Ascending placentitis is a significant cause of equine pregnancy loss, yet the upstream inflammatory triggers are poorly defined. Recently, we identified S100A8/S100A9 (S100A8/A9) alarmins as potential upstream regulators in a chronic equine placentitis model. The current study aimed to determine whether this upregulation is sustained in the acute model and in clinical cases, and to elucidate the expression of their downstream inflammatory mediators. Using an experimental model, we quantified S100A8/A9 mRNA expression in acute (n = 5) and chronic (n = 6) placentitis induced by Streptococcus equi ssp. zooepidemicus. We found mRNA expression of S100A8 and S100A9 was significantly upregulated in chorioallantois during both acute (p < 0.001) and chronic (p < 0.0001) disease compared to controls (n = 5), demonstrating their role is not limited to chronic pathology. A strong positive correlation (r = 0.945) underscored their coordinated expression. Immunohistochemistry revealed minimal staining in controls but dense infiltrations of S100A8/A9-positive neutrophils and macrophages in placentitis tissues. To define the clinical relevance of the downstream pathway, we analyzed RNA sequencing data from clinical placentitis cases (placentitis, n = 4) compared to normal postpartum placenta (control, n = 4). This confirmed upregulation of S100A8/A9 and revealed a concurrent increase in their receptors (TLR4, RAGE) and a spectrum of NF-κB-driven effectors, including pro-inflammatory cytokines (IL1β, IL6, TNF), chemokines (CXCL8, CCL2, CXCL10), and the apoptotic mediator CASP3. Our findings establish that S100A8/A9 upregulation is a sustained feature of equine placentitis and delineates a coherent S100A8/A9-TLR4/RAGE-NF-κB signaling axis that drives inflammation and tissue damage in clinical disease. These findings highlight the diagnostic potential of S100A8/A9 and position this alarmin system as a promising therapeutic target for mitigating infection-induced pregnancy loss.

Streptococcus equi subspecies zooepidemicus is an opportunistic pathogen. Human infections are rare and often linked to close contact with horses. Consumption of raw milk cheese has also been incriminated. We present two cases of S. equi ssp. zooepidemicus bacteremia diagnosed within a short space of time in the same hospital.

Uterine proteomic and cytokine profiling show that Streptococcus equi subsp. zooepidemicus induces immune-metabolic dysregulation and may predispose mares to endometrosis.

Infection of susceptible horses with Streptococcus equi subspecies equi (S. equi), the causative agent of strangles, is associated with commingling. Exposure may occur among horses at equestrian events, sales, or horses moved among different equine stabling environments. Strangles can affect all horses on a farm, leading to the death of up to 10% of cases depending on their immunity status at the time of infection, the development of complications, the success of biosecurity measures, and the use of vaccination. The current retrospective study used ELISAs to measure the exposure of horses to S. equi at a farm that experienced an outbreak of strangles shortly after the introduction of a new horse on the same day that the majority of the 17 resident horses were vaccinated with Strangvac for the first time. One vaccinated horse, which subsequently tested positive for S. equi and EHV-4, developed a cough, elevated body temperature, and nasal discharge 11 days after the first vaccination. Two other horses developed fever for one day at 22 days post-first vaccination, but only one had serological evidence of exposure to S. equi. All vaccinated horses had high antibody titres to vaccine components, whilst 7 of the 17 resident horses, and the new arrival, tested seropositive for exposure to S. equi. Although 3 out of the 17 vaccinated horses developed mild signs of disease before second vaccination, serological data support the effectiveness of vaccination in resident populations of horses to minimise the risk of strangles following the introduction of a new horse.

This work utilized an ORFeome phage display platform to systematically identify antigenic epitopes produced by Streptococcus equi subspecies equi (S. equi), an important equine pathogen and the causative agent of horses strangles. Three major S. equi surface proteins were identified: a novel proline-rich repeat domain protein, a serine peptidase, and the M-like protein SeM. The proline-rich repeat protein and serine peptidase were confirmed to be immunogenic in horses with strangles, and their sequences were shown to be conserved in global S. equi genomes, in contrast to their diversity in S. equi subsp. zooepidemicus. With the well-characterized S. equi immunogenic protein SeM, this paper identified an immunogenic region outside of the reported critical IgG-binding region. This work provides novel insights to the understanding of the S. equi immunogenic proteins and provides peptide regions that could serve as vaccine candidates against S. equi or as diagnostic markers to specifically identify S. equi infections.

