminal portion of SeM that varies in a nonsynonymous manner. This information should be useful in molecular epidemiologic studies of S equi. (Am J Vet Res 2005; 66:2167–2171)

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in American Journal of Veterinary Research