In vitro efficacy of 0.2% and 0.4% sodium oxychlorosene against meticillin-resistant Staphylococcus pseudintermedius.

BACKGROUND: There is a need for alternative topical therapies as a consequence of the increased prevalence of meticillin-resistant Staphylococcus pseudintermedius (MRSP) skin infections in dogs. Sodium oxychlorosene has been used as a topical antibacterial agent in human medicine since 1955. OBJECTIVES: To determine whether 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>3-log reduction) on MRSP strains isolated from canine skin infections. METHODS AND MATERIALS: A genetically heterogeneous collection of MRSP isolates from dogs was assembled from laboratories across the United States. Time-kill assays were performed with 0.2% and 0.4% sodium oxychlorosene on a 0.5 McFarland standard [approximately 108 colony-forming units (cfu/ml)] suspension of each strain. The average bacterial counts (cfu/ml) of each MRSP strain then were determined at 5, 10, 20 and 60 s after exposure to sodium oxychlorosene; cfu/ml data were converted to log10 scale to calculate microbial reduction. RESULTS: The average bacterial counts following exposure to the 0.2% solution at 5, 10, 20 and 60 s were 6.94 × 104 , 5.63 × 103 , 2.96 × 102 and 1.48 × 102  cfu/ml, respectively. For the 0.4% solution, the average bacterial count at 5 s was 2.12 × 103  cfu/ml. No bacterial growth was observed for any MRSP strain by 10 s. The greatest reduction in cfu/ml occurred within 5 s following exposure to each solution 3.4-log and 4.9-log reduction for 0.2% and 0.4%, respectively. CONCLUSIONS AND CLINICAL RELEVANCE: 0.2% and 0.4% sodium oxychlorosene solutions have a bactericidal effect (>99.9% reduction) against MRSP in vitro. Further in vivo studies are necessary to determine whether it is an appropriate alternative therapy for canine pyoderma.

Risk factors for meticillin-resistant Staphylococcus aureus (MRSA) carriage in MRSA-exposed household pets.

BACKGROUND: Household pets can carry meticillin-resistant Staphylococcus aureus (MRSA) introduced to the home by their human companions. Specific factors promoting pet carriage of this pathogen have not been fully elucidated. OBJECTIVE: This study evaluated MRSA cultured from pets and the home environment in households where a human infected with MRSA had been identified, and aimed to determine potential risk factors for pet MRSA carriage. MATERIALS AND METHODS: Humans diagnosed with community-associated MRSA (CA-MRSA) skin or soft-tissue infection (SSTI) in the mid-Atlantic United States were identified. One hundred forty-two dogs and cats from 57 affected households were identified of which 134 (94.4%) pets and the household environment were sampled for bacterial culture, PCR confirmation and spa-typing for MRSA strain determination. Samples were obtained 3 months later from 86 pets. RESULTS: At baseline, 12 (9.0%) pets carried MRSA. Potential risk factors associated with carriage included pet bed (environmental) MRSA contamination, flea infestation and prior antimicrobial use in the pet. Pets tended to carry human-adapted MRSA strains and spa-types of MRSA isolates cultured from pets were concordant with strains cultured from the home environment in seven of eight homes (87.5%) at baseline. CONCLUSIONS AND CLINICAL RELEVANCE: Results may inform risk-based veterinary clinical recommendations and provide evidence for selective pet testing as a possible alternative to early removal of pets from the homes of humans infected with MRSA. MRSA contamination of the home environment is likely an important risk factor for pet MRSA carriage, and household interventions should be considered to reduce risk of MRSA carriage in exposed pets.

Diagnostic clinical microbiology.

Technological advancements have changed the way clinical microbiology laboratories are detecting and identifying bacterial, viral, parasitic, and yeast/fungal pathogens. Such advancements have improved sensitivity and specificity and reduce turnaround time to reporting of clinically important results. This article discusses and reviews some traditional methodologies along with some of the technological innovations introduced into diagnostic microbiology laboratories. Some insight to what might be available in the coming years is also discussed.

Risk Factors for the Acquisition of a blaNDM-5 Carbapenem-Resistant Escherichia coli in a Veterinary Hospital.

