Risk factors for veterinary hospital environmental contamination with Salmonella enterica.

Healthcare-associated infections in veterinary hospitals are commonly attributed to Salmonella enterica, particularly in large animal facilities, and are characteristically associated with widespread environmental contamination. The objective of this study was to investigate factors influencing the likelihood of identifying environmental contamination of a veterinary hospital with S. enterica, while exploring different analytic methods to model complex factors that may influence this ecology. Environmental surveillance samples were collected in a large veterinary hospital as part of a long-term infection control programme. Data were collected retrospectively from the electronic medical records database. Many easily measured variables were complex in nature (i.e., they represented variance that is unmeasured or unidentified as a specific factor) necessitating the use of alternative analytic methods (variable cluster and principal components analyses) to provide perspective regarding the complex data structure and latent factors that may be contributing to this ecology. Subsequently, multivariable logistic regression was performed using generalised estimating equations. Results suggest the probability of detecting Salmonella in the environment increased as demand on personnel increased (e.g., in a busy hospital). Veterinary personnel need to remain vigilant in implementing practices that we believe empirically will mitigate risk for widespread environmental contamination and sustained transmission among patients (i.e., rigorous hygiene for personnel and the environment).

Factors associated with equine shedding of multi-drug-resistant Salmonella enterica and its impact on health outcomes.

BACKGROUND: Salmonella enterica is an important cause of healthcare-associated infections in veterinary hospitals - with outbreaks of multi-drug resistant (MDR) Salmonella among equine cases resulting in high case fatality rates and substantial financial cost. OBJECTIVES: Study objectives were to 1) investigate factors associated with shedding of MDR-Salmonella enterica and 2) evaluate the effect shedding may have on health outcomes of previously hospitalised horses and their stablemates. STUDY DESIGN: Retrospective case-control study with prospective cohort study. METHODS: Analysis of medical records (N = 373; 94 culture positive, 279 culture negative) was undertaken to determine factors associated with shedding of MDR-Salmonella. Additionally, a follow-up study was conducted to assess long-term outcomes associated with shedding among previously hospitalised horses and their stablemates. Data regarding exposures of interest were collected retrospectively from medical records. Information on long-term outcomes was obtained by phone interview of owners. Multivariable regression techniques were used to investigate factors associated with shedding and subsequent health outcomes. RESULTS: Horses experiencing diarrhoea during hospitalisation were more likely to shed Salmonella (OR 1.88; 95% CI 1.02, 3.45) compared with horses without diarrhoea, but isolates tended to be susceptible strains. Antimicrobial therapy during hospitalisation was not associated with shedding or recovery of MDR strains. Shedding did not increase long-term risk for non-survival, colic or abnormal faeces after hospital discharge; nor increase risk for hospitalisation or occurrence of abnormal faeces in stablemates. MAIN LIMITATIONS: Data collection was reliant upon the quality of medical records and owner recall, which may have led to information bias. The study population was derived from central Kentucky and may differ from horse populations in other regions. CONCLUSIONS AND CLINICAL IMPORTANCE: In general, Salmonella shedding was not associated with decreased average survival times or impacts to health of stablemates, perhaps due to owner implemented biosecurity precautions. Regardless, recently hospitalised horses should be segregated after discharge, in addition to employing rigorous hygiene practices. The Summary is available in Spanish - see Supporting Information.

Evaluation of the Efficacy of Disinfectant Footmats for the Reduction of Bacterial Contamination on Footwear in a Large Animal Veterinary Hospital.

