The pathogenic equine streptococci

Streptococci pathogenic for the horse include S. equi (S. equi subsp. equi), S. zooepidemicus (S. equi subsp. zooepidemicus), S. dysgalactiae subsp. equisimilis and S. pneumoniae capsule Type III. S. equi is a clonal descendent or biovar of an ancestral S. zooepidemicus strain with which it shares greater than 98% DNA homology and therefore expresses many of the same proteins and virulence factors. Rapid progress has been made in identification of virulence factors and proteins uniquely expressed by S. equi. Most of these are expressed either on the bacterial surface or are secreted. Notable examples include the antiphagocytic SeM and the secreted pyrogenic superantigens SePE-I and H. The genomic DNA sequence of S. equi will greatly accelerate identification and characterization of additional virulence factors and vaccine targets. Although it is the most frequently isolated opportunist pyogen of the horse, S. zooepidemicus has been the subject of few contemporary research studies. Variation in the protectively immunogenic SzP proteins has, however, been well characterized. Given its opportunist behavior, studies are urgently needed on regulation of virulence factors such as capsule and proteases. Likewise, information is also very limited on virulence factors and associated gene regulation of S. dysgalactiae subspecies equisimilis. It has recently been shown that equine isolates of Streptococcus pneumoniae are clonal, a feature shared with S. equi. All equine isolates express capsule Type III, are genetically similar, and have deletions in the genes for autolysin and pneumolysin. In summary, the evolving picture of the interaction of the equine pathogenic streptococci and their host is that of multiple virulence factors active at different stages of pathogenesis. The inherent complexity of this interaction suggests that discovery of effective combinations of immunogens from potential targets identified in genomic sequence will be laborious.     

Real-time PCR for detection and differentiation of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Strangles is a contagious equine disease caused by Streptococcus equi subsp. equi. In this study, clinical strains of S. equi (n=24) and Streptococcus equi subsp. zooepidemicus (n=24) were genetically characterized by sequencing of the 16S rRNA and sodA genes in order to devise a real-time PCR system that can detect S. equi and S. zooepidemicus and distinguish between them. Sequencing demonstrated that all S. equi strains had the same 16S rRNA sequence, whereas S. zooepidemicus strains could be divided into subgroups. One of these (n=12 strains) had 16S rRNA sequences almost identical with the S. equi strains. Interestingly, four of the strains biochemically identified as S. zooepidemicus were found by sequencing of the 16S rRNA gene to have a sequence homologous with Streptococcus equi subsp. ruminatorum. However, they did not have the colony appearance or the biochemical characteristics of the type strain of S. ruminatorum. Classification of S. ruminatorum may thus not be determined solely by 16S rRNA sequencing. Sequencing of the sodA gene demonstrated that all S. equi strains had an identical sequence. For the S. zooepidemicus strains minor differences were found between the sodA sequences. The developed real-time PCR, based on the sodA and seeI genes was compared with conventional culturing on 103 cultured samples from horses with suspected strangles or other upper respiratory disease. The real-time PCR system was found to be more sensitive than conventional cultivation as two additional field isolates of S. equi and four of S. zooepidemicus were detected.     

IdeE reduces the bactericidal activity of equine neutrophils for Streptococcus equi

Streptococcus equi (S. equi) causes equine strangles, a highly contagious and widespread purulent lymphadenitis of the head and neck. Highly resistant to phagocytosis, it produces long extracellular chains in affected lymph nodes. In a screen of clones reactive with convalescent serum from a gene library of S. equi CF32 we identified IdeE, an IgG-endopeptidase and homologue of the leucocyte receptor Mac-1 (CD11b). IdeE is expressed during S. equi infection eliciting both serum and mucosal antibody responses which persisted at significant levels in serum for over 200 days. Release from S. equi into culture medium was detected during the exponential phase of growth. The closely related Streptococcus zooepidemicus appeared to store the protein but not to release it. Antiphagocytic activity for equine neutrophils was dose-dependent and neutralized by IdeE-specific antiserum. Biotinylated IdeE bound weakly to about 77% of purified equine neutrophils and strongly to the remainder.     