Carbapenem-resistant Enterobacteriaceae (CRE) are an urgent antibiotic resistant threat. Only sporadic reports of CRE in companion animals have been described. Our objective was to identify risk factors associated with the acquisition of a blaNDM-5 CR-Escherichia coli strain as part of an outbreak investigation at a tertiary veterinary hospital in the United States. A matched case-control study was conducted among companion animals admitted during July 1, 2018, through June 30, 2019. The 15 identified blaNDM-5 CR-E coli cases were matched 1:2 with controls (culture negative for blaNDM-5 CR-E coli) based on species and number of days of hospitalization before bacterial culture sample collection. The association between exposure to various procedures and hospital services and the acquisition of blaNDM-5 CR-E. coli was assessed through conditional logistic regression. Case patients had significantly higher odds of exposure to the anesthesia service (odds ratio [OR] = 12.8, P = .017), the surgical service (OR = 4.0, P = .046), and to endotracheal intubation (OR = 10.0, P = .03). Veterinary hospitals should be aware of the potential for transmission of CRE via anesthetic and surgical procedures, especially those that require the placement of endotracheal tubes.

SODAPOP: A Metacognitive Mnemonic Framework to Teach Antimicrobial Selection.

Mnemonics are used widely throughout medical education to help manage large amounts of information and to promote a systematic approach to complex problems. SODAPOP is a metacognitive mnemonic that offers learners a framework for veterinary clinical decision making to support optimal antimicrobial selection. SODAPOP has students consider the source and organism before they decide to treat; then they consider the antimicrobials to which the organism is susceptible with regard to contraindications in the patient; and, ultimately, the options are weighed and a plan is formulated. A preliminary study showed that students' perception of SODAPOP was favorable and that exposure to SODAPOP improved student confidence levels. Further research is needed to determine whether SODAPOP improves students' optimal antimicrobial selection. SODAPOP could be a potentially helpful teaching tool because it can be mapped to the Association of American Veterinary Medical Colleges competency-based veterinary education framework under subcompetencies 1.3 and 4.2. A mnemonic such as SODAPOP could be integrated throughout the veterinary curriculum both in basic science courses (microbiology) and with real cases during clinical rotations.

New Delhi Metallo-ß-Lactamase-5-Producing Escherichia coli in Companion Animals, United States.

We report isolation of a New Delhi metallo-ß-lactamase-5-producing carbapenem-resistant Escherichia coli sequence type 167 from companion animals in the United States. Reports of carbapenem-resistant Enterobacteriaceae in companion animals are rare. We describe a unique cluster of blaNDM-5-producing E. coli in a veterinary hospital.

The otic microbiota and mycobiota in a referral population of dogs in eastern USA with otitis externa.

BACKGROUND: Canine otitis externa (OE) is a common inflammatory disease that is frequently complicated by secondary bacterial and/or yeast infections. The otic microbial population is more complex than appreciated by cytological methods and aerobic culture alone. HYPOTHESIS/OBJECTIVES: Differences in bacterial and fungal populations of the external ear canal will correlate with specific cytological and culture-based definitions of bacterial and Malassezia otitis. ANIMALS: Forty client-owned dogs; 30 with OE and 10 with healthy ears. METHODS AND MATERIALS: Prospective study comparing cytological samples, aerobic bacterial cultures and culture-independent sequencing-based analyses of the external ear canal. Subjects with OE included 10 dogs with only cocci [≥25/high power field (HPF)] on cytological evaluation and culture of Staphylococcus spp.; 10 dogs with rods (≥25/HPF) and exclusive culture of Pseudomonas aeruginosa; 10 dogs with only yeast on cytological results morphologically compatible with Malassezia spp. (≥5/HPF). RESULTS: Staphylococcus was the most abundant taxa across all groups. Ears cytologically positive for cocci had decreased diversity, and all types of OE were associated with decreased fungal diversity compared to controls. CONCLUSIONS AND CLINICAL IMPORTANCE: Cytological and culture-based assessment of the ear canal is not predictive of the diverse microbiota of the ear canal in cases of Pseudomonas or Malassezia otitis. Less abundant bacterial taxa in cases of staphylococcal OE are worth scrutiny for future biological therapy.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Short communication: Oral and intranasal administration of a modified-live Salmonella Dublin vaccine in dairy calves: Clinical efficacy and serologic response.

Our objectives were to evaluate the clinical efficacy of oral and intranasal administration of a commercial modified-live Salmonella Dublin vaccine in dairy calves and to determine the serologic response associated with these extralabel routes of administration. We conducted a randomized field trial with calves from a New York dairy farm following an outbreak of Salmonella Dublin. A total of 399 Holstein calves were allocated by pen to 3 treatment groups: oral vaccination, intranasal vaccination, and an unvaccinated control group. Administration of the vaccine through oral and intranasal routes did not have a significant effect on pneumonia incidence risk or weight gain; however, calves vaccinated orally and intranasally had lower mortality risk as compared with control calves. Among calves tested using a Salmonella Dublin ELISA, vaccination did not induce an increase in antibody production relative to control calves, indicating that oral and intranasal administration will not hinder diagnosis based on this assay.