BACKGROUND: Infection control is critical to providing high-quality patient care. Many veterinary teaching hospitals (VTHs) utilize footbaths or footmats at entrances and key control points throughout the facility to decrease trafficking of pathogenic microorganism on contaminated footwear. HYPOTHESIS/OBJECTIVES: To compare efficacy of 4 disinfectants used in footmats for decreasing bacterial contamination of footwear in a large animal hospital. ANIMALS: A single adult dairy cow was housed in a stall for 4 days to facilitate stall contamination with fecal material. METHODS: Overboots were experimentally contaminated with organic material in a standardized manner. Each boot was randomly assigned to 1 of 5 treatments (no treatment, or exposure to 1 of 4 disinfectants: an accelerated peroxygen [AHP], a peroxygen [VIRKON], a quaternary ammonium [QUAT], and a phenolic disinfectant [PHENOLIC]) by stepping on a soaked footmat and collecting samples from boot soles. Generalized linear modeling was used to analyze differences in bacterial counts. RESULTS: Reductions in colony-forming units (CFUs) on treated boots ranged from no detectable reduction to 0.45 log10 and varied by disinfectant. Percentage reductions in total bacterial counts generally were larger (albeit still modest) for AHP and QUAT disinfectants (range 37-45%) and smallest for the PHENOLIC (no detectable reduction). CONCLUSIONS AND CLINICAL IMPORTANCE: In general, use of disinfectant footmats was associated with significant reductions in viable bacteria on overboots-albeit with variable efficacy. Footmats may be useful adjuncts to cleaning and disinfection programs for decreasing trafficking of microorganisms throughout VTHs but should not be considered as a sole prevention method.

Comparison of disinfectant efficacy when using high-volume directed mist application of accelerated hydrogen peroxide and peroxymonosulfate disinfectants in a large animal hospital.

REASONS FOR PERFORMING STUDY: Effective decontamination of animal holding environments is critical for providing high quality patient care and maintaining a safe working environment. Disinfection of animal holding environments is a significant challenge during times of epidemic disease. OBJECTIVES: The purpose of this study was to evaluate the disinfectant efficacy of 3 strategies for high-volume directed mist application of accelerated hydrogen peroxide and peroxymonosulfate disinfectants; 4.25% accelerated hydrogen peroxide (Accel(®) ; AHP) at a 1:16 dilution and single and double applications of 2% peroxymonosulfate solution (Virkon-S(®) ; VIR-1 and VIR-2) for decontamination of a large animal hospital environment. STUDY DESIGN: Experiment. METHODS: After cleaning and disinfection of the hospital environment, transparencies experimentally contaminated with known concentrations of Staphylococcus aureus, Salmonella enterica and Pseudomonas aeruginosa were placed on vertical surfaces. Disinfectant solution was applied by directed mist application and, after 30 min of contact time, transparencies were collected and individually placed into tubes containing 10 ml Dey-Engley broth. The process was repeated for each disinfectant. Tenfold dilutions of each sample were plated onto tryptic soy blood agar with 5% sheep blood. Bacterial counts from transparencies exposed to disinfectants were compared with counts from control transparencies (unexposed to disinfectants) to evaluate reduction in colony forming units. RESULTS: The least squares mean reduction (log10 ) in colony forming units (CFUs) for S. aureus and P. aeruginosa was 1.5-2.5 logs and approximately 0.8-1.0 logs for S. enterica. Reductions were generally largest for VIR-2 and smallest for AHP, although these differences were not all statistically significant and the magnitude of differences may not be clinically relevant. CONCLUSIONS: For the organisms evaluated, all 3 disinfectants applied as a directed mist were effective at reducing CFUs in a veterinary hospital environment. Effective disinfection using this method of application is dependent on adequate cleaning prior to application, and use of adequate volumes of disinfectant.

Rapid Salmonella detection in experimentally inoculated equine faecal and veterinary hospital environmental samples using commercially available lateral flow immunoassays.

