Inhibition of collagenase breakdown of equine corneas by tetanus antitoxin,equine serum and acetylcysteine

Objective To determine whether tetanus antitoxin, equine serum, and acetylcysteine, which are currently used in the treatment of equine corneal ulcer, inhibit the digestion of equine corneal collagen when exposed to collagenase in vitro. Animals studied Corneas from 40 adult horses. Procedures Sections of equine corneas were incubated with saline, a solution of bacterial collagenase in saline, bacterial collagenase in saline plus equine tetanus antitoxin, bacterial collagenase in saline plus equine serum, or bacterial collagenase in saline plus acetylcysteine. Each one of the collagenase inhibitors was tested at different concentrations. The degree of corneal collagen digestion was determined by concentrations of hydroxyproline released into the incubation media and/or by weight loss of the cornea. Results Corneas exposed to collagenase released a significant (0.05 level) large amount of hydroxyproline (43.1 ± 2.3 µg/mL/100 mg cornea/5 h) and decreased cornea weight by up to 89%. Blood serum (200 µL/mL), purified albumin or globulin fractions of serum, tetanus antitoxin (120 units/mL), and acetylcysteine (20 mg/mL) when used at the highest concentrations blocked collagenase digestive activity by approximately 50%. Dilution of inhibitors decreased corneal protection and linearly increased corneal weight loss. Purified equine serum albumin and globulin fractions were equally effective in protecting corneas. Conclusions This experiment indicates that tetanus antitoxin, serum and acetylcysteine equally protected corneas from collagenase digestion, in vitro. However, a clinical trial is needed to establish relative therapeutic value.     

Theiler's disease associated with administration of tetanus antitoxin contaminated with nonprimate (equine) hepacivirus and equine parvovirus-hepatitis virus

An 11-year-old Trakehner gelding was presented for evaluation of lethargy, decreased appetite, mild icterus, and elevated hepatic enzyme activities. Physical examination, serum chemistry results, and liver biopsy histopathologic findings were supportive of Theiler's disease. Polymerase chain reaction (PCR) testing results of serum and liver tissue were positive for nonprimate (equine) hepacivirus (NPHV) and a novel equine parvovirus-hepatitis virus (EqPV-H). PCR testing of the lot of tetanus antitoxin administered to the gelding 3 months previously also yielded positive results for NPHV and EqPV-H. Treatment included supportive care and clinical signs resolved within 1 week, although hepatic enzyme activities remained elevated for several months. The horse successfully returned to work as a hunter/jumper for about 1 year until it developed a forelimb lameness and progressive atrophy of shoulder musculature (sweeney), prompting a decision for euthanasia 20 months after initial evaluation. Serial PCR testing of serum revealed persistent infection with both NPHV and EqPV-H and necropsy examination revealed chronic active hepatitis, mild liver atrophy, and positive PCR results for NPHV and EqPV-H in liver tissue. This case highlights the possible risk of administering potentially contaminated biologics of equine origin and the importance of screening for recently identified hepatic viruses in donors from which blood products are prepared.     

Thyroid function and serum hepatic enzyme activity in dogs after phenobarbital administration

Phenobarbital is the drug of choice for control of canine epilepsy. Phenobarbital induces hepatic enzyme activity, can be hepatotoxic, and decreases serum thyroxine (T4) concentrations in some dogs. The duration of liver enzyme induction and T4 concentration decreases after discontinuation of phenobarbital is unknown. The purpose of this study was to characterize the changes in serum total T4 (TT4), free T4 (FT4), thyroid-stimulating hormone (TSH), cholesterol and albumin concentrations, and activities in serum of alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyl transferase (GGT) after discontinuation of long-term phenobarbital administration in normal dogs. Twelve normal dogs were administered phenobarbital at a dosage of approximately 4.4-6.6 mg/kg PO q12h for 27 weeks. Blood was collected for analysis before and after 27 weeks of phenobarbital administration and then weekly for 10 weeks after discontinuation of the drug. The dogs were clinically normal throughout the study period. Serum ALT and ALP activity and TSH and cholesterol concentrations were significantly higher than baseline at week 27. Serum T4 and FT4 were significantly lower. Serum albumin and GGT were not changed from baseline at week 27. Changes in estimate of thyroid function (TT4, FT4, TSH) persisted for 1-4 weeks after discontinuation of phenobarbital, whereas changes in hepatic enzyme activity (ALT, ALP) and cholesterol concentration resolved in 3-5 weeks. To avoid false positive results, it is recommended that thyroid testing be performed at least 4 weeks after discontinuation of phenobarbital administration. Elevated serum activity of hepatic enzymes 6-8 weeks after discontinuation of phenobarbital may indicate hepatic disease.     

