Bacteriological and Molecular Detection of Streptococcus equi subsp. equi and Streptococcus equi subsp. zooepidemicus in Equines of Northern India

Present study was undertaken to study the prevalence of β-haemolytic streptococci in equine of northern temperate region of Jammu and Kashmir, India. One hundred and forty one samples were collected in duplicate from nasopharyngeal tract of diseased (53) and apparently healthy equine (88) for isolation and direct PCR. A total of 77 isolates of streptococci were recovered from 141 samples with an overall prevalence rate of 54.60%. Out of these 77 isolates, 52 were from diseased and 25 from apparently healthy animals. Of the 77 isolates, 4 were identified as Streptococcus equi subsp. equi, 56 as S. equi subsp. zooepidemicus and 17 as S. dysgalactiae subsp. equisimilis. Thus the overall prevalence of S. equi subsp. equi, S. equi subsp. zooepidemicus and S. dysgalactiae subsp. equisimilis was 2.83, 39.71 and 12.05% respectively. The sensitivity of the PCR for the detection of S. equi species was found higher when attempted from direct swab samples.     

Multiplex PCR-based identification of Streptococcus canis,Streptococcus zooepidemicus and Streptococcus dysgalactiae subspecies from dogs

Streptococcus canis (S. canis), Streptococcus equi subspecies zooepidemicus (S. zooepidemicus) and Streptococcus dysgalactiae subspecies (S. dysgalactiae subspecies) are β-haemolytic Gram positive bacteria infecting animals and humans. S. canis and S. zooepidemicus are considered as two of the major zoonotic species of Streptococcus, while more research is needed on S. dysgalactiae subspecies bacteria. In this work, a multiplex-PCR protocol was tested on strains and clinical samples to detect S. canis, S. dysgalactiae subspecies and S. equi subspecies bacteria in dogs. All strains were correctly identified as S. canis, S. equi subspecies or S. dysgalactiae subspecies by the multiplex-PCR. The main Streptococcus species isolated from symptomatic dogs were confirmed S. canis. The multiplex-PCR protocol described is a rapid, accurate and efficient method for identifying S. canis, S. equi subspecies and S. dysgalactiae subspecies in dogs and could be used for diagnostic purposes and for epidemiological studies.     

Etiologic and epidemiologic analysis of bacterial infectious upper respiratory disease in Thoroughbred horses at the Seoul Race Park

Infectious upper respiratory disease (IURD) of Thoroughbred racehorses has been a frequent problem (29.6% of incidence) at the Seoul Race Park (Korea). Risk factors for IURD include the season with a high transfer rate (summer and fall), the stabling period (≤ 3 months), and age (2 to 3 years old), suggesting that the movement and new environment may have depressed the immune system of the horses and decreased their ability to respond properly to pathogens. The bacterial strains (n = 98) isolated from IURD horses included Pseudomonas spp., Escherichia coli, Staphylococcus spp., Streptococcus equi subsp. equi and zooepidemicus.     

An outbreak of fatal hemorrhagic pneumonia caused by Streptococcus equi subsp. zooepidemicus in shelter dogs

An outbreak of fatal hemorrhagic pneumonia with 70~90% morbidity and 50% mortality occurred in an animal shelter in Yangju, Gyeonggi Province, Korea. Clinically, the affected dogs showed severe respiratory distress within 48 h after arriving in the shelter. The dead were found mainly with nasal bleeding and hematemesis. At necropsy, hemothorax and hemorrhagic pneumonia along with severe pulmonary consolidation was observed, though histopathological analysis showed mainly hemorrhagic bronchopneumonia. Lymphoid depletion was inconsistently seen in the spleen, tonsil and bronchial lymph node. Gram-positive colonies were shown in blood vessels or parenchyma of cerebrum, lung, liver, spleen, and kidney. Also, Streptococcus (S.) equi subsp. zooepidemicus was isolated from the various organs in which the bacterium was microscopically and histologically detected. In addition, approximately 0.9 Kb specific amplicon, antiphagocytic factor H binding protein, was amplified in the bacterial isolates. In this study, we reported an outbreak of canine hemorrhagic bronchopneumonia caused by S. equi subsp. zooepidemicus in an animal shelter in Yangju, Korea.     

