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Background Equine coronavirus (ECoV) is an emerging pathogen associated with fever and enteric disease in adult horses. Clinical features of ECoV infection have been described, but no study has compared these features to those of Salmonella infections. Objectives Compare the clinical features of ECoV infection with enteric salmonellosis and establish a disease signature to increase clinical suspicion of ECoV infection in adult horses. Animals Forty-three horses >1 year of age with results of CBC, serum biochemistry, and fecal diagnostic testing for ECoV and Salmonella spp. Methods Medical records of horses presented to the North Carolina State University Equine and Farm Animal Veterinary Center (2003-016) were retrospectively reviewed. Horses were divided into 3 groups based on fecal diagnostic test results: ECoV-positive, Salmonella-positive, or unknown diagnosis (UNK). Time of year presented, clinical signs, CBC, and serum biochemistry test results were recorded. Data were analyzed by 1-way analysis of variance, Kruskal-Wallis test, or Fisher's exact test with significance set at P Results Most common presenting complaints were fever and colic and were similar across groups. Horses with ECoV had significantly decreased neutrophil counts when compared to those with no diagnosis but were not different from horses with Salmonella. Horses with Salmonella had significantly lower mean leukocyte counts compared to those with UNK. No significant differences were found among groups for any other examined variable. Conclusions and clinical importance Equine coronavirus and Salmonella infections share clinical features, suggesting both diseases should be differential diagnoses for horses with fever and enteric clinical signs.

OBJECTIVE To evaluate the effects of vaporized hydrogen peroxide (VHP) sterilization on the in vitro antimicrobial efficacy of meropenem-impregnated polymethyl methacrylate (M-PMMA) beads. SAMPLE 6-mm-diameter polymethyl methacrylate beads that were or were not impregnated with meropenem. PROCEDURES Meropenem-free polymethyl methacrylate and M-PMMA beads were sterilized by use of an autoclave or VHP or remained unsterilized. To determine the antimicrobial efficacy of each bead-sterilization combination (treatment), Mueller-Hinton agar plates were inoculated with 1 of 6 common equine pathogens, and 1 bead from each treatment was applied to a sixth of each plate. The zone of bacterial inhibition for each treatment was measured after 24 hours. To estimate the duration of antimicrobial elution into a solid or liquid medium, 1 bead from each treatment was transferred every 24 hours to a new Staphylococcus aureus–inoculated agar plate or a tube with PBS solution, and an aliquot of the eluent from each tube was then applied to a paper disc on an S aureus–inoculated agar plate. All agar plates were incubated for 24 hours, and the zone of bacterial inhibition was measured for each treatment. RESULTS In vitro antimicrobial efficacy of M-PMMA beads was retained following VHP sterilization. The duration of antimicrobial elution in solid and liquid media did not differ significantly between unsterilized and VHP-sterilized M-PMMA beads. CONCLUSIONS AND CLINICAL RELEVANCE Results indicated that M-PMMA beads retained in vitro antimicrobial activity and eluted the drug for up to 2 weeks after VHP sterilization.

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival of Salmonella Typhimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potent Salmonella Typhimurium agonists of the neutrophil respiratory burst in vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identify Salmonella Typhimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with human neutrophils. We first screened Salmonella Typhimurium mutants lacking defined genomic regions and then tested single gene deletion mutants representing particular regions under selection. A subset of single gene deletion mutants were selected for further investigation. Mutants in four genes, STM1696 (sapF), STM2201 (yeiE), STM2112 (wcaD), and STM2441 (cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696 and ΔSTM2201). The ΔSTM2441 mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441 mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate that Salmonella Typhimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonella may alter human neutrophil antimicrobial defenses.

