Johanna R. Elfenbein

Effect of detomidine on visceral and somatic nociception and duodenal motility in conscious adult horses

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 microg 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 x treatment interaction was detected, differences were compared with a simple t-test or Bonferroni t-test. Significance was set at p < 0.05. 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 microg kg(-1) for 15 minutes and for at least 165 minutes with 20 microg kg(-1). Duodenal distension threshold was significantly increased at 15 minutes for the 20 microg 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.

Effect of acepromazine, butorphanol, or N-butylscopolammonium bromide on visceral and somatic nociception and duodenal motility in conscious horses

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 microg/kg) followed by constant rate infusion at 13 microg/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.

Systemic and anti-nociceptive effects of prolonged lidocaine, ketamine, and butorphanol infusions alone and in combination in healthy horses

BackgroundProlonged 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.MethodsEight 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.ResultsFour 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.ConclusionsDrug 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.

Systemic Effects of a Prolonged Continuous Infusion of Ketamine in Healthy Horses

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 Ringers 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.

Effect of butorphanol on thermal nociceptive threshold in healthy pony foals

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<0.05) increase in thermal threshold, relative to Time 0, following butorphanol (0.1 mg/kg bwt) administration in both age groups. No significant time or treatment effects were apparent for skin temperature. Significant time, but not treatment, effects were evident for behaviour score in both age groups. CONCLUSIONS Butorphanol (0.1 mg/kg bwt, but not 0.05 mg/kg bwt) significantly increased thermal nociceptive threshold in neonatal and older foals without apparent adverse behavioural effects. POTENTIAL RELEVANCE Butorphanol shows analgesic potential in foals for management of somatic painful conditions.

Multicopy single-stranded DNA directs intestinal colonization of enteric pathogens

Multicopy single-stranded DNAs (msDNAs) are hybrid RNA-DNA molecules encoded on retroelements called retrons and produced by the action of retron reverse transcriptases. Retrons are widespread in bacteria but the natural function of msDNA has remained elusive despite 30 years of study. The major roadblock to elucidation of the function of these unique molecules has been the lack of any identifiable phenotypes for mutants unable to make msDNA. We report that msDNA of the zoonotic pathogen Salmonella Typhimurium is necessary for colonization of the intestine. Similarly, we observed a defect in intestinal persistence in an enteropathogenic E. coli mutant lacking its retron reverse transcriptase. Under anaerobic conditions in the absence of msDNA, proteins of central anaerobic metabolism needed for Salmonella colonization of the intestine are dysregulated. We show that the msDNA-deficient mutant can utilize nitrate, but not other alternate electron acceptors in anaerobic conditions. Consistent with the availability of nitrate in the inflamed gut, a neutrophilic inflammatory response partially rescued the ability of a mutant lacking msDNA to colonize the intestine. These findings together indicate that the mechanistic basis of msDNA function during Salmonella colonization of the intestine is proper production of proteins needed for anaerobic metabolism. We further conclude that a natural function of msDNA is to regulate protein abundance, the first attributable function for any msDNA. Our data provide novel insight into the function of this mysterious molecule that likely represents a new class of regulatory molecules.

Novel Determinants of Intestinal Colonization of Salmonella enterica Serotype Typhimurium Identified in Bovine Enteric Infection

ABSTRACT 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.

The effects of deferoxamine mesylate on iron elimination after blood transfusion in neonatal foals.

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 dams 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.

Novel Two-Step Hierarchical Screening of Mutant Pools Reveals Mutants under Selection in Chicks

ABSTRACT Contaminated chicken/egg products are major sources of human salmonellosis, yet the strategies used by Salmonella to colonize chickens are poorly understood. We applied a novel two-step hierarchical procedure to identify new genes important for colonization and persistence of Salmonella enterica serotype Typhimurium in chickens. A library of 182 S. Typhimurium mutants each containing a targeted deletion of a group of contiguous genes (for a total of 2,069 genes deleted) was used to identify regions under selection at 1, 3, and 9 days postinfection in chicks. Mutants in 11 regions were under selection at all assayed times (colonization mutants), and mutants in 15 regions were under selection only at day 9 (persistence mutants). We assembled a pool of 92 mutants, each deleted for a single gene, representing nearly all genes in nine regions under selection. Twelve single gene deletion mutants were under selection in this assay, and we confirmed 6 of 9 of these candidate mutants via competitive infections and complementation analysis in chicks. STM0580, STM1295, STM1297, STM3612, STM3615, and STM3734 are needed for Salmonella to colonize and persist in chicks and were not previously associated with this ability. One of these key genes, STM1297 (selD), is required for anaerobic growth and supports the ability to utilize formate under these conditions, suggesting that metabolism of formate is important during infection. We report a hierarchical screening strategy to interrogate large portions of the genome during infection of animals using pools of mutants of low complexity. Using this strategy, we identified six genes not previously known to be needed during infection in chicks, and one of these (STM1297) suggests an important role for formate metabolism during infection.

A Salmonella Regulator Modulates Intestinal Colonization and Use of Phosphonoacetic Acid

Many microorganisms produce phosphonates, molecules characterized by stable carbon-phosphorus bonds that store phosphorus or act as antimicrobials. The role of phosphonates in the marine biosphere is well characterized but the role of these molecules in the intestine is poorly understood. Salmonella enterica uses its virulence factors to influence the host immune response to compete with the host and normal microflora for nutrients. Salmonella cannot produce phosphonates but encodes the enzymes to use them suggesting that it is exposed to phosphonates during its life cycle. The role of phosphonates during enteric salmonellosis is unexplored. We have previously shown that STM3602, encoding a putative regulator of phosphonate metabolism, is needed for colonization in calves. Here, we report that the necessity of STM3602 in colonization of the murine intestine results from multiple factors. STM3602 is needed for full activation of the type-3 secretion system-1 and for optimal invasion of epithelial cells. The ΔSTM3602 mutant grows poorly in phosphonoacetic acid (PA) as the sole phosphorus source, but can use 2-aminoethylphosphonate. PhnA, an enzyme required for PA breakdown, is not controlled by STM3602 suggesting an additional mechanism for utilization of PA in S. Typhimurium. Finally, the requirement of STM3602 for intestinal colonization differs depending on the composition of the microflora. Our data suggest that STM3602 has multiple regulatory targets that are necessary for survival within the microbial community in the intestine. Determination of the members of the STM3602 regulon may illuminate new pathways needed for colonization of the host.