Effects of a specific thromboxane synthetase inhibitor in equine endotoxaemia

Thromboxane A2 may play a major role in circulatory shock. In some species, thromboxane synthetase inhibitors have a beneficial effect on shock induced by endotoxin, trauma, sepsis and administration of arachidonate. In some shock models, however, results with thromboxane synthetase inhibitors have been conflicting. The effect of UK-38,485, a selective thromboxane inhibitor, was evaluated in ponies injected with endotoxin intraperitoneally. Four groups of ponies were used to compare the effects of endotoxin alone, UK-38,485 alone, treatment with UK-38,485 before endotoxin challenge and treatment with UK-38,485 after endotoxin challenge. Haematological, metabolic, eicosanoid and clinical responses in each group were evaluated. The results indicated that UK-38,485 is an effective inhibitor of thromboxane A2 generation following endotoxin challenge. Prostacyclin values were elevated compared with baseline in ponies administered UK-38,485 and endotoxin. However, prostacyclin values were not significantly different from those of ponies receiving endotoxin alone. Furthermore, UK-38,485 failed to attenuate the haematological, metabolic and clinical manifestations commonly seen in the pony after endotoxin challenge.     

Pattern recognition receptors in equine endotoxaemia and sepsis

Pattern recognition receptors (PRRs) on host cells detect pathogens to activate innate immunity which, in turn, initiates inflammatory and adaptive immune responses. Successful activation of PRRs is, therefore, critical to controlling infections and driving pathogen-specific adaptive immunity, but overactivity of PRRs causes systemic inflammation, which is detrimental to the host. Here we review the PRR literature as it relates to horses and speculate on the role PRRs may play in sepsis and endotoxaemia.     

Treatment of endotoxaemia and septicaemia in the equine patient

Endotoxins, constituents of the cell wall of gram‐positive and gram‐negative bacteria, regularly result in severe illness and death in horses. In endotoxaemia, these constituents are present in the systemic circulation; in septicaemia, whole microbes invade normally sterile parts of the body. Interaction of these endotoxins with pathogen recognition receptors leads to an inflammatory response that cannot always be sufficiently contained and hence needs direct treatment. Over the last decennia, our understanding of the pathophysiology of endotoxaemia and septicaemia has significantly increased. Based on improved understanding of the interaction between receptors and endotoxins as well as the subsequent downstream signalling pathways, new therapeutic targets have been identified in laboratory animal species and humans. Important species differences in the recognition of endotoxins and pathogens by their receptors as well as the inflammatory response to receptor activation hamper extrapolation of this information to the horse (and other species). Historically, horses with endotoxaemia and septicaemia have been treated mainly symptomatically and supportively. Based on the identified therapeutic targets, this review describes the current knowledge of the treatment for endotoxaemia and septicaemia in the horse with reference to the findings in other animal species and humans.     

Effects of endotoxaemia and carbohydrate overload on glucose and insulin dynamics and the development of laminitis in horses

Reasons for performing study: Insulin resistance (IR) is a risk factor for pasture‐associated laminitis in equids and alimentary carbohydrate overload may trigger laminitis. Whether glucose metabolism responses to carbohydrate overload are more pronounced in insulin‐resistant horses requires further study. Hypothesis: Horses pretreated with endotoxin to alter insulin sensitivity differ significantly in their glucose and insulin responses to carbohydrate overload. Methods: Horses (n = 24) were divided into 3 groups. A lipopolysaccharide (LPS; n = 8) group that received endotoxin as an 8 h 7.5 ng/kg bwt/h i.v. continuous rate infusion, an oligofructose (OF; n = 8) group that received an infusion of saline followed by 5 g/kg bwt OF via nasogastric intubation, and a LPS/OF (n = 8) group that received LPS followed 16 h later by OF. Glucose and insulin dynamics were evaluated at ‐24 h and 48 h using the frequently sampled i.v. glucose tolerance test and minimal model analysis. Physical examinations and haematology were performed and the severity of laminitis assessed. Results: Horses receiving LPS developed leucopenia and both LPS and OF induced clinical signs consistent with systemic inflammation. Insulin sensitivity significantly decreased (P<0.001) over time, but responses did not differ significantly among groups. Time (P<0.001) and treatment × time (P = 0.038) effects were detected for the acute insulin response to glucose, with mean values significantly increasing in LPS and LPS/OF groups, but not the OF group. Five horses in the LPS/OF group developed clinical laminitis compared with 0 and 2 horses in the LPS and OF groups, respectively. Conclusions: Endotoxaemia and carbohydrate overload reduce insulin sensitivity in horses. Endotoxin pretreatment does not affect the alterations in glucose metabolism induced by carbohydrate overload. Potential relevance: Insulin sensitivity decreases after carbohydrate overload in horses, which may be relevant to the development of pasture‐associated laminitis.     