REASONS FOR PERFORMING STUDY: Salmonella enterica is the most commonly reported cause of outbreaks of nosocomial infections in large animal veterinary teaching hospitals and the closure of equine hospitals. Rapid detection may facilitate effective control practices in equine populations. Shipping and laboratory testing typically require ≥48 h to obtain results. Lateral flow immunoassays developed for use in food-safety microbiology provide an alternative that has not been evaluated for use with faeces or environmental samples. OBJECTIVES: We aimed to identify enrichment methods that would allow commercially available rapid Salmonella detection systems (lateral flow immunoassays) to be used in clinical practice with equine faecal and environmental samples, providing test results in 18-24 h. STUDY DESIGN: In vitro experiment. METHODS: Equine faecal and environmental samples were inoculated with known quantities of S. enterica serotype Typhimurium and cultured using 2 different enrichment techniques for faeces and 4 enrichment techniques for environmental samples. Samples were tested blindly using 2 different lateral flow immunoassays and plated on agar media for confirmatory testing. RESULTS: In general, commercial lateral flow immunoassays resulted in fewer false-negative test results with enrichment of 1 g faecal samples in tetrathionate for 18 h, while all environmental sample enrichment techniques resulted in similar detection rates. The limit of detection from spiked samples, ∼4 colony-forming units/g, was similar for all methods evaluated. CONCLUSIONS: The lateral flow immunoassays evaluated could reliably detect S. enterica within 18 h, indicating that they may be useful for rapid point-of-care testing in equine practice applications. Additional evaluation is needed using samples from naturally infected cases and the environment to gain an accurate estimate of test sensitivity and specificity and to substantiate further the true value of these tests in clinical practice.

Detection of different serotypes of Salmonella enterica in experimentally inoculated equine fecal samples by commercially available rapid tests.

BACKGROUND: Salmonella enterica can significantly impact management of animal facilities. Comprehensive screening is essential for effective control in high-risk populations. Availability of reliable point-of-care diagnostic tests would facilitate these efforts. HYPOTHESIS/OBJECTIVES: Compare the ability of commercially available rapid diagnostic assays (2 lateral flow immunoassays [LFIs], DNA hybridization [DNAH], real-time PCR [qPCR]), and culture to detect common serotypes of S. enterica in feces. ANIMALS: n/a. METHODS: In an experimental study, 112 S. enterica isolates were randomly selected from the 10 most common serotypes recovered at a veterinary hospital. Archived isolates were amplified in broth and standardized inocula (100 colony forming units) were incubated with equine feces in tetrathionate broth (TET). Cultures were tested in a blinded fashion by using LFIs, DNAH, qPCR, and culture. RESULTS: The LFIs detected 84% and 67% of isolates, respectively, but reactivity varied among serotypes. Both reacted poorly with serotype Cerro (Group K) isolates, and 1 LFI did not react with any serotype Mbandaka (Group C1) or Montevideo (Group C1) isolates. DNAH detected 94% of isolates, whereas culture and qPCR most reliably detected all serotypes. False-positive results were obtained for 4 negative controls by using DNAH and 1 negative control by using qPCR, but LFIs and culture had no false-positive results. CONCLUSIONS AND CLINICAL IMPORTANCE: Culture, qPCR, and DNAH were effective in detecting most Salmonella isolates, but have limited application at point-of-care settings. LFIs are appealing as point-of-care tests because of low cost and ease of use, but limited detection of some serotypes needs to be evaluated with samples obtained from naturally infected animals.

Comparison of two sampling and culture systems for detection of Salmonella enterica in the environment of a large animal hospital.

REASONS FOR PERFORMING STUDY: Nosocomial salmonellosis is an important problem in veterinary hospitals that treat horses and other large animals. Detection and mitigation of outbreaks and prevention of healthcare-associated infections often require detection of Salmonella enterica in the hospital environment. OBJECTIVES: To compare 2 previously published methods for detecting environmental contamination with S. enterica in a large animal veterinary teaching hospital. STUDY DESIGN: Hospital-based comparison of environmental sampling techniques. METHODS: A total of 100 pairs of environmental samples were collected from stalls used to house large animal cases (horses, cows or New World camelids) that were confirmed to be shedding S. enterica by faecal culture. Stalls were cleaned and disinfected prior to sampling, and the same areas within each stall were sampled for the paired samples. One method of detection used sterile, premoistened sponges that were cultured using thioglycolate enrichment before plating on XLT-4 agar. The other method used electrostatic wipes that were cultured using buffered peptone water, tetrathionate and Rappaport-Vassiliadis R10 broths before plating on XLT-4 agar. RESULTS: Salmonella enterica was recovered from 14% of samples processed using the electrostatic wipe sampling and culture procedure, whereas S. enterica was recovered from only 4% of samples processed using the sponge sampling and culture procedure. There was test agreement for 85 pairs of culture-negative samples and 3 pairs of culture-positive samples. However, the remaining 12 pairs of samples with discordant results created significant disagreement between the 2 detection methods (P<0.01). CONCLUSIONS: Persistence of Salmonella in the environment of veterinary hospitals can occur even with rigorous cleaning and disinfection. Use of sensitive methods for detection of environmental contamination is critical when detecting and mitigating this problem in veterinary hospitals. These results suggest that the electrostatic wipe sampling and culture method was more sensitive than the sponge sampling and culture method.