Validation of a point-of-care polymerase chain reaction assay for detection of Streptococcus equi subspecies equi in rostral nasal swabs from horses with suspected strangles

This study aimed to validate a point-of-care polymerase chain reaction (PCR) assay for detection of Streptococcus equi subsp. equi (S. equi) in rostral nasal swabs from horses with suspected acute strangles and to compare the results against the molecular gold standard of quantitative polymerase chain reaction (qPCR). Two hundred thirty-two individual swabs of rostral nasal passages were characterized by qPCR as S. equi positive, S. equi subsp. zooepidemicus (S. zooepidemicus) positive, or S. equi and S. zooepidemicus negative. The specificity and sensitivity of the point-of-care PCR assay were 89% and 84%, respectively. The limits of detection of the qPCR assay and the point-of-care PCR analyzer were 3 and 277 eqbE target genes of S. equi, respectively. Overall agreement and short turnaround time make the point-of-care PCR assay a potential molecular diagnostic platform that will enhance the capability of equine veterinarians to timely support a diagnosis of strangles and institute proper biosecurity protocols.     

High prevalence of Mycoplasma equirhinis in Thoroughbred horses with respiratory symptoms in autumn 2018

Mycoplasma species are often isolated from horses with respiratory symptoms; however, the pathogenicity of Mycoplasma is still unclear. In autumn of 2018, we encountered an increase in cases with respiratory symptoms, mainly coughing, in a group of Thoroughbred racehorses in Japan. We examined tracheal wash samples obtained from 40 of those cases. Bacteria and viruses that commonly cause respiratory symptoms were investigated, and anaerobes were detected in only 5 cases and Streptococcus equi subsp. zooepidemicus (S. zooepidemicus) was detected in only 1 case of 40 cases with loop-mediated isothermal amplification assay. S. zooepidemicus and Streptococcus pneumoniae were isolated at a bacterial count of higher than 1.0 × 10⁴ CFU/ml from 5 and 2 cases of 28 cases cultured, respectively. None of the viruses investigated was detected in 40 cases. Mycoplasma equirhinis (M. equirhinis) was isolated from 40.0% (16/40) of the cases, which was higher than previously reported isolation rates. The rate of M. equirhinis isolation in the cases from 2018 was significantly higher than the isolation rates in the other horses: clinical cases with respiratory symptoms in 2019–2020 (13.6%, 3/22) and healthy horses (13.5%, 5/37) in Japan. In this study, the isolation rate of M. equirhinis from horse group with cough symptoms in 2018 was high and no other common etiological agents were detected. The pathogenesis of M. equirhinis is still unclear, however, M. equirhinis might have been associated with respiratory symptoms in the Thoroughbred horse cases in 2018.     

No evidence of horizontal infection in horses kept in close contact with dogs experimentally infected with canine influenza A virus (H3N8)

Background

Since equine influenza A virus (H3N8) was transmitted to dogs in the United States in 2004, the causative virus, which is called canine influenza A virus (CIV), has become widespread in dogs. To date, it has remained unclear whether or not CIV-infected dogs could transmit CIV to horses. To address this, we tested whether or not close contact between horses and dogs experimentally infected with CIV would result in its interspecies transmission.

Methods

Three pairs of animals consisting of a dog inoculated with CIV (10^8.3 egg infectious dose₅₀/dog) and a healthy horse were kept together in individual stalls for 15 consecutive days. During the study, all the dogs and horses were clinically observed. Virus titres in nasal swab extracts and serological responses were also evaluated. In addition, all the animals were subjected to a gross pathological examination after euthanasia.

Results

All three dogs inoculated with CIV exhibited clinical signs including, pyrexia, cough, nasal discharge, virus shedding and seroconversion. Gross pathology revealed lung consolidations in all the dogs, and Streptococcus equi subsp. zooepidemicus was isolated from the lesions. Meanwhile, none of the paired horses showed any clinical signs, virus shedding or seroconversion. Moreover, gross pathology revealed no lesions in the respiratory tracts including the lungs of the horses.

Conclusions

These findings may indicate that a single dog infected with CIV is not sufficient to constitute a source of CIV infection in horses.     

Adherence of Streptococcus equi on tongue,cheek and nasal epithelial cells of ponies

Streptococcus equi was found to adhere to tongue, cheek and nasal epithelial cells of ponies, in vitro. Maximum adherence was observed at pH 7.5 after one hour of incubation of bacteria with epithelial cells. This adherence was more on epithelial cells from adult animals than foals. Streptococci exposed to heat (60°C for 10 min) or treated with pepsin or trypsin showed a reduced adherence, whereas an increase occurred on treatment with hyaluronidase. Antibodies against whole S. equi cells or M-like protein blocked the adherence, whereas antibodies against group-specific carbohydrate or lipoteichoic acids did not. Pretreatment of epithelial cells with either the M-like protein or crude extract of S. equi lowered the adherence, whereas an extract of S. zooepidemicus did not. Adherence of S. equi to the epithelial cells was considered to be mediated by structures specific to S. equi.     

