Proline-glutamic acid-proline-lysine repetition peptide as an antigen for the serological diagnosis of strangles

The reactivity of the proline-glutamic acid-proline-lysine (PEPK) repetition peptide antigen in 3176 serum samples was investigated to evaluate its utility as an antigen for the serological diagnosis of strangles. The reactivity of the sera of horses infected with Streptococcus equi subspecies equi was high when the peptide had several PEPK repetitions. However, as the number of PEPK repetitions increased, the reactivity of the antigen with the sera of horses infected with Streptococcus equi subspecies zooepidemicus also increased. In horses infected experimentally with S equi, the reactivity of the PEPK antigen with five repetitions increased one week after inoculation and continued to increase during the following four weeks. The optical density (OD) values of test sera from horses infected experimentally with S equi and sera from horses that had recovered from strangles were high. The od values of sera from horses that had recovered from an experimental infection with S zooepidemicus and of sera from healthy horses were comparatively low.     

Se18.9, an anti-phagocytic factor H binding protein of Streptococcus equi

Evasion of phagocytosis is an important virulence determinant of Streptococcus equi (S. equi subsp. equi), the cause of equine strangles and distinguishes it from the closely related but much less virulent S. zooepidemicus (S. equi subsp. zooepidemicus). We describe Se18.9, a novel H factor binding protein secreted by S. equi but not by S. zooepidemicus that reduces deposition of C3 on the bacterial surface and significantly reduces the bactericidal activity of equine neutrophils suspended in normal serum for both S. equi and S. zooepidemicus. Se18.9 is secreted abundantly by actively dividing cells and is also bound to the bacterial surface. Strong serum and mucosal antibody responses are elicited in S. equi infected horses. Although a gene identical to se18.9 was not detected in S. zooepidemicus, sequences encoding proteins of similar size with similar signal peptide sequences were found in 3 of 12 randomly selected strains. Since Se18.9 is unique to S. equi, and immunoreactive with convalescent sera and mucosal IgA, it has potential for immunodiagnosis and for study of mucosal antibody response to S. equi.     

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.     

Tracing outbreaks of Streptococcus equi infection (strangles) in horses using sequence variation in the seM gene and pulsed-field gel electrophoresis

Strangles is a serious respiratory disease in horses caused by Streptococcus equi subspecies equi (S. equi). Transmission of the disease occurs by direct contact with an infected horse or contaminated equipment. Genetically, S. equi strains are highly homogenous and differentiation of strains has proven difficult. However, the S. equi M-protein SeM contains a variable N-terminal region and has been proposed as a target gene to distinguish between different strains of S. equi and determine the source of an outbreak. In this study, strains of S. equi (n=60) from 32 strangles outbreaks in Sweden during 1998-2003 and 2008-2009 were genetically characterized by sequencing the SeM protein gene (seM), and by pulsed-field gel electrophoresis (PFGE). Swedish strains belonged to 10 different seM types, of which five have not previously been described. Most were identical or highly similar to allele types from strangles outbreaks in the UK. Outbreaks in 2008/2009 sharing the same seM type were associated by geographic location and/or type of usage of the horses (racing stables). Sequencing of the seM gene generally agreed with pulsed-field gel electrophoresis profiles. Our data suggest that seM sequencing as a epidemiological tool is supported by the agreement between seM and PFGE and that sequencing of the SeM protein gene is more sensitive than PFGE in discriminating strains of S. equi.     

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.     

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.     

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.     

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.     

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.     

Identification of variations in SzP proteins of Streptococcus equi subspecies zooepidemicus and the relationship between protein variants and clinical signs of infection in horses

OBJECTIVE: To determine whether previously unidentified variations of the SzP protein of Streptococcus equi subsp zooepidemicus were present in horses with various clinical signs of infection and whether any relationship could be identified between SzP protein variants and naturally occurring clinical conditions. SAMPLE POPULATION: 23 isolates of S equi subsp zooepidemicus were recovered from specimens of horses with various clinical conditions and used as a representative population of isolates for evaluation of different SzP protein variants. PROCEDURE: Genetic heterogeneity of the isolates was demonstrated by repetitive extragenic palindromic-polymerase chain reaction analysis. The SzP gene was sequenced and the presumed protein sequence determined for each isolate. Characteristics of the SzP proteins were compared among the isolates and in relation to the clinical conditions of horses from which they were recovered. RESULTS: The signal peptide types, number of proline-glutamic acid-proline-lysine repeats, and anchor sequences were consistent with those previously described for the SzP protein. Many of the isolates clustered with 5 previously described types on the basis of the hypervariable region of the SzP protein. One additional variant, which represented 8 of the isolates, was identified. Particular motifs in the hypervariable region accounted for many of the differences among hypervariable types. CONCLUSIONS AND CLINICAL RELEVANCE: The SzP protein appears to be limited to a selected number of types. Variations in the SzP protein are frequently determined on the basis of different motifs rather than random amino acid substitutions. There does not appear to be any association of SzP protein variations and clinical manifestations of infection in horses.     

A case of zoonosis associated with Streptococcus equi ssp. zooepidemicus

A sexagenarian couple operating a dairy farm, where there were also many horses, suffered a severe respiratory infection associated with the bacterium Streptococcus equi ssp. zooepidemicus. The investigation revealed that the source of infection was due to contacts with a horse having an infected wound from which we isolated this zoonotic agent.     

