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The association of Streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease.

Canine infectious respiratory disease (CIRD) is a multi-factorial infection that affects many kennelled dogs despite the wide use of vaccination. Current vaccines aim to protect against viral agents and a single bacterial agent, Bordetella bronchiseptica. We sought to examine the role of streptococcal species in CIRD. The isolation and identification of streptococci in the lower respiratory tract of clinically healthy dogs and those with CIRD were used to correlate the presence of specific streptococcal species with respiratory disease. In this study we report that the presence of S. equi subsp. zooepidemicus is associated with increasing severity of disease in a population of kennelled dogs with endemic CIRD.

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Isolation and further characterization of phase variants of Streptococcus equi subsp. zooepidemicus.

In the present study the soft agar technique was used to isolate phase variants of S. equi subsp. zooepidemicus-cultures isolated from infections of horses. The phase variants were characterized by a compact or diffuse colony morphology in this media. The variants could be cultivated separately and further characterized genotypically by RAPD analysis and by macrorestriction analysis of their chromosomal DNA by pulsed-field gel electrophoresis, indicating the identity of both strains of each pair. The diffuse colony variants grew uniformly turbid after cultivation in fluid media, did not haemagglutinate rabbit erythrocytes, and displayed a reduced surface hydrophobicity in hexadecane and phenyl-sepharose adherence tests. The compact colony variants generally grew as sediment with clear supernatant in fluid media, haemagglutinated rabbit erythrocytes and showed an enhanced surface hydrophobicity in both hydrophobicity tests. The presented soft agar technique allowed a demonstration of phase variation of S. equi subsp. zooepidemicus and a subsequent isolation of the variants. This might be an important prerequisite to understanding the pathogenic importance of phase variation among isolates of this bacterial species.

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Determination of intraspecies variations of the V2 region of the 16S rRNA gene of Streptococcus equi subsp. zooepidemicus.

The 16S rRNA gene of 39 S. equi subsp. zooepidemicus strains and two S. equi subsp. equi strains was amplified by polymerase chain reaction and subsequently digested with the restriction enzyme Hinc II. A restriction profile with two fragments with sizes of 1250 bp and 200 bp could be observed for both S. equi subsp. equi strains and for 30 of the 39 S. equi subsp. zooepidemicus strains indicating a sequence variation within the V2 region of the 16S rRNA gene of the remaining nine S. equi subsp. zooepidemicus isolates. A segment of the 16S rRNA gene including the hypervariable V2 region of 11 S. equi subsp. zooepidemicus and two S. equi subsp. equi could be amplified by PCR and sequenced. The sequence of the V2 region of eight S. equi subsp. zooepidemicus strains appeared to be identical or almost identical to the sequence of the two S. equi subsp. equi strains. The sequence of the remaining three S equi subsp. zooepidemicus strains differed significantly from the sequence of S. equi subsp. equi. These differences allowed a division of S. equi subsp. zooepidemicus strains into two 16S rRNA types and might possibly have consequences for the taxonomic position of these phenotypically indistinguishable strains of one subspecies. A molecular typing could additionally be performed by amplification of the gene encoding the 16S-23S rRNA spacer region. A single amplicon of the spacer gene of 1100 bp could be observed for one S. equi subsp. zooepidemicus, an amplicon of 950 bp for two S. equi subsp. equi strains and 10 S. equi subsp. zooepidemicus strains, a amplicon of 780 bp for 27 S. equi subsp. zooepidemicus strains and a single amplicon of 600 bp for one S. equi subsp. zooepidemicus strain. The variations of the V2 region of the 16S rRNA gene and the size variations of the 16S-23S rRNA spacer gene were not related to each other. Both variations could be used for molecular typing of this species, possibly useful in epidemiological aspects.

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Molecular characterization of Streptococcus equi subspecies equi isolated from an Ethiopian camel by ribotyping and PCR-ribotyping.

The aim of this study was to characterize a S. equi subspecies equi strain isolated from an Ethiopian camel by different molecular techniques (Ribotyping and PCR-Ribotyping). We compared the results obtained with those generated from two strains of the Pasteur Collection. The ribotyping showed the highest power of differentiation, distinguishing between the strains analyzed, whereas PCR-Ribotyping was able only to differentiate the camel isolate but not the strains from the Pasteur Collection. The application of this technique will be very useful to establish a clonal relationship among equine and camelids strains and help the prevention and cure of the equine and camel pathology.

Animals↗

In vivo pathogenicity and resistance to phagocytosis of Streptococcus equi strains with different levels of capsule expression.

The glossy non-encapsulated strain of Steptococcus equi, NCTC 9682, was compared with the matt strain Hidaka/95/2 which expresses a medium sized capsule and with the mucoid CF32 which expresses a large sized capsule in phagocytosis assays and for virulence in inoculated horses. The three strains, NCTC 9682, Hidaka /95/2 and CF32 produced 2.0, 3.1, and 5.3 mg/g wet cells respectively after 3 h incubation, but similar amounts of M-like proteins, cytotoxin and mitogen. NCTC 9682 showed no resistance to phagocytosis by equine neutrophils regardless of the presence of opsonin while strains Hidaka /95/2 and CF32 showed almost complete resistance to phagocytosis. Furthermore, NCTC 9682 produced no clinical disease although it infected the guttural pouch and caused seroconversion. Typical strangles with guttural pouch invasion was observed in all horses infected with encapsulated strains.

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Immunity to Streptococcus equi.

Using the long chain test, and in some cases the bactericidal test, to measure antibody, the development of the immune response in horses to Str. equi has been followed. Long chain indices in excess of 5.0, accompanied by strong bactericidal capacity, were recorded in serums after the full 3-dose immunisation course with a commercial vaccine. The full course elicited the most satisfactory antibody titres declined within the 12 month post-vaccination period, thus providing support for the recommendation that yearly booster doses should be administered. The immune response in horses during 2 strangles outbreaks was compared with the response following vaccination. Recovered animals showed declining antibody levels 9 weeks after infection, and the evidence does not support longevity of the immune response after natural infection. Reaction to vaccination was observed in those animals with high initial antibody titre.

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