Strangles, caused by Streptococcus equi subspecies equi (S. equi), is a prevalent infectious disease of horses. This is the first report on the use of a new vaccine, Strangvac, in a natural outbreak of strangles. To measure the effects of Strangvac vaccination during an outbreak of strangles at a Swedish farm. Longitudinal cohort study. Healthy horses (n = 17) were vaccinated on day 0, which occurred 23 days after strangles was first confirmed in three unvaccinated horses on the same farm. Blood serum samples were collected on day 0, day 28 and day 489. A combined iELISA was used to measure total antibody titres towards the components of the vaccine. Specific antibody levels in blood sera to S. equi were also quantified using the antigen A/C iELISAs, which can differentiate infected from vaccinated animals. Clinical signs were monitored. All vaccinated horses had increased total antibody titres to CCE, Eq85 and/or IdeE from day 0 (2.50 ± 0.28) to day 28 (3.63 ± 0.31; p < 0.0001). Seropositivity in the antigen A/C iELISA was noted in 8/17 healthy horses on day 0 (prior to vaccination), in 9/16 vaccinates on day 28, and in 3/3 unvaccinated clinical cases on day 28. None of the vaccinated horses developed strangles, but 5/7 of the original vaccinated horses tested seropositive in the A/C iELISA on day 489, which was 377 days after their third vaccination. Aside from the three unvaccinated clinical cases, no unvaccinated control group was available. Despite adherence to recommended biosecurity measures, serological evidence of exposure to S. equi was demonstrated in half of the vaccinated horses. Notably, all horses vaccinated during the outbreak responded to the vaccine components and remained healthy, suggesting a protective effect of vaccination of healthy horses with Strangvac in outbreak situations.

Streptococcus equi ssp. zooepidemicus is a zoonotic bacterium known to cause severe respiratory tract infections in primates, and most documented mortality episodes occur in captivity. Here we report a mass mortality event in a wild population of long-tailed macaques (Macaca fascicularis) inhabiting an urban mosaic habitat in central Bali, Indonesia, and describe the demographic impact and clinical patterns of the outbreak. Mortality was observed in three of the nine social groups that shared overlapping ranges, and the infection spread progressively over time. Clinical signs included lethargy, respiratory distress, and locomotion disorders. Over a 2-mo period during March-May 2022, 170 carcasses were found. Demographic surveys revealed a mortality rate ranging from 16% to 53% among the three affected groups. Adult females and adult males were the most affected age-sex classes, representing 36% and 35% of the carcasses found, respectively. Necropsy findings from four individuals, combined with bacteriological culture, histopathology, qPCR, and 16S rRNA metabarcoding analysis, all suggested Streptococcus equi ssp. zooepidemicus as the most likely causative agent. Similar streptococcal outbreaks had occurred in this population in 1994 and 2012, raising concerns about the recurrent introduction of the bacterium or potential reservoirs either within or outside the macaque population. The recurrence of Streptococcus equi ssp. zooepidemicus outbreaks in this population highlights the impact of lethal bacterial epidemics in wild primates, which remain poorly documented. Strengthening long-term surveillance, including noninvasive serological monitoring, is essential for better understanding infection dynamics and improving conservation strategies.

To assess the antibacterial activity of manuka honey against bacterial isolates commonly associated with infected corneal ulcerations in horses,and to investigate possible combined effects of manuka honey and commonly prescribed topical antibiotics. Four Staphylococcus aureus, including three methicillin-resistant (MRSA), two methicillin-resistant coagulase-negative staphylococci (S. sciuri and S. haemolyticus), and two Streptococcus equi subspecies zooepidemicus isolates from horses with stromal ulcerative keratitis were selected. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of manuka honey were determined with microdilution assays and spectrophotometric analyses. Potential combined antibacterial effects were explored by assessing bacterial growth inhibition using disk diffusion and E-tests, both with and without a sub-inhibitory concentration of manuka honey. Tested antibiotics included chloramphenicol, tetracycline, gentamicin, ofloxacin, tobramycin, cloxacillin, and fusidic acid. The spectrophotometric MIC of manuka honey was 12% (w/v) for all tested isolates. MBC values ranged between 20% (w/v) and 28% (w/v) for all isolates, except S. sciuri. Combining 5% (w/v) manuka honey with tetracycline, chloramphenicol, or fusidic acid enhanced the antibacterial effect against Staphylococcus spp. (including methicillin-resistant isolates). The antibacterial effect of ofloxacin against S. aureus and streptococci and of cloxacillin against S. aureus appeared slightly reduced when combined with 5% manuka honey. No consistent difference was observed when manuka honey was combined with gentamicin or tobramycin. Manuka honey has invitro inhibitory and bactericidal activity against equine corneal surface pathogens including multi-resistant isolates. Further studies are required to assess potential synergistic and antagonistic effects of manuka honey in combination with antibiotics.