Treatment of tetanus in the horse by injections of tetanus antitoxin into the subarachnoid space.

In 40 horses with tetanus, large doses of tetanus antitoxin (TAT) were injected into the subarachnoid space. In all the horses that recovered, the disease stabilized immediately after the injection. The results (77.5% recovery) were much better than in a previous series of horses with tetanus (50% recovery), in which TAT was injected either intravenously, intramuscularly, or in the epidural space.     

Age- and sex-determined differences in the establishment of tetanus antitoxin production in guinea-pigs

For lack of relevant data of the literature, the tetanus immunisation results obtained in the two sexes were compared in an animal model. Complete immunisation series of weaned, adult and aged guinea-pigs (20-25 animals/group) were performed with aluminium phosphate (AlPO4) adsorbed purified tetanus toxoid (PTAP) as well as with typhoid-tetanus vaccine (TY-TE) containing lipopolysaccharide (LPS). Both vaccines contained 5.0 Lf (limes flocculans, Ramon) per single dose of tetanus toxoid, purity degree: 1500 Lf/mg protein nitrogen (PN). Tetanus antitoxin titres (TAT) were measured after the first shot, and subsequently before and after booster. Compared to TAT of male animals, significantly lower titres were found in female animals after basic immunisation with PTAP in all the three age groups: 1.03 vs. 0.57, 8.75 vs. 5.64, and 0.27 vs. 0.15 IU (international units, related to the Copenhagen International Standard) per ml (sex-chromosome-dependent differences?), as well as in adult animals immunised with TY-TE, before booster: 0.07 vs. 0.02 IU/ml (hormone-dependent differences?). In the latter case the TAT results after booster were 14.49 vs. 12.89 IU/ml. Thus, the lower female prebooster titres were counterbalanced by a quick and effective increase of titres following booster. These results are in accordance with our previous observations in humans (Réthy and Réthy, 1986). From our observations with tetanus immunisation series on guinea-pigs it can be concluded that TAT may be influenced by the effects of sex chromosomes as well as of sexual hormones. During active anti-tetanus immunisation with LPS-containing vaccine (TY-TE) the lower adult female prebooster titres are presumably counterbalanced by the better functionality of the female immune memory.     

Effects of oral administration of concentrated equine serum IgG to newborn foals on passive immunity

Thirteen newborn foals of Quarter Horse breeding were used to determine if oral administration of concentrated equine serum increases concentrations of IgG in foals allowed to naturally suckle colostrum. Foals were alternately assigned either to receive 300 ml of an oral equine serum IgG product or to serve as controls. Foals receiving the IgG product were given 150 ml orally at 10 hours and again at 12 hours after birth. All foals were allowed to suckle from their dams ad libitum. Jugular blood samples were obtained from foals at 10 hours and 24 hours of age for IgG determination. Colostrum samples from the dam were also obtained within 3 hours following parturition for determination of specific gravity. Plasma samples were analyzed for IgG level using a commercially available radial immunodiffusion kit. Oral administration of equine serum IgG had no significant effect on concentrations of plasma IgG in foals at 24 hours of age (p>.34). There was also no difference between control and treated foals in the rate of IgG absorption from 10-24 hours after birth (p>.34). In conclusion, oral administration of equine IgG to foals that ingest their dam's colostrum does not significantly increase concentrations of plasma IgG when compared to controls.     