Typical enteropathogenic E. coli (tEPEC) is a leading cause of diarrhea and associated death in children worldwide. Atypical EPEC (aEPEC) lacks the plasmid encoding bundle-forming pili and is considered less virulent, but the molecular mechanisms of virulence is poorly understood. We recently identified kittens as a host for aEPEC where intestinal epithelial colonization was associated with diarrheal disease and death. The purpose of this study was to (1) determine the genomic similarity between kitten aEPEC and human aEPEC isolates and (2) to identify genotypic or phenotypic traits associated with virulence in kitten aEPEC. We observed no differences between kitten and human aEPEC in core genome content or gene cluster sequence identities and no distinguishing genomic content was observed between aEPEC isolates from kittens with nonclinical colonization (NC) versus lethal infection (LI). Variation in adherence pattern and ability to aggregate actin in cultured cells mirrored descriptions of human aEPEC. The aEPEC isolated from kittens with LI were significantly more motile than isolates from kittens with NC. Kittens may serve as a reservoir for aEPEC that are indistinguishable from human aEPEC isolates and may provide a needed comparative animal model for the study of aEPEC pathogenesis. Motility seems to be an important factor in pathogenesis of LI associated with aEPEC in kittens.

During enteric salmonellosis, neutrophil generated reactive oxygen species alter the gut microenvironment favoring survival ofSalmonellaTyphimurium. While the type-3 secretion system-1 (T3SS-1) and flagellar motility are potentSalmonellaTyphimurium agonists of the neutrophil respiratory burstin vitro, neither of these pathways alone are responsible for stimulation of a maximal respiratory burst. In order to identifySalmonellaTyphimurium genes that impact the magnitude of the neutrophil respiratory burst, we performed a two-step screen of defined mutant libraries in co-culture with neutrophils. We first screenedSalmonellaTyphimurium mutants lacking defined genomic regions, followed by the individual mutants mapping to genomic regions under selection. Mutants in four genes,STM1696(sapF),STM2201(yeiE),STM2112(wcaD), andSTM2441(cysA), induced an attenuated respiratory burst. We linked the altered respiratory burst to reduced T3SS-1 expression and/or altered flagellar motility for two mutants (ΔSTM1696and ΔSTM2201). The ΔSTM2441mutant, defective for sulfate transport, formed aggregates in minimal media and adhered to surfaces in rich media, suggesting a role for sulfur homeostasis in regulation of aggregation/adherence. We linked the aggregation/adherence phenotype of the ΔSTM2441mutant to biofilm-associated protein A and flagellins and hypothesize that aggregation caused the observed reduction in the magnitude of the neutrophil respiratory burst. Our data demonstrate thatSalmonellaTyphimurium has numerous mechanisms to limit the magnitude of the neutrophil respiratory burst. These data further inform our understanding of how Salmonellamay alter neutrophil antimicrobial defenses.

Biofilms formed by Salmonella enterica are a frequent source of food supply contamination. Since biofilms are inherently resistant to disinfection, new agents capable of preventing biofilm formation are needed. Synthetic analogs of 4-oxazolidinone containing natural products have shown promise as antibiofilm compounds against Gram-positive bacteria. The purpose of our study was 2-fold: to establish the antibiofilm effects and mechanism of action of a synthetic 4-oxazolidinone analog (JJM-ox-3-70) and to establish mechanisms of resistance to this compound in Salmonella enterica serovar Typhimurium (S. Typhimurium). JJM-ox-3-70 inhibited biofilm formation but had no effect on cell growth. The antibiofilm effects were linked to disruption of curli fimbriae and flagellar gene expression and alteration in swimming motility, suggesting an effect on multiple cellular processes. Using a 2-step screening approach of defined multigene and single-gene deletion mutant libraries, we identified 3 mutants that produced less biofilm in the presence of JJM-ox-3-70 than the isogenic WT, with phenotypes reversed by complementation in trans. Genes responsible for S. Typhimurium resistance to the compound included acrB, a component of the major drug efflux pump AcrAB-TolC, and two genes of unknown function (STM0437 and STM1292). The results of this study suggest that JJM-ox-3-70 inhibits biofilm formation by indirect inhibition of extracellular matrix production that may be linked to disruption of flagellar motility. Further work is needed to establish the role of the newly characterized genes as potential mechanisms of biofilm intrinsic antimicrobial resistance. IMPORTANCE Biofilms are resistant to killing by disinfectants and antimicrobials. S. enterica biofilms facilitate long-term host colonization and persistence in food processing environments. Synthetic analogs of 4-oxazolidinone natural products show promise as antibiofilm agents. Here, we show that a synthetic 4-oxazolidinone analog inhibits Salmonella biofilm through effects on both motility and biofilm matrix gene expression. Furthermore, we identify three genes that promote Salmonella resistance to the antibiofilm effects of the compound. This work provides insight into the mechanism of antibiofilm effects of a synthetic 4-oxazolidinone analog in Gram-negative bacteria and demonstrates new mechanisms of intrinsic antimicrobial resistance in Salmonella biofilms.

