Tetanus prophylaxis in horses: guidelines for New Zealand and Australia based on a critical appraisal of the evidence.

Horses are exquisitely sensitive to tetanus neurotoxin and are exposed to the risk of infection with Clostridium tetani throughout life. The vaccine against tetanus is highly effective at preventing disease, whereas tetanus in unvaccinated populations is associated with high mortality rates. Current guidelines in New Zealand and Australia for the available vaccine contain contradictions and limitations surrounding the optimal tetanus immunisation protocols for both adult horses and foals. This review critically evaluates the scientific literature on tetanus prophylaxis in horses within the context of equine practice and available products in New Zealand and Australia. The review was conducted by a panel of industry and specialist veterinarians to obtain agreement on nine equine tetanus prophylaxis guidelines for practising veterinarians. The primary protocol for tetanus toxoid (TT) immunisation consists of a three-dose series IM for all horses ≥ 6 months of age, and a four-dose series IM is proposed if commencing vaccination in foals between 3 and 6 months of age. Tetanus prophylaxis in foals < 3 months of age relies on passive immunity strategies. Following the completion of the primary protocol, a TT booster dose IM should be administered within 5 years, and every 5 years thereafter. When followed, these protocols should provide adequate protection against tetanus in horses. Additional tetanus prophylaxis guidelines are provided for veterinarians attending a horse experiencing a known "risk event" (e.g. wound, hoof abscess, surgery, umbilical infection). When a correctly vaccinated horse experiences a risk event, pre-existing immunity provides protection against tetanus. When an unvaccinated horse or one with unknown vaccination status, or a foal born to an unvaccinated dam, experiences a risk event, TT IM and tetanus antitoxin (TAT) 1,500 IU SC should be administered simultaneously at separate sites, and the TT primary immunisation protocol should subsequently be completed for the horse's respective age. In previously immunised pregnant broodmares, a TT booster dose administered 4-8 weeks prior to parturition optimises the transfer of passive immunity against tetanus to the newborn foal via colostrum; provided that post-natal IgG concentration in serum is > 800 mg/dL (8 g/L), such foals should be passively protected against tetanus up to 6 months of age. Survivors of clinical tetanus must still receive the primary protocol for vaccination against tetanus. In summary, all horses in New Zealand and Australia should be vaccinated against tetanus with protection maintained throughout life via TT booster doses, facilitated by accurate medical record keeping and client education.

The effects of dose and diet on the pharmacokinetics of omeprazole in the horse.

This study aimed to investigate the effect of diet and dose on the pharmacokinetics of omeprazole in the horse. Six horses received two doses (1 and 4 mg/kg) of omeprazole orally once daily for 5 days. Each dose was evaluated during feeding either a high-grain/low-fibre (HG/LF) diet or an ad libitum hay (HAY) diet in a four-way crossover design. Plasma samples were collected for pharmacokinetic analysis on days 1 and 5. Plasma omeprazole concentrations were determined by ultra-high pressure liquid chromatography-mass spectrometry. In horses being fed the HG/LF diet, on day 1, the area under the curve (AUC) and maximal plasma concentration (Cmax ) were higher on the 4 mg/kg dose than on the 1 mg/kg dose. The AUC was higher on day 5 compared to day 1 with the 4 mg/kg dose on the HG/LF diet. On days 1 and 5, the AUC and Cmax were higher in horses being fed the HG/LF diet and receiving the 4 mg/kg dose than in horses being fed the HAY diet and receiving the 1 mg/kg dose. These findings suggest that both dose and diet may affect pharmacokinetic variables of omeprazole in the horse.

Pharmacokinetics and bioequivalence testing of five commercial formulations of omeprazole in the horse.

