ISCOM-matrix-based equine influenza (EIV) vaccine stimulates cell-mediated immunity in the horse

The humoral immune response induced by ISCOM-matrix (Immuno Stimulating COMplex-Matrix)-adjuvanted equine influenza virus (EIV) vaccine is well documented in horses. ISCOM-matrix adjuvanted vaccines against human influenza are strong inducers of cell-mediated immunity (CMI), including T cell proliferation and virus-specific cytotoxic T cell. In the horse, the CMI response to equine influenza vaccination is less well characterised. An ISCOM-based vaccine has been shown to induce interferon gamma (IFN-γ) synthesis, a CMI marker, in the horse, but this has not been shown for the ISCOM-matrix vaccine, which is a different formulation. The objective of this study was to measure EIV-specific IFN-γ synthesis after vaccination with an ISCOM-matrix-adjuvanted EIV vaccine. Equilis Prequenza is a commercialised inactivated EIV vaccine containing purified haemagglutinin (HA) and neuraminidase (NA) subunits adjuvanted with ISCOM-matrix. Six influenza-naïve Welsh mountain ponies were vaccinated twice with Equilis Prequenza at an interval of four weeks. Six control ponies received a placebo of physiological water. EIV-specific IFN-γ synthesis by peripheral blood lymphocytes and the antibody response to a panel of representative EIV isolates were measured prior to and after both injections. Immunisation with the ISCOM-matrix-based EIV vaccine stimulated significant EIV-specific IFN-γ synthesis and EIV-specific single radial haemolysis (SRH) antibody. In conclusion, EIV vaccine adjuvanted with ISCOM-matrix stimulates both antibody and a cellular immune response in the horse.     

The effect of orally administered marbofloxacin on the pharmacokinetics of theophylline

As certain quinolones can interfere with the metabolism of theophylline by competitive inhibition of the hepatic microsomal cytochrome P450 system, concomitant use of these drugs with theophylline could result in theophylline toxicity. This study investigated the effect of orally administered marbofloxacin (2 and 5 mg/kg each once daily) on steady-state plasma pharmacokinetics of theophylline after concomitant oral administration of a sustained release theophylline preparation in dogs. Marbofloxacin caused some alteration in theophylline metabolism. A 2 mg/kg dose of marbofloxacin did not clearly result in an increased area under the concentration--time curve (AUC) or decreased clearance of theophylline, but at a dose of 5 mg/kg, a statistically significant increase in AUC and a decrease in the total clearance of theophylline was found. The 26% reduction in theophylline clearance is probably not clinically significant in healthy dogs, but for dogs with renal impairment, there might be a chance of theophylline accumulation when dosed concomitantly with marbofloxacin.     

An outbreak of equine influenza at a harness horse racetrack

An outbreak of an influenza-like illness affected approximately 1/3 of the 1050 race horses stabled at a standardbred racetrack and resulted in a 3-day suspension of racing. A/Equi-2 influenza virus was isolated from 1 affected horse and 8 of 10 horses sampled seroconverted. Threshold protective levels of HI antibody against A/Equi-2 influenza virus were not demonstrated in unaffected horses. Resistance in unaffected horses was assumed to result from other factors following previous exposure. Few of the horses had been vaccinated against equine influenza. It was felt that an outbreak of this magnitude might have been prevented if a vaccination program had been followed.     

The pharmacokinetics of meclofenamic acid in the horse

The pharmacokinetics of meclofenamic acid were studied in Thoroughbred horses and in ponies. After intravenous (i.v.) administration of either 2 mg/kg or 4 mg/kg sodium meclofenamate the elimination half-life was of the order of 0.9 h while the volume of distribution was found to be 0.128 litre/kg. Elimination was in accordance with a one-compartment model. Following oral administration of either meclofenamic acid (4 mg/kg) or sodium meclofenamate (4 mg/kg) a much longer terminal half-life than that calculated for K _el from i.v. data was found. This anomaly indicated that the 'flip-flop' phenomenon was present, i.e. K _a exceeded K _el. More rapid and higher peak levels occurred following sodium meclofenamate than meclofenamic acid, although total bioavailability was similar. Studies in ponies with meclofenamic acid showed a lower absorption than that found in Thoroughbreds. Overnight fasting before meclofenamic acid administration did not alter the rate or extent of absorption. Intramuscular administration of sodium meclofenamate resulted in low plasma concentrations and after 25 h only 46% of the drug had been absorbed.     