Retrons are genetic retroelements, commonly found in bacterial genomes and recently repurposed as genome editing tools. Their encoded reverse transcriptase (RT) produces a multi-copy single-stranded DNA (msDNA). Despite our understanding of their complex biosynthesis, the function of msDNAs and therefore, the physiological role of retrons has remained elusive. We establish that the retron-Sen2 inSalmonellaTyphimurium encodes a toxin, which we have renamed as RcaT (Retron cold-anaerobic Toxin). RcaT is activated when msDNA biosynthesis is perturbed and its toxicity is higher at ambient temperatures or during anaerobiosis. The RT and msDNA form together the antitoxin unit, with the RT binding RcaT, and the msDNA enabling the antitoxin activity. Using anotherE. coliretron, we establish that this toxin/antitoxin function is conserved, and that RT-toxin interactions are cognate. Altogether, retrons constitute a novel family of tripartite toxin/antitoxin systems.

Retrons are prokaryotic genetic retroelements encoding a reverse transcriptase that produces multi-copy single-stranded DNA

Neutrophils are innate immune response cells designed to kill invading microorganisms. One of the mechanisms neutrophils use to kill bacteria is generation of damaging reactive oxygen species (ROS) via the respiratory burst. However, during enteric salmonellosis, neutrophil-derived ROS actually facilitates Salmonella expansion and survival in the gut. This seeming paradox led us to hypothesize that Salmonella may possess mechanisms to influence the neutrophil respiratory burst. In this work, we used an in vitro Salmonella-neutrophil co-culture model to examine the impact of enteric infection relevant virulence factors on the respiratory burst of human neutrophils. We report that neutrophils primed with granulocyte-macrophage colony stimulating factor and suspended in serum containing complement produce a robust respiratory burst when stimulated with viable STm. The magnitude of the respiratory burst increases when STm are grown under conditions to induce the expression of the type-3 secretion system-1. STm mutants lacking the type-3 secretion system-1 induce less neutrophil ROS than the virulent WT. In addition, we demonstrate that flagellar motility is a significant agonist of the neutrophil respiratory burst. Together our data demonstrate that both the type-3 secretion system-1 and flagellar motility, which are established virulence factors in enteric salmonellosis, also appear to directly influence the magnitude of the neutrophil respiratory burst in response to STm in vitro.

Summary Salmonella enterica is an important foodborne pathogen that uses secreted effector proteins to manipulate host pathways to facilitate survival and dissemination. Different S. enterica serovars cause disease syndromes ranging from gastroenteritis to typhoid fever and vary in their effector repertoire. We leveraged this natural diversity to identify stm2585 , here designated sarA ( Salmonella anti-inflammatory response activator ), as a Salmonella effector that induces production of the anti-inflammatory cytokine IL-10. RNA-seq of cells infected with either Δ sarA or wild-type S. Typhimurium revealed that SarA activates STAT3 transcriptional targets. Consistent with this, SarA is necessary and sufficient for STAT3 phosphorylation, STAT3 inhibition blocks IL-10 production, and SarA and STAT3 interact by co-immunoprecipitation. These effects of SarA contribute to intracellular replication in vitro and bacterial load at systemic sites in mice. Our results demonstrate the power of using comparative genomics for identifying effectors and that Salmonella has evolved mechanisms for activating an important anti-inflammatory pathway.