Omeprazole is widely used in the treatment of equine gastric ulcer syndrome. To date, little is known about the relative pharmacokinetics of the different formulations making comparisons between products difficult. The objectives of the study were to investigate the relative pharmacokinetics of five commercially available formulations of omeprazole in the horse and to test for bioequivalence of four of the formulations using one of the formulations as a reference standard. Twelve mature Thoroughbred horses were fasted for 16 h then administered 2 g of each formulation in a cross-over design. Serial blood samples were collected and plasma omeprazole concentration was determined by ultra high-performance liquid chromatography-mass spectrometry (UHPLC-MS). No significant differences were present between three of the formulations and the reference formulation, while the fourth formulation had a lower Cmax and longer Tmax than the reference formulation. Bioequivalence against the reference formulation could not be demonstrated for any of the formulations tested. The findings of the study suggested that the method of protection utilised by different formulations of omeprazole (enteric-coated granules vs. buffering) does not significantly alter the pharmacokinetics of the drug. Further work to establish bioequivalence is needed before direct comparisons can be drawn between different formulations.

Pharmacokinetics of intravenous, plain oral and enteric-coated oral omeprazole in the horse.

The objectives were to document the pharmacokinetics of intravenous, enteric-coated oral and plain oral omeprazole in fasted horses and to investigate the impact of feeding on the bioavailability of an enteric-coated omeprazole. Twelve horses received four treatments: intravenous omeprazole (0.5 mg/kg) in the fasted state (IV-Fasted), enteric-coated omeprazole (4 mg/kg) orally in the fasted state (ECO-Fasted), enteric-coated omeprazole (4 mg/kg) orally in the fed state (ECO-Fed) and plain omeprazole (4 mg/kg) orally in the fasted state (PL-Fasted). Plasma omeprazole concentrations were determined by UHPLC-MS. Bioavailability was higher (P = 0.038) in the ECO-Fasted group (21.5 [9.0-27.7]%) than the PL-Fasted group (10.1 [7.7-13.3]%). Similarly, AUC0-∞ was higher in the ECO-Fasted group than the PL-Fasted group (P = 0.027). No significant differences were present between the ECO-Fasted and ECO-Fed groups with regards to bioavailability, Cmax , Tmax or AUC0-∞ . When the half-life data from the oral formulations was pooled, it was longer than that observed in the IV-Fasted group (100 [73-118] min) and 35 [34-39] min, respectively; P < 0.0001). Bioavailability of enteric-coated omeprazole was higher than previously reported and feeding had minimal impact. Bioavailability of plain omeprazole was approximately half that of enteric-coated omeprazole. The longer half-life observed following oral administration was consistent with the flip-flop effect and has not previously been described for omeprazole in the horse.

The effect of feeding on the pharmacokinetic variables of two commercially available formulations of omeprazole.

The objectives of this study were to investigate the impact of formulation (enteric coated and buffered) and feeding on pharmacokinetic variables associated with the oral administration of omeprazole in the horse. Six thoroughbred racehorses were studied in a crossover design. Each received 2 g of an enteric coated or buffered formulation in both the fed and fasted state. Plasma omeprazole concentrations were determined by UHPLC-MS. The effects of feeding or formulation on AUC0-inf_obs, half-life, Tmax or Cmax were not statistically significant. However, a wider-than-expected degree of variation was present and examination of the raw data suggests that an effect of feeding, wherein the bioavailability of omeprazole may be reduced in the fed animal, may be present. Further investigation in a larger population of animals to assess the factors that contribute to the wide degree of absorption observed is warranted.

Metabolomic profiling in multiple sclerosis: insights into biomarkers and pathogenesis.

Metabolomics enables the provision of sensitive bio-markers of disease. We performed 800 MHz (1)H-nuclear magnetic resonance (NMR) spectroscopic analyses of cerebrospinal fluid (CSF) specimens to identify biomarkers of multiple sclerosis (MS), yielding reproducible detection of 15 metabolites from MS (n=15) and non-MS (n=17) patients. Mean levels of choline, myo-inositol and threonate were increased, whereas 3-hydroxybutyrate, citrate, phenylalanine, 2-hydroxyisovalerate and mannose were decreased in MS-derived CSF (p<0.05), suggesting alterations to energy and phospholipid metabolism. Multivariate hierarchal cluster analysis indicated a high correlation within the metabolite profiles, significantly clustering samples into the two clinical groups, which was corroborated using principal components analysis. CSF metabolomics have the capacity to yield quantitative biomarkers and insights into the pathogenesis of MS.

