The host-restricted bacterium Streptococcus equi is the causative agent of equine strangles, the most frequently diagnosed infectious disease of horses worldwide. The disease is characterized by abscessation of the lymph nodes of the head and neck, leading to significant welfare and economic cost. S. equi is believed to have evolved from an ancestral strain of Streptococcus zooepidemicus, an opportunistic pathogen of horses and other animals. Comparison of the genome of S. equi strain 4047 with those of S. zooepidemicus identified examples of gene loss due to mutation and deletion, and gene gain through the acquisition of mobile genetic elements that have probably shaped the pathogenic specialization of S. equi. In particular, deletion of the CRISPR (clustered regularly interspaced short palindromic repeats) locus in the ancestor of S. equi may have predisposed the bacterium to acquire and incorporate new genetic material into its genome. These include four prophages and a novel integrative conjugative element. The virulence cargo carried by these mobile genetic elements is believed to have shaped the ability of S. equi to cause strangles. Further sequencing of S. zooepidemicus has highlighted the diversity of this opportunistic pathogen. Again, CRISPRs are postulated to influence evolution, balancing the need for gene gain over genome stability. Analysis of spacer sequences suggest that these pathogens may be susceptible to a limited range of phages and provide further evidence of cross-species exchange of genetic material among Streptococcus pyogenes, Streptococcus agalactiae and Streptococcus dysgalactiae.

Introduction

The acquisition of new traits through horizontal gene transfer has played an important role in the evolution of pathogenic bacteria [13]. In addition to the effects that insertion into the bacterial genome may have through inactivation or modification of host genes, the acquisition of cargo genes carried by MGEs (mobile genetic elements) can increase the survival fitness and influence niche adaptation of the recipient strain [35].

However, the gain of MGEs can also exert a fitness cost, and a trade-off between the benefit of new traits against genome stability may ensue [6,7]. Furthermore, phages can cause lytic infection of susceptible bacterial hosts, which can impose stringent selective pressure on bacterial populations [8]. To maintain genome integrity and survive phage attack, bacterial populations have evolved an array of innate immune systems that include restriction modification systems, abortive infection systems and the production of a hyaluronic acid capsule. More recently, the CRISPR (clustered regularly interspaced short palindromic repeats)–Cas (CRISPR-associated) adaptive immune system has become increasingly recognized as playing an important role in immunity to invading DNA [911].

Streptococcus zooepidemicus is an opportunistic pathogen of horses, associated with inflammatory airway disease in thoroughbred racehorses [12,13], uterine infections in mares [14,15] and ulcerative keratitis [16]. However, it is also associated with disease, which can be life-threatening, in a wide range of other animal hosts, including dogs [17,18], cattle [19], sheep [20,21], pigs [22,23], monkeys [22,23] and humans [2427]. A biovar of S. zooepidemicus, Streptococcus equi, is host-restricted and the causative agent of equine strangles, one of the most frequently diagnosed and important infectious diseases of horses worldwide [28,29]. The disease is characterized by abscessation of the lymph nodes of the head and neck. As abscesses form and the lymph nodes swell, they may restrict the airway, and it is from this clinical condition that the disease is named. S. equi and S. zooepidemicus strains share approximately 80% DNA sequence identity with Streptococcus pyogenes, an important host-restricted pathogen of humans [30], which is associated with cases of pharyngitis, tonsillitis, scarlet fever, toxic shock syndrome and necrotizing fasciitis [31].

An MLST (multilocus sequence typing) scheme has been developed for S. zooepidemicus and S. equi, which currently identifies 308 different STs (sequence types) (http://pubmlst.org/szooepidemicus/ last accessed 16 July 2013) on the basis of the sequences of seven housekeeping genes [32]. Five genomes of S. zooepidemicus, including the S. equi biovar, have been published to date. S. equi strain 4047 (Se4047) of ST-179 was isolated from a horse with strangles in the New Forest, U.K., in 1990 [30]. S. zooepidemicus strain H70 (SzH70) of ST-1 was isolated from a nasal swab taken from a healthy thoroughbred racehorse in Newmarket during 2000. Strain MGCS10565 (Sz10565) of ST-72 was isolated from a human case of nephritis in Brazil in 1998 [33], which was part of a large outbreak of 133 confirmed cases associated with the consumption of unpasteurized goat's cheese that hospitalized 96 people and killed three [34]. Strain ATCC35246 (Sz35246) of ST-194 was isolated from a dead pig in Sichuan province, China [35]. In China, S. zooepidemicus is a major cause of disease in pigs and was responsible for the deaths of 300000 pigs during a large outbreak in 1975 [35]. Strain BHS5 (SzBHS5) of ST-123 was isolated from a dog with acute fatal haemorrhagic pneumonia during an outbreak in London, U.K., during 2001 [36]. None of the currently sequenced strains of S. zooepidemicus are closely related genetically by MLST, and so the analysis of their complement of MGEs and CRISPR–Cas systems was predicted to provide an insight into the wider diversity of this opportunistic pathogen.