Nasal shedding of equine herpesvirus-1 from horses in an outbreak of equine herpes myeloencephalopathy in Western Canada.

BACKGROUND: There is little information on the duration of nasal shedding of EHV-1 from horses with naturally occurring equine herpesvirus myeloencephalopathy (EHM). OBJECTIVES: To evaluate the duration of nasal shedding of EHV-1 in horses affected by EHM. ANIMALS: One hundred and four horses naturally exposed to EHV-1, 20 of which had clinical signs of EHM. METHODS: All horses on affected premises were monitored. Those horses developing EHM were sampled in a longitudinal outbreak investigation. Nasal swabs were collected daily from 16 of 20 horses affected by EHM. A qPCR was performed on 98 of 246 nasal swab samples to determine nasal shedding duration. Historical and clinical information was analyzed to evaluate potential risk factors for developing EHM and duration of shedding during this outbreak. RESULTS: The last day shedding was detected in any horse was Disease Day 9. EHV-1 was detected in two-thirds of horses tested on Disease Days 0-3. The amount of EHV-1 DNA found in nasal swabs varied markedly and was not associated with disease severity or age. The odds of developing EHM were greater for febrile horses (OR = 20.3; 95% CI 3.4-390.3; P = .01) as well as for horses attending the riding clinic (OR = 4.1; 95% CI 0.84-21.65; P = .08). CONCLUSIONS AND CLINICAL IMPORTANCE: Biosecurity measures should be implemented for a minimum of 14 days beyond the onset of clinical signs of EHM. Animal managers cannot rely on the severity of clinical signs to predict the duration of EHV-1 shedding.

1-Methyl-2-pyrrolidone (NMP): reproductive and developmental toxicity study by inhalation in the rat.

A two-generation reproduction study with a developmental toxicity component was conducted. For the reproduction phase, male and female rats inhaled 0, 10, 51, or 116 ppm NMP daily for 6 hr/day, 7 days/week from 34 days of age to the end of the mating period for the males (100 exposure days) and till weaning for the females (about 143 exposure days, but interrupted from Day 20 of gestation to Day 4 Postpartum). On Day 70 postpartum, one male and one female selected from each litter later mated with newly obtained, nonexposed adults of the opposite sex to produce an F2 generation. For the developmental phase, rats of both sexes inhaled 0 or 116 ppm NMP as outlined above, but euthanization of the females occurred on Day 21 of gestation followed by fetal examination for structural alterations. The indices of reproductive performance for the NMP-exposed rats did not differ significantly from those obtained for the control rats. Rats exposed to 116 ppm had a detectable decrease in response to sound. No other signs of NMP-related toxicity were detected among the parental rats. An exposure-related but slight decrease in fetal weight was detected only among the F1 offspring whose parents both inhaled NMP at 116 ppm. This slight effect also appeared at birth among the pups of the reproductive phase where it persisted till 21 days after birth when NMP inhalation by the mother ceased. Thereafter, the body weight of the offspring was comparable to the control values. No detectable or developmental effects appeared in the 10 or 51 ppm groups.

Inhalation studies in rats exposed to dimethylacetamide (DMAc) from 3 to 12 hours per day.