Streptococcus equi bacteriophage SeP9 binds to group C carbohydrate but is not infective for the closely related S. zooepidemicus

Streptococcus equi (S. equi subsp. equi) is widely believed to have evolved from an ancestral strain of S. zooepidemicus (S. equi subsp. zooepidemicus) based on high sequence homology. A striking difference is the absence of phage sequences from S. zooepidemicus. In this study we show that the receptor for SeP9, a temperate bacteriophage of S. equi, is the Lancefield group C carbohydrate. However, although SeP9 binds to group C carbohydrate from S. zooepidemicus, it appears not to replicate and produce plaques.     

Pulsed-field gel electrophoresis and distribution of the genes zag and fnz in isolates of Streptococcus equi

Streptococcus equi subsp. equi and subsp. zooepidemicus are important pathogens of the equine respiratory tract. Isolates of both subspecies were examined by pulsed-field gel electrophoresis (PFGE). With the exception of eight isolates, a unique band pattern was displayed for each of the 48 subsp. zooepidemicus isolates tested. A method to distinguish isolates of the genetically very homogeneous subsp. equi has hitherto not been available, although several methods have been tested. By the use of PFGE, 50 isolates of subsp. equi could be divided into eleven groups, each with a unique pulsotype. In addition, the recently characterised genes encoding the cell-wall proteins ZAG and FNZ of S. equi subsp. zooepidemicus strain ZV were shown by Southern blots to be present in all 98 tested isolates, including the type strains of the two subspecies. Binding assays showed that the expression of the two genes clearly differentiate between the two subspecies.     

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.     

Results of bacteriological and cytological examinations of the endometrium of subfertile mares in stud farms in Serbia

Uterine microbiology, antimicrobial susceptibility and endometrial cytology were investigated in a total of 51 mares with fertility problems from 16 different stud farms in Serbia. Uterine cultures were performed after collection with a double guarded uterine swab, and endometrial cytology was evaluated after collection of endometrial cells with a special device (cytology brush). In 21 of 51 mares, at least one bacterial species was isolated from the uterus; the most frequent were Streptococcus equi subsp. zooepidemicus (13 isolates) and E. coli (four isolates). All isolates of Streptococcus equi subsp. zooepidemicus were susceptible to penicillin. Results from endometrial cytology were inconsistent; in 17 animals with positive bacteriological culture, cytology was not altered. It can be concluded that in Serbia, as in many other contries, Streptococcus equi subsp. zooepidemicus is the main cause for equine endometritis. It can be easily diagnosed by uterine culture but endometrial cytology does not always prove the existence of an endometrial infection with this agent.     

Dissemination of the superantigen encoding genes seeL, seeM, szeL and szeM in Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus

Bacterial superantigens are one of the major virulence factors produced by Streptococcus pyogenes and Staphylococcus aureus. The two novel superantigen encoding genes seeM and seeL were described for S. equi subsp. equi which is known as the causative agent of strangles in equids. In the present study previously characterized S. equi subsp. equi strains and strains of various other animal pathogenic streptococcal species and subspecies were investigated for the presence of the superantigen encoding genes seeM and seeL by polymerase chain reaction. According to these studies seeL and seeM appeared to be a constant characteristic of all investigated S. equi subsp. equi strains. Surprisingly, one S. equi subsp. zooepidemicus strain (S.z. 122) was also positive for both genes. The species identity of this S. equi subsp. zooepidemicus strain could additionally be confirmed by sequencing the 16S rRNA gene and the 16S-23S rDNA intergenic spacer region. The superantigen encoding genes could not be found among additionally investigated S. equi subsp. zooepidemicus strains or among strains of seven other streptococcal species. The seeL and seeM genes of the S. equi subsp. equi strain S.e. CF32 and the genes szeL and szeM of the S. equi subsp. zooepidemicus strain S.z. 122 were cloned and sequenced. A sequence comparison revealed a high degree of sequence homology between seeL, szeL, speL and seeM, szeM and speM, respectively. The superantigenic toxins L and M seemed to be widely distributed virulence factors of S. equi subsp. equi, rare among S. equi subsp. zooepidemicus but did not occur among a number of other animal pathogenic streptococcal species.     