Balancing gene gain

The ability of S. zooepidemicus to infect a wide variety of hosts and tissues poses significant questions in balancing the need to adapt to diverse environments, potentially through the acquisition of MGEs, while resisting phages encountered in these environments and maintaining genome stability. Of the four sequenced strains of S. zooepidemicus, three (SzBHS5, Sz35246 and SzH70, but not Sz10565) contain prophages and/or ICEs (integrative and conjugative elements) [30,33,35,36] (Table 1). These S. zooepidemicus prophages do not appear to carry virulence cargo and ICE cargo appears to be limited to toxin–antitoxin and restriction modification systems that may enhance the overall innate immunity of recipient strains to incoming MGEs and phages [37].

Table 1
Summary of CRISPR–Cas systems and mobile genetic elements within the S. zooepidemicus and S. equi genomes sampled

An asterisk (*) indicates that a complete genome sequence is available [30,33,35]

      Number of CRISPR–Cas spacer sequences Number of putative ICEs/prophages 
Strain ST Host Disease Country Year isolated Subtype I-C Subtype II-A ICE Prophage 
SzH70* Horse No disease U.K. 2000 18 – 
SzMGCS10565* 72 Human Nephritis Brazil 1998 17 
SzBHS5 123 Dog Lower respiratory U.K. 2001 26 – 
Se4047* 179 Horse Strangles U.K. 1990 – – 
SzATCC35246* 194 Pig Septicaemia China 1977 18 – 
      Number of CRISPR–Cas spacer sequences Number of putative ICEs/prophages 
Strain ST Host Disease Country Year isolated Subtype I-C Subtype II-A ICE Prophage 
SzH70* Horse No disease U.K. 2000 18 – 
SzMGCS10565* 72 Human Nephritis Brazil 1998 17 
SzBHS5 123 Dog Lower respiratory U.K. 2001 26 – 
Se4047* 179 Horse Strangles U.K. 1990 – – 
SzATCC35246* 194 Pig Septicaemia China 1977 18 – 

The relatively low frequency of prophages in S. zooepidemicus genomes is in stark contrast with the genome of Se4047, which contains two ICEs and four prophages [30]. The gain of MGEs by Se4047 may be influenced by the loss of nine putative competence genes that are intact in SzH70, SzBHS5 and Sz10565 (a deletion in comD of Sz35246 is likely to induce the premature termination of this protein [35]). The competence system has been postulated to provide resistance to the uptake and incorporation of foreign DNA and may coincidentally prevent stable prophage integration [30,33]. However, an alternative explanation of the proliferation of prophages in Se4047 can be found in genome comparisons between Se4047 and strains of S. zooepidemicus [30]. The SzH70, Sz10565, Sz35246 and SzBHS5 genomes contain one or two loci encoding CRISPR arrays and Cas genes. However, the Se4047 genome lacks both CRISPR–Cas loci, the CRISPR–Cas subtype I-C locus having been lost through recombination between ISSeq11 elements (Figure 1). The CRISPR–Cas subtype II-A locus of Sz10565 is flanked by IS (insertion sequence) elements. However, the lack of this locus in the other sequenced strains of S. zooepidemicus suggests that it may have been horizontally acquired by Sz10565 rather than lost from each of the other genomes [33] (Figure 2).

Comparison of the CRISPR–Cas subtype I-C locus of Se4047, SzH70 and Sz10565 viewed using the Artemis comparison tool

Comparison of the region containing the CRISPR–Cas subtype II-A locus of Sz10565 with Se4047 and SzH70 viewed using the Artemis comparison tool

The CRISPR–Cas loci of S. zooepidemicus

The SzH70 CRISPR–Cas subtype I-C locus contains 18 spacer sequences, of which ten have no significant database matches, three [spacers #3, #15 (targeting speL) and #17] share >94% identity with prophage sequences present in the published genomes of S. pyogenes. Four spacers have identical matches with prophage target protospacer sequences found in the Se4047 genome [#6 with SEQ0163 of ϕSeq1, #7 with SEQ1743 of ϕSeq3, #8 with SEQ1745 of ϕSeq3 and #15 with SEQ1727 of ϕSeq3 (seeM)] (Figure 3). Spacer #18 has a near identical protospacer sequence match with the Se4047 prophage coding sequences SEQ0190 of ϕSeq1 and SEQ1729 of ϕSeq3. This latter spacer is the only exact match with the spacer sequences of Sz10565 CRISPRs (spacer #9 of the CRISPR–Cas subtype I-C locus) or indeed any of the other S. zooepidemicus strains sequenced.