Sustained-release theophylline pharmacokinetics in the cat

Theophylline was administered in a three-way crossover design study to six cats intravenously (Aminophylline USP, Invenex Laboratories, Chagrin Falls, OH) and orally as two sustained-release formulations (Slo-bid Gyrocaps (SB), William H. Rorer, Inc., Fort Washington, PA; Theo-Dur Tablets (TD), Key Pharmaceuticals, Miami, FL). Values were determined for mean residence time (SB = 19.4 +/- 3.2 h; TD = 15.8 +/- 4.8 h), mean absorption time (SB = 8.0 +/- 2.3 h; TD = 4.8 +/- 2.3 h), absolute bioavailability (SB = 82 +/- 27%; TD = 76 +/- 38%), and time to peak plasma concentrations (SB = 8 h; TD = 8 h). After normalization to a dose of 25 mg/kg, the average peak plasma concentrations were also predicted (SB = 10.5 +/- 3.4 micrograms/ml; TD = 14.3 +/- 6.7 micrograms/ml). Slo-bid was predicted to provide the least peak:trough fluctuation in theophylline concentrations. Slo-bid and Theo-Dur appear to have pharmacokinetic characteristics which, if given once-daily, would maintain plasma theophylline concentrations of 5-20 micrograms/ml in the cat.     

Formulation with CpG ODN enhances antibody responses to an equine influenza virus vaccine

Previous studies have shown that protection against equine influenza virus (EIV) is partially mediated by virus-specific IgGa and IgGb. In this study we tested whether addition of a CpG ODN formulation to a commercial killed virus vaccine would enhance EIV-specific IgGa and IgGb antibody responses, and improve protection against an experimental EIV challenge. Thirty na?ve horses were assigned to one of three groups and vaccinated as follows: 10 were given vaccine (Encevac TC4, Intervet Inc.) alone, 10 were given vaccine plus 0.25 mg CpG ODN 2007 formulated with 30% Emulsigen (CpG/Em), and 10 controls were given saline. All horses were challenged with live virus 12 weeks after the final vaccination. Antibody responses were tested by single radial hemolysis (SRH) and ELISA, and protection was evaluated by determination of temperature, coughing, and clinical scores. Killed virus vaccine combined with CpG/Em induced significantly greater serologic responses than did the vaccine alone. All antibody isotypes tested increased after the addition of CpG/Em, although no shift in relative antibody isotypes concentrations was detected. Vaccination significantly improved protection against challenge but the differences between the two vaccine groups were not statistically significant. This study is the first demonstration that CpG/Em enhances antigen-specific antibody responses in horses and supports its potential to be used as an adjuvant for vaccines against equine infections.     

The pharmacokinetics of some aminoglycoside antibiotics in the horse

The disposition kinetics and bioavailability of streptomycin, kanamycin and neomycin were determined following their administration as parenteral preparations to horses. Single doses (10 mg/kg) of each aminoglycoside were given by the intravenous (i.v.) and intramuscular (i.m.) routes and, at a later time, seven intramuscular doses were injected at 12-h intervals. The pharmacokinetic behaviour of the three aminoglycosides was similar, in that a rapid distribution phase was followed by a relatively short half-life. The half-life (mean ± SD, n= 6) of kanamycin (1.80 ± 0.17 h) was significantly (P<0.01; t test, 10 d.f.) shorter than that of streptomycin (3.40 ± 0.42 h), while neomycin half-life (2.10 ± 0.97 h) was of an intermediate length. The apparent volume of distribution of neither kanamycin nor neomycin varied significantly (P > 0.05) from that of streptomycin and numerically (V¹ d = 230 ml/kg) was the same as the extracellular fluid volume. The body clearance of kanamycin (88.5 ± 11.3 ml/kg.h) was significantly (P < 0.01) larger than that of streptomycin (47.5 ± 7.9 ml/kg.h), while a significant difference in this parameter did not exist (P > 0.05) between neomycin and streptomycin. Following intramuscular injection, each aminoglycoside was rapidly and completely absorbed from the injection site, although neomycin showed wide individual variation in the fraction absorbed. The administration of multiple doses did not change either the bioavailability or the apparent half-life from the values obtained after a single dose. The only pharmacokinetic difference between these aminoglycosides that is of clinical importance lies in the rate of their elimination. A dosage interval of 8 h would be appropriate for kanamycin compared with a 12-h interval for streptomycin. The dosage interval for neomycin based on half-life should be 8 h but, due to the relatively greater toxicity of this aminoglycoside, an interval of 12 h might be recommended. The height of the peak serum concentration is determined by the size of the dose.     