BackgroundIntravenous regional limb perfusion (IVRLP) of antibiotics is a therapeutic method used to treat distal limb infections in horses. The objectives of this study were to determine the pharmacokinetic parameters and tolerance of sodium benzylpenicillin (NaBP) administered via IVRLP; to compare the feasibility, tolerance, and efficacy of performing IVRLP on a flexed limb versus a weightbearing limb; and to predict the efficacy of IVRLP using gentamicin or NaBP. A prospective crossover study was conducted with six horses, each undergoing four phases of antibiotic infusion (gentamicin at 2.2 mg/kg vs. NaBP at 7,333 IU/kg of BP) and limb positioning (weightbearing vs. flexed). Each antibiotic administration was followed by serial synovial and blood sampling to assess antibiotic concentrations. Inflammation and sensitivity to palpation were evaluated at both the injection site and the sampled joint. A pharmacokinetic/pharmacodynamic analysis was carried out to predict the efficacy of IVRLP by estimating area under the concentration–time curve to minimum inhibitory concentration (AUC/MIC) index values for various minimum inhibitory concentrations (MICs).ResultsHigh synovial concentrations of NaBP and gentamicin were achieved, with notable variability between horses. NaBP IVRLP was well tolerated, although cephalic vein inflammation scores were significantly higher following NaBP infusion than gentamicin (p = 3.4 × 10− 11). For all horses and both antibiotics, synovial antibiotic exposures were significantly greater in weightbearing limbs than in flexed limbs (p = 0.04 and 0.02 for NaBP and gentamicin, respectively). Cephalic vein inflammation scores were also significantly lower in weightbearing limbs than in flexed limbs (p = 2.5 × 10− 5). In terms of predicted efficacy, a daily dose of 2.2 mg/kg gentamicin should be adequate to treat synovial infections involving Escherichia coli or Staphylococcus aureus, including resistant strains. For NaBP, a daily or every-other-day dose of 7,333 IU/kg of BP should be adequate to treat infections caused by Streptococcus equi and sensitive Staphylococcus aureus.ConclusionsThese findings suggest that daily IVRLP with NaBP (7,333 IU/kg of BP) could be an effective treatment for distal limb infections caused by susceptible bacteria with a MIC of ≤ 2 µg/mL, with only mild local inflammation at the injection site. IVRLP with 2.2 mg/kg gentamicin appears effective for treating infections involving susceptible bacteria with an MIC of ≤ 16 µg/mL. Performing IVRLP on a weightbearing limb resulted in higher antibiotic exposure in the metacarpophalangeal joint and better tolerance than the flexed limb method.

Prosecco grape pomace: a new feedstock for high molecular weight hyaluronic acid.

ABSTRACTMembers of the genus Streptococcus are gram‐positive bacteria, commonly found in humans and animals. This group of microorganisms has been associated with severe infections and the development of resistance to antibiotics and production of bacteriocins. Many class I bacteriocins, especially lanthipeptides, have been described for streptococci; however, only one circular bacteriocin has been described for the group (uberolysin). The objective of this study was to characterize streptocyclin BTW, a 64 amino acid circular bacteriocin produced by Streptococcus devriesei DSM 19639, as well as to assess the diversity of circular bacteriocins within the genus. Streptocyclin BTW displayed a broad inhibitory spectrum with pronounced activity against strains of streptococci. A colony mass spectrum of the strain revealed a 6118.75 Da mass, which matches the predicted mass from sequencing. The gene cluster is composed of five genes: a core peptide (stpA), a membrane protein (stpB), a DUF95 family protein (stpC), an ATP‐binding protein (stpD), and a putative immunity protein (stpE). Sequence Similarity Network (SSN) analysis revealed that streptocyclin BTW‐like circular bacteriocins were found in Streptococcus orisasini, Streptococcus equi, Streptococcus pneumoniae, Streptococcus pseudopneumoniae, Streptococcus mitis, and Streptococcus bouchesdurhonensis species. This is the first report of a bacteriocin produced by S. devriesei.