Streptococcus equi subsp. zooepidemicus isolates from equine infectious endometritis belong to a distinct genetic group

Streptococcus equi subsp. zooepidemicus is the pathogen most commonly isolated from the uterus of mares. S. zooepidemicus is an opportunistic pathogen and part of the resident flora in the caudal reproductive tract. The aim of this study was to investigate whether a genotypically distinct subpopulation of S. zooepidemicus is associated with endometritis in the mare, by genotyping and comparing uterine S. zooepidemicus strains with isolates from the vagina and clitoral fossa. Mares with (n = 18) or without (n = 11) clinical symptoms of endometritis were included. Uterine samples were obtained using a guarded endometrial biopsy punch, whereas a swab was used to recover samples from the cranial vagina and the clitoral fossa. If S. zooepidemicus was present, up to three colonies were selected from each anatomical location (max. 9 isolates per mare). Bacterial isolates were characterized by pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). S. zooepidemicus was isolated from the endometrium of 12 mares. A total of 88 isolates were analyzed by PFGE: 31 from the endometrium, 26 from the cranial vagina and 31 isolates from the clitoral fossa. For MLST 21 isolates were chosen. Results demonstrated a higher genetic similarity of the isolates obtained from infectious endometritis compared to isolates obtained from the caudal reproductive tract. In conclusion, we demonstrate for the first time that a genetically distinct group of S. zooepidemicus is associated with infectious endometritis in the mare.     

Changes in antibiotic resistance in equine bacterial ulcerative keratitis (1991-2000): 65 horses

Objective To document changes in antibiotic resistance of organisms in cases of equine bacterial ulcerative keratitis over a 10‐year time period. Design A retrospective study. Participants Medical records of equine patients with bacterial ulcerative keratitis seen at the University of Florida's VMTH for the years 1991–2000 were reviewed. Materials and methods All cases of equine bacterial ulcerative keratitis for the above mentioned years were examined. Bacterial isolates were identified and subjected to Kirby‐Bauer disc diffusion method sensitivity tests. Antibiotics used in the sensitivity tests included bacitracin, ampicillin, gentamicin, chloramphenicol, polymyxin B, trimethoprim‐sulfa, neomycin, kanamycin, carbenicillin, tobramycin and enrofloxacin. Results A total of 65 bacterial isolates were subjected to sensitivity testing. Of these isolates, Pseudomonas aeruginosa accounted for 14 of the bacterial isolates (22%), Streptococcus equi subspecies zooepidemicus accounted for 13 of the bacterial isolates (20%), and Staphylococcus aureus accounted for four of the isolates (6%). A statistically significant increase in resistance of Pseudomonas aeruginosa isolates to the antibiotics gentamicin and tobramycin was found between the isolates from 1992 to 1998 and those from 1999 to 2000. An increase in resistance of Streptococcus equi subspecies zooepidemicus to gentamicin was found between the isolates from 1993 to 1997 and those from 1998 to 2000. Conclusions Streptococcus equi subspecies zooepidemicus and Pseudomonas aeruginosa were the most common organisms isolated from cases of equine bacterial keratitis referred to the University of Florida's VMTH for the years 1991–2000. There appears to be an increase in resistance of Streptococcus equi subspecies zooepidemicus to gentamicin over the past 10 years. In addition, there is a significant increase in resistance of Pseudomonas aeruginosa to both gentamicin and tobramycin over the same time period.     

Detection of Streptococcus equi subspecies equi using a triplex qPCR assay

Genome sequencing data for Streptococcus equi subspecies equi and zooepidemicus were used to develop a novel diagnostic triplex quantitative PCR (qPCR) assay targeting two genes specific to S. equi (eqbE and SEQ2190) and a unique 100 base pair control DNA sequence (SZIC) inserted into the SZO07770 pseudogene of S. zooepidemicus strain H70. This triplex strangles qPCR assay can provide results within 2 h of sample receipt, has an overall sensitivity of 93.9% and specificity of 96.6% relative to the eqbE singlex assay and detects S. equi at levels below the threshold of the culture assay, even in the presence of contaminating bacteria.     