猪链球菌病双重PCR诊断方法的建立

根据猪链球菌2型荚膜多糖和马链球菌兽疫亚种类M蛋白的保守区序列分别设计了2对简并引物,建立了一种能同时检测猪链球菌2型和马链球菌兽疫亚种的双重PCR方法。结果显示,该双重PCR能从100个细菌的混合纯培养物中扩增出2条目的片段。而且可以直接从病料组织中检测到相应的病原菌。用建立的双重PCR方法和细菌分离培养法平行检测人工感染的组织病料,PCR方法与细菌培养法的阳性检出率基本一致,但PCR方法的特异性好、敏感性高,简便易行,可以用于猪链球菌病的流行病学调查和实验室的快速鉴别诊断。     

Streptococcus equi but not Streptococcus zooepidemicus produces potent mitogenic responses from equine peripheral blood mononuclear cells

Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity was neutralised by Proteinase K and a post-strangles convalescent serum, and evidence for the presence of both thermo-resistant and thermo-labile mitogenic factors was obtained. Release of proteinaceous immunogenic mitogens in combination with the antiphagocytic protein SeM unique to S. equi may therefore contribute to some of the severe clinical manifestations of acute strangles in the horse.     

兽药典收载羊败血性链球菌病疫苗生产检验用菌种的系统分类鉴定

根据现有细菌分类鉴定方法,采用血清学、Biolog、16S rRNA序列分析和全基因组序列分析等方法对羊败血性链球菌病疫苗生产检验用菌种CVCC 553、55001和55002进行系统鉴定。结果显示,菌种CVCC 553、55001和55002血清群为兰氏C群;Biolog鉴定为马链球菌反刍亚种;16S rRNA序列分析与马链球菌反刍亚种模式菌株CECT 5772的同源性最高(均为99.72%),且在系统发育树中位于同一分支;与马链球菌反刍亚种模式菌株CCUG 47520的DNA-DNA的杂交值大于70%,分别为80.5%、80.2%、80.5%,与马链球菌反刍亚种模式菌株CECT 5772的平均核苷酸同源性分别为97.73%、97.64%、97.77%。因此本研究认为羊败血性链球菌病疫苗生产用菌种CVCC 553、55001和55002应为马链球菌反刍亚种。     

Bacterial isolates and antimicrobial susceptibilities in equine bacterial ulcerative keratitis (1993--2004)

REASONS FOR PERFORMING STUDY: Bacterial ulcerative keratitis is a common and often vision-threatening problem in horses. Emerging bacterial resistance to commonly used topical antibiotics has been demonstrated. Previous antibiotic use may alter the antimicrobial susceptibility of bacterial isolates. OBJECTIVES: To document aerobic bacterial isolates and associated bacterial susceptibilities from horses with ulcerative keratitis treated at the University of Tennessee between January 1993 and May 2004 and determine whether prior antibiotic therapy affected antimicrobial susceptibility of the isolates. METHODS: Medical records from horses with ulcerative keratitis and positive aerobic bacterial cultures and antimicrobial susceptibility were evaluated. Clinical history regarding antibiotic therapy prior to culture was documented. RESULTS: Fifty-one aerobic bacterial isolates from 43 horses were identified. Streptococcus equi subspecies zooepidemicus was the most commonly isolated organism, accounting for 33.3% of all isolates, followed by Pseudomonas aeruginosa (11.8%), Staphylococcus spp. (11.8%) and Gram-negative nonfermenting rods (7.8 %). No resistance was noted amongst S. equi ssp. zooepidemicus to cephalothin, chloramphenicol or ciprofloxacin. Only 64 % of S. equi ssp. zooepidemicus isolates were sensitive to bacitracin. No resistance was noted among P. aeruginosa to gentamicin, tobramycin or ciprofloxacin. Antibiotic therapy with neomycin-polymixin B-bacitracin prior to presentation and culture was documented in 11/17 horses in which S. equi ssp. zooepidemicus was isolated and in 4/6 horses in which P. aeruginosa was isolated. Three horses received topical corticosteroids prior to culture, of which 2 had polymicrobial infections. CONCLUSIONS: S. equi ssp. zooepidemicus and P. aeruginosa were the most frequently isolated bacterial organisms in equine ulcerative keratitis. No significant trends in aminoglycoside or fluoroquinolone resistance were noted among these organisms. However, the resistance of S. equi ssp. zooepidemicus to bacitracin with common use of this antibiotic suggests that previous antibiotic therapy probably affects antimicrobial resistance. POTENTIAL RELEVANCE: Therapy prior to culture may play an important role in antimicrobial susceptibility of corneal bacterial isolates. Corticosteroid use may increase the risk of polymicrobial infections of corneal ulcers, leading to a worse prognosis. Although significant fluoroquinolone resistance has not been documented in the veterinary literature, these antimicrobials should be reserved for known infected corneal ulcers and not used for prophylaxis. Empirical antibiotic therapy should not only be guided by clinical signs, but also take into consideration previous antimicrobial and corticosteroid therapy.     