Background: Streptococcus equi subspecies equi (S. equi) is the cause of strangles, one of the most prevalent diseases of horses worldwide. The disease is characterised by fever and the formation of abscesses in the lymph nodes of the head and neck, which can restrict the airway. A multicomponent subunit vaccine, Strangvac, has been shown to effectively reduce clinical signs of strangles and to reduce its incidence. Objective: The aim of this study was to determine the immune response against the immunoglobulin-cleaving endopeptidase IdeE, a key protective component within the vaccine and the ability of antibodies to neutralize the proteolytic activity of IdeE. Methods: An in vitro assay was developed to measure the functional inhibition of recombinant IdeE by horse sera pre- and post-vaccination. The IdeE-neutralising titres were compared to the corresponding IdeE-specific antibody titres measured by iELISA (indirect Enzyme-Linked Immunosorbent Assay). Results: A significant IdeE-specific antibody response in blood serum collected from ponies was induced after Strangvac vaccinations. Concomitantly, significant increases in the neutralising activity of IdeE occurred, persisting for at least 12 months post-second vaccination. IdeE-neutralising activity was further increased significantly after a third vaccination, even when the third dose was administered 12 months after the second dose, demonstrating that immunological memory to the vaccine persisted for 12 months. There was a significant correlation between the IdeE-neutralising activity of blood sera and the level of IdeE-specific antibodies. Conclusions: These data provide insights into one potential mechanism by which this vaccine protects Equids against or during S. equi infection.

Antimicrobial resistance is a major global health issue requiring a coordinated response. This study investigated for the first time the prevalence, antimicrobial resistance phenotypes of bacteria causing infections in Arabian horses, and the potential of bacteriophage therapy for wound treatment. One hundred clinical samples from infected Arabian horses, presenting respiratory disorders, diarrhea, abortion, wound, and ocular infection, were examined using direct sample multiplex PCR and phenotypic methods. Antimicrobial susceptibility testing of the recovered isolates was performed using panels of 37 antibiotics and broth microdilution method. Bacteriophages were isolated from horse manure. A bacteriophage cocktail was used for treating infected wounds in Arabian horses. Streptococcus equi was the most predominant pathogen isolated from respiratory infections (17/29, 58.6%), followed by Klebsiella pneumoniae and Pseudomonas aeruginosa (9/29, 31.03%, each), and Escherichia coli (7/29, 24.13%). Staphylococcus aureus and Corynebacterium ovis biovar equi were the most frequently isolated bacteria from pyogenic infections. All isolated bacteria showed resistance to multiple antibiotics. Streptococcus spp. exhibited extensive drug resistance (XDR) with complete resistance to amoxicillin-clavulanic acid, amikacin, kanamycin, streptomycin, and cefotaxime. All Staphylococcus spp. displayed multidrug resistance (MDR) phenotype. Staphylococci isolates were highly resistant to fusidic acid, β-lactams, and tetracyclines. Amoxicillin-clavulanic acid, fosfomycin, and cephalosporines were ineffective against Enterobacteriaceae isolates. Ticarcillin, clavulanic acid, and colistin were ineffective against P. aeruginosa and Acinetobacter baumannii. Pan-drug-resistant (PDR) P. aeruginosa isolate was detected in the infected wound. Two lytic bacteriophages (vB_Pae_LP125 and vB_Pae_LS225) from the Podoviridea and Siphoviridea families were isolated from the horse manure. Both phages were stable across various temperatures and pH levels. In vitro tests showed significant lytic activity against a wide range of bacterial strains. The DNA genomes of all phages displayed distinctive restriction fragment length polymorphism. A bacteriophage cocktail (vB_Pae_LP125 and vB_Pae_LS225), when combined with gentamicin, improved wound healing in infected horses. There were significant differences (p < 0.05) in the wound closure % among the gentamicin group and phage cocktaoil+gentamicin groups on days 3, 5, 7, 10, and 14. This study highlights the widespread antibiotic resistance in bacteria infecting Arabian horses and posing significant challenges to equine infection management. Bacteriophage therapy shows promise as a potential treatment for wound infections.