Evaluation of the effects of medium-term (57-day) omeprazole administration and of omeprazole discontinuation on serum gastrin and serum chromogranin A concentrations in the horse.

BACKGROUND: Rebound gastric hyperacidity (RGH) secondary to hypergastrinemia has been suggested to contribute to the rapid recurrence of equine squamous gastric disease (ESGD) in horses after discontinuation of omeprazole. HYPOTHESIS/OBJECTIVES: To evaluate changes in serum gastrin and chromogranin A (CgA) concentrations in response to medium-term (57-day) omeprazole treatment and after omeprazole discontinuation. ANIMALS: Fourteen mature Thoroughbred racehorses in simulated race training. METHODS: Horses received 2.28 g of oral omeprazole PO q24h for 57 days within a 61-day period, excluding a withholding period applied mid-protocol during which treatment was stopped as part of a concurrent study. Serum samples were collected on day 0 before omeprazole treatment, on day 1 of each week of the treatment period, and for an additional 5 weeks after discontinuation of treatment. Serum gastrin and CgA concentrations were analyzed using radioimmunoassay (RIA) and ELISA, respectively. RESULTS: Median serum gastrin concentrations increased 2.5-fold from baseline to day 7 (P < .001) but did not increase further during the omeprazole treatment period. Median serum gastrin concentrations returned to baseline within 2 to 4 days after administration of the last dose of omeprazole. No effect of treatment or discontinuation was seen in serum CgA concentrations. CONCLUSIONS AND CLINICAL IMPORTANCE: Serum gastrin concentrations increased in response to omeprazole treatment but returned to baseline within 2 to 4 days after the last dose of omeprazole. No effect of treatment or discontinuation was seen in serum CgA concentrations. Our results do not support the use of tapering protocols in horses.

Caenorhabditis elegans diet significantly affects metabolic profile, mitochondrial DNA levels, lifespan and brood size.

Diet can have profound effects on an organism's health. Metabolic studies offer an effective way to measure and understand the physiological effects of diet or disease. The metabolome is very sensitive to dietary, lifestyle and genetic changes. Caenorhabditis elegans, a soil nematode, is an attractive model organism for metabolic studies because of the ease with which genetic and environmental factors can be controlled. In this work, we report significant effects of diet, mutation and RNA interference on the C.elegans metabolome. Two strains of Escherichia coli, OP50 and HT115 are commonly employed as food sources for maintaining and culturing the nematode. We studied the metabolic and phenotypic effects of culturing wild-type and mutant worms on these two strains of E. coli. We report significant effects of diet on metabolic profile, on mitochondrial DNA copy number and on phenotype. The dietary effects we report are similar in magnitude to the effects of mutations or RNA interference-mediated gene suppression. This is the first critical evaluation of the physiological and metabolic effects on C.elegans of two commonly used culture conditions.

Calcium-induced peptide association to form an intact protein domain: 1H NMR structural evidence.

The 70-residue carboxyl-terminal domain of the muscle contractile protein troponin-C contains two helix-loop-helix calcium (Ca)-binding sites that are related to each other by approximate twofold rotational symmetry. Hydrophobic residues from the helices and a short three residue beta sheet at the interface of the two sites act to stabilize the protein domain in the presence of Ca. A synthetic 34-residue peptide representing one of these sites (site III) has been synthesized and studied by H-1 nuclear magnetic resonance (NMR) spectroscopy. In solution this peptide undergoes a Ca-induced conformational change to form the helix-loop-helix Ca-binding motif. Two-dimensional nuclear Overhauser effect spectra have provided evidence for the formation of a beta sheet and interactions between several hydrophobic residues from opposing helices as found in troponin-C. It is proposed that a symmetric two-site dimer similar in tertiary structure to the carboxyl-terminal domain of troponin-C forms from the assembly of two site III peptides in the Ca-bound form.