新疆地区3株驴源马链球菌马亚种新SeM基因型的鉴定与进化分析

从新疆地区某驴养殖场获得了3株驴腺疫链球菌分离株HTP133、HTP123和HTP232.为了解这3株驴腺疫链球菌的生物学特性和确定其分子分型,本研究对其进行了生化特性、药敏特性的检测,对16S rRNA进行序列对比分析,并利用PCR扩增SeM等位基因和测序鉴定其基因型.研究结果表明3株分离菌均为马链球菌马亚种.药敏试...     

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.     

The role of hyaluronic acid capsular material of Streptococcus equi subsp. zooepidemicus in mediating adherence to HeLa cells and in resisting phagocytosis.

Hyaluronic acid is thought to be one of the critical virulence factors of Streptococcus equi subsp. zooepidemicus. The present study was designed to study the role of hyaluronic acid capsular material in mediating adherence and to resist the phagocytosis of the host's immune defence. The studies were performed with two encapsulated S. equi subsp. zooepidemicus and two unencapsulated phase variants. The bacteria had been previously isolated from diseased pigs and monkeys in Indonesia. The presence of capsular material was determined using the hyaluronic acid decapsulation test and by electron microscopic studies. Both encapsulated bacteria showed mucoid colonies after cultivation on blood agar, grew with diffuse colonies in soft agar media and reacted negatively in the salt aggregation test. The unencapsulated bacteria grew with small colonies on blood agar, formed compact colonies in soft agar media and reacted positively in the salt aggregation test. Adherence and phagocytosis studies revealed that the encapsulated bacteria adhered significantly more to HeLa cells and were less phagocytosed by murine macrophages compared to unencapsulated bacteria. Pretreatment of the HeLa cells using hyaluronic acid or pretreatment of the bacteria by hyaluronidase decreased the adherence value of encapsulated bacteria. Pretreatment of bacteria with pronase had no effect. The presented results strongly indicate that the hyaluronic acid capsular material contributes to adherence properties of S. equi subsp. zooepidemicus and might help the bacteria to resist phagocytosis by macrophages.     

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。     

The equine immune response to Streptococcus equi subspecies zooepidemicus during uterine infection

The purpose of this study was to describe strain-specific immune responses to Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) during uterine infection in horses. Five isolates of S. zooepidemicus were differentiated into four strains antigenically by bactericidal testing in blood of 12 horses, and genetically by pulsed-field gel electrophoresis. Eight healthy mares were then divided into two groups, each inoculated with one strain intrauterinely on three successive oestrous cycles followed by a second strain for three successive cycles, first and second strains being reversed for each group. Immune responses to both strains were assessed by bactericidal testing and immunoblotting over eight cycles. Both techniques indicated that immune responses to each strain arose at different times. Immunoblots showed greater binding to the first inoculated strain than to the second (P < 0.05). These data confirm that immune responses to S. zooepidemicus during uterine infection are partly strain-specific.     

Description of an epizootic and persistence of Streptococcus equi infections in horses

The age-specific attack rates of Streptococcus equi infections of the upper respiratory tract and lymph nodes (strangles) in horses for the different age groups were 17.6% for broodmares, 47.5% for 1-year-old horses, and 37.5% for foals. Streptococcus equi was isolated from nasal, pharyngeal, or lymph node specimens in 31 (60.8%) of 51 sick horses. A male 1-year-old horse, shipped from Kentucky to farm A, was considered to be the index case. Six (19.4%) of 31 horses with strangles remained as shedders of S equi after clinical signs of the disease had ended. Shedders of S equi were not identified from horses that were exposed to infected horses but never developed strangles.     

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.     

马源马链球菌兽疫亚种新疆株的分离鉴定及遗传特性分析

为了解马源马链球菌兽疫亚种（S.zooepidemicus）新疆分离株马链球菌兽疫亚种类M蛋白（SzM）基因的分子进化与变异情况,为该菌引起的感染性疾病的防控提供依据,本试验对新疆地区某马场采集的病马淋巴结样品进行病原菌的分离培养和生化鉴定,并对分离菌株进行药物敏感性试验。根据已发表的马源SzM基因序列设计引物,对其SzM基因进行PCR扩增及序列测定。将获得的SzM序列与GenBank中不同动物源马链球菌兽疫亚种分离株序列进行同源性比对和遗传进化分析。结果显示,分离得到了一株革兰氏阳性链球菌,将其命名为马链球菌兽疫亚种ZMSY15-1。药物敏感性试验结果表明,分离菌株对青霉素、磺胺嘧啶钠耐药,对其他14种药物均敏感。序列分析结果显示,马链球菌兽疫亚种ZMSY15-1与国内外不同动物源分离株SzM基因氨基酸同源性为56.0%~70.0%。遗传进化分析结果显示,这些菌株可分为4个群。马链球菌兽疫亚种ZMSY15-1与猪源分离株SzM蛋白的氨基酸同源性为59.9%,分别属于2个不同的群,其与美国马源分离株NH55426亲缘关系最近。本试验结果可丰富国内马源马链球菌兽疫亚种SzM基因的信息数据,为马链球菌兽疫亚种的致病机制研究和预防控制提供参考依据。