Male rats were exposed by inhalation from 10 to 300 ppm Dimethylacetamide (DMAc) for either 3, 6, or 12 hrs/day for a total of 10 exposures (5 exposures, 2 rest days, 5 exposures). Rats were observed daily for signs of DMAc-related effects, growth was monitored by body weights, clinical laboratory tests and microscopic examination of the liver, testes epididymides, and nasal passages were conducted. One half of the rats in each group was allowed a 14-day post-exposure period to evaluate the reversibility of DMAc-induced changes. No clinical signs of toxicity or DMAc-related gross changes at necropsy were seen in any of the rats although 1 rat exposed to 300 ppm for 12 hours per day died following the seventh exposure. Slight (< 5%) decreases in body weight gain were seen in rats exposed to 300 ppm for 6 or 12 hrs/day. Serum cholesterol levels were elevated in rats exposed to either 100 or 300 ppm (all exposure durations) and in rats exposed to 30 ppm for 12 hours. Total serum protein concentrations were increased in rats exposed for 12 hours/day to either 30, 100, or 300 ppm. Hepatocellular hypertrophy together with margination of hepatocellular cytoplasmic contents and lipid-like cytoplasmic vacuolation in hepatocytes were seen microscopically only in rats exposed for 12 hours/day to 300 ppm. Recovery from these liver changes was not complete after 14-day post-exposure period. No evidence of either testicular damage or irritation to the upper respiratory tract was seen.

2-week inhalation study of N-monomethylformamide in rats.

N-Monomethylformamide (MMF) is a chemical intermediate with potential for inhalation exposure in humans. Human exposures to MMF have occurred in cancer chemotherapy but have been limited due to liver damage. To assess the toxicity of MMF, groups of 15 male rats each were exposed by nose-only inhalation, 6 hr/day, 5 days/week, for 2 weeks to either 0 (control), 50, 130, or 400 ppm MMF. Five rats per group were killed following the 10th exposure, five were killed after a 14-day postexposure recovery period, and five rats were used to determine urinary MMF excretion. Parameters investigated were clinical observations and body weights, clinical pathology, and gross and microscopic pathology including organ weights. Liver damage occurred in rats exposed to either 130 or 400 ppm. This was detected both by increases in serum enzyme activity indicative of liver injury and by microscopic changes in the liver. The changes were more severe in the 400-ppm rats and were partially reversible. Other organs were not adversely affected by inhalation of MMF. The amount of MMF excreted in the urine was dependent on the exposure concentration and MMF was present 14 days postexposure at the higher exposure levels. The no-observed-effect level under the conditions of this experiment was 50 ppm.

Characterization of technetium-99m-L,L-ECD for brain perfusion imaging, Part 1: Pharmacology of technetium-99m ECD in nonhuman primates.

Technetium-99m ethyl cysteinate dimer ([99mTc]ECD) is a neutral, lipophilic complex which rapidly crosses the blood-brain barrier. Brain retention and tissue metabolism of [99mTc]ECD is dependent upon the stereochemical configuration of the complex. While both L,L and D,D enantiomers are extracted by the brain, only the L,L but not the D,D form, is metabolized and retained in the monkey brain (4.7% injected dose initially, T 1/2 greater than 24 hr). Dynamic single photon emission computed tomography imaging studies in one monkey indicates 99mTc-L,L-ECD to be distributed in a pattern consistent with regional cerebral blood flow for up to 16 hr postinjection. Dual-labeled 99mTc-L,L-ECD and [14C]iodoantipyrine autoradiography studies performed 1 hr after administration show cortical gray to white matter ratios of both isotopes to be equivalent (approximately 4-5:1). These data suggest that 99mTc-L,L-ECD will be useful for the scintigraphic assessment of cerebral perfusion in humans.

Inhalation toxicity of methyl difluoromalonyl fluoride in rats.

Methyl 2,2-difluoromalonyl fluoride (MMF) is highly toxic by inhalation producing mortality in rats exposed for 4 hours to 0.55 mg/L. Repeated inhalation exposures of rats to 0.009 mg/L produced irritation but no other signs of a toxic response. Mortality was encountered following repeated exposures to 0.066 mg/L.