The nonhelical structure of antifreeze protein type III.

Antifreeze proteins (AFPs) are present in the blood of some marine fishes and inhibit the growth of ice crystals at subzero temperatures by adsorption to the ice lattice. The solution structure of a Type III AFP was determined by two-dimensional nuclear magnetic resonance spectroscopy. These measurements indicate that this 66-residue protein has an unusual fold in which eight beta strands form two sheets of three antiparallel strands and one sheet of two antiparallel strands, and the triple-stranded sheets are packed orthogonally into a beta sandwich. This structure is completely different from the amphipathic, helical structure observed for Type I AFPs.

Neutrophil activation by monomeric interleukin-8.

Interleukin-8 (IL-8), a pro-inflammatory protein, has been shown by nuclear magnetic resonance (NMR) and x-ray techniques to exist as a homodimer. An IL-8 analog was chemically synthesized, with the amide nitrogen of leucine-25 methylated to selectivity block formation of hydrogen bonds between monomers and thereby prevent dimerization. This analog was shown to be a monomer, as assessed by analytical ultracentrifugation and NMR. Nevertheless, it was equivalent to IL-8 in assays of neutrophil activation, which indicates that the monomer is a functional form of IL-8.

Antifreeze proteins.

Antifreeze proteins comprise a structurally diverse class of proteins that inhibit the growth of ice. Recently, new AFP types have been discovered; more active AFPs have been isolated; antecedents have been recognized supporting the notion of recent, multiple origins; and detailed structures have emerged leading to models for their adsorption to ice.

Thermodynamic insights into proteins from NMR spin relaxation studies.

NMR spin relaxation measurements of picosecond to nanosecond timescale backbone and sidechain fluctuations of protein molecules, and subsequent entropic interpretation yield interesting, but sometimes counterintuitive, insights into proteins. The stabilities of proteins and protein interactions are achieved through enthalpy-entropy compensation, which is partitioned between the backbone and sidechains depending on the nature of the system.

The effects of dose and diet on the pharmacodynamics of esomeprazole in the horse.

BACKGROUND: Esomeprazole warrants further investigation as a treatment for equine gastric ulcer syndrome. OBJECTIVES: To investigate the duration of intraday acid suppression achieved with two doses of esomeprazole under two dietary conditions. STUDY DESIGN: A four way crossover design. METHODS: Six adult Thoroughbreds instrumented with percutaneous gastrotomy tubes were used. Intragastric pH was measured for continuous 23 h periods (08.00-07.00 h) for 6 consecutive days (Days 0-5). Baseline data was recorded on Day 0 and esomeprazole was administered on Days 1-5. Two doses (0.5 and 2.0 mg/kg bwt/day per os once daily) and two diets (a high grain/low fibre (HG/LF) and ad libitum hay (HAY) diet) were studied. Data for the percentage of time pH was above 4 (%tpH>4) and median intraday pH was reported for two measurement points and analysed using generalised estimating equations. RESULTS: An inconsistent effect of both diet and dose was evident with mean %tpH>4 and mean of the median intraday pHs typically higher at the 2.0 mg/kg bwt dose and in HG/LF diet. A cumulative effect of dosing was present with the magnitude of acid suppression observed on Day 5 consistently higher than that observed on Day 1. The magnitude of acid suppression, at measurement point 1, compared favourably with previous reports on omeprazole and exceeded human therapeutic breakpoints for the 0.5 mg/kg bwt dose in the HG/LF diet and 2.0 mg/kg bwt dose in the HAY diet. MAIN LIMITATIONS: Instrumentation may have modified gastric function and horses were not fasted or exercised. CONCLUSIONS: The findings of the present study suggested that both dose and diet affect the response to esomeprazole in the horse and that a cumulative effect is present over the first 5 days of treatment. Further investigation into the clinical efficacy of esomeprazole and trials directly comparing esomeprazole and omeprazole appear to be warranted.

