Differences in the Accessory Genomes and Methylomes of Strains of Streptococcus equi subsp. equi and of Streptococcus equi subsp. zooepidemicus Obtained from the Respiratory Tract of Horses from Texas.

Streptococcus equi subsp. equi (SEE) is a host-restricted equine pathogen considered to have evolved from Streptococcus equi subsp. zooepidemicus (SEZ). SEZ is promiscuous in host range and is commonly recovered from horses as a commensal. Comparison of a single strain each of SEE and SEZ using whole-genome sequencing, supplemented by PCR of selected genes in additional SEE and SEZ strains, was used to characterize the evolution of SEE. But the known genetic variability of SEZ warrants comparison of the whole genomes of multiple SEE and SEZ strains. To fill this knowledge gap, we utilized whole-genome sequencing to characterize the accessory genome elements (AGEs; i.e., elements present in some SEE strains but absent in SEZ or vice versa) and methylomes of 50 SEE and 50 SEZ isolates from Texas. Consistent with previous findings, AGEs consistently found in all SEE isolates were primarily from mobile genetic elements that might contribute to host restriction or pathogenesis of SEE. Fewer AGEs were identified in SEZ because of the greater genomic variability among these isolates. The global methylation patterns of SEE isolates were more consistent than those of the SEZ isolates. Among homologous genes of SEE and SEZ, differential methylation was identified only in genes of SEE encoding proteins with functions of quorum sensing, exopeptidase activity, and transitional metal ion binding. Our results indicate that effects of genetic mobile elements in SEE and differential methylation of genes shared by SEE and SEZ might contribute to the host specificity of SEE. IMPORTANCE Strangles, caused by the host-specific bacterium Streptococcus equi subsp. equi (SEE), is the most commonly diagnosed infectious disease of horses worldwide. Its ancestor, Streptococcus equi subsp. zooepidemicus (SEZ), is frequently isolated from a wide array of hosts, including horses and humans. A comparison of the genomes of a single strain of SEE and SEZ has been reported, but sequencing of further isolates has revealed variability among SEZ strains. Thus, the importance of this study is that it characterizes genomic and methylomic differences of multiple SEE and SEZ isolates from a common geographic region (viz., Texas). Our results affirm many of the previously described differences between the genomes of SEE and SEZ, including the role of mobile genetic elements in contributing to host restriction. We also provide the first characterization of the global methylome of Streptococcus equi and evidence that differential methylation might contribute to the host restriction of SEE.

Globetrotting strangles: the unbridled national and international transmission of Streptococcus equi between horses.

The equine disease strangles, which is characterized by the formation of abscesses in the lymph nodes of the head and neck, is one of the most frequently diagnosed infectious diseases of horses around the world. The causal agent, Streptococcus equi subspecies equi, establishes a persistent infection in approximately 10 % of animals that recover from the acute disease. Such 'carrier' animals appear healthy and are rarely identified during routine veterinary examinations pre-purchase or transit, but can transmit S. equi to naïve animals initiating new episodes of disease. Here, we report the analysis and visualization of phylogenomic and epidemiological data for 670 isolates of S. equi recovered from 19 different countries using a new core-genome multilocus sequence typing (cgMLST) web bioresource. Genetic relationships among all 670 S. equi isolates were determined at high resolution, revealing national and international transmission events that drive this endemic disease in horse populations throughout the world. Our data argue for the recognition of the international importance of strangles by the Office International des Épizooties to highlight the health, welfare and economic cost of this disease. The Pathogenwatch cgMLST web bioresource described herein is available for tailored genomic analysis of populations of S. equi and its close relative S. equi subspecies zooepidemicus that are recovered from horses and other animals, including humans, throughout the world. This article contains data hosted by Microreact.

Cases of high mortality in cull sows and feeder pigs associated with Streptococcus equi subsp. zooepidemicus septicemia.