Presence of Bacteria on the External Genitalia of Healthy Stallions and its Transmission to the Mare at the Time of Breeding by Live Cover

The current field study used thoroughbred stallions and mares from central Kentucky to investigate the occurrence of potentially pathogenic bacteria on the stallion's external genitalia, based on cultures, and investigated the occurrence of bacteria and type of isolate in the mare's uterus after breeding by live cover to stallions with or without positive bacterial cultures. Fifteen thoroughbred stallions and 206 mares from two central Kentucky thoroughbred farms were used during the 2010 and 2011 breeding seasons. Samples for bacteriological evaluation were taken from the prepuce and postejaculate urethra (n = 201) of stallions. Uterine swabs (n = 264) were collected 12-18 hours postbreeding. For statistical analyses, a chi-squared test was used to test the relationship between stallion culture results and postbreeding uterine culture results, as well as the effect of bacterial types found on the stallion cultures with bacterial types found on the postbreeding uterine cultures. Of stallion cultures, 22.4% were positive for potentially pathogenic bacteria, with Streptococcus equi subspecies zooepidemicus (51.1%) being the most common isolate. Uterine cultures resulted in a 29.2% positive rate for potentially pathogenic bacteria, with S. equi subsp. zooepidemicus (90.9%) being the most common. There was no difference (P > .05) in the occurrence of bacteria or type of isolate found on uterine cultures after breeding stallions with or without positive cultures. In conclusion, potentially pathogenic bacteria found on the stallion's external genitalia did not affect the occurrence and type of bacterial isolate found in the mare's uterus after breeding by live cover.     

Efficacy and safety of lomefloxacin on bacterial extraocular disease in the horse

Lomefloxacin is a broad-spectrum fluoroquinolone antibiotic used for the treatment of bacterial extraocular disease. This study investigated the efficacy and safety of lomefloxacin eye drops for bacterial extraocular disease in horses. Lomefloxacin ophthalmic solution (0.3%) was instilled three times daily for 2–5 days in 65 horses diagnosed with bacterial extraocular disease based on clinical findings. Clinical observations and bacteriological examinations were performed at the start of treatment, 2 and 5 days after the start of treatment, and at the discontinuation or termination of treatment. Of the 65 horses, 64 were positive for bacteria, and 22 bacterial genera and 47 bacterial species were identified. The efficacy of lomefloxacin was evaluated in 63 horses; one horse with a negative culture and another with suspected bacterial contamination were excluded. Lomefloxacin was considered to be clinically effective in 54 horses. The major bacterial species identified were Staphylococcus aureus, Streptococcus equi subsp. zooepidemicus, Acinetobacter lwoffii, Staphylococcus xylosus, Staphylococcus vitulinus, Enterobacter agglomerans, Flavimonas oryzihabitans and Staphylococcus sciuri, with a cumulative disappearance rate of 80% or more at the termination of instillation. Excluding one horse that did not undergo a bacteriological examination, the remaining 62 horses were assessed for bacteriological outcome. Full or partial bacterial clearance was detected in 95% or more of the 62 horses. One of the 65 horses reported adverse events that had no causal relation with the eye drops. Our results showed that lomefloxacin is safe and effective for the treatment of bacterial extraocular disease in horses.     

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.     

Infectious disease surveillance of apparently healthy horses at a multi-day show using a novel nanoscale real-time PCR panel

In the United States, horses are used for a variety of purposes including recreation, exhibition, and racing. As farm, performance, and companion animals, horses are a unique species from a zoonotic disease risk perspective, and the risks of subclinical infections spreading among horses can pose challenges. Using a nanoscale real-time PCR platform, we investigated the prevalence of 14 enteric pathogens, 11 Escherichia coli genes, and 9 respiratory pathogens in fecal samples from 97 apparently healthy horses at a multi-day horse event. In addition, sugar flotation test was performed for fecal parasites. E. coli f17 was commonly detected, prevalent in 59% of horses, followed closely by Streptococcus equi subsp. zooepidemicus (55%). Additional pathogens recognized included betacoronavirus, Campylobacter jejuni, Cryptosporidium sp., E. coli O157, equine adenovirus 1, equine rhinitis B virus, and others. The use of PCR data may overestimate the true prevalence of these pathogens but provides a sensitive overview of common pathogens present in healthy horses. Our results prompt the continued need for practical biosecurity measures at horse shows, both to protect individuals interacting with these horses and to minimize transmission among horses.     

Septic tendonitis of the deep digital flexor tendon in a Thoroughbred horse referred for weight loss and intermittent fever

This case report describes the history, clinical and diagnostic findings, treatment and outcome of a 3‐year‐old Thoroughbred gelding. The horse was examined for chronic weight loss, intermittent fever of 4 months' duration and acute lameness of 2 weeks' duration, and was finally diagnosed with septic tendonitis of the deep digital flexor tendon and digital flexor tendon sheath caused by Streptococcus equi ssp. zooepidemicus. Investigation of the lower respiratory tract also allowed isolation of S. equi ssp. zooepidemicus. Although surgical treatment was recommended, because of the severity of the lesion and the guarded prognosis for a return to full athletic function, the owner declined surgery. The horse was therefore treated aggressively with antimicrobials using systemic, local and regional approaches, and with anti‐inflammatory drugs. At 20 months after discharge, the horse was able to race, and he is now in full work with 20 races finished successfully.     