Quantification of Staphylococcus aureus adhesion to equine bone surfaces passivated with Plasmalyte and hyperimmune plasma

OBJECTIVE: To quantify the adhesion of Staphylococcus aureus to 4 equine bone surfaces passivated in a balanced polyionic solution (Plasmalyte) or hyperimmune equine plasma (Polymune plasma). STUDY DESIGN: In vitro comparative study. SAMPLE POPULATION: Third metacarpal bone (MC3) surface explants from 9 equine cadavers. METHODS: Approximately 1 cm(2) sections of periosteum were removed from MC3 and stapled to sterile stainless steel screens. Three bone surface explants were cut using a surgical saw to present 1 cm(2) surfaces of subperiosteal bone, cut cortical bone, or endosteum. Duplicate explants of each surface were immersed for 1 hour in Plasmalyte or hyperimmune equine plasma. Each explant was then placed in a well of a 6-well sterile tissue culture plate with the surface of interest exposed. Each surface was inoculated with approximately 100 colony-forming units of S. aureus in 10 microL of Mueller Hinton broth and incubated for 6 hours at 37 degrees C. After gentle rinsing to remove non-adherent bacteria, samples were sonicated for 5 minutes at 60 kHz to loosen adhered bacteria. The number of adherent bacteria was determined by serial dilutions and incubation of the sonicate. Scanning electron microscopy (SEM) was performed on samples identically treated from an additional horse to confirm bacterial removal by sonication from all surfaces and support quantitative culture results. RESULTS: Less S. aureus adhered to periosteum than to cortical bone, cut cortical bone, and endosteal surfaces, which were all similar. Exposure of all surfaces to hyperimmune plasma reduced S. aureus adherence compared with Plasmalyte exposure; SEM supported these conclusions. CONCLUSION: Less bacteria adhere to periosteum than other bone surfaces. Hyperimmune plasma reduces bacterial adhesion to all bone tissue surfaces. CLINICAL RELEVANCE: Understanding the factors that affect bacterial adhesion to bone will facilitate development of improved intraoperative lavage solutions to reduce the morbidity and mortality associated with postoperative infection.     

Low dose flunixin meglumine: effects on eicosanoid production and clinical signs induced by experimental endotoxaemia in horses

The efficacy of low doses of flunixin meglumine in reducing eicosanoid generation and clinical signs in response to experimentally induced endotoxaemia was investigated. Thromboxane B2 and 6-keto-prostaglandin F1 alpha were measured in serum and plasma by radioimmunoassay. Plasma flunixin concentrations were determined by high performance liquid chromatography and pharmacokinetic parameters derived non-compartmentally. In horses administered flunixin meglumine before endotoxin challenge, a significant suppression in plasma thromboxane B2 and 6-keto-prostaglandin F1 alpha generation was observed. Elevations in blood lactate were significantly suppressed in horses pretreated with 0.25 mg/kg bodyweight flunixin meglumine. Reduction of the clinical signs of endotoxaemia by flunixin meglumine was dose dependent. Low doses of flunixin inhibited eicosanoid production without masking all of the physical manifestations of endotoxaemia necessary for accurate clinical evaluation of the horse's status.     

Antibody responses of horses to equine influenza viruses during a postepizootic period in Japan.

The antibody responses to equine influenza viruses were investigated during a postepizootic period of the disease. Serum samples were collected from a total of 128 horses on three occasions during the years 1967-77. No significant increase of hemagglutination-inhibition antibody titers to subtypes 1 and 2 of equine influenza virus were detected in any of the sera tested. The maternal hemagglutination-inhibition antibody titers of foals decreased over a four month interval. A marked increase of the titers was recognized in only the equine influenza virus vaccinated horses. These findings suggest that equine influenza virus was not prevalent in the horse populations during the observation period. In such conditions, the dissemination of equine influenza viruses in the horses is discussed in relation to introduction of the disease from abroad. We also examined whether the doctrine of original antigenic sin, an immunological phenomenon recognized in human influenza, was applicable for equine influenza. However, no marked increase of hemagglutination-inhibition antibody titer to the primary infecting subtype in the 44 horses was observed after administration of the heterologous subtype vaccine.     

