Molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of Thoroughbred racehorses in training

The objective of this study was to characterise the molecular epidemiology of Streptococcus zooepidemicus isolated from the respiratory tracts of 198 Thoroughbred racehorses based at three Newmarket training yards over a 10 month period. Typing utilised two separate PCR procedures targeting the M-like protein hypervariable and the 16S–23S RNA gene intergenic spacer regions of the bacterium. S. zooepidemicus, isolated from 23% (224/983) of study samples, comprised 24 different types of varying prevalence. The four most common types, A1HV4, A1HV2, C1HVu and D1HV1, accounted for 45% of all the typed isolates. Overall, the findings were similar to those reported in a study of Welsh Mountain ponies and confirm that in equids, S. zooepidemicus is not a homogeneous, clonal population but instead represents a wide diversity of strain types. This diversity also highlights potential difficulties in using vaccines to control S. zooepidemicus infections in horses as it is likely that the induction of a heterologous, cross-protective immunity will be required.     

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.     

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.     

Studies into the prevalence of Mycoplasma species in small ruminants in Benue State,North-central Nigeria

The indicative prevalence of respiratory Mycoplasma species in small ruminants (SR) was determined in North-central Nigeria. Nasal swabs from 172 sheep and 336 goats from the Northeast, Northwest and South Senatorial Districts of Benue State were examined. Initial Mycoplasma isolation used Mycoplasma culture techniques followed by digitonin sensitivity testing. Species identification was done using polymerase chain reaction (PCR) and denaturing gradient gel electrophoresis (DGGE). Overall, Mycoplasma organisms were isolated from 131 (25.8 %) of the 508 SR examined. Prevalence rates of 18.1 and 29.8 % were recorded for sheep and goats, respectively. A total of 135 isolates of Mycoplasma belonging to three different species were identified: Mycoplasma ovipneumoniae (127), Mycoplasma arginini (7) and Mycoplasma mycoides subspecies capri (1). More than one Mycoplasma species were detected in four (3.1 %) of the 131 confirmed Mycoplasma positive cultures. Mycoplasma was isolated from 16.2 and 29.1 % of animals with and without respiratory signs, respectively. The high isolation rate of mycoplasmas in apparently healthy and clinically sick sheep and goats in this study indicates a carrier status in these SR which may constitute a serious problem in disease control.     

Comparison of Serum Amyloid A in Horses With Infectious and Noninfectious Respiratory Diseases

The acute phase protein serum amyloid A (SAA) has been shown to be a useful inflammatory parameter in the horse, but studies showing SAA responses to specific respiratory disease etiologies are limited. The goal of this study was to evaluate SAA responses in horses with infectious and noninfectious respiratory diseases as well as healthy, control horses. Two hundred seven horses were grouped into the following categories: equine influenza virus (EIV), equine herpesvirus-4 (EHV-4), Streptococcus equi subspecies equi (S. equi ss equi), inflammatory airway disease (IAD), and healthy controls. Serum amyloid A concentrations were determined for all horses on serum using a stall-side lateral flow immunoassay test. Serum amyloid A levels were found to be significantly greater for infectious respiratory diseases (EIV, EHV-4, S. equi ss equi) and horses with IAD when compared to control horses. There was a significant difference between viral and bacterial infections and IAD. Although SAA values from horses with S. equi ss equi were significantly greater when compared to horses with viral infections (EIV/EHV-4), the wide range of SAA values precluded accurate classification of the infectious cases. In conclusion, SAA is more reliably elevated with infections of the respiratory tract rather than noninfectious airway conditions. This can facilitate early detection of respiratory infections, help track disease progression, and aid practitioners in making recommendations about proper biosecurity and isolation of potentially contagious horses.     

A case-control study of respiratory disease in Thoroughbred racehorses in Sydney, Australia

In order to investigate the role of infectious agents in the aetiology of lower respiratory tract disease in Thoroughbred racehorses, a matched case-control study was conducted. Cases were identified by the presence of coughing, and were compared to a control population matched on time of sample collection and location within the same training establishment. Tracheal wash samples were collected from 100 cases and 148 controls. Case horses were more likely than controls to have endoscopic and cytological evidence of airway inflammation. There was no significant association between serological evidence of infection by commonly implicated respiratory viruses and coughing. Similarly, mycoplasma were rarely isolated and were not associated with disease. In contrast, there was a strong association between isolation of greater than a total of 10(3) colony-forming units/ml of tracheal wash and coughing. Individual bacterial species associated with disease included Streptococcus zooepidemicus, Streptococcus pneumoniae, Streptococcus suis, Streptococcus sanguis, Pasteurella spp and Bordetella bronchiseptica. This study provides evidence of the role of bacterial infection in the aetiology of lower respiratory tract inflammation in racehorses. However, in 58% of cases, few or no bacteria were isolated. Hence, at the time of identification of disease, there was no evidence of viral, bacterial or mycoplasmal infection in the majority of coughing horses. The aetiology of the signs observed in these horses requires further investigation.     