Location of putative protospacer sequences within the prophage of Se4047

Figure 3
Location of putative protospacer sequences within the prophage of Se4047

An asterisk (*) indicates that a single spacer sequence partially matches putative protospacers in both ϕSeq1 and ϕSeq3.

Figure 3
Location of putative protospacer sequences within the prophage of Se4047

An asterisk (*) indicates that a single spacer sequence partially matches putative protospacers in both ϕSeq1 and ϕSeq3.

Sz10565 is the only strain of S. zooepidemicus sequenced to date to have two CRISPR–Cas systems [33] (Figures 1 and 2). The subtype I-C system has nine spacer sequences. The final spacer sequence is identical with spacer #18 of the SzH70 subtype I-C locus. Spacer #8 partly matches SEQ0158 of Se4047 ϕSeq1, and spacer #7 is an exact protospacer match to Spy0796 of S. pyogenes strain MGAS9429. The subtype II-A locus contains 17 spacer sequences, including four spacers that share sequence identity with prophages of Se4047 [#1 with SEQ0167 of ϕSeq1, #5 with SEQ0801 of ϕSeq2, #8 (a duplicate of spacer #5) and #13 with SEQ0194 of ϕSeq1]. Spacer #6 (no database match) is a duplicate of spacer #9, and spacer #7 (no database match) is a duplicate of #10, suggesting that these duplications may have been due to a single genetic event. Spacers #4 and #15 have partial matches (93% sequence identity) with S. pyogenes prophage sequences, and spacer #12 partly matches SEQ0179 of ϕSeq1 in Se4047. Spacer #14 is a duplication of spacer #11 and matches a putative protospacer target sequence in Streptococcus dysgalactiae subspecies equisimilis strain GGS124 [38]. Spacer #17 shares 97% sequence identity (one base mismatch) with an intergenic region of Se4047, Sz35246 and the host Sz10565 genome. However, the single point mutation relative to the host protospacer sequence suggests that this spacer is unlikely to induce cytotoxicity by targeting the Sz10565 genome [39].

The CRISPR–Cas subtype I-C locus of SzBHS5 contains 26 spacer sequences, five of which share 100% sequence identity with S. equi prophages (#7 with SEQ0180 of ϕSeq1, #9 to SEQ0195 of ϕSeq1, #12 with SEQ0802 of ϕSeq2, #14 with an intergenic region of ϕSeq1 and #24 with SEQ0171 of ϕSeq1). Four other spacers share >98% sequence identity with prophages within the Se4047 genome (spacer #6 has a partial match to SEQ1765 of ϕSeq3, #11 with SEQ0143 of ϕSeq1, #19 partially matches SEQ0175 of ϕSeq1 and #26 partially matches SEQ0799 of ϕSeq2). There are three spacers that have partial matches to MGEs of S. pyogenes or Streptococcus agalactiae (spacers #8, #13 and #22).

Finally, strain Sz35246 contains 18 spacer sequences in its subtype 1-C CRISPR–Cas locus. Spacer #12 is an exact match of an intergenic protospacer sequence within ϕSeq2. Spacer #1 partially matches an intergenic region of ϕSeq1, spacer #6 partially matches SEQ1737 of ϕSeq3, spacer #8 partially matches SEQ0136 of ϕSeq1, spacer #9 partially matches a protospacer sequence at the very end of ϕSeq3 in Se4047, and spacer #13 partially matches SEQ0159 of ϕSeq1. Spacers #10 and #17 match and spacers #2 and #7 partially match sequences within prophages of S. pyogenes. Spacer #16 partially matches protospacer sequences in the streptococcal phages JX01 and LYGO9. Spacer #18 matches a protospacer sequence within parB of S. pyogenes strain alab49, but none of the strains of S. zooepidemicus sequenced.

Overall, 30 of the 88 (34%) spacer sequences either exactly or partially match protospacer sequences in three of the Se4047 prophages (Figure 3). A further 15 spacers (17%) exactly or partially match protospacer sequences in S. pyogenes, S. agalactiae or S. dysgalactiae. One spacer partially matches the genomes of streptococcal phages JX01 and LYGO9 and one partially matches the host genome of Sz10565 (Table 2). Only two spacer sequences at the trailer end of the subtype I-C CRISPR–Cas systems, spacers #18 of SzH70 and #9 of Sz10565, match each other. The strains of S. zooepidemicus that have been sequenced to date were isolated from diverse mammalian hosts, geographical regions and over a 24-year period. Therefore the low number of matches in the spacer sequences between strains is not unexpected. However, it is surprising that so many of the spacer sequences share similarity to putative protospacers within ϕSeq1, ϕSeq2 or ϕSeq3 of Se4047 (Table 2). These data suggest that the global population of S. zooepidemicus may be susceptible to a relatively small population of phages and provide further evidence that S. zooepidemicus and S. equi share a common phage pool with S. pyogenes, S. dysgalactiae and S. agalactiae [30].