Multilaboratory Survey To Evaluate Salmonella Prevalence in Diarrheic and Nondiarrheic Dogs and Cats in the United States between 2012 and 2014.

Eleven laboratories collaborated to determine the periodic prevalence of Salmonella in a population of dogs and cats in the United States visiting veterinary clinics. Fecal samples (2,965) solicited from 11 geographically dispersed veterinary testing laboratories were collected in 36 states between January 2012 and April 2014 and tested using a harmonized method. The overall study prevalence of Salmonella in cats (3 of 542) was <1%. The prevalence in dogs (60 of 2,422) was 2.5%. Diarrhea was present in only 55% of positive dogs; however, 3.8% of the all diarrheic dogs were positive, compared with 1.8% of the nondiarrheic dogs. Salmonella-positive dogs were significantly more likely to have consumed raw food (P = 0.01), to have consumed probiotics (P = 0.002), or to have been given antibiotics (P = 0.01). Rural dogs were also more likely to be Salmonella positive than urban (P = 0.002) or suburban (P = 0.001) dogs. In the 67 isolates, 27 unique serovars were identified, with three dogs having two serovars present. Antimicrobial susceptibility testing of 66 isolates revealed that only four of the isolates were resistant to one or more antibiotics. Additional characterization of the 66 isolates was done using pulsed-field gel electrophoresis and whole-genome sequencing (WGS). Sequence data compared well to resistance phenotypic data and were submitted to the National Center for Biotechnology Information (NCBI). This study suggests an overall decline in prevalence of Salmonella-positive dogs and cats over the last decades and identifies consumption of raw food as a major risk factor for Salmonella infection. Of note is that almost half of the Salmonella-positive animals were clinically nondiarrheic.

Two-Stage Isothermal Enzymatic Amplification for Concurrent Multiplex Molecular Detection.

BACKGROUND: The wide array of pathogens responsible for infectious diseases makes it difficult to identify causative pathogens with single-plex tests. Although multiplex PCR detects multiple targets, it is restricted to centralized laboratories, which delays test results or makes multiplexing unavailable, depriving healthcare providers of critical, real-time information. METHODS: To address the need for point-of-care (POC) highly multiplexed tests, we propose the 2-stage, nested-like, rapid (<40 min) isothermal amplification assay, dubbed rapid amplification (RAMP). RAMP's first-stage uses outer loop-mediated isothermal amplification (LAMP) primers to amplify all targets with recombinase polymerase amplification (RPA). First-stage amplicons are aliquoted to second stage reactors, each specialized for a specific target, to undergo LAMP. The assay is implemented in a microfluidic chip. LAMP amplicons are detected in situ with colorimetric dye or with a fluorescent dye and a smartphone. RESULTS: In experiments on a benchtop and in a microfluidic format, RAMP demonstrated high level of multiplexing (≥16); high sensitivity (i.e., 1 plaque-forming unit of Zika virus) and specificity (no false positives or negatives); speed (<40 min); ease of use; and ability to cope with minimally processed samples. CONCLUSIONS: RAMP is a hybrid, 2-stage, rapid, and highly sensitive and specific assay with extensive multiplexing capabilities, combining the advantages of RPA and LAMP, while circumventing their respective shortcomings. RAMP can be used in the lab, but one of its distinct advantages is amenability to simple implementation in a microfluidic format for use at the POC, providing healthcare personnel with an inexpensive, highly sensitive tool to detect multiple pathogens in a single sample, on site.

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

BACKGROUND: Streptococcus 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. METHODS: A 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). RESULTS: One 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). CONCLUSION: This 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.

Molecular and epidemiological characterization of canine Pseudomonas otitis using a prospective case-control study design.