Comparison of the bacterial and fungal flora in the pharynx of normal horses and horses affected with pharyngitis

A total of 43 horses were used for the study of the pharyngeal bacterial flora. The median value of the number of bacteria in the group of 19 normal horses was 3.8 × 10⁴ cfu/g of secretions. This value was 6.4 × 10⁴cfu/g in horses with grade I pharyngitis, 1.3 × 10⁵ cfu/g in horses with grade II pharyngitis and 3.5 × 10⁶ cfu/g in horses affected with grades III and IV pharyngitis. Corynebacterium spp, coagulase-negative staphylococci, Nocardia spp, Moraxella spp and Enterobacter spp were the most frequently encountered bacteria in the normal animals as well as in horses affected with pharyngitis of grades I or II. Moraxella spp were isolated in 87.5% of the horses with pharyngitis of grades III and IV, followed by Streptococcus zooepidemicus, Pseudomonas aeruginosa, coagulase-negative staphylococci and Enterobacter spp. No fastidious bacteria, nor strict anaerobes were isolated from any of the 43 horses. None of the microorganisms were found in 100% of the animals and the majority of the isolates were opportunistic bacteria. These results demonstrate that the isolation of Moraxella spp and S. zooepidemicus in large numbers is frequent in horses with lymphoid follicular hyperplasia grades III and IV. Fungi were isolated in small numbers from two or three horses in each group.     

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.     

Haemorrhagic pneumonia in sled dogs caused by Streptococcus equi subsp. zooepidemicus - one fatality and two full recoveries: a case report

In spite of yearly vaccination, outbreaks of canine infectious respiratory disease are periodically seen amongst domestic dogs. These infections compromise host defense mechanisms, and, when combined with other stressful events, allow opportunistic pathogens like Streptococcus equi subsp. zooepidemicus to create serious disease. Early recognition and treatment are tremendously important for a successful outcome in these cases. A polyvalent vaccine was given to 22 racing dogs three days after a competition, followed by two days of rest, and then the dogs were returned to regular training. Coughing was noticed among the dogs four days after immunisation. Three days after this outbreak one of the dogs was unusually silent and was found dead the next morning. Simultaneously two other dogs developed haemorrhagic expectorate, depression and dyspnea and were brought in to the veterinary hospital. Streptococcus equi subsp. zooepidemicus was isolated in pure culture from all three cases. They were treated and rehabilitated successfully, and won a sledge race three months later. This paper discusses the necropsy results, treatment regime, rehabilitation and the chronology of vaccination, stressful events and disease.     

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.     

A Multiplex Polymerase Chain Reaction Assay for Direct Detection and Differentiation of β-Hemolytic Streptococci in Clinical Samples from Horses

Streptococcus equi subspecies equi, S equi subspecies zooepidemicus, and S dysgalactiae subspecies equisimilis are β-hemolytic Streptococci, often isolated from horses with respiratory or genital diseases. The aim of this study was (i) defining and validating a multiplex polymerase chain reaction (PCR) protocol for identifying these Streptococci in bacterial cultures and for detecting them directly in equine clinical specimens, and (ii) defining and validating a cheap DNA extraction protocol for clinical specimens. When respiratory and genital samples from symptomatic and asymptomatic horses were tested by bacterial culture and by multiplex PCR, all the 150 samples culture-positive for S equi, S zooepidemicus, or S equisimilis were also positive by PCR. Of 150 culture-negative samples, 143 were negative by PCR. Seven samples were positive by PCR but negative by bacteriology. The multiplex PCR protocol described in this study is proven suitable for a sensitive, specific, and rapid detection and identification of S equi, S zooepidemicus, and S equisimilis in cultured bacterial colonies, as well as in clinical specimens from symptomatic or asymptomatic horses. The inclusion of internal control primers in the PCR protocol excludes false-negative results. A cheap DNA extraction method has been also validated for swabs, tracheal aspirates, bronchoalveolar lavage, and guttural pouches lavage samples.     

