Sequence variation of the SeM gene of Streptococcus equi allows discrimination of the source of strangles outbreaks

Abstract

Improved understanding of the epidemiology of Streptococcus equi transmission requires sensitive and portable subtyping methods that can rationally discriminate between strains. S. equi is highly homogeneous and cannot be distinguished by multilocus enzyme electrophoretic or multilocus sequence-typing methods that utilize housekeeping genes. However, on sequence analysis of the N-terminal region of the SeM genes of 60 S. equi isolates from 27 strangles outbreaks, we identified 21 DNA codon changes. These resulted in the nonsynonymous substitution of 18 amino acids and allowed the assignment of S. equi strains to 15 distinct subtypes. Our data suggest the presence of multiple epitopes across this region that are subjected to selective immune pressure (nonsynonymous-synonymous substitution rate [d(N)/d(S)] ratio = 3.054), particularly during the establishment of long-term S. equi infection. We further report the application of SeM gene subtyping as a method to investigate potential cases of disease related to administration of a live attenuated S. equi vaccine. SeM gene subtyping successfully differentiated between the vaccine strain and field strains of S. equi responsible for concurrent disease. These results were confirmed by the development and application of a PCR diagnostic test, which identifies the aroA partial gene deletion present in the Equilis StrepE vaccine strain. Although the vaccine strain was found to be responsible for injection site lesions, all seven outbreaks of strangles investigated in recently vaccinated horses were found to be due to concurrent infection with wild-type S. equi and not due to reversion of the vaccine strain.

FIG. 1.

Schematic representation of the SeM protein of S. equi. Amino acids 37 to 352 are required for fibrinogen binding (16). Amino acids 1 to 37 contain the M-protein signal sequence (ss) (31). Amino acids 37 to 184 (deleted) were found to be absent from 24% of S. equi strains isolated from outwardly healthy horses (3). Amino acids 226 to 406 (central repeat region) contain the A and B repeat regions (31). Amino acids 482 to 534 contain the wall-spanning region and “LPSTG” cell wall anchor (cwa) (31). Amino acids 71 to 421 are required for IgG binding (17).

FIG. 2.

Maximum likelihood tree showing the relationships of the 15 different SeM gene sequence types of S. equi generated using PAUP* version 4.0 (27). Bootstrap values are shown for bipartitions supported by >50% of replicate trees (1,000 replicates were performed).

FIG. 3.

Alignment of S. equi 4047 SeM and S. zooepidemicus H70 SzM amino acid sequences. The dashes indicate a gap inserted to optimize sequence alignment. The asterisks indicate identical amino acid residues. The colons indicate closely related nonidentical amino acid residues, and the periods indicate similar amino acid residues. The signal sequence of S. equi is shown in boldface, the repeat domain is in italics, and the LPSTG cell wall anchor motif is underlined.