Identification of a novel collagen-like protein, SclC, in Streptococcus equi using signal sequence phage display

Strangles is a serious disease in horses caused by Streptococcus equi subspecies equi. In this study, genes encoding putative extracellular proteins in this subspecies have been identified using signal sequence phage display. Among these, one showed similarities to the SclB protein, a member of the collagen-like proteins of Streptococcus pyogenes. The novel gene denoted sclC encodes a protein, SclC, of 302 amino acids, containing typical features found in cell wall-anchored proteins in Gram-positive bacteria. Based on similarities to the S. pyogenes collagen-like proteins the mature SclC protein can be divided into various domains: an N-terminal non-repetitive region (A), a highly repetitive collagen-like region (CL), and a C-terminal proline-rich wall-associated region (W). Using PCR, the sclC gene was detected in all studied strains of S. equi subsp. equi and S. equi subsp. zooepidemicus. Further, antibodies against recombinant SclC were detected in a collection of sera from horses with no history of strangles as well as horses previously infected with S. equi subsp. equi. Interestingly, the sera from convalescence horses were found to have significantly increased antibody titers against the SclC protein indicating that this protein is expressed during infection of S. equi subsp. equi.     

Multiplex polymerase chain reaction for identification and differentiation of Streptococcus equi subsp. zooepidemicus and Streptococcus equi subsp. equi

The closely related streptococcal species Streptococcus equi subsp. zooepidemicus and S. equi subsp. equi were identified by polymerase chain reaction using oligonucleotide primers designed according to species-specific parts of the superoxide dismutase A encoding gene sodA. A further differentiation of both subspecies could be performed by amplification of the genes seeH and seeI encoding the exotoxins SeeH and SeeI, respectively, which could be detected for S. equi subsp. equi but not for S. equi subsp. zooepidemicus. A further simplification of the identification and differentiation of both subspecies was conducted by sodA-seeI multiplex polymerase chain reaction.     

Clinical and epidemiological investigation of chronic upper respiratory diseases caused by beta-haemolytic Streptococci in horses

An outbreak of strangle-like disease involving 26 horses farmed in central Italy was investigated by clinic examination, endoscopy, cytology, bacteriology and polymerase chain reaction (PCR). At weekly interval, a total of three nasal swabs and one guttural pouches lavage fluid (GPLF) were collected, and no Streptococcus equi subsp. equi carrier was found. Some horses showed upper airways disease and endoscopic signs of pharyngeal lymphoid hyperplasia of different grade and/or abnormal endoscopic appearance of guttural pouches. Streptococcus dysgalactiae subsp. equisimilis was isolated from 14 horses while S. equi subsp. zooepidemicus was isolated from six horses. PCR confirmed the biochemical and serological identification of all isolates and was positive in 10 bacteriological negative samples. The absence of S. equi and the frequent detection of S. equisimilis and S. zooepidemicus suggest that beta-haemolytic streptococci other than S. equi could be the causative agent of strangle-like disease.     