Cattle are naturally infected with Salmonella enterica serotype Typhimurium and exhibit pathological features of enteric salmonellosis that closely resemble those in humans. Cattle are the most relevant model of gastrointestinal disease resulting from nontyphoidal Salmonella infection in an animal with an intact microbiota. We utilized this model to screen a library of targeted single-gene deletion mutants to identify novel genes of Salmonella Typhimurium required for survival during enteric infection. Fifty-four candidate mutants were strongly selected, including numerous mutations in genes known to be important for gastrointestinal survival of salmonellae. Three genes with previously unproven phenotypes in gastrointestinal infection were tested in bovine ligated ileal loops. Two of these mutants, STM3602 and STM3846 , recapitulated the phenotype observed in the mutant pool. Complementation experiments successfully reversed the observed phenotypes, directly linking these genes to the colonization defects of the corresponding mutant strains. STM3602 encodes a putative transcriptional regulator that may be involved in phosphonate utilization, and STM3846 encodes a retron reverse transcriptase that produces a unique RNA-DNA hybrid molecule called multicopy single-stranded DNA. The genes identified in this study represent an exciting new class of virulence determinants for further mechanistic study to elucidate the strategies employed by Salmonella to survive within the small intestines of cattle.

Summary Reasons for performing study Pain management is an important component of foal nursing care, and no objective data currently exist regarding the analgesic efficacy of opioids in foals. Objectives To evaluate the somatic antinociceptive effects of 2 commonly used doses of intravenous (i.v.) butorphanol in healthy foals. Our hypothesis was that thermal nociceptive threshold would increase following i.v. butorphanol in a dose-dependent manner in both neonatal and older pony foals. Methods Seven healthy neonatal pony foals (age 1–2 weeks), and 11 healthy older pony foals (age 4–8 weeks). Five foals were used during both age periods. Treatments, which included saline (0.5 ml), butorphanol (0.05 mg/kg bwt) and butorphanol (0.1 mg/kg bwt), were administered i.v. in a randomised crossover design with at least 2 days between treatments. Response variables included thermal nociceptive threshold, skin temperature and behaviour score. Data within each age period were analysed using a 2-way repeated measures ANOVA, followed by a Holm–Sidak multiple comparison procedure if warranted. Results There was a significant (P

Summary Reason for performing study: Gastric ulcer disease is reported to be a significant cause of morbidity in foals, but the prevalence of ulcers in this population has not recently been evaluated. Objectives: To determine the prevalence of gastric and duodenal ulceration in nonsurviving foals, and the association of ulceration with the body system of primary diagnosis. Secondary objectives were to evaluate a potential association between age and ulcer prevalence and to evaluate the use of antacid medication in the neonatal hospital population during the study years. Methods: Necropsy records were searched for all equine accessions from 1995 to 2006. Foals aged from one day to 6 months were included. Year, age, breed, sex, diagnosis and the presence of glandular, nonglandular and/or duodenal ulceration were recorded. Diagnoses were divided into groups based on the body system of primary diagnosis, with multiple diagnoses possible. A computerised database was searched for antacid treatment of all neonatal admissions. Results: The overall prevalence of ulcers was 22%, with nonglandular ulcers predominating. Ulceration was significantly associated with gastrointestinal disease. There was no significant change in ulcer prevalence over time, although there was a significant decrease in the use of antacid medications in the later study years. Neonatal foals (

