Ashley G. Boyle

Saccharomyces boulardii viability and efficacy in horses with antimicrobial-induced diarrhoea

Saccharomyces boulardii has been successfully used in the prevention and treatment of antimicrobial-associated diarrhoea in humans. We hypothesised that a viable, dried lyophilised preparation of S boulardii would survive in the gastrointestinal tract of horses with antimicrobial-associated enterocolitis, and significantly decrease the duration of diarrhoea. Twenty-one horses, over one year of age, with antimicrobial-associated diarrhoea of up to 72 hours duration, were consecutively randomised in a controlled prospective study. The treatment group received S boulardii (25 g, orally, every 12 hours) until the cessation of clinical signs. S boulardii was successfully cultured in 58.3 per cent of treatment horses on day 3. No statistically significant differences were found in days to return to normal faecal consistency; resolution of watery diarrhoea; return to normal heart rate, respiratory rate and temperature; resolution of leucopaenia; attitude improvement; appetite improvement; and survival at discharge. This is the first study to demonstrate survival of S boulardii in horses with gastrointestinal illness. Further study of the efficacy and safety of S boulardii in horses with antimicrobial-associated diarrhoea in a larger group is warranted.

Streptococcus equi Detection Polymerase Chain Reaction Assay for Equine Nasopharyngeal and Guttural Pouch Wash Samples.

Background Bacterial culture and polymerase chain reaction (PCR) assays for the detection of Streptococcus equi in nasopharyngeal washes (NPW) and guttural pouch lavage (GPL) samples have low sensitivity. In human diagnostics, processing of samples with flocked swabs has improved recovery rates of bacterial agents because of improved surface area and elution factors. Hypothesis For S. equi subsp. equi (S. equi) detection in NPW and GPL samples we hypothesized that: direct‐PCR would be more reliable than flocked swab culture (FS culture); flocked swab PCR (FS‐PCR) would be equivalent to direct‐PCR; and FS culture would be more reliable than traditional culture. Samples A total of 193 samples (134 NPW and 59 GPL) from 113 horses with either suspected S. equi infection, convalescing from a known S. equi infection, or asymptomatic horses screened for S. equi. Methods Prospective study. Samples were submitted for S. equi direct‐PCR. Using logistic regression, direct‐PCR (gold standard) was compared to FS culture, traditional culture, and FS‐PCR also performed. Results Direct‐PCR was statistically more sensitive than FS‐PCR, FS culture, and traditional culture (P < .001). All methods had sensitivities <70% relative to the direct‐PCR. FS culture had a similar sensitivity relative to traditional culture. The odds of GPL samples being positive on direct‐PCR (P = .030) and FS‐PCR were greater than those for NPW samples (P = .021). Conclusions and Clinical Importance Use of flocked swabs during laboratory preprocessing did not improve detection of S. equi via either PCR or bacterial culture from samples. Direct‐PCR is the preferred method of detection of S. equi.

Optimization of an in vitro assay to detect Streptococcus equi subsp. equi

Streptococcus equi is the etiologic agent of a highly infectious upper respiratory disease of horses known as strangles. Bacterial culture methods and polymerase chain reaction (PCR) of nasopharyngeal washes and guttural pouch lavages are used routinely to test clinical and carrier animals for the presence of S. equi but no definitive or gold standard test method has been shown to be optimal. We hypothesized that (i) a flocked swab submerged in ten-fold serial dilution suspensions of S. equi prepared in 0.9% NaCl would detect more colony forming units (CFU) than a rayon swab when used to inoculate a blood agar plate, (ii) centrifugation of a 1 ml aliquot of each suspension would improve the limit of detection (LOD) by bacterial culture and PCR compared to the culture or PCR of submerged swab samples, (iii) PCR of the centrifuged samples from each suspension would be more sensitive than aerobic culture alone, and (iv) PCR of a 1 ml aliquot directly from a sample would be more sensitive than PCR of a sample following submersion of a flocked swab in 1 ml saline. Using 7 ten-fold serial dilutions of S. equi in 0.9% NaCl, the LOD for 4 bacterial culture methods and 3 PCR methods were compared. The LOD of direct PCR and flocked swab culture was determined at 1 cfu/ml. All PCR methods were equivalent to each other and were more sensitive than any of the culture methods at the lower dilutions. At higher cell densities (>100 cfu/ml) flocked swab culture was not statistically better than rayon swab culture, but it was superior to all other methods tested.

