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Strangles, caused by Streptococcus equi subspecies equi , is a highly infectious disease of equines causing major health issues and financial losses. The aim of the study was to detect the presence of the SeM gene in Streptococcus equi isolated from equine suspected of having strangles. A cross-sectional study design was conducted from July to December 2022 in five districts of the central Gondar zone, Ethiopia. One-hundred sixty swab samples were taken from animals that had been clinically suspected. The SeM gene was detected using polymerase chain reaction, and the antimicrobial susceptibility test was performed using the Kirby-Bauer disc diffusion method. The binary logistic regression model was employed to test for statistical significance. In 31.87% (51/160) of the samples, Streptococcus equi species were isolated, and 31.37% (16/51) of these species carried the SeM gene. There was a significant amount of tetracycline (81.5%), erythromycin (81.5%), and vancomycin (75.5%) resistance among the 16 isolates. Strangles were more likely to be present in animals who shared feed containers (AOR = 7.59; 95% CI = 1.44–39.93), drank from the same water troughs (AOR = 7.74; 95% CI = 1.44–41.01), and spent the night together (AOR = 5.97; 95% CI 1.41–25.37). The findings of this study showed that the research areas harboured Streptococcus equi subspecies equi . Sharing feed containers and water troughs were potential sources of strangles infection; thus, these containers need to be cleaned regularly.

Strangles, a highly contagious disease caused by Streptococcus equi subspecies equi ( S.equi ), significantly impacts horse populations worldwide, with Iceland as the only exception. This disease poses serious threats to equine health and results in considerable economic losses. Consequently, the accurate, sensitive, and rapid detection of S.equi from clinical samples is essential for early warning and effective disease management. This study introduces a novel detection method that integrates recombinase-aided amplification (RAA) with CRISPR/Cas12a technologies. We specifically designed RAA primers and CRISPR RNA to target the eqbE gene of S.equi , and we have carefully optimized the reaction systems for this purpose. The newly established visual diagnostic method has shown to be highly effective, demonstrating 97.14% specificity and 100% sensitivity, with the capability to detect as few as 5.6×10 0 copies of the target. This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi . This is the first study to propose the combined application of RAA and CRISPR/Cas12a for the on-site rapid detection of S.equi , which enables visual point-of-care diagnosis of Strangles.

Hyaluronic acid (HA) based biomaterials have several biomedical applications. HA biosynthesis is catalysed by hyaluronan synthase (HAS). The unavailability of 3-D structure of HAS and gaps in molecular understanding of HA biosynthesis process pose challenges in rational engineering of HAS to control HA molecular weight and titer. Using in-silico approaches integrated with mutation studies, we define a dictionary of sub-structural elements (SSE) of the Class I Streptococcal HAS (SeHAS) to guide rational engineering. Our study identifies 9 SSE in HAS and elucidates their role in substrate and polymer binding and polymer biosynthesis. Molecular modelling and docking assessment indicate a single binding site for two UDP-substrates implying conformationally-driven alternating substrate specificities for this class of enzymes. This is the first report hypothesizing the involvement of sites from SSE5 in polymer binding. Mutation at these sites influence HA production, indicating a tight coupling of polymer binding and synthase functions. Mutation studies show dispensable role of Lys-139 in substrate binding and a key role of Gln-248 and Thr-283 in HA biosynthesis. Based on the functional architecture in SeHAS, we propose a plausible three-step polymer extension model from its reducing end. Together, these results open new avenues for rational engineering of Class I HAS to study and regulate its functional properties and enhanced understanding of glycosyltransferases and processive enzymes.

