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.

STREPTOCOCCUS EQUI SSP. ZOOEPIDEMICUS MENINGOENCEPHALOMYELITIS IN A SOUTHERN TAMANDUA (TAMANDUA TETRADACTYLA) ASSOCIATED WITH A RAW HORSEMEAT DIET.

A 1-y-old female southern tamandua (Tamandua tetradactyla) presented with vomiting, hyporexia, and neurologic signs. Magnetic resonance imaging revealed contrast-enhancing material within the lateral and fourth ventricles and a T2 hyperintense cerebellar lesion, consistent with meningoencephalitis. The tamandua rapidly declined and was euthanatized. On gross postmortem exam, the tamandua had diffusely injected leptomeninges, opaque fluid in the fourth ventricle, and subdural brainstem and spinal cord hemorrhage. Histologically, there was regionally hemorrhagic and multifocal fibrinosuppurative meningoencephalomyelitis, ventriculitis, choroid plexitis, cerebellar folia necrosis, ependymitis, radiculoneuritis, and abundant intralesional gram-positive cocci. Streptococcus equi ssp. zooepidemicus was cultured from brain, cardiac blood clot, and multiple samples of horsemeat collected from the animal's diet. This is the first report of streptococcal meningoencephalomyelitis in a southern tamandua. The route of infection was likely gastrointestinal inoculation, which may have implications for the routine practice of feeding diets containing raw meat to insectivores.

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.

Glomerulonefritis aguda postinfecciosa por Streptococcus equi en paciente pediátrico / Acute post infectious glomerulonephritis by Streptococcus equi in a pediatric patient

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Infección diseminada por Streptococcus equi subespecie zooepidermicus en un paciente inmunocompetente / Disseminated Streptococcus equi subspecies zooepidermicus infection in an immunocompetent patient

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Diagnosis, Surveillance and Management of Streptococcus equi subspecies zooepidemicus Infections in Chinchillas (Chinchilla lanigera).

During a 6-mo period, two 5-6 mo old female chinchillas (Chinchilla lanigera) were examined at the University of Colorado Anschutz Medical Campus after the discovery of firm, nonmobile masses in the left ventral cervical and left axillary region. Other than these findings and mild weight loss, both chinchillas' physical exams were normal. Bloodwork revealed an inflammatory leukogram characterized by leukocytosis, toxic neutrophils, lymphopenia, and monocytosis with mild, nonregenerative anemia. At necropsy, both masses were identified as abscesses. Streptococcus equi, subspecies zooepidemicus (S. zooepidemicus) was isolated in pure culture. Histology of the lungs, liver, spleen, and kidneys showed a marked increase in the numbers of both polymorphonuclear leukocytes and lymphocytes. Both animals were deemed unsuitable for research and were euthanized under isoflurane anesthesia by an intracardiac injection of pentobarbital sodium solution. S. zooepidemicus is an opportunistic, commensal organism found in the upper respiratory tract of horses. This organism has been documented to cause disease in other species and is zoonotic. Infections in humans have been reported, resulting in glomerulonephritis, endocarditis, septic arthritis, osteomyelitis, meningitis, and death. To aid in diagnosis and prospective surveillance of this bacteria, oral and nasal swabs were collected from the remaining cohort of chinchillas, and a qPCR screening assay was implemented. Within 12 mo, 4 of 41 additional females tested positive by culture or qPCR, resulting in a disease prevalence of 14% (6 of 43). However, only 2 of the additional 4 S. zooepidemicus positive animals developed clinical signs. The potential for the spread of infection, zoonosis, and adverse effects on research demonstrate that surveillance for S. zooepidemicus should be considered in a biomedical research environment.

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.

Disseminated septic arthritis caused by Streptococcus equi infection.