Factors associated with likelihood of horses having a high serum Streptococcus equi SeM-specific antibody titer

OBJECTIVE To identify factors associated with an increased likelihood that horses would have a serum Streptococcus equi SeM-specific antibody titer > or = 1:1,600. DESIGN Cross-sectional study. ANIMALS 188 healthy client-owned horses. PROCEDURES A single serum sample from each horse was tested for SeM-specific antibody titer with an ELISA. Multivariate logistic regression was used to identify factors associated with having a titer > or = 1:1,600. RESULTS Age, breed, and vaccination status were significantly associated with the likelihood of having a titer > or = 1:1,600. The odds of having a titer > or = 1:1,600 increased by a factor of 1.07 with each 1-year increase in age. Quarter Horses and horses of other breeds were 4.08 times as likely as were Thoroughbreds and warmbloods to have a titer this high. Horses that had previously received an intranasal S equi vaccine were 4.7 times as likely as were horses without any history of vaccination to have a titer this high. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that older horses, horses other than Thoroughbreds and warmbloods, and horses that had been vaccinated with an attenuated-live intranasal S equi vaccine between 1 and 3 years previously had an increased likelihood of having a serum SeM-specific antibody titer > or = 1:1,600.

Predictor variables for and complications associated with Streptococcus equi subsp equi infection in horses

OBJECTIVE To evaluate predictor variables for and complications associated with Streptococcus equi subsp equi infection (strangles) in horses. DESIGN Retrospective case-control study. ANIMALS 108 horses with strangles (cases) and 215 horses without strangles (controls). PROCEDURES Medical records from January 2005 through July 2012 were reviewed. Cases were defined as horses with clinical signs of strangles (pyrexia, retropharyngeal lymphadenopathy, and mucopurulent nasal discharge) that were associated with a confirmed strangles outbreak or had positive results for S equi on PCR assay or bacteriologic culture. Controls were defined as horses with pyrexia that did not meet the other criteria for cases. Data compared between cases and controls included signalment, clinical signs, diagnostic test results, and disease complications and outcome. Logistic regression was used to identify variables associated with strangles and its complications. RESULTS Clinical signs of strangles were not evident in 12 of 25 cases classified as S equi carriers (infected > 40 days). Predictor variables associated with strangles included mucopurulent nasal discharge and external abscesses in the pharyngeal region. Strangles was more likely to be diagnosed in the spring than in the summer. Cases with anemia were more likely to develop purpura hemorrhagica than were cases without anemia. No risk factors were identified for the development of guttural pouch empyema or metastatic abscesses. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that not all horses infected with S equi develop clinical signs of strangles. We recommend that guttural pouch endoscopy and lavage with PCR assay of lavage fluid samples be performed to identify S equi carrier horses.

Comparison of nasopharyngeal and guttural pouch specimens to determine the optimal sampling site to detect Streptococcus equi subsp equi carriers by DNA amplification

BackgroundStreptococcus equi subsp equi (S. equi) is the cause of “equine strangles” which is a highly infectious upper respiratory disease. Detection of S. equi is influenced by site of specimen collection, method of sampling, and type of diagnostic test that is performed. We hypothesized i) that a loop-mediated isothermal amplification (LAMP) assay that targets the S. equi-specific eqbE gene would be more sensitive than a realtime PCR assay that targets the S. equi-specific seeI gene and ii) that LAMP of specimens obtained by guttural pouch lavage (GPL) would be more sensitive than LAMP of nasopharyngeal specimens to identify S. equi carriers.MethodsA nasopharyngeal flocked swab, nasopharyngeal wash, and GPL specimen was collected from 44 convalescent horses and the eqbE LAMP assay was performed. The seeI realtime PCR assay and aerobic culture were also performed on the GPL specimen. Logistic regression was performed to compare sampling sites and test methods (P-values ≤0.05 were considered significant).ResultsOne of 41 nasopharyngeal flocked swabs, 6/38 nasopharyngeal wash and 24/44 GPL specimens were positive by eqbE LAMP. 18/44 GPL specimens were positive by seeI PCR and S. equi was isolated from 4/44 of these specimens. Detection of S. equi DNA was 51 times more likely from the GPL samples than nasopharyngeal samples (OR 51.0, P < 0.0001). When eqbE LAMP GPL samples were positive, it was eight times more likely that the guttural pouch had any abnormality on endoscopy (OR 8.2, P ≤ 0.005), almost 20 times more likely that mild empyema was found (OR 19.7, P ≤ 0.002), and eight times more likely that the SeeI PCR was positive for S. equi DNA (OR 8.1, P ≤ 0.006).ConclusionThis study demonstrates that guttural pouch lavage specimens should be used to detect S. equi and that the eqbE LAMP assay was comparable to the seeI PCR.

Streptococcus equi Infections in Horses: Guidelines for Treatment, Control, and Prevention of Strangles—Revised Consensus Statement

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.