Table 2
Summary of the CRISPR–Cas spacer sequence matches to putative protospacers

An asterisk (*) indicates putative protospacer partial matches in both ϕSeq1 and ϕSeq3

   Host ϕSeq1 ϕSeq2 ϕSeq3 S. pyogenes, S. agalactiae or S. dysgalactiae Phage 
Sequence CRISPR–Cas type Total # spacers Partial Identical Partial Identical Partial Identical Partial Identical Partial Partial 
SzH70 I-C 18  1*   1*   
Sz10565 I-C        
 II-A 17      
SzBHS5 I-C 26     
Sz35246 I-C 18    
Total  88 10 
   Host ϕSeq1 ϕSeq2 ϕSeq3 S. pyogenes, S. agalactiae or S. dysgalactiae Phage 
Sequence CRISPR–Cas type Total # spacers Partial Identical Partial Identical Partial Identical Partial Identical Partial Partial 
SzH70 I-C 18  1*   1*   
Sz10565 I-C        
 II-A 17      
SzBHS5 I-C 26     
Sz35246 I-C 18    
Total  88 10 

The CRISPR–Cas subtype I-C locus was present in 94% (131/140) of a larger collection of diverse S. zooepidemicus isolates (as determined by MLST) that were examined by quantitative PCR, but was absent from all strains of S. equi tested [30]. The loss of the CRISPR locus from the ancestor of Se4047 may have resulted in increased genome instability and illustrates that, in some circumstances, gene loss may in turn influence the subsequent rate of gene gain. It will be interesting to sequence the genomes of those strains that lack a subtype I-C CRISPR–Cas locus to determine whether these strains have also acquired new MGEs.

The consequences of CRISPR–Cas deletion: evolution of S. equi

The increased size of the Se4047 genome (2253793 bp) relative to that of SzH70 (2149866 bp) is due to an increased content of MGEs, which together account for 16.4% of the Se4047 genome compared with 7.5% of the SzH70 genome [30]. Of the four prophage elements, ϕSeq1 contains no cargo sequences, but the orthologues of the phospholipase A2 toxin, SlaA, encoded on ϕSeq2 and the superantigens SeeL and SeeM, SeeH and SeeI encoded on ϕSeq3 and ϕSeq4 respectively have been associated with increased morbidity and mortality in humans following infection with strains of S. pyogenes [4042]. The S. equi superantigens have been produced in recombinant form and shown to induce a mitogenic response in equine and asinine peripheral blood mononuclear cells [43,44].

An ICE, ICESe2, increases the ability of S. equi to import iron [45]. The NRPS (non-ribosomal peptide synthesis system) encoded within ICESe2 is predicted to synthesize the first example of a streptococcal siderophore, equibactin, and the acquisition of this locus is postulated to represent the speciation event in the evolution of S. equi from an ancestral S. zooepidemicus strain [30].

The loss of the subtype I-C CRISPR–Cas system may have accelerated or stabilized the acquisition of MGEs that probably influence the ability of S. equi to cause strangles. Several of the MGEs acquired by Se4047 encode systems that putatively reduce the ability of invading DNA to infect S. equi, which may compensate for the loss of CRISPR function and combine to increase the resistance of this bacterium to phage attack, leading to a rebalancing of the susceptibility of S. equi to invading DNA. ICESe2 contains coding sequences SEQ1257 and SEQ1258 that together encode a putative abortive infection system, with homology with AbiEii and AbiEi of Lactococcus lactis respectively [30]. SEQ1974, SEQ1975 and SEQ1976 within a novel genomic element encode a putative RSM (restriction site mutation) Type I restriction-modification system, and SEQ0757 and SEQ0758 of ICESe1 encode a putative Type II restriction-modification system. The coding sequences SEQ1570 and SEQ1571 within the core genome of Se4047 encode a putative restriction-modification system with 48% amino acid identity with FokI of Flavobacterium okeanokoites. These coding sequences are located between two IS elements and may also have been acquired via a horizontal gene transfer event.