Investigations of 2 cases of high mortality in cull sows and feeder pigs from a buying station in Ohio and cull sows at an abattoir in Tennessee were conducted at the Iowa State University Veterinary Diagnostic Laboratory. The animals were presented as weak, lethargic, and some with high fever. Rapidly escalating mortality was reported to be as high as 30-50% within groups at the buying station over 8-10 d, and 30-40% over 5-7 d at the abattoir. Splenomegaly and red lymph nodes were the most consistent macroscopic findings, with scant fibrinous polyserositis observed in one sow. The microscopic lesions of vasculitis, fibrin thrombi, fibrinosuppurative polyserositis, and intralesional bacteria were consistent with acute bacterial septicemia. Bacterial culture isolated Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) from multiple organs, including spleen, lung, and kidney. PCR tests were negative for African swine fever virus, classical swine fever virus, Erysipelothrix rhusiopathiae, porcine reproductive and respiratory syndrome virus, porcine circovirus 2, and Salmonella spp. Porcine circovirus 3 was inconsistently detected at low levels by PCR, with a lack of associated lesions. Next-generation sequencing identified S. zooepidemicus and porcine partetravirus in the serum sample of the feeder pig from the buying station. Phylogenetic analysis of the szP gene indicated that the S. zooepidemicus isolates from Ohio and Tennessee are in genotype VI. We conclude that the cause of these high mortality events in swine was S. zooepidemicus septicemia.

Genetic diversity of Streptococcus equi subsp. zooepidemicus isolated from horses.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic and zoonotic pathogen of horses. In this study, genetic intraspecies variability of SEZ obtained mainly from respiratory and genital samples of horses was investigated by analysis of the 16S-23S rRNA intergenic spacer region (ISR) and of the 16S rRNA gene. 16S-23S ISR rRNA type A1 was predominant, although a high rate of multiple products (30.5%) was obtained. Phylogenetic analysis of the 16S rRNA gene detected three genogroups (I, II and III). 16S rRNA variable regions V1 and V2 are the most important regions for evaluating SEZ intraspecies variability, but at least V1-V5 regions should be considered to avoid mistakes. Analysis of all 16S rRNA sequences available in databases assigned human SEZ to groups I and III but not to group II. These results show a high genetic variability in SEZ collected from different specimens of horses from various regions of Italy.

Outbreak of Glomerulonephritis Caused by Streptococcus zooepidemicus SzPHV5 Type in Monte Santo de Minas, Minas Gerais, Brazil.

Streptococcus zooepidemicus is an emerging and opportunistic zoonotic pathogen which plays an important role in the development of severe and life-threatening diseases and is potentially capable of triggering large glomerulonephritis outbreaks. Between December 2012 and February 2013, 175 cases of glomerulonephritis were confirmed in the town of Monte Santo de Minas, MG, Brazil. During the outbreak, 19 isolates of S. zooepidemicus were recovered, 1 from ice cream, 2 from the oropharynx of food handlers, and 16 from patients affected by acute poststreptococcal glomerulonephritis (APSGN). All S. zooepidemicus isolates involved in the outbreak amplified the same sequence of the hypervariable region of the SzP protein (SzPHV5) and presented indistinguishable banding patterns with high similarity (>99%) to each other by the repetitive element sequence-based PCR (rep-PCR) technique. Inspection programs on the milk supply chain should be strengthened and continuously encouraged so that the health of consumers is preserved.

Multi-locus sequence typing of Streptococcus equi subspecies zooepidemicus strains isolated from cats.

Streptococcus equi subspecies zooepidemicus ( S. zooepidemicus) causes outbreaks of fatal respiratory disease in dog shelters and fatal respiratory and neurologic disease in cat shelters. We conducted multi-locus sequence typing analysis on S. zooepidemicus isolates from 5 Canadian and 3 Israeli cats with severe respiratory and neurologic disease, plus 1 isolate from a clinically normal shelter cat. Our aim was to determine if feline outbreaks are clonal and whether there is commonality between feline and canine strains. ST363 was identified as the causative strain of a Canadian outbreak of S. zooepidemicus-linked disease, and is a double-locus variant of ST173, which was isolated from one of the Israeli cats. ST363 was also isolated from the clinically normal cat, indicative of the potential for enzootic infection in shelters. Strains within the ST173 clonal complex were responsible for 2 large canine outbreaks in the United States and the United Kingdom, as well as the death of 1 cat in the United States outbreak. ST215 was isolated from 2 cats in the Israeli outbreak, and is unrelated to the ST173 complex. We conclude that S. zooepidemicus outbreaks in cat shelters are clonal and that strains within the ST173 clonal complex are pathogenic for both dogs and cats.