Apparent ELISA detection times for albuterol after administration with the torpex equine inhaler device

Single doses of one, three, and six actuations (120 micro g albuterol/actuation) and multiple daily doses (six actuations per dose four times daily) for 5 days of aerosol albuterol sulfate were sequentially administered to each of six horses using an equine inhaler device (Torpex, 3M Animal Care Products, St. Paul, MN [corrected] and Boehringer Ingleheim Vetmedica, Inc., St. Joseph, MO [corrected]). A 2-week washout period was allowed between each dose. ELISA testing revealed no evidence of albuterol in urine at 24 hours after any single-dose administration. Results indicated that 48 hours or longer should be allowed for albuterol to be cleared from urine after single doses. When given at the maximum recommended rate of six actuations per dose four times a day for 5 days, urine samples tested by ELISA showed no evidence of albuterol at 48 hours after the final dose. Testing of nasal swabs by ELISA demonstrated the presence of albuterol for 8 hours after each single dose, and some horses might have detectable levels of albuterol in nasal swabs for several days following administration of multiple doses. As a guideline for withdrawal time, 72 hours or longer should be allowed after administration of aerosol albuterol sulfate to horses before participation in equestrian competitions that are regulated for detection of certain performance-enhancing substances. However, these recommendations were based on a small sample of horses and the specific ELISA test used and interpreted as described. Factors specific to individual horses may influence these detection times.     

Kinetic study of serum gentamicin concentrations in baboons after single-dose administration

This study establishes preliminary pharmacokinetic data on the use of gentamicin sulfate administered IM to baboons. Serum concentrations greater than or equal to 12 micrograms/ml are generally agreed to cause toxicosis in human beings. On the basis of preliminary test results suggesting that the manufacturer's recommended dosage for dogs of 4.4 mg/kg of body weight caused potentially toxic serum concentrations, a dosage of 3 mg/kg was chosen to conduct a single-dose kinetic study in 6 baboons. Using a single-compartment model, the gentamicin serum half-life for IM administration of 3 mg of gentamicin/kg was 1.58 hours, and serum concentrations remained below the potentially toxic concentrations reported for human beings. We suggest that a dosage of 3 mg/kg is safer than a dosage of 4.4 mg/kg administered IM to baboons. Minimal inhibitory concentrations for 2 Pseudomonas aeruginosa isolates were less than or equal to 1 micrograms/ml. On the basis of our measured elimination half-life of 1.58 hours, it is reasonable to suppose that dosing q24 h will be inadequate to maintain therapeutic serum concentrations. We calculate that serum concentrations will remain at or above our measured minimal inhibitory concentration for P aeruginosa (1 micrograms/ml) for 100% of the treatment time if the animal is dosed q 6h, 78% for dosing q 8h, and 52% for dosing q 12h. Therefore, we suggest 3 mg/kg, q 8h or q 6h as appropriate dosing schedules for the use of gentamicin sulfate administered IM to baboons.     

Duration of immunity induced by an adjuvanted and inactivated equine influenza, tetanus and equine herpesvirus 1 and 4 combination vaccine