Lysis of S. equi by circulating phages may be inhibited by the increased quantities of hyaluronic acid capsules on its surface. The Se4047 genome contains a 4 bp deletion in the hylA gene that disrupts function of this hyaluronate lyase. The reduced ability of S. equi to degrade hyaluronic acid and chondroitins provides one explanation for its lack of ability to invade beyond the lymphatic system of horses [46]. Novel deletions in hylA of S. zooepidemicus ST-57 and ST-236 strains similarly explain their increased levels of hyaluronic acid capsules. However, typical of the phage/bacterial host arms race, SEQ2045 on ϕSeq4 encodes a phage-encoded hyaluronate lyase. Although phage-encoded hyaluronate lyases typically have lower activity and reduced substrate range relative to HylA [47], they are incorporated into the structure of the phage tail and may assist invading phages in overcoming the physical barrier to attachment of a phage to its cognate receptor presented by the hyaluronic acid capsule [48].

Conclusions

The CRISPR–Cas systems of S. zooepidemicus are likely to play an important role in stabilizing the host genome and are believed to provide adaptive immunity to novel genetic elements and phages that may be encountered through the opportunistic lifestyle of this pathogen. The origins of the currently sequenced strains of S. zooepidemicus are diverse on the basis of the animal host and tissue infected, geography, and date of isolation. They differ in their complement of CRISPR–Cas loci and only two trailer spacer sequences are conserved between strains. However, one-third of the putative protospacer targets share sequence similarity or identity with only three prophages of Se4047, suggesting that there may be a limited number of phages that are capable of infecting this bacterial species. The similarity of 17% of spacer sequences with prophages of S. pyogenes, S. agalactiae or S. dysgalactiae provides additional evidence that these bacterial species share a common phage pool, which may permit the cross-species exchange of genetic material.

S. equi is believed to have evolved from an ancestral strain of S. zooepidemicus through a process of gene loss and gain. The loss of a CRISPR–Cas system is likely to have altered the balance of genome stability over gene gain in favour of the acquisition of new genetic material. The acquisition of virulence cargo carried on MGEs is believed to have played a key role in the evolution of S. equi as a distinct host-restricted pathogen of horses. The gain of innate immune systems that are also carried on these MGEs may have helped to partly restore balance and stabilize the S. equi genome once more. However, it is likely that the lack of an adaptive immune system will continue to influence gene gain in S. equi, permitting new strains to emerge as this pathogen continues to accessorize its genome.

CRISPR Evolution, Mechanisms and Infection: A Biochemical Society Focused Meeting held at the University of St Andrews, U.K., 17–19 June 2013. Organized and Edited by Emmanuelle Charpentier (Laboratory for Molecular Infection Medicine Sweden, Sweden), John van der Oost (Wageningen University, The Netherlands) and Malcolm White (University of St Andrews, U.K.).

Abbreviations

     
  • Cas

    CRISPR-associated

  •  
  • CRISPR

    clustered regularly interspaced short palindromic repeats

  •  
  • ICE

    integrative and conjugative element

  •  
  • IS

    insertion sequence

  •  
  • MGE

    mobile genetic element

  •  
  • MLST

    multilocus sequence typing

  •  
  • ST

    sequence type

Funding

We acknowledge the Horse Trust, Horserace Betting Levy Board and the Royal College of Veterinary Surgeons (RCVS) Charitable Trust who funded the sequencing of Se4047, SzH70 and SzBH5S respectively.

References

References
1
de la Cruz
F.
Davies
J.
Horizontal gene transfer and the origin of species: lessons from bacteria
Trends Microbiol.
2000
, vol. 
8
 (pg. 
128
-
133
)
2
Gogarten
J.P.
Townsend
J.P.
Horizontal gene transfer, genome innovation and evolution
Nat. Rev. Microbiol.
2005
, vol. 
3
 (pg. 
679
-
687
)
3
Brussow
H.
Canchaya
C.
Hardt
W.D.
Phages and the evolution of bacterial pathogens: from genomic rearrangements to lysogenic conversion
Microbiol. Mol. Biol. Rev.
2004
, vol. 
68
 (pg. 
560
-
602
)
4
Beres
S.B.
Musser
J.M.
Contribution of exogenous genetic elements to the group A Streptococcus metagenome
PLoS ONE
2007
, vol. 
2
 pg. 
e800
 
5
Ochman
H.
Lawrence
J.G.
Groisman
E.A.
Lateral gene transfer and the nature of bacterial innovation
Nature
2000
, vol. 
405
 (pg. 
299
-
304
)
6
Starikova
I.
Harms
K.
Haugen
P.
Lunde
T.T.
Primicerio
R.
Samuelsen
O.
Nielsen
K.M.
Johnsen
P.J.
A trade-off between the fitness cost of functional integrases and long-term stability of integrons
PLoS Pathog.
2012
, vol. 
8
 pg. 
e1003043
 