Discrimination of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Accurate and timely identification of infectious etiologies is of great significance in veterinary microbiology, especially for critical diseases such as strangles, a highly contagious disease of horses caused by Streptococcus equi subsp. equi. We evaluated a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) platform for use in species- and subspecies-level identification of S. equi isolates from horses and compared it with an automated biochemical system. We used 25 clinical isolates each of S. equi subsp. equi and S. equi subsp. zooepidemicus. Using the MALDI-TOF MS platform, it was possible to correctly identify all 50 isolates to the species level. Unique mass peaks were identified in the bacterial peptide mass spectra generated by MALDI-TOF MS, which can be used for accurate subspecies-level identification of S. equi. Mass peaks (mass/charge, m/ z) 6,751.9 ± 1.4 (mean ± standard deviation) and 5,958.1 ± 1.3 were found to be unique to S. equi subsp. equi and S. equi subsp. zooepidemicus, respectively. The automated biochemical system correctly identified 47 of 50 of the isolates to the species level as S. equi, whereas at the subspecies level, 24 of 25 S. equi subsp. equi isolates and 22 of 25 S. equi subsp. zooepidemicus isolates were correctly identified. Our results indicate that MALDI-TOF MS can be used for accurate species- and subspecies-level identification of S. equi.

Clinical and microbiological features of bacteremia with Streptococcus equi.

Streptococcus equi (SE) rarely causes human infections. We identified 18 SE isolates from blood cultures. The focus of infection was unknown (n = 5), arthritis (n = 3), catheter-related (n = 2), pneumonia (n = 2), or other (n = 6). There were no fatalities. Several patients had animal contacts but there were no indications of clonal outbreaks.

Detection and differentiation of wild-type and a vaccine strain of Streptococcus equi ssp. equi using pyrosequencing.

Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is an important equine pathogen. Strangles is a highly contagious disease and a commercial modified live vaccine (MLV) is used for protection, which although effective, may also result in clinical signs of the disease. A rapid means to differentiate between the MLV and wild-type infection is crucial for quarantine release and limiting the disease spread. This study describes the use of a pyrosequencing assay targeting a single nucleotide deletion upstream of the SzPSe gene to distinguish between the wild-type and vaccine strains. A set of 96 characterized clinical specimens and isolates were tested using the assay. The assay was successful in differentiating between wild-type S. equi and the vaccine strains and in discriminating S. equi from other Streptococci. The vaccine strain was identified in 61.7% (29/47) of the strangles cases in horses with a history of MLV vaccination.

Predictor variables for and complications associated with Streptococcus equi subsp equi infection in horses.

OBJECTIVE: To evaluate predictor variables for and complications associated with Streptococcus equi subsp equi infection (strangles) in horses. DESIGN: Retrospective case-control study. ANIMALS: 108 horses with strangles (cases) and 215 horses without strangles (controls). PROCEDURES: Medical records from January 2005 through July 2012 were reviewed. Cases were defined as horses with clinical signs of strangles (pyrexia, retropharyngeal lymphadenopathy, and mucopurulent nasal discharge) that were associated with a confirmed strangles outbreak or had positive results for S equi on PCR assay or bacteriologic culture. Controls were defined as horses with pyrexia that did not meet the other criteria for cases. Data compared between cases and controls included signalment, clinical signs, diagnostic test results, and disease complications and outcome. Logistic regression was used to identify variables associated with strangles and its complications. RESULTS: Clinical signs of strangles were not evident in 12 of 25 cases classified as S equi carriers (infected > 40 days). Predictor variables associated with strangles included mucopurulent nasal discharge and external abscesses in the pharyngeal region. Strangles was more likely to be diagnosed in the spring than in the summer. Cases with anemia were more likely to develop purpura hemorrhagica than were cases without anemia. No risk factors were identified for the development of guttural pouch empyema or metastatic abscesses. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that not all horses infected with S equi develop clinical signs of strangles. We recommend that guttural pouch endoscopy and lavage with PCR assay of lavage fluid samples be performed to identify S equi carrier horses.

Comparison of specificities of serum antibody responses of horses to clinical infections caused by Streptococcus equi or zooepidemicus.

PROBLEM ADDRESSED: Streptococcus zooepidemicus (Sz) and its clonal derivative Streptococcus equi (Se) share greater than 96% DNA identity and elicit immune responses to many shared proteins. Identification of proteins uniquely targeted by the immune response to each infection would have diagnostic value. OBJECTIVE: The aim of the study was to compare serum antibody responses of horses infected by Se or Sz. METHODS AND APPROACH: Antibody levels were measured to panels of recombinant proteins of Sz and Se in sera of horses and ponies before and after experimental and naturally occurring invasive infections by these organisms. Antibody responses to an Se extract vaccine were also measured. Sera diluted 1:200 were assayed in triplicate using optimum concentrations of 9 and 14 immunoreactive proteins of Se and Sz, respectively. Bound IgG was detected using HRP-Protein G conjugate. RESULTS: Antibodies specific for SeM-N2, IdeE2, Se42.0 and Se75.3 (SEQ2190) were elicited by Se but not by Sz infection. Commercial Se extract vaccine did not elicit responses to IdeE2 or Se75.3. Sz infections resulted in significant (p<0.01) responses to Sz115, SzM, ScpC, SzP, MAP and streptokinase an indication these proteins are expressed during opportunistic invasions of the respiratory tract. FSR and HylC specific responses were unique to infections by Sz. CONCLUSIONS: The data indicate antibodies to IdeE2, Se75.3 and SeM-N2 may be used to distinguish infection by Se from that caused by the closely related Sz. Se infection, but not vaccination with Se extract elicits antibody to IdeE2 and Se75.3.