Background: Ketamine as continuous rate infusion (CRI) provides analgesia in hospitalized horses. Objective: Determine effects of prolonged CRI of ketamine on gastrointestinal transit time, fecal weight, vital parameters, gastrointestinal borborygmi, and behavior scores in healthy adult horses. Animals: Seven adult Thoroughbred or Thoroughbred cross horses, with permanently implanted gastric cannulae. Methods: Nonblinded trial. Random assignment to 1 of 2 crossover designed treatments. Ketamine (0.55 mg/kg IV over 15 minutes followed by 1.2 mg/kg/h) or lactated Ringer's solution (50 mL IV over 15 minutes followed by 0.15 mL/kg/h) treatments. Two hundred 3 × 5 mm plastic beads administered by nasogastric tube before drug administration. Every 2 hours vital parameters, behavior scores recorded, feces collected and weighed, and beads retrieved. Every 6 hours gastrointestinal borborygmi scores recorded. Study terminated upon retrieval of 180 beads (minimum 34 hours) or maximum 96 hours. Nontransit time data analyzed between hours 0 and 34. Results: No significant (P < .05) differences detected between treatments in vital signs or gastrointestinal borborygmi. Significant (P = .002) increase in behavior score during ketamine infusion (0.381) from hours 24–34 compared with placebo (0). Ketamine caused significant delay in passage of 25, 50, and 75% of beads (ketamine = 30.6 ± 5.3, 41.4 ± 8.4, 65.3 ± 13.5 hours versus placebo = 26.8 ± 7.9, 34.3 ± 11.1, 45.8 ± 19.4 hours), and significant (P < .05) decrease in fecal weight from hours 22 (12.6 ± 3.2 versus 14.5 ± 3.8 kg) through 34 (18.5 ± 3.9 versus 12.8 ± 6.4 kg) of infusion. Conclusions and Clinical Importance: Ketamine CRI delayed gastrointestinal transit time in healthy horses without effect on vital parameters.

Background: Hepatic failure is one of the more common complications in foals requiring blood transfusion to treat neonatal isoerythrolysis. Iron intoxication is likely the cause of hepatic injury. Objectives: To determine the effects of deferoxamine on iron elimination in normal foals. Animals: Thirteen neonatal foals. Methods: Randomized-controlled trial. At 1–3 days of age, foals received either 3 L of washed packed dam's red blood cells (RBC) or 3 L of saline IV once. Foals were treated with deferoxamine (1 g) or saline (5 mL) SC twice daily for 14 days. Foals were randomly assigned to 1 of 3 groups: RBC/deferoxamine (deferoxamine), RBC/saline (placebo), or saline/saline (control). Blood and urine samples and liver biopsy specimens were collected for measurement of hematological, biochemical, and iron metabolism variables. Results: There was a significant (P < .05) increase in hematocrit, RBC count, and hemoglobin in the groups transfused with packed RBC as compared with controls at all times. Biochemical variables and liver biopsy scores were not significantly different between groups at any time. Urine iron concentrations and fractional excretion of iron were significantly higher in deferoxamine treated foals. By 14 days after transfusion, liver iron concentrations in foals treated with deferoxamine (79.9 ± 30.9 ppm) were significantly lower than that of foals receiving placebo (145 ± 53.0 ppm) and similar to that of controls (44.8 ± 4.09 ppm). Conclusions and Clinical Importance: Deferoxamine enhances urinary iron elimination and decreases hepatic iron accumulation after blood transfusion in foals.

Background Prolonged drug infusions are used to treat horses with severe signs of pain, but can be associated with altered gastrointestinal transit. The purpose of this study was to determine the effects of prolonged constant rate infusions (CRI) of lidocaine (L), butorphanol (B), and ketamine (K) alone and in combination on gastrointestinal transit, behavior, and thermal nociceptive threshold in healthy horses. Methods Eight healthy adult horses were used in a randomized, cross-over, blinded, prospective experimental trial. Interventions were saline, L, K, B, LK, LB, BK, and LBK as an intravenous CRI for 96 hours. Drugs were mixed or diluted in saline; following a bolus, CRI rate was 0.15mL/kg/hr with drug doses as follows: L – 1.3 mg/kg then 3 mg/kg/hr; B – 0.018 mg/kg then 0.013 mg/kg/hr; K – 0.55 mg/kg then 0.5 mg/kg/hr. Two-hundred plastic beads were administered intragastrically by nasogastric tube immediately prior to the bolus. Feces were collected every 2 hours, weighed, and beads manually retrieved. Behavior was scored every 2 hours, vital parameters every 6 hours, and thermal nociceptive threshold every 12 hours for 96 hours. Drug concentrations in the LBK solution were tested every 6 hours for 72 hours. Results Four of 64 trials (3 LBK, 1 BK) were discontinued early due to signs of abdominal discomfort. There were no apparent differences between groups in vital parameters or thermal threshold. Transit time was delayed for LB and LBK with a corresponding decrease in fecal weight that was most severe in the final 24 hours of infusion. Significant changes in behavior scores, vital parameters, or thermal threshold were not observed. The concentration of each drug in the combined solution declined by less than 31% over the sampling period. Conclusions Drug combinations containing butorphanol cause an apparent delay in gastrointestinal transit in healthy horses without substantially affecting somatic nociception at the doses studied. Combinations of lidocaine and ketamine may have less impact on gastrointestinal transit than infusions combined with butorphanol. Further work is needed to determine the effects of these drugs in painful or critically ill patients.