The objective of this study was to determine the prevalence of Streptococcus equi (S. equi subsp equi and S. equi subsp zooepidemicus) in the state of Santa Catarina and evaluate the antimicrobial susceptibility of the isolates. For this, 420 nasal swab samples were collected from randomly selected horses. Isolation and phenotypic characterization of the bacteria were performed by sowing on 5% sheep blood agar, followed by analysis of morphotinctorial characteristics and biochemical analysis. To differentiate the main beta-hemolytic Streptococcus in horses, the fermentation profiles of the sugar’s lactose, maltose, sorbitol, and trehalose were used, which were confirmed at the subspecies level by the PCR technique. The antimicrobial susceptibility panel was defined by the disk diffusion method, testing 13 antimicrobials from ten different classes, all regularly used in equine medical clinics, followed by the calculation of the multiple antimicrobial resistance index. Ten strains of S. equi were isolated, with a prevalence of 2.38% (10/420). Of the total positive samples, 3% (3/10) were confirmed as belonging to S. equi subsp equi and 70% (7/10) were confirmed as belonging to S. zooepidemicus. Multidrug resistance was observed in 60% (6/10) of isolates. The antimicrobial with the greatest resistance was clindamycin with 70% (7/10), followed by beta-lactams, with 40% (4/10) resistance to penicillin and 30% (3/10) to ceftiofur. The isolates were 100% (10/10) sensitive to gentamicin, chloramphenicol, levofloxacin, and vancomycin. This was the first study carried out in the state, and based on these data, it can be said that Santa Catarina has a low prevalence of S. equi and the presence of multi-resistant strains of S. equi was confirmed in the equine herd in Santa Catarina.

The continued evolution of bacterial pathogens has major implications for both human and animal disease, but the exchange of genetic material between host-restricted pathogens is rarely considered. Streptococcus equi subspecies equi (S. equi) is a host-restricted pathogen of horses that has evolved from the zoonotic pathogen Streptococcus equi subspecies zooepidemicus (S. zooepidemicus). These pathogens share approximately 80% genome sequence identity with the important human pathogen Streptococcus pyogenes. We sequenced and compared the genomes of S. equi 4047 and S. zooepidemicus H70 and screened S. equi and S. zooepidemicus strains from around the world to uncover evidence of the genetic events that have shaped the evolution of the S. equi genome and led to its emergence as a host-restricted pathogen. Our analysis provides evidence of functional loss due to mutation and deletion, coupled with pathogenic specialization through the acquisition of bacteriophage encoding a phospholipase A(2) toxin, and four superantigens, and an integrative conjugative element carrying a novel iron acquisition system with similarity to the high pathogenicity island of Yersinia pestis. We also highlight that S. equi, S. zooepidemicus, and S. pyogenes share a common phage pool that enhances cross-species pathogen evolution. We conclude that the complex interplay of functional loss, pathogenic specialization, and genetic exchange between S. equi, S. zooepidemicus, and S. pyogenes continues to influence the evolution of these important streptococci.

•Strangles infection caused by Streptococcus equi subspecies equi (S. equi), is an important equine respiratory infection.•Vaccine strains (Pinnacle I.N.) sometimes can cause infection and hence differentiation of strains is important.•This study describes the differentiation of vaccine from wild type strains using pyrosequencing rapidly with a single nucleotide polymorphism upstream of the SzPSe gene. 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.

Direct interaction between pathogens and host cells often is a prerequisite for colonization, infection and dissemination. Regulated production of capsular polysaccharide (CPS), which is made of hyaluronic acid, is essential for the pathogenicity of Streptococcus equi subsp. Zooepidemicus (SEZ). Here, we constructed a CPS-deleted mutant and analyzed it along with the parental wild-type strain in attachment and invasion of mammalian epithelial and endothelial cell lines. The CPS-deleted mutant exhibited significant increase in adherence and invasion by several orders of magnitude compared with the wild-type strain through quantitative analysis and electron microscopy observation. After the wild-type strain was recovered from invaded cells, its morphology was analyzed by visual methods and scanning electron microscopy, which revealed that its capsule was almost completely absent. Capsule measurements showed a similar result in which CPS production was nearly attenuated to the same extent as in the CPS-deleted mutant. qPCR assays revealed a marked reduction in the transcriptional levels of the CPS biosynthesis genes, has operon. Moreover, the repression in capsular production was stable inheritance. Our findings indicate that SEZ is a facultative intracellular bacterium, capsule attenuation in SEZ contributes to attachment and invasion in interactions with host cells, and the active regulation of capsule breakdown is controlled by SEZ during internalization. Capsule breakdown in cell invasion by Streptococcus equi subsp. zooepidemicus.