An adjuvanted vaccine containing inactivated equine influenza, herpesvirus antigens, and tetanus toxoid was administered to young seronegative foals of 8 months of age by deep intramuscular injection in the neck (Group A). The first two vaccinations were given 4 weeks apart. The third was administered 6 months later. Another group of foals (Group B) was vaccinated according to the same scheme at the same time with monovalent equine herpes virus (EHV) vaccine (EHV1.4) vaccine. Antibody responses to the equine influenza (single radial haemolysis; SRH) and tetanus (ToBi ELISA) components of the vaccines were examined from first vaccination until 1 year after the third vaccination. The influenza components of the combination vaccine induced high antibody titres at two weeks after the second vaccination whereafter titres declined until the time of the third vaccination. After the third vaccination, the titres rose rapidly again to remain high for at least 1 year. Antibody titres against tetanus peaked only after the third vaccination but remained high enough to offer protective immunity for at least 1 year. Foals vaccinated with monovalent EHV1.4 remained seronegative for influenza and tetanus throughout the study. Four and a half months after the third vaccination of groups A and B, a third group of animals was vaccinated twice with monovalent EHV1.4 vaccine 4 weeks apart (Group C). Two weeks after the administration of the second dose in the later group, all groups (A, B, C and an unvaccinated control group D) were challenged with EHV-4. Vaccinated foals (Group A, B, C) showed a clear reduction of clinical symptoms and virus excretion after EHV-4 challenge compared with the unvaccinated control foals. No difference could be demonstrated among the vaccinated groups, suggesting that the combination vaccine protects as well as the monovalent vaccine. In EHV1.4-vaccinated foals both antigenic fractions induced clear protection up to 6 months after vaccination (9). It can therefore be anticipated that the efficacy of the combination vaccine against EHV-1 challenge is similar to the efficacy against EHV-1 induced by EHV1.4 vaccination.     

Determination of concentration of hyaluronate in equine serum

Concentration of hyaluronate (HA) in equine serum was determined by a recently developed specific radioassay. The mean +/- SD HA concentration in equine serum was 288 +/- 145 micrograms/L, was age dependent, and varied widely between horses (range, 190 to 760 micrograms/L). Light or moderate exercise increased serum HA concentration from baseline values by 1.5- to 3-fold. In all horses, serum HA concentration returned to or below the original resting values 1 and 2 hours after exercise.     

Kinetic analysis of 5 sugar probes in dog serum after orogastric administration

The objective of this study was to describe the kinetics of orally administered sugar probes in serum for the assessment of gastrointestinal permeability and intestinal absorptive capacity in dogs. Eight healthy dogs received lactulose (L), rhamnose (R), methylglucose (M), xylose (X), and sucrose (S) by orogastric intubation. Baseline blood samples and subsequently timed blood samples were taken during 24 hours. Sugars were analyzed by gas chromatography-mass spectrometry (GC-MS). Statistical analysis was performed using a Friedman test with Dunn’s multiple comparison post test and a Kruskal-Wallis test. Statistical significance was set at a P-value < 0.05. Sugars in serum were detected after orogastric administration. Concentrations of L and R were significantly different from the baseline from 90 to 240 and 60 to 300 min, respectively, and those of X, M, and S were different from 30 to 240 min post-dosing (P < 0.05 for all 5 probes). Maximum concentrations of L and R were obtained at 180 min, while X, M, and S reached their maximum concentrations at 90 min post-dosing. For all sugars, no statistically significant differences were found between concentrations at 90, 120, and 180 min or between the coefficients of variation (CV%) of those mean concentrations for these 3 time points. Based on these data, the collection of 2 blood samples, one taken at baseline and the other obtained between 90 and 180 minutes after dosing, might be sufficient for the determination of gastrointestinal permeability and mucosal absorptive capacity using these 5 sugar probes in canine serum.     

Vitamin E serum levels in calves after various methods of administration]

We observed the levels of vitamin E in the blood serum of calves after peroral application of Combinal E (1 ml contains 20 mg of tocopherol acetate in water solution), after application through a fistula into the rennet stomach and after an intramuscular injection of Erevit (1 ml contains 300 mg of tocopherol acetate in vegetable oil). The fastest increase in the vitamin E level was recorded after the application of Combinal E directly into the rennet stomach. The application of Combinal E per os resulted in the same level of vitamin E in the ninth hour after application as in the third hour after application into the rennet stomach, the intramuscular injection of Erevit had a much lower effect on raising the vitamin E level in the blood serum of calves. It was confirmed that for a faster supplementing of vitamin E to the organism it is more suitable to give Combinal E perorally.     