Recurrent Streptococcus equi subsp. zooepidemicus Bacteremia in an Infant.

We describe a case of an infant with recurrent bacteremia caused by Streptococcus equi subsp. zooepidemicus, likely transmitted from mother to infant. Our case highlights the importance of an epidemiological history and molecular diagnostics in ascertaining insights into transmission, pathogenesis, and optimal management.

Genome specialization and decay of the strangles pathogen, Streptococcus equi, is driven by persistent infection.

Strangles, the most frequently diagnosed infectious disease of horses worldwide, is caused by Streptococcus equi. Despite its prevalence, the global diversity and mechanisms underlying the evolution of S. equi as a host-restricted pathogen remain poorly understood. Here, we define the global population structure of this important pathogen and reveal a population replacement in the late 19th or early 20th Century. Our data reveal a dynamic genome that continues to mutate and decay, but also to amplify and acquire genes despite the organism having lost its natural competence and become host-restricted. The lifestyle of S. equi within the horse is defined by short-term acute disease, strangles, followed by long-term infection. Population analysis reveals evidence of convergent evolution in isolates from post-acute disease samples as a result of niche adaptation to persistent infection within a host. Mutations that lead to metabolic streamlining and the loss of virulence determinants are more frequently found in persistent isolates, suggesting that the pathogenic potential of S. equi reduces as a consequence of long-term residency within the horse post-acute disease. An example of this is the deletion of the equibactin siderophore locus that is associated with iron acquisition, which occurs exclusively in persistent isolates, and renders S. equi significantly less able to cause acute disease in the natural host. We identify several loci that may similarly be required for the full virulence of S. equi, directing future research toward the development of new vaccines against this host-restricted pathogen.

Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS.

Streptococcus equi includes very important animal and human pathogens. S. equi subsp. equi (SEE) is a highly pathogenic equine specific subspecies, while S. equi subsp. zooepidemicus (SEZ) and S. equi subsp. ruminatorum are opportunistic pathogens of various animal species and humans. Due to great phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies in horses. Superspectra created in this study enabled correct identification of 86 strains belonging to different subspecies of S. equi, isolated from various hosts, infection sites and years. In general, higher average identification accuracy was achieved for SEE (99.0±3.0%) than for SEZ (93.3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method with spectra analyses using the SARAMIS database. Additionally, first results on subtyping of SEZ indicated that a more refined discrimination, for example for epidemiological surveys, may be possible.

Update on Streptococcus equi subsp equi infections.

There are few diseases that ignite as much fervor among horse owners as strangles. Streptococcus equi subsp equi (strangles) infections frequently require the treating veterinarian to manage not only the clinical cases but also the biosecurity and provision of information to all involved parties. Although the disease is typically characterized by low mortality and high morbidity, restrictions of horse movement that result from appropriate quarantine procedures often frustrate the involved parties. The aims of this article are to provide clinically relevant information for diagnosis, treatment, and biosecurity management of strangles infection.

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

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.

Illustration of the difficulty of identifying Streptococcus equi strains at the subspecies level through a case of endocarditis in an immunocompetent man.

We report a case of endocarditis caused by Streptococcus equi in an immunocompetent patient who was subsequently cured after appropriate antibiotherapy and cardiac surgery. However, it was challenging to identify the strain to the subspecies level, which highlights the necessity of developing reliable molecular tools to discriminate between the subspecies.

Evidence of lateral gene transfer among strains of Streptococcus zooepidemicus in weanling horses with respiratory disease.