Streptococcus equi subsp. zooepidemicus (SEZ) is an opportunistic pathogen of both humans and animals. Quorum sensing (QS) plays an important role in the regulation of bacterial group behaviors. The aim of this study was to characterize the LuxS in SEZ and evaluate its impact on biofilm formation, pathogenesis and gene expression. The wild-type SEZ and its LuxS mutant (Δ luxS ) were examined for growth, biofilm formation, virulence factors, and transcriptomic profiles. Our results showed that LuxS deficiency did not affect SEZ hemolytic activity, adhesion or capsule production. For biofilm assay demonstrated that mutation in the luxS gene significantly enhances biofilm formation, produced a denser biofilm and attached to a glass surface. RAW264.7 cell infection indicated that Δ luxS promoted macrophage apoptosis and pro-inflammatory responses. In mice infection, there was no significant difference in mortality between SEZ and Δ luxS . However, the bacterial load in the spleen of mice infected with Δ luxS was significantly higher than in those infected with SEZ. And the pathological analysis further indicated that spleen damage was more severe in the Δ luxS group. Moreover, transcriptomics analysis revealed significant alterations in carbon metabolism, RNA binding and stress response genes in Δ luxS . In summary, this study provides the first evidence of AI-2/LuxS QS system in SEZ and reveals its regulatory effects on biofilm formation, pathogenicity and gene expression.

This consensus statement update reflects our current published knowledge and opinion about clinical signs, pathogenesis, epidemiology, treatment, complications, and control of strangles. This updated statement emphasizes varying presentations in the context of existing underlying immunity and carrier states of strangles in the transmission of disease. The statement redefines the “gold standard” for detection of possible infection and reviews the new technologies available in polymerase chain reaction diagnosis and serology and their use in outbreak control and prevention. We reiterate the importance of judicious use of antibiotics in horses with strangles. This updated consensus statement reviews current vaccine technology and the importance of linking vaccination with currently advocated disease control and prevention programs to facilitate the eradication of endemic infections while safely maintaining herd immunity. Differentiation between immune responses to primary and repeated exposure of subclinically infected animals and responses induced by vaccination is also addressed.

Comparison of Streptococcus zooepidemicus ( S. zooepidemicus ) as a natural strain for hyaluronic acid (HA) production with Corynebacterium glutamicum ( C. glutamicum ) as a recombinant host is a valuable tool to identify key metabolic targets for enhanced HA production. For this purpose, a comparative analysis based on the genome-scale metabolic networks of native and recombinant HA producers was conducted. In this study, the first genome-scale metabolic model (GEM) of S. zooepidemicus , was reconstructed and named i ZN522 . This systematically curated model was validated using various biochemical data, consisting of growth rate prediction, amino acids auxotrophy, and different carbon sources consumption. Following the validation, key genes for HA overproduction in both bacteria were identified through iterative single-gene deletion analysis. Comparing optimal flux distributions predicted by models of S. zooepidemicus strain with recombinant C. glutamicum revealed that removing the oxidative pentose-phosphate pathway in C. glutamicum effectively increases the hyaluronan synthase flux. Additionally, improvement strategies in C. glutamicum primarily rely on up- and down-regulation, while in S. zooepidemicus , gene deletions can optimize the HA production rate. By comparing the two metabolic models, it can be inferred that eliminating of glucose-6-phosphate dehydrogenase enzymatic reaction in C. glutamicum results in greater similarity in the central carbon metabolisms of C. glutamicum and S. zooepidemicus , thereby enhancing HA production.