7
Foucault
M.L.
Depardieu
F.
Courvalin
P.
Grillot-Courvalin
C.
Inducible expression eliminates the fitness cost of vancomycin resistance in enterococci
Proc. Natl. Acad. Sci. U.S.A.
2010
, vol. 
107
 (pg. 
16964
-
16969
)
8
Rodriguez-Valera
F.
Martin-Cuadrado
A.B.
Rodriguez-Brito
B.
Pasic
L.
Thingstad
T.F.
Rohwer
F.
Mira
A.
Explaining microbial population genomics through phage predation
Nat. Rev. Microbiol.
2009
, vol. 
7
 (pg. 
828
-
836
)
9
Barrangou
R.
Fremaux
C.
Deveau
H.
Richards
M.
Boyaval
P.
Moineau
S.
Romero
D.A.
Horvath
P.
CRISPR provides acquired resistance against viruses in prokaryotes
Science
2007
, vol. 
315
 (pg. 
1709
-
1712
)
10
Labrie
S.J.
Samson
J.E.
Moineau
S.
Bacteriophage resistance mechanisms
Nat. Rev. Microbiol.
2010
, vol. 
8
 (pg. 
317
-
327
)
11
Bikard
D.
Marraffini
L.A.
Innate and adaptive immunity in bacteria: mechanisms of programmed genetic variation to fight bacteriophages
Curr. Opin. Immunol.
2011
, vol. 
24
 (pg. 
15
-
20
)
12
Wood
J.L.
Newton
J.R.
Chanter
N.
Mumford
J.A.
Association between respiratory disease and bacterial and viral infections in British racehorses
J. Clin. Microbiol.
2005
, vol. 
43
 (pg. 
120
-
126
)
13
Wood
J.L.
Burrell
M.H.
Roberts
C.A.
Chanter
N.
Shaw
Y.
Streptococci and Pasteurella spp. associated with disease of the equine lower respiratory tract
Equine Vet. J.
1993
, vol. 
25
 (pg. 
314
-
318
)
14
Hong
C.B.
Donahue
J.M.
Giles
R.C.
Jr
Petrites-Murphy
M.B.
Poonacha
K.B.
Roberts
A.W.
Smith
B.J.
Tramontin
R.R.
Tuttle
P.A.
Swerczek
T.W.
Etiology and pathology of equine placentitis
J. Vet. Diagn. Invest.
1993
, vol. 
5
 (pg. 
56
-
63
)
15
Smith
K.C.
Blunden
A.S.
Whitwell
K.E.
Dunn
K.A.
Wales
A.D.
A survey of equine abortion, stillbirth and neonatal death in the UK from 1988 to 1997
Equine Vet. J.
2003
, vol. 
35
 (pg. 
496
-
501
)
16
Brooks
D.E.
Andrew
S.E.
Biros
D.J.
Denis
H.M.
Cutler
T.J.
Strubbe
D.T.
Gelatt
K.N.
Ulcerative keratitis caused by β-hemolytic Streptococcus equi in 11 horses
Vet. Ophthalmol.
2000
, vol. 
3
 (pg. 
121
-
125
)
17
Pesavento
P.A.
Hurley
K.F.
Bannasch
M.J.
Artiushin
S.
Timoney
J.F.
A clonal outbreak of acute fatal hemorrhagic pneumonia in intensively housed (shelter) dogs caused by Streptococcus equi subsp. zooepidemicus
Vet. Pathol.
2008
, vol. 
45
 (pg. 
51
-
53
)
18
Chalker
V.J.
Brooks
H.W.
Brownlie
J.
The association of Streptococcus equi subsp. zooepidemicus with canine infectious respiratory disease
Vet. Microbiol.
2003
, vol. 
95
 (pg. 
149
-
156
)
19
Sharp
M.W.
Prince
M.J.
Gibbens
J.
S. zooepidemicus infection and bovine mastitis
Vet. Rec.
1995
, vol. 
137
 pg. 
128
 