An investigation of the movement patterns and biosecurity practices on Thoroughbred and Standardbred stud farms in New Zealand

Utilising a face-to-face interview of stud managers, a survey was conducted to investigate the movement patterns around, and biosecurity practices on, Thoroughbred and Standardbred stud farms. Eligible stud farms (n = 60) were identified from the 2009 Thoroughbred stallion register and 2009 Standardbred stallion register, and stud managers were asked to participate in the interview. In total, 27 stud managers agreed to participate in the study, and participating stud farms included 38% of Thoroughbred and 60% of Standardbred mares bred in 2009. All stud managers reported the movement of horses to and from their stud farm. The median number of movement events per year was 127 (interquartile range 83–300). The frequency of movement events from a stud farm was not associated with the breed of horse managed on the stud farm, however Thoroughbred horses travelled further than Standardbred horses during these movement events (P < 0.001). The movement patterns of horses around stud farms showed a strong seasonality associated with the commercial breeding season of each breed. While 26 (96%) of stud managers reported having procedures in place for checking newly arriving horses, only 6 (22%) stud managers reported isolating horses on arrival as a standard protocol. The main reason for isolating horses on properties, where isolation was not a standard procedure, was in response to strangles outbreaks on other stud farms (n = 10). Only 2 (7%) stud managers reported implementing visitor protocols, and these protocols only applied to visiting veterinarians, but not to farriers. These findings have important implications for the control of both endemic and exotic infectious disease outbreaks within the New Zealand breeding population as the high frequency of movement around stud farms, the high number of visitors to stud farms and the lack of effective biosecurity practices or visitor protocols will be critical factors in the spread of equine influenza during an outbreak.     

Polymorphism Analysis of Microsatellites Associated with Seven Candidate Genes for Equine Cryptorchidism

Association of seven candidate genes with cryptorchidism was investigated in the Thoroughbred. A pedigree composed of 23 cryptorchids and 24 nonaffected horses, sharing a common ancestor, was constituted. Sixteen microsatellite markers were developed either from bacterial artificial chromosomes (BAC) isolated for each candidate gene or by in silico screening. DNA from our pedigree was genotyped for these microsatellites. Statistical analysis of the allelic and genotypic frequencies observed with these markers did not reveal any association between the candidate genes and the cryptorchidism phenotype in our horse panel.     

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.     

Subepiglottic cysts in 15 horses

Subepiglottic cysts (SECs) are an infrequent cause of upper respiratory tract noise and exercise intolerance in horses. They may also be associated with no clinical signs and be an incidental finding during routine upper airway endoscopy. The aim of this study was to assess the effect on performance of horses undergoing surgical removal of SECs. The case records of 15 horses (1995–2009) diagnosed with SECs were retrieved. Eleven (73%) of the 15 horses included in the study were Thoroughbred racehorses. Eleven (73%) of the 15 horses had no preoperative clinical signs related to the SECs, with the remaining 4 (27%) having a respiratory noise (n = 4), nasal discharge (n = 1), difficulty swallowing (n = 1) or a cough (n = 1). Endoscopic examination in the standing horse was diagnostic in 93% (n = 14) of horses. Nine (82%) of the 11 Thoroughbred horses were yearlings, of which only one horse (11%) presented with clinical signs consisting of a respiratory noise and nasal discharge. Four of the 11 (36%) Thoroughbred horses were found to have concurrent epiglottic entrapment. Surgical removal was successful in all cases. Eight of the 11 (73%) Thoroughbred horses in this study raced following SEC removal. The majority of SECs are identified during routine endoscopic examinations and are not associated with clinical signs. The prognosis following surgical removal of SECs is good and future performance does not appear to be affected.     