Streptococcus zooepidemicus (Sz) is a tonsillar commensal of healthy horses but with potential to opportunistically invade the lower respiratory tract. Sz is genetically variable and recombinogenic based on analysis of gene sequences including szp, szm and MLST data. Although a variety of serovars of the protective SzP are commonly harbored in the tonsils of the same horse, lower respiratory infections usually involve a single clone. Nevertheless, isolation of specific clones from epizootics of respiratory disease has been recently reported in horses and dogs in N. America, Europe and Asia. In this report, we provide evidence suggestive of lateral gene exchange and recombination between strains of Sz from cases of respiratory disease secondary to experimental equine herpes 1 virus infection in an isolated group of weanling horses and ponies. Nasal swabs of 13 of 18 weanlings with respiratory disease yielded mucoid colonies of Sz following culture. Comparison of arcC, nrdE, proS, spi, tdk, tpi and yqiL of these Sz revealed 3 Clades. Clade-1 (ST-212) and 2 (ST-24) were composed of 7 and 3 isolates, respectively. ST-24 and 212 differed in all 7 housekeeping as well as szp and szm alleles. Two isolates of Clade-1 were assigned to ST-308, a single locus variant of ST-212 that contained the proS-16 allele sequenced in ST-24. One isolate of ST-308 contained szm-2, the same allele sequenced in Clade 2 isolates; the other was positive for the szp-N2HV2 allele of Clade 2. These observations are consistent with gene transfer between Sz in the natural host and may explain formation of novel clones that invade the lower respiratory tract or cause epizootics of respiratory disease in dogs and horses.

The molecular identification of Streptococcus equi subsp. equi strains isolated within New Zealand.

AIMS: To identify Streptococcus equi subsp. equi (S. equi) by PCR analysis and obtain isolates by culture, in order to investigate the strains of S. equi infecting horses within New Zealand. METHODS: A diagnostic PCR, based on the amplification of the seeI gene for S. equi, was used on 168 samples submitted from horses with and without clinical signs of strangles. Samples were also processed and cultured on selective media for the isolation of ß-haemolytic colonies. In addition, the hypervariable region of the seM gene of S. equi was amplified and then sequenced for strain typing purposes. RESULTS: Of the 168 samples, 35 tested positive for S. equi using PCR. Thirty-two confirmed samples were from horses with a clinical diagnosis of strangles and three were from horses where clinical information was unavailable. Only 22/35 (63%) confirmed S. equi samples were successfully isolated following culture. Strain typing demonstrated that two novel seM alleles of S. equi were found in New Zealand with SeM-99 strains being restricted to the North Island while SeM-100 strains were found in both North and South Islands. CONCLUSIONS: The application of PCR for the laboratory confirmation of strangles allowed for a rapid and sensitive identification of S. equi. Moreover, seM typing revealed that within the samples examined two strains of S. equi co-circulated within the North Island of New Zealand but only one strain in the South Island. CLINICAL RELEVANCE: PCR reduces the time required to obtain laboratory confirmation of strangles compared with culture methods. It also has greater sensitivity in detecting S. equi infections, which is of particular importance in the detection of carrier animals which normally shed low numbers of bacteria. Additionally, seM molecular typing can differentiate between bacterial strains, assisting in the monitoring of local strains of S. equi subsp. equi causing disease.

Development of a real-time PCR to detect Streptococcus equi subspecies equi.

REASONS FOR PERFORMING STUDY: Infection with Streptococcus equi subspecies equi (S. equi) is endemic in the UK. A proportion of horses serve as long-term carriers and act as a reservoir of infection. Detection of these persistently infected horses is difficult using standard culture techniques owing to a lack of sensitivity and overgrowth by contaminating bacteria. In addition, differentiation of this causative bacterium from the closely related S. equi zooepidemicus has made the development of reliable and accurate diagnostic tests difficult. OBJECTIVE: To develop and validate a sensitive and specific real-time PCR assay to detect S. equi and to compare the results with traditional culture techniques. STUDY DESIGN: Retrospective cross-sectional study. METHODS: The assay was validated using a panel of 92 samples from suspected clinical cases of strangles. These were cultured using microbial techniques and tested using the S. equi real-time PCR. The results of the 2 methods were compared, and the diagnostic sensitivity and specificity of the real-time PCR were calculated. The real-time PCR was tested for cross-reactivity with horse commensal bacteria, and the efficiencies and limits of detection were established. RESULTS: The assay had a diagnostic sensitivity of 95% and specificity of 86%. No cross-reactivity was observed with any of the bacterial species tested, including S. equi zooepidemicus. The assay detected as few as 3 gene copies. CONCLUSION: The assay is fast, sensitive and specific and will detect S. equi DNA directly from a crude extract of clinical material on a swab. POTENTIAL RELEVANCE: This assay could aid in the rapid detection of subclinical shedders of S. equi, enabling quicker treatment and helping to limit the spread of strangles in equine populations.