20
Las Heras
A.
Vela
A.I.
Fernandez
E.
Legaz
E.
Dominguez
L.
Fernandez-Garayzabal
J.F.
Unusual outbreak of clinical mastitis in dairy sheep caused by Streptococcus equi subsp. zooepidemicus
J. Clin. Microbiol.
2002
, vol. 
40
 (pg. 
1106
-
1108
)
21
Stevenson
R.G.
Streptococcus zooepidemicus infection in sheep
Can. J. Comp. Med.
1974
, vol. 
38
 (pg. 
243
-
250
)
22
Soedarmanto
I.
Pasaribu
F.H.
Wibawan
I.W.
Lammler
C.
Identification and molecular characterization of serological group C streptococci isolated from diseased pigs and monkeys in Indonesia
J. Clin. Microbiol.
1996
, vol. 
34
 (pg. 
2201
-
2204
)
23
Salasia
S.I.
Wibawan
I.W.
Pasaribu
F.H.
Abdulmawjood
A.
Lammler
C.
Persistent occurrence of a single Streptococcus equi subsp. zooepidemicus clone in the pig and monkey population in Indonesia
J. Vet. Sci.
2004
, vol. 
5
 (pg. 
263
-
265
)
24
Hashikawa
S.
Iinuma
Y.
Furushita
M.
Ohkura
T.
Nada
T.
Torii
K.
Hasegawa
T.
Ohta
M.
Characterization of group C and G streptococcal strains that cause streptococcal toxic shock syndrome
J. Clin. Microbiol.
2004
, vol. 
42
 (pg. 
186
-
192
)
25
Bradley
S.F.
Gordon
J.J.
Baumgartner
D.D.
Marasco
W.A.
Kauffman
C.A.
Group C streptococcal bacteremia: analysis of 88 cases
Rev. Infect. Dis.
1991
, vol. 
13
 (pg. 
270
-
280
)
26
Downar
J.
Willey
B.M.
Sutherland
J.W.
Mathew
K.
Low
D.E.
Streptococcal meningitis resulting from contact with an infected horse
J. Clin. Microbiol.
2001
, vol. 
39
 (pg. 
2358
-
2359
)
27
Abbott
Y.
Acke
E.
Khan
S.
Muldoon
E.G.
Markey
B.K.
Pinilla
M.
Leonard
F.C.
Steward
K.
Waller
A.
Zoonotic transmission of Streptococcus equi subsp. zooepidemicus from a dog to a handler
J. Med. Microbiol.
2010
, vol. 
59
 (pg. 
120
-
123
)
28
Timoney
J.F.
Strangles
Vet. Clin. North Am.: Equine Pract.
1993
, vol. 
9
 (pg. 
365
-
374
)
29
Ivens
P.A.
Matthews
D.
Webb
K.
Newton
J.R.
Steward
K.
Waller
A.S.
Robinson
C.
Slater
J.D.
Molecular characterisation of ‘strangles’ outbreaks in the UK: the use of M-protein typing of Streptococcus equi ssp. equi
Equine Vet. J.
2011
, vol. 
43
 (pg. 
359
-
364
)
30
Holden
M.T.
Heather
Z.
Paillot
R.
Steward
K.F.
Webb
K.
Ainslie
F.
Jourdan
T.
Bason
N.C.
Holroyd
N.E.
Mungall
K.
, et al. 
Genomic evidence for the evolution of Streptococcus equi: host restriction, increased virulence, and genetic exchange with human pathogens
PLoS Pathog.
2009
, vol. 
5
 pg. 
e1000346
 
31
Wong
C.J.
Stevens
D.L.
Serious group a streptococcal infections
Med. Clin. North Am.
2013
, vol. 
97
 (pg. 
721
-
736
)
32
Webb
K.
Jolley
K.A.
Mitchell
Z.
Robinson
C.
Newton
J.R.
Maiden
M.C.
Waller
A.
Development of an unambiguous and discriminatory multilocus sequence typing scheme for the Streptococcus zooepidemicus group
Microbiology
2008
, vol. 
154
 (pg. 
3016
-
3024
)
33
Beres
S.B.
Sesso
R.
Pinto
S.W.
Hoe
N.P.
Porcella
S.F.
Deleo
F.R.
Musser
J.M.
Genome sequence of a Lancefield group C Streptococcus zooepidemicus strain causing epidemic nephritis: new information about an old disease
PLoS ONE
2008
, vol. 
3
 pg. 
e3026
 
34
Balter
S.
Benin
A.
Pinto
S.W.
Teixeira
L.M.
Alvim
G.G.
Luna
E.
Jackson
D.
LaClaire
L.
Elliott
J.
Facklam
R.
Schuchat
A.
Epidemic nephritis in Nova Serrana, Brazil
Lancet
2000
, vol. 
355
 (pg. 
1776
-
1780
)
35
Ma
Z.
Geng
J.
Zhang
H.
Yu
H.
Yi
L.
Lei
M.
Lu
C.P.
Fan
H.J.
Hu
S.
Complete genome sequence of Streptococcus equi subsp. zooepidemicus strain ATCC 35246
J. Bacteriol.
2011
, vol. 
193
 (pg. 
5583
-
5584
)
36
Paillot
R.
Darby
A.C.
Robinson
C.
Wright
N.L.
Steward
K.F.
Anderson
E.
Webb
K.
Holden
M.T.
Efstratiou
A.
Broughton
K.
, et al. 
Identification of three novel superantigen-encoding genes in Streptococcus equi subsp. zooepidemicus, szeF, szeN, and szeP
Infect. Immun.
2010
, vol. 
78
 (pg. 
4817
-
4827
)
37
Ma
Z.
Geng
J.
Yi
L.
Xu
B.
Jia
R.
Li
Y.
Meng
Q.
Fan
H.
Hu
S.
Insight into the specific virulence related genes and toxin-antitoxin virulent pathogenicity islands in swine streptococcosis pathogen Streptococcus equi ssp. zooepidemicus strain ATCC35246
BMC Genomics
2013
, vol. 
14
 pg. 
377
 