Lipid Utilization Pathways Induced by Early Training in Standardbred Trotters and Thoroughbreds

Controversial results on lipid utilization as an energy source during training in horses are found in the literature. The objective of this study was to assess blood lipid profile during different training programs in horses. Seventeen Standardbred horses (400 ± 50 kg) and 17 Thoroughbred horses (380 ± 15 kg) followed different training programs. Blood lipid profile, including triglycerides (TGs), total cholesterol, high-density lipoprotein (HDL), and low-density lipoprotein (LDL), was analyzed, and very-low-density lipoprotein (VLDL) concentration was calculated. Data were analyzed using Student t test, and linear regressions were done. Cholesterol and LDL decreased during training programs in Standardbred trotters (P = .0001 and P = .0053, respectively), and VLDL was found to be close to the significance level (P = .053). Blood lipid profile, including TGs (P = .0014), cholesterol (P = .0057), HDL (P = .0128), LDL (P = .0091), and VLDL (P = .0014), varied throughout the training program in Thoroughbred horses. Negative slope of blood lipids and positive slope of TG linear regression in Standardbred trotters were significant for all parameters (P ≤ .05), whereas cholesterol and LDL regression showed poor P and r² values and HDL P value was slightly above the significance level (P = .069) in Thoroughbred horses. TGs and VLDL showed a positive linear trend in Thoroughbred horses (P = .002). Exercise and different training programs lead to significant variations of lipid profile and lipid utilization in horses. Lipid utilization as an energy source improved with training in Standardbred trotters, whereas this was not the case in Thoroughbred horses. Further studies on the effect of training programs using different conditions and horse breeds would be necessary to understand lipid utilization as an energy source in athletic horses.     

Pneumocystis Pneumonia in a Thoroughbred Racehorse

Pneumocystis pneumonia is an opportunistic respiratory infection that occurs in immunocompromised animals. In horses, pneumocystic pneumonia is observed mostly in foals and often progresses rapidly. Here, we report pneumocystic pneumonia in a Thoroughbred racehorse. A 3-year-old Thoroughbred racehorse colt had marked respiratory symptoms for 3 weeks and was unresponsive to antibiotic treatment. At necropsy, firm, tan, patchy lesions were scattered diffusely in the lungs. Microscopically, alveolar septa thickened by proliferation of collagen fibers and infiltration of inflammatory cells were observed. In the alveolar spaces, many brown-black yeast-like organisms similar to cystic forms of Pneumocystis carinii were recognized by staining with Gomori's methenamine silver. Bronchoalveolar lavage fluid (BALF) obtained before necropsy included macrophages engulfing the fungus bodies. Amplified products were obtained from BALF and lung tissue samples by Pneumocystis-specific nested PCR. Phylogenetic analysis based on the 18S rRNA gene sequence revealed that the P. carinii organism from BALF was related to the Pneumocystis spp. detected in other animals and was especially close to P. carinii derived from ferrets. This is a rare case of pneumocystic pneumonia in a colt with chronic pulmonary lesions.     

Optimized protocol for multiplex nested polymerase chain reaction to detect and differentiate Haemophilus parasuis,Streptococcus suis,and Mycoplasma hyorhinis in formalin-fixed,paraffin-embedded tissues from pigs with polyserositis

An optimized protocol was developed for the simultaneous detection and differentiation of Haemophilus parasuis, Streptococcus suis, and Mycoplasma hyorhinis in formalin-fixed, paraffin-embedded (FFPE) tissues with multiplex nested polymerase chain reaction (PCR). This method also determines the prevalence of these bacteria in pigs with polyserositis. DNA extraction with a combination of a commercial reagent and proteinase K resulted in more frequent detection of the pathogens than DNA extraction with proteinase K alone. Among FFPE tissue samples from 312 cases of polyserositis in which at least 1 bacterial species was detected, multiplex nested PCR detected H. parasuis in 239 (77%), S. suis in 124 (40%), and M. hyorhinis in 40 (13%). The disease was caused by a single pathogen in 224 (72%) of the cases and multiple pathogens in 88 (28%). Among the pigs positive for H. parasuis, S. suis, and M. hyorhinis by multiplex nested PCR, the pathogen was isolated from only 11%, 35%, and 28%, respectively. Therefore, the PCR protocol developed in this study is a useful diagnostic method when samples are negative after isolation methods and even for samples in which only 1 pathogen was isolated.     

Feedtrough dirt as a source of Clostridium botulinum type C intoxication in a group of farm horses

Four horses from the same farm developed clinical signs of botulism during the winter months; three of these horses died. One horse survived an initial attack and recovered over a three-week period, but died during a second attack. The horse that survived took six weeks to recover. Clinical and postmortem examination ruled out other causes of disease. Confirmation of the diagnosis was made by isolation of Clostridium botulinum type C toxin from the dirt in the bottom of an oak feedtrough used by all horses, and from the colonic contents of one of the horses that died. To our knowledge, this is the second case of C. botulinum type C intoxication reported in horses in North America. In both cases, soil and sand near aquatic environments were identified as the source of toxin.