Estimation of acute oral toxicity in rats by determination of the approximate lethal dose rather than the LD50.

The approximate lethal dose (ALD) to rats following oral administration of nine chemicals was determined. The ALD (lowest dose at which death was produced) for each chemical was used to define the general order of toxicity as being either extremely, highly, moderately, slightly, or practically non-toxic. For each of the nine chemicals tested, the category indicated by the ALD was identical with the category determined in the more extensive LD50 studies. In this study, an average of 6.8 rats was needed to determine the ALD while an average of 56.3 rats was needed to determine the LD50.

Sensory irritation potential of selected nasal tumorigens in the rat.

Several important chemicals, including formaldehyde, 1,4-dichloro-2-butene, bis-chloromethyl ether, hexamethylphosphoramide, and epichlorohydrin have been shown to produce nasal tumours in rats following repeated or continuous inhalation exposures. Some of these compounds are respiratory irritants. To determine whether there is a correlation between the ability of a chemical to produce sensory irritation and to elicit nasal tumours, the atmospheric concentration causing a 50% decrease in the respiratory rate (RD50) of male rats was determined. Three other nasal tumorigens, dimethylcarbamoyl chloride, 2,3,4-trichloro-1-butene and 1,2-ethoxy-3-phenoxypropane, were also studied. No correlation between sensory irritation potency and nasal tumorigenic potential was observed. The most potent nasal tumorigen hexamethylphosphoramide, which produces tumours in rats following 12 months' continuous exposure to 50 ppb, failed to cause any decrease in respiratory rate when tested at 351 ppm (an aerosol exposure level which exceeds atmospheric saturation by approximately ten times).

The embryo-fetal toxicity and teratogenic potential of ammonium perfluorooctanoate (APFO) in the rat.

Ammonium perfluorooctanoate (APFO, greater than 95% pure) was administered to Sprague-Dawley rats from Days 6 through 15 of gestation by inhalation as a dust (whole body exposure) for 6 hr/day at 0, 0.1, 1, 10, and 25 mg/m3, or by gavage at 100 mg/kg body wt/day in corn oil. Maternal deaths occurred in the groups given the highest level of APFO by each route and overt toxicity was evident among the surviving dams of these groups and among those of the 10-mg/m3 group. The fetuses were examined for external, visceral, and skeletal alterations and for APFO-related macroscopic and microscopic alterations of the eyes. In the postpartum period, pups from additional control and experimental dams were examined externally and ophthalmoscopically, and the usual fertility and viability indices were calculated. A teratogenic response was not demonstrated. Toxic effects on the conceptus were noted only in the groups given the highest level of APFO by each route. Hence, APFO was not demonstrated to represent a unique hazard to the conceptus of the rat.

The influence of enterogastric reflux on gastric juice bacterial growth, nitrite, and N-nitroso compound concentrations following gastric surgery.

Detailed analyses were carried out in 207 fasting gastric juice samples obtained at endoscopy or with a nasogastric tube from 50 patients with partial gastrectomy, 43 with vagotomy, 20 with gastric carcinoma and 50 controls. Significantly higher mean pH, nitrite and N-nitroso compound (N-NO) concentrations and nitrate-reducing bacterial cultures were noted following partial gastrectomy compared with normal controls and comparable to findings in gastric cancer. A highly significant relationship (p less than 10(-6)) was also demonstrated between pH and N-NO concentrations, highest levels of which were seen following Billroth II gastrectomy, significantly higher (p = 0.02) than Billroth I, whereas the only change observed following proximal gastric vagotomy was a nearly threefold rise in N-NO compared with normal controls. However, vagotomy and pyloroplasty produced gastric juice changes comparable to those seen with gastrectomy and gastric carcinoma. Thus, the most marked changes were observed following surgical procedures involving increased enterogastric reflux and these findings lend further support to the possible involvement of N-nitroso compounds in the development of gastric cancer following both Billroth I and II gastrectomy and vagotomy with pyloroplasty.