We describe a rare case of bloodstream infection and disseminated septic arthritis in a relatively fit and well 73-year-old retired farmer and gamekeeper, due to the zoonotic organism Streptococcus equi The presence of the organism in multiple joints led to slow clinical response to treatment and was complicated by relapse of infection and lengthy disability. Source control was achieved with multiple joint washouts and spinal cord decompression. Following this, a 6-week course of intravenous antibiotics was required for complete clearance of infection. After a long period of rehabilitation, the patient made a good recovery. This case demonstrates that S. equi can cause life threatening and difficult to treat sepsis in humans and requires a high index of suspicion in people who have regular contact with equine species, cattle and unpasteurised milk.

SpeS: A Novel Superantigen and Its Potential as a Vaccine Adjuvant against Strangles.

Bacterial superantigens (sAgs) are powerful activators of the immune response that trigger unspecific T cell responses accompanied by the release of proinflammatory cytokines. Streptococcus equi (S. equi) and Streptococcus zooepidemicus (S. zooepidemicus) produce sAgs that play an important role in their ability to cause disease. Strangles, caused by S. equi, is one of the most common infectious diseases of horses worldwide. Here, we report the identification of a new sAg of S. zooepidemicus, SpeS, and show that mutation of the putative T cell receptor (TCR)-binding motif (YAY to IAY) abrogated TCR-binding, whilst maintaining interaction with major histocompatibility complex (MHC) class II molecules. The fusion of SpeS and SpeSY39I to six S. equi surface proteins using two different peptide linkers was conducted to determine if MHC class II-binding properties were maintained. Proliferation assays, qPCR and flow cytometry analysis showed that SpeSY39I and its fusion proteins induced less mitogenic activity and interferon gamma expression when compared to SpeS, whilst retaining Antigen-Presenting Cell (APC)-binding properties. Our data suggest that SpeSY39I-surface protein fusions could be used to direct vaccine antigens towards antigen-presenting cells in vivo with the potential to enhance antigen presentation and improve immune responses.

Genetic characterization of Streptococcus equi subspecies zooepidemicus associated with high swine mortality in the United States.

High mortality events due to Streptococcus equi subspecies zooepidemicus (Streptococcus zooepidemicus) in swine have not previously been reported in the United States. In September and October 2019, outbreaks with swine mortality up to 50% due to S. zooepidemicus septicaemia were reported in Ohio and Tennessee. Genomic epidemiological analysis revealed that the eight outbreak isolates were clustered together with ATCC 35246, a Chinese strain caused outbreaks with high mortality, also closely related to three isolates from human cases from Virginia, but significantly different from an outbreak-unrelated swine isolate from Arizona and most isolates from other animal species. Comparative genomic analysis on two outbreak isolates and another outbreak-unrelated isolate identified several genomic islands and virulence genes specifically in the outbreak isolates only, which are likely associated with the high mortality observed in the swine population. These findings have implications for understanding, tracking and possibly preventing diseases caused by S. zooepidemicus in swine.

Smartphone-based multiplex 30-minute nucleic acid test of live virus from nasal swab extract.

Rapid, sensitive and specific detection and reporting of infectious pathogens is important for patient management and epidemic surveillance. We demonstrated a point-of-care system integrated with a smartphone for detecting live virus from nasal swab media, using a panel of equine respiratory infectious diseases as a model system for corresponding human diseases such as COVID-19. Specific nucleic acid sequences of five pathogens were amplified by loop-mediated isothermal amplification on a microfluidic chip and detected at the end of reactions by the smartphone. Pathogen-spiked horse nasal swab samples were correctly diagnosed using our system, with a limit of detection comparable to that of the traditional lab-based test, polymerase chain reaction, with results achieved in ∼30 minutes.

Novel Streptococcus equi strains causing strangles outbreaks in Arabian horses in Egypt.