BACKGROUND: Pseudomonas aeruginosa is an opportunistic pathogen of the canine ear canal and occupies aquatic habitats in the environment. Nosocomial and zoonotic transmission of P. aeruginosa have been documented, including clonal outbreaks. HYPOTHESIS/OBJECTIVES: The primary objective of this study was to assess various environmental exposures as potential risk factors for canine Pseudomonas otitis. It was hypothesized that isolates derived from infected ears would be clonal to isolates derived from household water sources and the mouths of human and animal companions of the study subjects. ANIMALS: Seventy seven privately owned dogs with otitis were enrolled, along with their human and animal household companions, in a case-control design. METHODS: Data on potential risk factors for Pseudomonas otitis were collected. Oral cavities of all study subjects, their human and animal companions, and household water sources were sampled. Pulsed field gel electrophoresis was used to estimate clonal relatedness of P. aeruginosa isolates. RESULTS: In a multivariate model, visiting a dog park was associated with 77% increased odds of case status (P = 0.048). Strains clonal to the infection isolates were obtained from subjects' mouths (n = 18), companion pets' mouths (n = 5), pet owners' mouths (n = 2), water bowls (n = 7) and water taps (n = 2). Clonally related P. aeruginosa isolates were obtained from dogs that had no clear epidemiological link. CONCLUSIONS AND CLINICAL IMPORTANCE: Genetic homology between otic and environmental isolates is consistent with a waterborne source for some dogs, and cross-contamination with other human and animal members within some households.

Comparison of Culture-Based Methods for Identification of Colonization with Methicillin-Resistant and Methicillin-Susceptible Staphylococcus aureus in the Context of Cocolonization.

Two screening methods to detect staphylococcal colonization in humans were compared. Direct plating to CHROMagar (BD Diagnostics) was compared to a broth preenrichment followed by plating to Baird-Parker agar. The broth-enrichment method was comparable to CHROMagar for methicillin-resistant Staphylococcus aureas (MRSA) detection, but the enrichment method was optimum for recovery of coagulase-positive Staphylococcus spp.

Salmonella Prevalence and Antimicrobial Susceptibility Among Dairy Farm Environmental Samples Collected in Texas.

Dairy cattle are a reservoir of several Salmonella serovars that are leading causes of human salmonellosis. The objectives of this study were to estimate the environmental prevalence of Salmonella on dairy farms in Texas and to characterize the antimicrobial susceptibility of the isolates. Eleven dairy farms throughout Texas were sampled from August through October 2013, using a cross-sectional approach. Samples were collected from four locations within each farm (hospital pen, maternity pen, cow housing area, and calf housing area), and feces were collected from cull cows as available. Environmental and fecal samples were processed for Salmonella, and isolates were tested for susceptibility to 15 antimicrobial agents. Serovar characterization was performed on a subset of these isolates. Salmonella was isolated from 67.0% (236/352) of the environmental samples and 64.2% (43/67) of the cull cow fecal samples. Environmental samples from the maternity pen were significantly more likely to be Salmonella positive than samples from the cow and calf housing areas. Multidrug resistance was evident in 11.9% (27/226) of environmental isolates and 19.5% (8/41) of fecal isolates. Salmonella isolates from the calf housing area and maternity pen were significantly more likely to be multidrug resistant (MDR) than isolates from the cow housing area. The most common serovars found among the MDR isolates were Newport, Muenchen, and Typhimurium. These results help provide a focus for efforts to mitigate the burden of antimicrobial-resistant Salmonella at the preharvest level.

Genotypic relatedness and antimicrobial resistance of Staphylococcus schleiferi in clinical samples from dogs in different geographic regions of the United States.

BACKGROUND: Staphylococcus schleiferi is a known pathogen that can cause canine skin and ear infections. The aim of this study was to determine the molecular epidemiology and antimicrobial susceptibility of clinical veterinary isolates from different geographic regions in the United States. HYPOTHESIS: It was hypothesized that S. schleiferi would maintain genotypic homogeneity across the different geographic regions and that meticillin-resistant (MR) isolates of S. schleiferi would predominate. METHODS: Isolates were identified as S. schleiferi by a commercial microbiology identification system and confirmed by nuc gene PCR. Antibiotic susceptibility data were collected and PBP2a latex agglutination testing was performed on MR isolates. Pulsed-field gel electrophoresis (PFGE) was performed and clonal clusters were identified with a Dice coefficient similarity of >80%. RESULTS: There were 116 isolates from the Mid-Atlantic region and 101 from across the United States. Of these 217 isolates, 209 (96%) were obtained from cutaneous sites. Of the Mid-Atlantic isolates, 62% (72 of 116) were MR and 16% (18 of 116) were multidrug-resistant (MDR). Of the isolates from the other geographic regions, 73% (74 of 101) were MR and 24% (24 of 101) were MDR. All MR isolates were positive by PBP2a latex agglutination. PFGE identified 155 individual pulsed-field profiles and three major pulsed-field types (PFT) that contained 61% (133 of 217) of the isolates. These pulsed-field types were geographically heterogeneous. CONCLUSIONS: This study demonstrates the dissemination of successful MR pulsed-field types of S. schleiferi across the United States.

Use of a chromogenic medium with and without selective enrichment to screen for carbapenemase-producing Enterobacterales (CPE) from canine and feline fecal specimens during an outbreak of NDM-5-producing Escherichia coli.