Preliminary investigations into a novel, long-acting, injectable, intramuscular formulation of omeprazole in the horse.

BACKGROUND: Pilot investigations have suggested that a novel, long-acting, injectable i.m. formulation of omeprazole (LA-OMEP) can induce acid suppression for up to 7 days following a single injection. OBJECTIVES: To investigate the pharmacodynamics and assess the clinical efficacy of the LA-OMEP formulation. STUDY DESIGN: Part A comprised a pharmacodynamic study. Part B consisted of a pilot clinical trial. METHODS: Part A enrolled six adult Thoroughbred horses with percutaneous gastrotomy tubes. Intragastric pH was measured for continuous 23-h periods (08.00-07.00 h) for eight consecutive days (days 0-7). A single 2.0-g dose of a 100 mg/mL LA-OMEP formulation was administered at 08.00 h on day 1. In Part B, 26 horses with squamous or glandular gastric disease were enrolled based on routine gastroscopic evaluation. Once enrolled, horses received 2.0 g of the 100 mg/mL LA-OMEP formulation by i.m. injection on days 0 and 7. Repeat gastroscopy was performed on days 14 (23 horses) or 16 (one horse). RESULTS: In Part A, the percentage of time during which pH was above 4 exceeded 66% for days 1-4 in all horses and days 1-7 in four of the six horses studied. In Part B, healing was observed in all 22 (100%, 95% confidence interval [CI] 89-100%) horses with squamous disease and in nine of 12 (75%, 95% CI 47-92%) horses with glandular disease. Improvement, by at least one grade, was observed in all 22 (100%, 95% CI 89-100%) horses with squamous disease and in all 12 (100%, 95% CI 81-100%) horses with glandular disease. No worsening of lesions was observed. Lesion grade decreased over time in both the squamous (P<0.0001) and glandular (P = 0.0024) mucosa. MAIN LIMITATIONS: Small sample sizes. CONCLUSIONS: The results of the present study compare favourably with previous reports on the pharmacodynamics of omeprazole and the clinical outcomes of trials reporting response to oral omeprazole therapy.

The effects of dose and diet on the pharmacodynamics of omeprazole in the horse.

BACKGROUND: Conflicting data are presented in the current literature regarding the efficacy of omeprazole for suppressing gastric acidity in the horse. OBJECTIVES: The objective of this study was to investigate the duration of intraday acid suppression achieved with two doses of omeprazole under two different dietary conditions. STUDY DESIGN: A four-way crossover design. METHODS: Six adult Thoroughbred horses instrumented with percutaneous gastrotomy tubes were used. Intragastric pH was measured for continuous 23 h periods (08.00-07.00 h) for six consecutive days (Days 0-5). Baseline data was recorded on Day 0 and omeprazole administered on Days 1-5. Two doses (1 mg/kg and 4 mg/kg bwt per os once a day) and two diets (a high grain/low fibre [HG/LF] and ad libitum hay [HAY)] diet) were studied. Data for the percent (%) time pH was above 4 (%tpH>4) and median intraday pH was reported for two measurement locations and analysed using generalised estimating equations. RESULTS: An effect of both diet and dose was evident with mean %tpH>4 and the mean of the median intraday pHs typically higher at the higher (4 mg/kg bwt) dose and in HG/LF diet. The overall efficacy of omeprazole in raising intragastric pH was good under the HG/LF conditions but relatively poor in the HAY diet. A cumulative effect of dosing, not previously reported in the horse, was observed. CONCLUSIONS: The overall efficacy of omeprazole in raising ventral gastric pH was less than previously reported. Both dose and diet may play a role in the efficacy of omeprazole in the horse. Therefore, the use of singular dosing recommendations that encompass all horse types and management conditions may not be appropriate and dosing recommendations that take into account the diet of the horse may be advantageous.