Strangles displays a major challenge to veterinary medicine worldwide. However, no data on Streptococcus equi subsp. equi (S. equi) M protein alleles have been reported so far from Arabian horses. We report here for the first time the S. equi SeM alleles causing strangles in Arabian horses, and the associated risk factors for the disease. Duplicate samples from one hundred Arabian horses with acute strangles in confirmed outbreaks and sporadic cases were analysed by phenotypic methods and multiplex polymerase chain reaction (PCR) targeting streptokinase precursor, seeI and sodA genes. PCR and sequencing of S. equi SeM gene were employed for strains typing, and the four superantigens were determined among the allelic variants. Direct-sample PCR confirmed and highly positively correlated (r = .85) with the phenotypic results, and detected S. equi in five samples more than the conventional culture. A combination of multiplex PCR from samples and culture could successfully identify S. equi (92%), S. zooepidemicus (5%) and S. equisimilis (3%). SeM typing demonstrated five SeM alleles, including four previously unidentified alleles that were deposited in the PubMLST-SeM database. SeM-139 and SeM-141 are related to some strains that were recently recovered from donkeys in China. SeM-140 and SeM-199 are related to a group of alleles from horses in Europe. Variation in the presence of seeM, seeH and seeL superantigens was found across the four novel alleles without interference with the severity of strangles and clinical presentation seen in different outbreaks. Horse age was the most important factor in developing strangles, followed by seasonality and the diagnosis of strangles in the previous year. These new findings comprise a significant contribution to the horse industry through the identification of novel S. equi SeM types that may bolster measures for strangles control as the identified SeM alleles will certainly help in the development of SeM-containing vaccine.

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.

Streptococcus equi subsp. zooepidemicus and 'Neighbourhood syndrome' -extra-orbitocranial rhinosinusitis with reversible sudden loss of vision- a case report.

BACKGROUND: Rhinosinusitis is a common condition which may present with complications commonly involving the orbit and the intracranial space. Loss of vision in the absence of clinical or radiological signs of involvement of the orbit and intracranium is rare and carries a high morbidity rate. Streptococcus equi subspecies zooepidemicus is not a well-documented cause of paranasal sinus infection. CASE PRESENTATION: We present a case of a 16 year old female patient who had unilateral loss of vision with signs of retrobulbar optic neuritis and no other neurological signs. We isolated an unusual organism- Streptococcus equi subspecies zooepidemicus from the maxillary sinus. Emergency endoscopic sinus surgery and antibiotic treatment resulted in complete reversal of the loss of vision. CONCLUSION: The presence of paranasal sinus disease in association with loss of vision even in the absence of a clear link between the two should be treated as an emergency with surgical drainage and or appropriate antibiotic therapy. In patients presenting with suspected inflammatory orbital involvement, imaging of the orbit and paranasal sinuses should be considered early.

Genetic analysis of Streptococcus equi subsp. equi isolated from horses imported into Japan.

Strangles is a commonly diagnosed and important infectious disease of equids worldwide, caused by Streptococcus equi subsp. equi. We determined the SeM genotypes of S. equi isolated from imported horses at the Japanese border within the past 8 years, which allowed us to classify 12 strains isolated from these horses from each exporter into four allelic groups. These alleles were different from the alleles of past isolates found in Japan. Furthermore, four strains classified into the same allele were isolated from horses from one exporter over several years. In this study, S. equi isolates from different exporters had different SeM alleles. Attention to the hygiene status of farms will be necessary to prevent the incursion of strangles.

Long term silent carriers of Streptococcus equi ssp. equi following strangles; carrier detection related to sampling site of collection and culture versus qPCR.