Serologic responses to eastern and western equine encephalomyelitis vaccination in previously vaccinated horses

A prospective study was performed to determine the serologic response of previously vaccinated horses to revaccination against eastern and western equine encephalomyelitis (EEE and WEE). Horses responded variably to each antigen, and some horses had low or undetectable antibodies 6 months after vaccination. Some horses did not develop increasing titers to EEE or WEE despite recent vaccination. Geometric mean titers peaked 2 weeks after revaccination and were significantly increased from before revaccination. Except for one horse, EEE:WEE titer ratios ranged from 0.25 to 2.0. Regular vaccination against EEE and WEE did not interfere with testing for Saint Louis encephalitis.     

Rapid infusion of a phospholipid emulsion attenuates the effects of endotoxaemia in horses

REASONS FOR PERFORMING STUDY: Endotoxaemia currently is associated with a poor prognosis in horses. The results of recent trials in other species indicate that phospholipid emulsions reduce the deleterious effects of endotoxin (LPS). However, in a previous study in horses, a 2 h infusion of emulsion caused an unacceptable degree of haemolysis. HYPOTHESIS: Rapid administration of a lower total dose of emulsion would reduce the effects of LPS and induce less haemolysis; the emulsion would reduce inflammatory effects of LPS in vitro. METHODS: Twelve healthy horses received an i.v. infusion either of saline or a phospholipid emulsion (100 mg/kg), followed immediately by E. coli 055:B5 LPS (30 ng/kg). Clinical parameters, haematological profiles, serum tumour necrosis factor (TNF) activity, serum lipid profiles, urine analyses and severity of haemolysis were monitored before and at selected times after LPS. Monocytes were also incubated in vitro with LPS in the presence or absence of emulsion, after which TNF and tissue factor activities were determined. RESULTS: Clinical signs of endotoxaemia were reduced in horses receiving the emulsion, including clinical score, heart rate, rectal temperature, serum TNF activity, and the characteristic leucopenic response to LPS, when compared to horses not receiving the emulsion. Three horses receiving the emulsion had none, 2 had mild and one had moderate haemolysis. There were no differences in urinalysis results and creatinine concentrations, either within the groups over time or between the groups. Serum concentrations of phosphatidylcholine, bile acids and triglycerides peaked immediately after the infusion; there were no significant changes in concentrations of nonesterified fatty acids or cholesterol. Incubation of equine monocytes with emulsion prevented LPS-induced TNF and tissue factor activities. CONCLUSIONS: Rapid administration of emulsion significantly reduced inflammatory effects of LPS in vivo and caused a clinically insignificant degree of haemolysis. The results of the in vitro studies indicate that emulsion prevents not only LPS-induced synthesis of cytokines, but also expression of membrane-associated mediators (i.e. tissue factor). POTENTIAL RELEVANCE: Rapid i.v. administration of emulsions containing phospholipids that bind endotoxin may provide a clinically useful method of treating endotoxaemia in horses.     

A serological and clinical follow-up in horses with confirmed equine granulocytic ehrlichiosis

For diagnosis of equine granulocytic ehrlichiosis (EGE) serological testing of antibodies to Ehrlichia equi is frequently used. An elevated antibody level is often misinterpreted as confirmative of active infection and results in treatment with antibiotics. If only seropositivity is considered as the diagnostic criterium, many horses showing convalescence titres will be treated. This study was undertaken to obtain information about the kinetics of antibodies during the course of infection and, for this purpose, 45 horses with clinical signs of EGE and confirmed ehrlichiaemia were monitored serologically and clinically over time. For a correct handling of cases with suspected EGE, the following results should be helpful: (i) 44% of the horses in the acute ehrlichiaemic stage were found to be serologically positive to E. equi; (ii) all horses showed a rapid increase in antibody titre, reaching maximum value within a month after the ehrlichiaemic stage; (iii) when 8 months had passed, titres had decreased, but 18 of 24 examined horses were still serologically positive; (iv) after 12-15 months most of the horses (n = 10) were serologically negative; and (v) the period required for complete clinical recovery varied from one day up to 6 months after antibiotic treatment.     