Refined solution structure of type III antifreeze protein: hydrophobic groups may be involved in the energetics of the protein-ice interaction.

BACKGROUND: Antifreeze proteins are found in certain fish inhabiting polar sea water. These proteins depress the freezing points of blood and body fluids below that of the surrounding sea water by binding to and inhibiting the growth of seed ice crystals. The proteins are believed to bind irreversibly to growing ice crystals in such a way as to change the curvature of the ice-water interface, leading to freezing point depression, but the mechanism of high-affinity ice binding is not yet fully understood. RESULTS: The solution structure of the type III antifreeze protein was determined by multidimensional NMR spectroscopy. Twenty-two structures converged and display a root mean square difference from the mean of 0.26 A for backbone atoms and 0.62 A for all non-hydrogen atoms. The protein exhibits a compact fold with a relatively large hydrophobic core, several short and irregular beta sheets and one helical turn. The ice-binding site, which encompasses parts of the C-terminal sheet and a loop, is planar and relatively nonpolar. The site is further characterized by the low solvent accessibilities and the specific spatial arrangement of the polar side-chain atoms of the putative ice-binding residues Gln9, Asn14, Thr15, Thr18 and Gln44. CONCLUSIONS: In agreement with the adsorption-inhibition mechanism of action, interatomic distances between active polar protein residues match the spacing of water molecules in the prism planes (¿10&1macr;0¿) of the hexagonal ice crystal. The particular side-chain conformations, however, limit the number and strength of possible proten-ice hydrogen bonds. This suggests that other entropic and enthalpic contributions, such as those arising from hydrophobic groups, could play a role in the high-affinity protein-ice adsorption.

Improved synthetic methods for the selective deuteration of aromatic amino acids: applications of selective protonation towards the identification of protein folding intermediates through nuclear magnetic resonance.

In this report we describe several novel methods for the preparation of selectively deuterated aromatic amino acids. New syntheses for [2,3,5,6-2H4]phenylalanine and [2,4,6,7-2H4]tryptophan, as well as improved catalytic exchange methods for [2,3,5,6-2H4]tyrosine and [2,3,4,5,6-2H5]phenylalanine are presented. Isotopic substitution levels for all compounds are generally found to be greater than 95%. Biosynthetic incorporation of these amino acids is also shown to be possible with little or no evidence of isotopic scrambling. The products from these new syntheses, in combination with other selectively deuterated aromatic amino acids, are found to permit group-specific 'single-proton' labelling of proteins. This highly-efficient and very cost-effective method of selective protonation is shown to produce greatly simplified 1H-NMR spectra of the aromatic region of proteins. The utility of this approach to isotopic editing is demonstrated with the identification of a transient folding intermediate of Escherichia coli thioredoxin which is undetectable by standard 2-D NMR techniques.

Interaction of troponin I and troponin C: use of the two-dimensional transferred nuclear Overhauser effect to determine the structure of a Gly-110 inhibitory troponin I peptide analog when bound to cardiac troponin C.

The structure of a peptide analog of the inhibitory region of cardiac troponin-I (N-acetyl-G110-TnI(104-115) amide) when bound to cardiac troponin-C has been determined by 2-dimensional 1H-NMR techniques. The bound structure determined for this peptide is similar to that determined previously for the skeletal peptide (which has a proline at position 110) bound to skeletal troponin-C (Campbell and Sykes (1991) J. Mol. Biol. 222, 405-421). This structure shows a helical like peptide backbone 'bent' around P109-G110 to bring the hydrophobic residues F106, L111 and V114 closer together. The other 'side' of this structure is surrounded by the basic residues extending outwards towards the protein or solution. While the bound structures of the cardiac and skeletal peptides are shown to be quite similar, the cardiac peptide appears more flexible near the central glycine residue.