After strangles outbreaks, Streptococcus equi ssp. equi (S. equi) can persist in clinically normal silent carriers for months to years. Two naturally occurring outbreaks of strangles with 53 and 100% morbidity, respectively, were followed longitudinally to assess occurrence of carrier state and optimal detection methods Outbreak A involved 98 yearling warmbloods, and outbreak B 38 mature Icelandic horses. Fully recovered horses were sampled at least 6 months after index cases using nasal swabs (one sampling occasion only) nasopharyngeal lavage and guttural pouch visualisation and lavages for culture and qPCR to S. equi. Any horse with at least a single sample positive was deemed a carrier. Descriptive statistics and sensitivity and negative predictive values were calculated. Comparisons were made with McNemars and Fishers exact tests. Carrier rates in outbreak A were 3% based on culture and 15% based on qPCR and for outbreak B 13% based on culture and 37% based on qPCR. All culture positives were also qPCR positive. One carrier culture negative sampled after an additional 8 months was culture positive to S. equi, indicating that qPCR positives should be suspected to carry live bacteria. Findings indicate that reliance on guttural pouch sampling and appearance does not capture all silent carriers. All culture positives were identified by qPCR and even horses positive by qPCR but culture negative should be suspected carriers of live bacteria.

Zoonotic Endocarditis in a Man, the Netherlands.

In 2017, endocarditis caused by Streptococcus equi subspecies zooepidemicus was diagnosed in a man in the Netherlands who had daily contact with horses. Whole-genome sequencing of isolates from the man and his horses confirmed the same clone, indicating horse-to-human transmission. Systematic reporting of all zoonotic cases would help with risk assessment.

Denaturing Gradient Gel Electrophoresis Analysis of Bacteria in Italian Ticks and First Detection of Streptococcus equi in Rhipicephalus bursa from the Lazio Region.

Tick-borne diseases are an increasing problem for the community. Ticks harbor a complex microbial population acquired while feeding on a variety of animals. Profiling the bacterial population by 16S rDNA amplification and denaturing gradient gel electrophoresis enables detection of the broad spectrum of bacteria that settles in the ticks. This study identified known and unknown tick-infecting bacteria in samples from Italy. Seven adult ticks from different hosts and origins were analyzed: two Rhipicephalus sanguineus ticks from dogs (Lombardia), two Rhipicephalus bursa ticks from bovines (Lazio), and three Ixodes ricinus ticks from humans (Marche). The major result was the first report of the zoonotic agent Streptococcus equi in ticks. S. equi is a species complex of highly contagious pathogens. Subsequent to S. equi detection in a R. bursa tick removed from a bovine of Lazio in 2012, we studied 95 R. bursa samples collected from 3 bovines, 3 ponies, and 1 sheep grazing in the same area in 2012 and from 6 ponies grazing there in 2017. The results of a specific PCR assay indicated a not sporadic occurrence of S. equi in ticks. This finding provides a basis for assessing the potential of ticks to harbor and disperse S. equi.

Long term dynamics of a Streptococcus equi ssp equi outbreak, assessed by qPCR and culture and seM sequencing in silent carriers of strangles.

The aim of the study was to use culture, qPCR and seM sequencing to map Streptococcus equi subspec. equi (S.equi) isolates in long term carrier animals. A strangles outbreak affecting 41 Icelandic horses was followed to determine strangles free status using nasal and/or guttural pouch lavages collected serially on eleven separate occasions over 13 months. Ten persistent carriers, of which eight had repeated culture positive samples for S. equi, were selected for the study. Of 115 samples collected, 61 were S. equi positive on qPCR; from which 32 were also culture positive. Amplification of parts of the gene encoding the M-protein seM was performed on isolated colony material (n = 32) or, where only PCR product was obtained, directly on the DNA sample (n = 29) with a nested amplification approach. The seM sequence could be determined for six of the 29 samples that were solely qPCR positive. The outbreak was due to a S. equi strain of seM type 72. Three months after initial sampling isolates from two horses had seM gene sequences with one amino acid change. After six months S. equi with truncated seM genes were found in two horses; one variant in a single horse once, and in the other horse a variant that persisted and that was later identified in two additional horses. Non- mucoid S. equi colonies were found in two horses. Importantly, after acute strangles outbreaks many horses not only remain persistently qPCR positive for S. equi but are also intermittently culture positive.

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.