Carbapenemase-producing Enterobacterales (CPE) are one of the most urgent threats to human healthcare globally. Descriptions of CPE outbreaks in veterinary hospitals suggest the need for screening strategies for CPE from companion animals. Our aim was to optimize a chromogenic agar method with and without selective enrichment to isolate CPE from companion animal feces in an ongoing outbreak of New Delhi metallo-ß-lactamse-5 Escherichia coli. A limit of detection (LOD) assay for spiked canine and feline feces was performed for both methods using a carbapenamase-producing E. coli (24213-18); the LOD (1.5 × 103 cfu/g of feces) was equivalent to that reported for human fecal specimens. We screened 1,247 companion animal fecal specimens for carriage of CPE by 1) direct plating to chromogenic agar and 2) plating to chromogenic agar following selective enrichment. Twenty-one specimens were positive for CPE by both direct culture and enrichment culture. No specimens were positive with selective enrichment and negative by direct culture. A selective enrichment step did not result in any increased recovery of CPE from companion animals, which suggests that enrichment broth may not be necessary for outbreak surveillance testing. It is important to continue to validate methods for the detection of CPE in companion animals as outbreaks become more common in veterinary facilities.

Evaluation of the affinity of various species and strains of Staphylococcus to adhere to equine corneocytes.

BACKGROUND: Meticillin-resistant Staphylococcus aureus (MRSA) strain USA 500 predominately colonizes horses and people working with them. Previous studies demonstrate that some Staphylococcus species exhibit higher affinity for corneocytes of specific mammalian species. HYPOTHESIS/OBJECTIVES: The objective was to determine the relative affinities of various MRSA strains, meticillin-susceptible S. aureus (MSSA) strains and a meticillin-susceptible Staphylococcus pseudintermedius (MSSP) for equine corneocytes. We hypothesized that MRSA strain USA 500 would exhibit greater adhesion than other staphylococcal strains tested. METHODS: Epidemic MRSA strains (USA 100, USA 300, USA 500 and USA 800), two MSSA control strains and an MSSP field strain were tested on corneocytes from 15 client-owned horses. Isolates were incubated with corneocytes in conditions (bacterial concentration of 10(8) colony-forming units/mL for 45 min) recently shown to maximize adherence of S. aureus without competitive interference. A validated image-analysis system was used to quantify the cell surface density of bacterial adhesion. RESULTS: The MSSP strain adhered with significantly higher affinity (P < 0.0015) to corneocytes than did MSSA strains. All MRSA strains other than USA 500 had significantly higher affinity than MSSA strains (P range <0.03 to <0.0015). There were no statistical differences in adhesion between strain USA 500 and the other MRSA strains tested. CONCLUSIONS AND CLINICAL IMPORTANCE: Meticillin-resistant S. aureus strain USA 500 did not adhere more robustly than other strains of Staphylococcus; therefore, its affinity to colonize horses may not be solely attributed to corneocyte adhesion. Additional studies are required to explain the epidemiological role of this strain as the predominant cause of colonization and infections of horses in North America.

Detection of Viable Streptococcus equi equi Using Propidium Monoazide Polymerase Chain Reaction.

There is debate around the clinical significance of Streptococcus equi subsp. equi detection in low numbers using quantitative real-time PCR (qPCR). Propidium monoazide (PMA) qPCR has been used to differentiate DNA from viable and nonviable bacterial cells. The aim of this study was to evaluate the ability of PMA eqbE SEQ2190 triplex qPCR to differentiate DNA from viable and nonviable S. equi in positive and suspect positive clinical specimens. Fifty-seven stored (frozen and refrigerated) positive (36) or suspect positive (21) clinical specimens (determined via SeeI qPCR as the gold standard) were tested using eqbE SEQ2190 triplex qPCR with (+) and without (-) PMA pretreatment. Cycle thresholds were higher when using PMA indicating a mixture of heat killed and viable cells. Number of S. equi positive specimens were as follows: 6/57 eqbE + PMA, 13/57 eqbE -PMA (Chi- squared 3.1, p = .079); 10/57 SEQ2190 +PMA, 53/57 SEQ2190 -PMA (Chi- squared 65.6, p < .0001). The mean cycle thresholds were as follows: 23.88 eqbE -PMA, 29.89 eqbE + PMA (p = .04); 24.9 SEQ2190 -PMA, 31.9 SEQ2190 +PMA (p < .0001). PMA qPCR can be used to determine S. equi viability, but testing should be performed on fresh specimens.