Genetic structure of populations of β-haemolytic Lancefield group C streptococci from horses and their association with disease

The genetic structure of β-haemolytic Lancefield group C streptococci isolated from horses in Australia was examined by multilocus enzyme electrophoresis. The 249 isolates comprised 70 classified phenotypically as Streptococcus equi subspecies equi, 177 classified as S equi subspecies zooepidemicus and two which were unclassifiable. Forty-one electrophoretic types were identified which could be classified into three major clusters, A, B and C. Of the isolates, 178 fell into cluster B (types 4 to 22) and lay within a genetic distance of 0·36. Sixty-nine of the 70 S equi subspecies equi isolates fell into type 12, which suggests that they were members of a single clone, and the isolates from abscesses were significantly more likely to belong to type 12 than those from horses with no clinical signs (P<0·001). There were no other significant associations between electrophoretic types or clusters and the isolation of the organism from particular sites. These data suggested that S zooepidemicus may be the archetypal species from which the clone designated subspecies equi has been derived. If isolates of the subspecies equi from other geographical regions also prove to be members of electrophoretic type 12, this hypothesis would be strengthened.     

First Report of Molecular Characterization of Argentine Isolates of Streptococcus equi subsp. equi by Pulsed-Field Gel Electrophoresis

Strangles is one of the most frequently diagnosed equine respiratory infectious diseases in the world. It is caused by Streptococcus equi subsp. equi (S. equi), and it is an acute infection characterized by pyrexia, nasal discharge, pharyngitis, and abscessation of lymph nodes. Frequently, healthy horses might continue to harbor S. equi after clinical recovery. Although the genetic distance between S. equi isolates is short, strains can be differentiated by pulsed-field gel electrophoresis (PFGE) and single locus sequence typing for epidemiological studies. The aim of this study was to characterize by PFGE Argentine isolates of S. equi obtained from horses with acute strangles and those that had recovered. Bacterial isolation and identification of 80 S. equi isolates by phenotypic and genotypic tests were performed using samples from 29 horses with acute strangles and 95 from healthy animals. Also, the isolates were characterized by PFGE using Bsp120I and SmaI. Visual comparison of macrorestriction patterns generated with both enzymes revealed three different DNA fragment profiles with variations of one or two bands. Interestingly, an identical profile was found in isolates from the same horse and from horses that were infected at the same time, and the horses recovered from strangles continue to carry the same strain. Some vaccinated horses have been mild infected for a different strain from that of carriers suggesting other source of infection. This is the first molecular characterization of Argentine isolates of S. equi, which shows the presence of three strains between 2010 and 2013 in Buenos Aires.     

Survival of Streptococcus equi on surfaces in an outdoor environment

Management practices to prevent or control outbreaks of Streptococcus equi subsp. equi often include consideration of environment survival, but limited objective data are available. This study involved evaluation of S. equi persistence following inoculation of wood, metal, and rubber surfaces in an outdoor environment. Survival was short, ranging from < 1 to 3 d. There was no effect of rain (P = 0.33) or surface type (P = 0.95), but there was an effect of sunlight (P = 0.002). Outdoor survival of S. equi is poor, and prolonged quarantine of outdoor areas, particularly areas exposed to the sun, is probably unnecessary.     

Diagnostic testing patterns for Streptococcus equi subsp. equi in Ontario horses during the years 2008 to 2018

This retrospective study describes testing patterns and the incidence of Streptococcus equi subsp. equi in Ontario to assess the utility of laboratory data for surveillance purposes. Laboratory records for equine infectious disease test submissions were extracted from the Animal Health Laboratory (AHL) at the University of Guelph for the years 2008 to 2018. Yearly and seasonal trends in S. equi testing and the proportion of tests that returned positive results were assessed. The number of samples submitted for S. equi testing decreased over the 11-year period (odds ratio = 0.96, 95% confidence interval: 0.92 to 0.999; P = 0.04). A generalized linear model identified a significant seasonal effect for animals recognized as clinically ill, with the highest test positivity noted in the winter. Although this study identified important trends in the incidence of S. equi in Ontario, the variability in information accompanying test submissions made the data challenging to interpret, highlighting the need for more complete diagnostic submission data for S. equi.