Effect of acute sublethal endotoxaemia on in vitro digital vascular reactivity in horses

Endotoxaemia is a syndrome linked to the development of equine laminitis; however, the relationship between them is uncertain. The aim of this experiment was to evaluate the effect of an experimental acute sublethal endotoxaemia model on in vitro equine palmar digital vascular reactivity. Rings of arteries and veins of each forelimb were obtained from 11 clinically healthy horses submitted to two surgical procedures, 3 weeks apart. Before the second surgery, 0.25 microg/kg of lipopolysaccharide from Escherichia coli O55:B5 in saline, was administered i.v. in 30 min. After 3 h, the vessels were harvested and submitted to in vitro vascular reactivity experiments and histopathology. The response to depolarizing Krebs solution (DKS, 40 mm), phenylephrine (PHE), acetylcholine (ACh) and sodium nitroprusside (SNP) were evaluated. All horses showed colic pain and watery diarrhoea, tachycardia, tachypnea, hyperthermia and leucopenia. Concentration-response curve (CRC) to PHE was shifted to the left in arteries rings from endotoxemic horses without any effect on vein rings. The CRC to ACh was shifted to the right with a reduction in the maximal response. The response to SNP and DKS was similar between groups. There was no evidence of histopathological effects. The increased response to PHE in digital arteries together with a reduction of the endothelium-dependent response to ACh in arteries and veins, confirm the existing reports where endotoxaemia was found to modify the digital vascular reactivity during the acute phase. As the digital endothelial function is impaired, there may be an increased potential to develop a digital prothrombotic state with a reduced vasodilatory capacity.     

Effects of two large doses of equine recombinant growth hormone on clinical, haematological and serum biochemical variables in adult horses

Objective To evaluate the clinical, haematological, and serum biochemical effects of two large doses of recombinant equine growth hormone.
Design Duplicated Latin square.
Sample population
Three Thoroughbred and three Standardbred mares aged between 12 and 17 years.
Procedure Two horses were randomly assigned into one of three groups. On each of three successive days, each horse pair received one of two dosages of growth hormone or a saline placebo so that by the end of the experiment all three horse pairs had received both dosages and the saline placebo. Dose rates selected were 50 μg/kg, and 100 μg/kg. A clinical examination was performed and a venous blood sample drawn for a complete blood count and serum biochemical analysis before administration of growth hormone and at 1, 2, 3, 4, 6, 8 and 24 h after injection. Data were analysed by a repeated measures analysis of variance assessing the effects of dose and time.
Results There was an effect of time on a number of clinical, haematological, and serum biochemical variables. There were significant effects of growth hormone on heart rate and serum glucose concentration but values for both variables remained within the reference range.
Conclusion The results of the present study suggest that equine recombinant growth hormone has a wide margin of safety and show that the single administration of up to five times the recommended dose rate has no significant effects on clinical, haematological, or serum biochemical variables.     

Lyophilized hyperimmune equine serum as a source of antibodies for neonatal foals

In a study with 15 neonatal foals (5 per treatment group), foals were fed within 4 hours of birth as follows: 250 ml of colostrum, 250 ml of lyophilized serum reconstituted at 5 times the original concentration, or 250 ml of a mixture (1:1) of colostrum and lyophilized serum. Foal serum samples were tested for immunoglobulin (Ig)G concentration and titrated for anti-equine rhinovirus 1 and anti-equine influenza A1 and A2 antibodies at 0 and 24 hours after foals were born. Except in a foal which had suckled the dam before treatment, there was no evidence of IgG or specific viral antibodies in the samples taken at birth. There were no significant differences found in the serum IgG concentrations and antibody titers among the 3 treatment groups. Seemingly, IgG was absorbed efficiently from both serum and colostrum, so that the use of reconstituted lyophilized serum as a prophylactic measure of conferring passive immunity to a newborn foal deserves serious consideration.     

Effects of exogenous equine somatotropin on mineral balance in two-year-old horses in race training

This study examined the effects of exogenous equinesomatotropin (eST — Equigen) administration on mineral absorption and retention in two-year-old horses in race training. Sixteen Quarter Horse geldings were paired by age (ave.age=794 d), and one horse from each pair was assigned at random to either the eST treatment group or the control. The experiment was conducted over 112 days during which the horses were gentled to ride and trained on a dirt track in a regimen typical for race horses in training. At 28-day intervals, collections of total fecal and urinary output were made to determine effects on Ca, inorganic P, Mg, Cu and Zn mineral balance.Due to marginal and slightly deficient amounts (P<01)of Ca, P, Mg and Zn intake by days 84 and 112, a result of reduced feed intake to maintain a constant body condition in the presence of a decreased workload, mineral balance for the aforementioned minerals was only examined through day 56. In contrast to the previous minerals, Cu intake appeared adequate. Ca, P, Mg, Cu and Zn intake did not differ (P>.05) by treatment. The eST-treated horses increased (P<.05) Ca retention as a percent of Ca absorbed, had greater (P<.05) overall apparent efficiency of P absorption and greater (P<.05) apparent retention of P on day 56 than the control horses. The eST-treated horses had greater (P<.05) apparent Cu absorption on day 56, greater (P<.05) apparent Cu absorption efficiency on days 56 and 84, and there was a trend (P<.1) for the eST-treated horses to retain more Cu on day 56 than the control horses. Finally, the eST-treated horses retained a greater percent (P<.1) of absorbed Zn on day 56.     

