Clones of Streptococcus zooepidemicus from outbreaks of hemorrhagic canine pneumonia and associated immune responses

Abstract

Acute hemorrhagic pneumonia caused by Streptococcus zooepidemicus has emerged as a major disease of shelter dogs and greyhounds. S. zooepidemicus strains differing in multilocus sequence typing (MLST), protective protein (SzP), and M-like protein (SzM) sequences were identified from 9 outbreaks in Texas, Kansas, Florida, Nevada, New Mexico, and Pennsylvania. Clonality based on 2 or more isolates was evident for 7 of these outbreaks. The Pennsylvania and Nevada outbreaks also involved cats. Goat antisera against acutely infected lung tissue as well as convalescent-phase sera reacted with a mucinase (Sz115), hyaluronidase (HylC), InlA domain-containing cell surface-anchored protein (INLA), membrane-anchored protein (MAP), SzP, SzM, and extracellular oligopeptide-binding protein (OppA). The amino acid sequences of SzP and SzM of the isolates varied greatly. The szp and szm alleles of the closely related Kansas clone (sequence type 129 [ST-129]) and United Kingdom isolate BHS5 (ST-123) were different, indicating that MLST was unreliable as a predictor of virulence phenotype. Combinations of conserved HylC and serine protease (ScpC) and variable SzM and SzP proteins of S. zooepidemicus strain NC78 were protectively immunogenic for mice challenged with a virulent canine strain. Thus, although canine pneumonia outbreaks are caused by different strains of S. zooepidemicus, protective immune responses were elicited in mice by combinations of conserved or variable S. zooepidemicus proteins from a single strain.

FIG 1

Immunoreactive secreted proteins from clones of S. zooepidemicus from outbreaks of pneumonia in dogs and greyhounds in different regions of the United States. Proteins secreted in overnight cultures in THB were precipitated with 50% ammonium sulfate, dissolved in PBS, and transferred to nitrocellulose membranes following SDS-PAGE (12%). The blot was probed with convalescent-phase serum (1:200) from Angel (Pennsylvania), infected with ST-315. Lane 1, S. zooepidemicus strain 007 (Kansas); lane 2, S. zooepidemicus strain 985 (Kansas); lane 3, S. zooepidemicus strain 43 (Florida); lane 4, S. zooepidemicus strain 2201 (Texas); lane 5, S. zooepidemicus strain 2295 (Texas); lane 6, S. zooepidemicus strain 800 (New Mexico); lane 7, S. zooepidemicus strain 1150K (Nevada); lane 8, S. zooepidemicus strain 653 (Pennsylvania). Molecular masses (kDa) are shown on the left. The positions of metalloproteinase/mucinase (115 kDa) and enolase (47 kDa) are shown on the right. The positions of metalloproteinase/mucinase (115 kDa) and enolase (47 kDa) are shown on the right.

FIG 2

(Upper) Levels of antibodies (ELISA OD values) reactive with each of 16 recombinant proteins of S. zooepidemicus strain NC78 in 1:200 dilutions of convalescent-phase sera from Angel (▲) and Lockjaw (△), which survived infection in the Pennsylvania shelter outbreak. Each symbol represents the mean OD value of 3 replications of the ELISA for that protein. (Lower) Antibody levels for 23 dogs that remained healthy in the shelter for 11 months during the pneumonia epizootic. Mut., mutanolysin-extracted surface proteins of S. zooepidemicus; CSP, culture supernatant secreted proteins of S. zooepidemicus. ●, outlier values. Horizontal lines, median values; error bars, 90th and 10th percentiles.

FIG 3

Tandem repeats in SzM proteins of S. zooepidemicus clones from outbreaks of canine pneumonia. Sig.pep, N-terminal signal peptide.