Relatedness of Streptococcus equi subsp. zooepidemicus strains isolated from harbour seals (Phoca vitulina) and grey seals (Halichoerus grypus) of various origins of the North Sea during 1988-2005

The present study was designed to identify 15 beta-hemolytic streptococci isolated during a period between 1988 and 2005 from nine harbour seals and six grey seals from various origins of the North Sea. All isolates were identified as Streptococcus equi subsp. zooepidemicus. The bacteria were additionally investigated for relatedness by restriction fragment length polymorphism analysis of PCR amplified 16S-23S rDNA intergenic spacer region and gene szp and by macrorestriction analysis of chromosomal DNA of the strains by pulsed field gel electrophoresis. The molecular analysis yielded identical or closely related patterns within the strains of the present study and with the S. equi subsp. zooepidemicus strains isolated from harbour seals of German North Sea which were investigated previously [Akineden, O., Hassan, A.A., Alber, J., El-Sayed, A., Estoepangestie, A.T.S., L?mmler, C., Weiss, R., Siebert, U., 2005. Phenotypic and genotypic properties of S. equi subsp. zooepidemicus isolated from harbor seals (Phoca vitulina) from the German North Sea during the phocine distemper outbreak in 2002. Vet. Microbiol. 110, 147-152]. This indicates that this single or closely related bacterial clone existed during both phocine distemper virus epidemics in 1988 and 2002 and that a direct transmission of the strains has occurred between two seal species and between seal populations of far distant regions possibly with grey seals as a vector.     

Proline-glutamic acid-proline-lysine peptide set as a specific antigen for the serological diagnosis of strangles

The reactivity of synthesised peptide sets for the M-like proteins SeM and SzPSe with sera from horses infected with Streptococcus equi or Streptococcus zooepidemicus, or control horses, was investigated by an ELISA. Seventeen horses were infected experimentally with S equi or S zooepidemicus, convalescent sera were obtained from 25 horses and control sera were obtained from 1945 horses. The serum antibody responses of individual horses to the peptide sets were highly variable. Some of the peptide sets for SeM reacted strongly with the sera from the horses infected experimentally with S equi, but also reacted with sera from some of the horses infected experimentally with S zooepidemicus. However, the proline-glutamic acid-proline-lysine (PEPK) repeats peptide set, synthesised from the PEPK repeats areas of SzPSe, reacted most strongly with the sera from the horses infected experimentally with S equi and the horses convalescing from strangles, and reacted only minimally with the sera from the horses infected experimentally with S zooepidemicus and the control horses.     

Characteristics of an R antigen common to Streptococcus equi and zooepidemicus

An R antigen of the group C streptococcus S. equi that cross reacts with a similar antigen of S. zooepidemicus has been identified and characterized. It is acid, heat and trypsin resistant, but pepsin sensitive and has an isoelectric point of 4.8. The amino acids in highest concentration are glutamic, aspartic, alanine, leucine, and valine. Bacterial components released in a French Press contain large amounts of R antigen, which is present also in culture supernatants and acid extracts. It has a molecular weight of about 82,000. Trypsin extraction of cells yields molecules of predominantly 56,000 and 25,000 molecular weight that appeared by immunoblotting to be similar to those obtained by trypsinization of purified R protein from preparations derived from the French Press. Although horses naturally infected with S. equi or S. zooepidemicus develop cross reacting R antibodies, the role of the R antigen in pathogenesis is unknown. Purified R protein does not stimulate bactericidal antibody in horses nor is it protective for mice. Its occurrence in antigen preparations in assays for S. equi antibody could be a source of interpretive errors because of the presence in many sera of antibody to the immunologically similar R antigen of S. zooepidemicus, a normal nasopharyngeal commensal of Equidae.     

Validation of a point-of-care polymerase chain reaction assay for detection of Streptococcus equi subspecies equi in rostral nasal swabs from horses with suspected strangles

This study aimed to validate a point-of-care polymerase chain reaction (PCR) assay for detection of Streptococcus equi subsp. equi (S. equi) in rostral nasal swabs from horses with suspected acute strangles and to compare the results against the molecular gold standard of quantitative polymerase chain reaction (qPCR). Two hundred thirty-two individual swabs of rostral nasal passages were characterized by qPCR as S. equi positive, S. equi subsp. zooepidemicus (S. zooepidemicus) positive, or S. equi and S. zooepidemicus negative. The specificity and sensitivity of the point-of-care PCR assay were 89% and 84%, respectively. The limits of detection of the qPCR assay and the point-of-care PCR analyzer were 3 and 277 eqbE target genes of S. equi, respectively. Overall agreement and short turnaround time make the point-of-care PCR assay a potential molecular diagnostic platform that will enhance the capability of equine veterinarians to timely support a diagnosis of strangles and institute proper biosecurity protocols.