38
Shimomura
Y.
Okumura
K.
Murayama
S.Y.
Yagi
J.
Ubukata
K.
Kirikae
T.
Miyoshi-Akiyama
T.
Complete genome sequencing and analysis of a Lancefield group G Streptococcus dysgalactiae subsp. equisimilis strain causing streptococcal toxic shock syndrome (STSS)
BMC Genomics
2011
, vol. 
12
 pg. 
17
 
39
Vercoe
R.B.
Chang
J.T.
Dy
R.L.
Taylor
C.
Gristwood
T.
Clulow
J.S.
Richter
C.
Przybilski
R.
Pitman
A.R.
Fineran
P.C.
Cytotoxic chromosomal targeting by CRISPR/Cas systems can reshape bacterial genomes and expel or remodel pathogenicity islands
PLoS Genet.
2013
, vol. 
9
 pg. 
e1003454
 
40
Beres
S.B.
Sylva
G.L.
Barbian
K.D.
Lei
B.
Hoff
J.S.
Mammarella
N.D.
Liu
M.Y.
Smoot
J.C.
Porcella
S.F.
Parkins
L.D.
, et al. 
Genome sequence of a serotype M3 strain of group A Streptococcus: phage-encoded toxins, the high-virulence phenotype, and clone emergence
Proc. Natl. Acad. Sci. U.S.A.
2002
, vol. 
99
 (pg. 
10078
-
10083
)
41
Sitkiewicz
I.
Nagiec
M.J.
Sumby
P.
Butler
S.D.
Cywes-Bentley
C.
Musser
J.M.
Emergence of a bacterial clone with enhanced virulence by acquisition of a phage encoding a secreted phospholipase A2
Proc. Natl. Acad. Sci. U.S.A.
2006
, vol. 
103
 (pg. 
16009
-
16014
)
42
Ikebe
T.
Wada
A.
Inagaki
Y.
Sugama
K.
Suzuki
R.
Tanaka
D.
Tamaru
A.
Fujinaga
Y.
Abe
Y.
Shimizu
Y.
Watanabe
H.
Dissemination of the phage-associated novel superantigen gene speL in recent invasive and noninvasive Streptococcus pyogenes M3/T3 isolates in Japan
Infect. Immun.
2002
, vol. 
70
 (pg. 
3227
-
3233
)
43
Paillot
R.
Robinson
C.
Steward
K.
Wright
N.
Jourdan
T.
Butcher
N.
Heather
Z.
Waller
A.S.
Contribution of each of four superantigens to Streptococcus equi-induced mitogenicity, γ interferon synthesis, and immunity
Infect. Immun.
2010
, vol. 
78
 (pg. 
1728
-
1739
)
44
Artiushin
S.C.
Timoney
J.F.
Sheoran
A.S.
Muthupalani
S.K.
Characterization and immunogenicity of pyrogenic mitogens SePE-H and SePE-I of Streptococcus equi
Microb. Pathog.
2002
, vol. 
32
 (pg. 
71
-
85
)
45
Heather
Z.
Holden
M.T.
Steward
K.F.
Parkhill
J.
Song
L.
Challis
G.L.
Robinson
C.
Davis-Poynter
N.
Waller
A.S.
A novel streptococcal integrative conjugative element involved in iron acquisition
Mol. Microbiol.
2008
, vol. 
70
 (pg. 
1274
-
1292
)
46
Waller
A.S.
Strangles: taking steps towards eradication
Vet. Microbiol.
2013
 
doi:10.1016/j.vetmic.2013.03.033
47
Lindsay
A.M.
Zhang
M.
Mitchell
Z.
Holden
M.T.
Waller
A.S.
Sutcliffe
I.C.
Black
G.W.
The Streptococcus equi prophage-encoded protein SEQ2045 is a hyaluronan-specific hyaluronate lyase that is produced during equine infection
Microbiology
2009
, vol. 
155
 (pg. 
443
-
449
)
48
Smith
N.L.
Taylor
E.J.
Lindsay
A.M.
Charnock
S.J.
Turkenburg
J.P.
Dodson
E.J.
Davies
G.J.
Black
G.W.
Structure of a group A streptococcal phage-encoded virulence factor reveals a catalytically active triple-stranded β-helix
Proc. Natl. Acad. Sci. U.S.A.
2005
, vol. 
102
 (pg. 
17652
-
17657
)