Streptococcus equi subsp. equi (S. equi) is the etiological agent of strangles, a contagious equine disease characterized by lymph node abscess and respiratory complications. To clarify the epidemiology and virulence factors of isolates, this study demonstrated phenotypic and genotypic differences between S. equi obtained from nasal secretions and lymph node aspirates of clinical strangles cases. Additionally, circulating alleles were differentiated through sequencing of the 5' end of the seM gene. A total of 23 clinical isolates collected from horses with strangles over the past decade were analyzed for phenotypic characteristics such as colony morphology, sugar fermentation, capsule production, biofilm formation, and antimicrobial susceptibility, as well as genotypic features. The analysis revealed phenotypic variability, particularly differences in sugar metabolism and capsule expression associated with colony morphology. Most isolates exhibited weak biofilm formation and susceptibility to cephalothin, ceftiofur, and streptomycin, while resistance to tetracycline was most common. Sequencing of the N-terminal region of the seM gene identified four alleles: seM-115, seM-158, seM-270, and seM-271. Of these, only seM-115 had previously been reported in Rio Grande do Sul State (southern Brazil). Phylogenetic analysis showed distinct clustering patterns, especially among the newly detected alleles (seM-270 and seM-271). These findings highlight the importance of integrated phenotypic and genotypic analyses to understand the diversity and potential virulence of circulating S. equi strains.

Infection with Streptococcus equi subspecies equi (S. equi) is characterised by acute disease, with about 10% of infected animals remaining persistently infected. Clinically, infection with S. equi cannot readily be distinguished from infection caused by other respiratory pathogens, including Streptococcus equi subspecies zooepidemicus (S. zooepidemicus), equine influenza virus, and equine herpes virus. Screening protocols, with appropriate quarantining facilities, are important to detect horses infected with S. equi and avoid strangles outbreaks. Virulent strains of S. zooepidemicus can also cause strangles-like presentations. To evaluate the effectiveness of the screening process implemented at a UK welfare centre to prevent the introduction of strangles and strangles-like presentations. Retrospective cross-sectional study. Clinical records of 626 equids admitted to a UK welfare centre between 2017 and 2021 and from horses that developed respiratory signs after admission were reviewed. The screening protocol, which included a clinical examination, paired serology samples (iELISA) taken 6 weeks apart, and bilateral guttural pouch endoscopy to identify abnormalities such as chondroids with lavage for qPCR and culture analysis for S. equi (and often S. zooepidemicus) was implemented during this time. There were 34 screening-positive equids. Of these, 24 (3.8%) were qPCR-positive for S. equi, 8 were qPCR/culture positive for S. zooepidemicus, and 2 were qPCR/culture negative but had chondroids. Bilateral guttural pouch endoscopy, with qPCR analysis of lavage material, was an effective method of screening equids. There were no cases of strangles or strangles-like presentations within the main herds after screening and admission of new horses. Variation in the level of detail of clinical records. The screening process resulted in the identification of screening-positive equids and maintained a strangles-free herd. Further research is required to elucidate the significance of S. zooepidemicus infection in the guttural pouch.

Infectious diseases significantly impact equine health and welfare, causing illness and death, and loss of productivity globally. One such disease is 'strangles', a highly contagious upper respiratory condition in horses caused by Streptococcus equi subspecies equi (SEE). Diagnostic methods for this pathogen include sensitive molecular assays and less reliable bacterial isolation and biochemical testing. However, the presence of closely related streptococci, such as Streptococcus equi subspecies zooepidemicus (SZOO), may confound diagnosis. Rapid assays for SEE are crucial for outbreak control. This study aimed to validate species-specific loop-mediated isothermal amplification (LAMP) assays for SEE and SZOO using a portable real-time fluorometer performed in a clinical setting and to compare their performance with laboratory-based conventional API20 Strep strip and qPCR assays. Rapid sample processing methods were also evaluated with a range of clinical samples, including nasopharyngeal and guttural pouch lavage, purulent exudate, mucosal swabs and bacterial isolates. The LAMP assays demonstrated an analytical sensitivity of 50 genome copies per reaction and showed high congruence with external diagnostic methods. For SEE, the agreement was 96.55% (Kappa 0.88), while for SZOO, it was 94.87% (Kappa 0.87). When applied to rapidly processed clinical samples, SEE and SZOO LAMPs achieved agreements of 97.01% (Kappa 0.70) and 94.94% (Kappa 0.87), respectively, compared with qPCR assays. Testing in an equine clinical setting revealed a 93.84% agreement between LAMP and qPCR results, and 86.96% between LAMP and API20 Strep assays. This study highlights LAMP assays as effective, rapid diagnostic tools, offering reliable options for clinical settings and enabling appropriate and more timely biosecurity interventions.