Fractionation of calcium and magnesium in equine serum

OBJECTIVE: To establish reference values for protein-bound, ionized, and weak-acid complexed fractions of calcium and magnesium in equine serum and determine stability of ionized calcium (iCa) and ionized magnesium (iMg) in serum samples kept under various storage conditions. ANIMALS: 28 clinically normal horses. PROCEDURE: Total calcium (tCa) and magnesium (tMg) in equine serum were fractionated by use of a micropartition system that allows separation of protein-bound calcium (pCa) and magnesium (pMg) and ultrafiltrable calcium (microCa) and magnesium (microMg) fractions. Serum concentrations of iCa and iMg were measured in the ultrafiltrate by use of selective electrodes. Serum concentration of complexed calcium (cCa) or magnesium (cMg) was calculated by subtracting iCa or iMg from microCa or microMg, respectively. RESULTS: Mean +/-SE serum tCa concentration was 3.26 +/- 0.06 mmol/L. Calcium fractions were as follows: pCa, 1.55 +/- 0.03 mmol/L (47.4 +/- 0.9%); iCa, 1.58 +/- 0.03 mmol/L (48.5 +/- 0.7%); and cCa, 0.13 +/- 0.02 mmol/L (4.1 +/- 0.9%). Serum tMg concentration was 0.99 +/- 0.04 mmol/L. Magnesium fractions were as follows: pMg, 0.33 +/- 0.04 mmol/L (33.3 +/- 4.2%); iMg, 0.57 +/- 0.02 mmol/L (57.6 +/- 1.7%); and cMg, 0.09 +/- 0.02 mmol/L (9.1 +/- 1.9%). Refrigeration (4 degrees C) did not affect iCa values, whereas iMg declined by 8% after 120 hours. Neither iCa nor iMg was affected by freezing (-20 degrees C). CONCLUSIONS AND CLINICAL RELEVANCE: In equine serum, iMg is less stable than iCa; thus, when serum samples are not going to be analyzed promptly, freezing may be preferable to refrigeration for storage.     

Production of an equine anti-bovine leukocyte serum.

A method is described for production of an equine anti-bovine leukocyte serum (EABLS). Leukocytes were harvested from the milk of cow's udders which had been irritated with endotoxin. The washed leukocytes as antigens were administered to 2 horses in a series of subcutaneous and intravenous injections. There was a variably progressive increase in total serum proteins and a decrease in albumin/blobulin ratios, but the most pronounced change was an increase in beta2-globulins. Accompanying these changes was an increase in the number of precipitin lines as shown by Ouchterlony analysis. Four old cows and 2 calves were intravenously given a 50- and 20-ml dose, respectively, of the EABLS. Two of the cows and 1 of the calves were given the EABLS which had been heated to 56 C for 30 minutes. Both calves and the 2 cows given heated EABLS and 1 cow given unheated EABLS developed marked neutropenia within the 1st hour. The 4th cow given unheated EABLS developed only slight neutropenia. It was concluded that there may be variation in the individual animal's susceptibility to the neutropenia-inducing activity of EABLS.     

Effect of day of transfer and treatment administration on the recipient on pregnancy rates after equine embryo transfer

Veterinary Research Communications -     

Alterations in selected serum biochemical constituents in equids after induced hepatic disease

Effects of induced cholestasis and hepatocellular necrosis and of fasting on serum biochemical constituents including bile acids, IgA, bilirubin, alkaline phosphatase, gamma-glutamyltransferase (GGT), arginase, and the clearance of sodium sulfobromophthalein were studied in 4 groups of equids. The reference value for serum bile acids, as determined by an enzymatic colorimetric procedure for horses and ponies was 5.94 +/- 2.72 mumol/L, there being no statistical difference for horses and ponies. Sample collection at time of feeding had no effect on serum bile acid concentration. Seemingly, serum bile acids, arginase, and GGT were the most sensitive indicators of cholestasis and/or hepatocellular necrosis and would form an essential minimum effective battery of tests to diagnose and prognose hepatic disease in equids. These tests provided a measure of hepatobiliary transport function (bile acids), cell necrosis (arginase), and cholestasis (GGT and bile acids).