Pharmacokinetics of low-dose methotrexate in horses

This study aimed to investigate both the pharmacokinetic behavior and tolerance of methotrexate (MTX) in horses to design a specific dosing regimen as a new immunomodulatory drug for long-term treatment. To determine the primary plasma pharmacokinetic variables after single intravenous, subcutaneous or oral administration, six horses were administered 0.3 mg/kg MTX in a crossover design study. After a 10-week washout, MTX was administered subcutaneously to three of the six previously treated horses at a dose of 0.3 mg/kg once per week for 3 months. In both studies, MTX and metabolite concentrations were measured using LC-MS/MS. The absolute bioavailability of MTX was 73% following subcutaneous administration but less than 1% following oral administration. The plasma clearance was 1.54 ml min⁻¹ kg⁻¹ (extraction ratio = 2%). After 24 hr, plasma concentrations were below the LOQ. No adverse effects were noted except for a moderate reversible elevation in liver enzymes (GLDH). With regards to the main metabolites of MTX, very low concentrations of 7-hydroxy-MTX were found, whereas polyglutamated forms (mainly short chains) were found in red blood cells. A subcutaneous dose of 0.2 mg kg⁻¹ week⁻¹ may be safe and relevant in horses, although this has yet to be clinically confirmed.     

Effects of multiple intramuscular injections and doses of dexamethasone on plasma cortisol concentrations and adrenal responses to ACTH in horses

Adrenocortical function was assessed in horses given multiple IM doses of dexamethasone to determine the duration of adrenocortical suppression and insufficiency caused by 2 commonly used dosages of dexamethasone (0.044 and 0.088 mg/kg of body weight). Dexamethasone was administered at 5-day intervals for a total of 6 injections. Daily blood samples were collected. The plasma was frozen and later assayed for cortisol. An ACTH response test was determined 2 days before the first injection of dexamethasone and again 8 days after the last dexamethasone injection. Maximum suppression of plasma cortisol was observed in horses given both dosages of dexamethasone (0.044 and 0.088 mg/kg). Plasma cortisol concentrations returned to base-line values in all horses by 4 days after dexamethasone injection. Normal ACTH responses observed after 6 dexamethasone injections given at 5-day intervals indicated that measurable adrenal atrophy did not develop under the conditions of this study.     

Effects of corn processing on the glycaemic and insulinaemic responses in horses

This study was conducted to evaluate the effects of different corn processing techniques on the glycaemic and insulinaemic responses in horses. It was hypothesized that the changes in pre-caecal starch digestibility caused by various types of corn processing would alter the post-prandial glycaemic and/or insulinaemic response. Six horses were fed in random order: untreated, finely ground, steamed, micronized, steam-flaked and popped corn. The total corn intake was adjusted to 630 g starch/horse/day (1.2-1.5 g starch/kg BW/day). During a stabilization period of 10 days, horses also received 6 kg grass hay/horse/day. At blood collection day horses were fed their test diet (exclusively corn), and blood samples were taken at defined times. Corn feeding resulted in a significant increase in mean plasma glucose and insulin concentration, but glucose and insulin peaks as well as areas under the curve (AUC) were not clearly influenced by corn processing. The glycaemic index (in which each test diet's plasma glucose AUC was expressed relative to untreated corn) varied between 91.4 +/- 9.4% (steamed corn) and 108.4 +/- 11.8% (popped corn, treatment n.s.), the insulinaemic index (in which each test diet's plasma insulin AUC was expressed relative to untreated corn) ranged between 98.2 +/- 12.6% (steamed corn) and 121.0 +/- 29.9% (micronized corn, treatment n.s.). However, the well-established improvement in pre-caecal starch digestibility was not reflected by differences in the glucose or insulin responses.