Objective To evaluate the effects of detomidine on visceral and somatic nociception, heart and respiratory rates, sedation, and duodenal motility and to correlate these effects with serum detomidine concentrations. Study design Nonrandomized, experimental trial. Animals Five adult horses, each with a permanent gastric cannula weighing 534 ± 46 kg. Methods Visceral nociception was evaluated by colorectal (CRD) and duodenal distension (DD). The duodenal balloon was used to assess motility. Somatic nociception was assessed via thermal threshold (TT). Nose–to–ground (NTG) height was used as a measure of sedation. Serum was collected for pharmacokinetic analysis. Detomidine (10 or 20 μg kg −1 ) was administered intravenously. Data were analyzed by means of a three–factor anova with fixed factors of treatment and time and random factor of horse. When a significant time × treatment interaction was detected, differences were compared with a simple t –test or Bonferroni t –test. Significance was set at p Results Detomidine produced a significant, dose–dependent decrease in NTG height, heart rate, and skin temperature and a significant, nondose–dependent decrease in respiratory rate. Colorectal distension threshold was significantly increased with 10 μg kg −1 for 15 minutes and for at least 165 minutes with 20 μg kg −1 . Duodenal distension threshold was significantly increased at 15 minutes for the 20 μg kg −1 dose. A significant change in TT was not observed at either dose. A marked, immediate decrease in amplitude of duodenal contractions followed detomidine administration at both doses for 50 minutes. Conclusions and clinical relevance Detomidine caused a longer period of visceral anti–nociception as determined by CRD but a shorter period of anti–nociception as determined by DD than has been previously reported. The lack of somatic anti–nociception as determined by TT testing may be related to the marked decrease in skin temperature, likely caused by peripheral vasoconstriction and the low temperature cut–off of the testing device.

Objective—To evaluate effects of butorphanol, acepromazine, and N-butylscopolammonium bromide (NBB) on visceral and somatic nociception and duodenal motility in conscious, healthy horses. Animals—6 adult horses. Procedures—Visceral nociception was evaluated by use of colorectal distention (CRD) and duodenal distention (DD) threshold. Somatic nociception was evaluated via thermal threshold (TT). Nose-to-ground height, heart rate, and respiratory rate were also measured. Each horse received each treatment in randomized order; investigators were not aware of treatments. Butorphanol was administered IV as a bolus (18 μg/kg) followed by constant rate infusion at 13 μg/kg/h for 2 hours, whereas acepromazine (0.04 mg/kg), NBB (0.3 mg/kg), and saline (0.9% NaCl) solution (2 mL) were administered IV as a bolus followed by constant rate infusion with saline solution (10 mL/h) for 2 hours. Variables were measured before and for 3 hours after treatment. Data were analyzed by use of a 3-factor ANOVA followed by a Bonferroni t test for multiple comparisons. Results—Nose-to-ground height decreased after acepromazine. Respiratory rate decreased after acepromazine and increased after butorphanol. Heart rate increased briefly after NBB. Some horses had an increase in TT after butorphanol and acepromazine, but there was not a significant treatment effect over time. Drug effect on DD or motility was not evident. The CRD threshold increased significantly at 5, 65, 155, and 185 minutes after acepromazine and from 5 to 65 minutes after NBB. Conclusions and Clinical Relevance—Each drug caused predictable changes in sedation and vital signs, but consistent anti-nociceptive effects were not evident.