Detection of clade 2 equine influenza virus in an adult horse recently imported to the USA

A 4‐year‐old Warmblood mare presented to the William R. Pritchard Veterinary Medical Teaching Hospital, School of Veterinary Medicine, University of California at Davis with bilateral mucoid nasal discharge and pyrexia. The mare had recently been imported from Germany, arriving at a quarantine holding facility 72 h prior to presentation. Based on clinical presentation and culture results of tracheal fluid, the mare was diagnosed with bacterial bronchopneumonia secondary to equine influenza. The equine influenza virus (EIV) identified in the imported mare displayed 99.1% nucleotide homology of the HA1 gene to the prototype Florida sublineage clade 2 isolate A/equine/Richmond/1/2007 (H3N8). This case illustrates the risk of introducing a clade 2 EIV in North America.     

Derivation and characterization of a live attenuated equine influenza vaccine virus

OBJECTIVE: To develop and characterize a cold-adapted live attenuated equine-2 influenza virus effective as an intranasal vaccine. ANIMALS: 8 ponies approximately 18 months of age. PROCEDURES: A wild-type equine-2 virus, A/Equine/Kentucky/1/91 (H3N8), was serially passaged in embryonated chicken eggs at temperatures gradually reduced in a stepwise manner from 34 C to 30 C to 28 C to 26 C. At different passages, infected allantoic fluids were tested for the ability of progeny virus to replicate in Madin-Darby canine kidney (MDCK) cells at 34 C and 39.5 C. Virus clones that replicated at 26 C in eggs and at 34 C in MDCK cells, but not at 39.5 C in MDCK cells, were tested for stability of the cold-adapted, temperature-sensitive (ts), and protein synthesis phenotypes. A stable clone, P821, was evaluated for safety, ability to replicate, and immunogenicity after intranasal administration in ponies. RESULTS: Randomly selected clones from the 49th passage were all ts with plaquing efficiencies of < 10(-6) (ratio of 39.5 C:34 C) and retained this phenotype after 5 serial passages at 34 C in either embryonated eggs or MDCK cells. The clone selected as the vaccine candidate (P821) had the desired degree of attenuation. Administered intranasally to seronegative ponies, the virus caused no adverse reactions or overt signs of clinical disease, replicated in the upper portion of the respiratory tract, and induced a strong serum antibody response. CONCLUSION AND CLINICAL RELEVANCE: A candidate live attenuated influenza vaccine virus was derived by cold-adaptation of a wild-type equine-2 influenza virus, A/Equine/Kentucky/1/91, in embryonated eggs.     

Studies on the antigenicity of an inactivated, aluminum hydroxide adjuvant equine influenza vaccine.

An inactivated, aluminum hydroxide adjuvant equine influenza vaccine was tested in horses and guinea pigs to determine the levels of antigen that would elicit maximum serological responses. Vaccine containing serial twofold increments of A/Equi-1/Prague and A/Equi-2/Miami strains of equine influenza virus was administered to random groupings of both types of test animals. The hemagglutination inhibition antibody response for each group was then measured. Results in horses and guinea pigs were compared to determine if the equine serological values could be related to a potency test in laboratory animals. The highest mean hemagglutination inhibition antibody response in horses occurred in groups vaccinated, respectively, with 128 or 256 hemagglutination units of A/Equi-1 and 512 or 1024 hemagglutination units of A/Equi-2 antigen. Groups vaccinated with further two- or fourfold increases in these antigens had mean hemagglutination inhibition titers that were somewhat lower than the maximum levels. When graded doses of vaccine were given to guinea pigs, their hemagglutination inhibition antibody titers reached a plateau of maximum values, similar to the serological response in vaccinated horses. Test horses remained clinically free from signs of equine influenza during the year following vaccination and no untoward post-vaccination reactions were observed.