This study presents an ordinary differential equation (ODE) based hybrid kinetic-metabolic model to predict the time evolution of biomass, glucose, hyaluronic acid (HA), and lactic acid during fermentation by Streptococcus equi subsp. zooepidemicus. The model incorporates simplified metabolic pathways and estimates the qualitative dynamics of internal, unmeasured metabolites involved in glycolysis, biomass synthesis, and HA production. Special emphasis is placed on the energetic molecules ATP/ADP, as well as the coenzymes NADH/NAD+, which are involved in redox reactions. These molecules have been shown to play regulatory roles in metabolism. The model predictions closely match the experimental data and provide insights into how varying glucose levels affect intracellular metabolic fluxes.

Streptococcus equi subsp. zooepidemicus (SEZ) is a bacterium that primarily affects horses but can also infect other animals and humans. In 2021-2022, two SEZ outbreaks occurred in Abruzzo, Italy: one in humans linked to unpasteurized cheese (37 cases) and another in donkeys (4 deaths). These events led researchers to investigate SEZ in horses, donkeys, and a mule in the regions of Abruzzo and Molise, focusing on antibiotic resistance and genetic traits. A total of 490 nasal and genital swabs were collected from equids and analyzed for SEZ presence, with 61 positive samples. Isolated strains underwent antimicrobial susceptibility testing to assess their resistance to various antibiotics. Genetic analysis was performed to detect antibiotic resistance and virulence genes. Multi-locus sequence typing (MLST) was used to compare SEZ strains and identify potential epidemiological links. The study found that 37.7% of SEZ strains were resistant to at least one antibiotic, with tetracycline and trimethoprim/sulfamethoxazole being the most common. Additionally, 8.2% of strains showed MDR. Genetic analysis identified resistance genes such as tet(W) and erm(B), along with virulence genes like Fbp54. No major genetic clusters were detected, but ST61 was identified, similar to the human outbreak. The presence of antibiotic-resistant SEZ highlights the importance of monitoring and controlling its spread under a One Health approach, considering human, animal, and environmental health.

From January 2022 to September 2022, a total of 58 swab samples were collected from an industrial pig farm located in South Bulgaria - 54 samples from vaginal/cervical discharge of breeding sows and nasal mucosa of neonatal piglets (44 samples from sexually mature sows; 10 samples from suckling piglets) and 4 tonsillar samples from dead or culled pigs. Out of the 58 swabs, 13 (22.4%) Streptococcus suis strains were isolated: 9 (20.4%) isolates from vaginal/cervical discharge of sows, 1 (10%) isolate from the nasal mucosa of a suckling piglet, and 2 strains (50%) from tonsils of dead or culled sows. One isolate (2.3%) from the vaginal/cervical discharge of breeding sows was determined as serotype 2-1/2. Also, 15 strains (34.1%) identified as Streptococcus equi subsp. zooepidemicus were isolated from vaginal/cervical discharge of breeding sows, and one tonsillar isolate (25%) was identified as Streptococcus dysgalactiae subsp. equisimilis. The resistant Streptococcus suis strains to tetracycline (91.7%), florphenicol (91.7%), enrofloxacin (91.7%) and colistin (75%) were more prevalent among streptococcal isolates. A multiresistant pattern including the nine tested antimicrobial drugs was demonstrated for three Streptococcus equi subsp. zooepidemicus strains (20%) isolated from vaginal/cervical discharge of breeding sows.

Novel primers for the detection and differentiation of S. zooepidemicus and S. equi by PCR.