A case-control study developing a model for predicting risk factors for high SeM-specific antibody titers after natural outbreaks of Streptococcus equi subsp equi infection in horses

OBJECTIVE To develop a risk prediction model for factors associated with an SeM-specific antibody titer ≥ 3,200 in horses after naturally occurring outbreaks of Streptococcus equi subsp equi infection and to validate this model. DESIGN Case-control study. ANIMALS 245 horses: 57 horses involved in strangles outbreaks (case horses) and 188 healthy horses (control horses). PROCEDURES Serum samples were obtained from the 57 cases over a 27.5-month period after the start of outbreaks; serum samples were obtained once from the 188 controls. A Bayesian mixed-effects logistic regression model was used to assess potential risk factors associated with an antibody titer ≥ 3,200 in the case horses. A cutoff probability for an SeM-specific titer ≥ 3,200 was determined, and the model was externally validated in the control horses. Only variables with a 95% credibility interval that did not overlap with a value of 1 were considered significant. RESULTS 9 of 57 (6%) case horses had at least 1 titer ≥ 3,200, and 7 of 188 (3.7%) of control horses had a titer ≥ 3,200. The following variables were found to be significantly associated with a titer ≥ 3,200 in cases: farm size > 20 horses (OR, 0.11), history of clinically evident disease (OR, 7.92), and male sex (OR, 0.11). The model had 100% sensitivity but only 24% specificity when applied to the 188 control horses (area under the receiver operating characteristic curve = 0.62.) CONCLUSIONS AND CLINICAL RELEVANCE Although the Bayesian mixed-effects logistic regression model developed in this study did not perform well, it may prove useful as an initial screening tool prior to vaccination. We suggest that SeM-specific antibody titer be measured prior to vaccination when our model predicts a titer ≥ 3,200.

Gastrointestinal spindle cell tumor of the rumen with metastasis to the liver in a goat

Many neoplasms have been reported in goats; however, neoplasia of the rumen is rarely reported. A 9-y-old castrated male pygmy goat was presented with a history of respiratory stertor, fever, and anorexia. A respiratory diagnostic work-up including skull and thorax radiographs and endoscopy revealed minor enlargement of the arytenoids but no other abnormal findings. After a month of little improvement on symptomatic treatment and worsening general health, the goat was euthanized. On autopsy, the forestomachs, liver, spleen, diaphragm, and the ventral and lateral aspects of the cranial third of the walls of the peritoneal cavity were adhered to one another by fibrinous and fibrous adhesions. Numerous firm, white, up to 2 cm diameter nodules were found throughout the liver. A large sessile mass extended from the rumen wall into the lumen. The rumen mass was a gastrointestinal stromal tumor with metastasis to the liver.

Prevalence of Methicillin-Resistant Staphylococcus aureus from Equine Nasopharyngeal and Guttural Pouch Wash Samples.

Background Methicillin‐resistant Staphylococcus aureus (MRSA) is recognized as a cause of nosocomial infections in both human and veterinary medicine. Studies that examine the nasopharynx and guttural pouches of the horse as carriage sites for MRSA have not been reported. Hypothesis/Objective MRSA colonizes the nasopharynx and guttural pouch of horses. To determine the prevalence of MRSA in equine nasopharyngeal wash (NPW) and guttural pouch lavage (GPL) samples in a field population of horses. Samples One hundred seventy‐eight samples (123 NPW and 55 GPL) from 108 horses. Methods Prospective study. Samples were collected from a convenience population of clinically ill horses with suspected Streptococcus equi subsp. equi (S. equi) infection, horses convalescing from a known S. equi infection, and asymptomatic horses undergoing S. equi screening. Samples were submitted for S. aureus aerobic bacterial culture with mannitol salt broth and two selective agars (cefoxitin CHROMagar as the PBP2a inducer and mannitol salt agar with oxacillin). Biochemical identification of Staphylococcus species and pulsed‐field gel electrophoresis (PFGE), to determine clonal relationships between isolates, were performed. Results Methicillin‐resistant Staphylococcus (MRS) was isolated from the nasopharynx of 7/108 (4%) horses. Three horses had MRSA (2.7%), and 4 had MR‐Staphylococcus pseudintermedius (MRSP). MRSA was isolated from horses on the same farm. PFGE revealed the 3 MRSA as USA 500 strains. Conclusions and Clinical Importance Sampling the nasopharynx and guttural pouch of community‐based horses revealed a similarly low prevalence rate of MRSA as other studies sampling the nares of community‐based horses. More study is required to determine the need for sampling multiple anatomic sites when screening horses for MRSA.