Oxygen affinity in stored canine blood: the effect of prednisolone.

Erythrocyte 2,3 diphosphoglycerate (DPG) levels and P450 measurements were made on canine blood stored in acid citrate dextrose solution at weekly intervals for four weeks. A negative correlation (-0.904) was found between weekly DPG and P50 values. The mean P50 measurement of 28.2 mm Hg on day 0 was significantly (P less than 0.001) greater than values measured on days 14, 21 and 28. Mean DPG values in the blood at the time of collection of 8.59 micrometers/gHB were only significantly (P less than 0.005) greater compared to values measured on day 28. On day 27, 0.5 mg of prednisolone sodium succinate per ml of blood was added to certain blood samples. This treatment did not result in an increase in P50 and DPG values on day 28.     

Immune responses to commercial equine vaccines against equine herpesvirus-1, equine influenza virus, eastern equine encephalomyelitis, and tetanus

Horses are commonly vaccinated to protect against pathogens which are responsible for diseases which are endemic within the general horse population, such as equine influenza virus (EIV) and equine herpesvirus-1 (EHV-1), and against a variety of diseases which are less common but which lead to greater morbidity and mortality, such as eastern equine encephalomyelitis virus (EEE) and tetanus. This study consisted of two trials which investigated the antigenicity of commercially available vaccines licensed in the USA to protect against EIV, EHV-1 respiratory disease, EHV-1 abortion, EEE and tetanus in horses. Trial I was conducted to compare serological responses to vaccines produced by three manufacturers against EIV, EHV-1 (respiratory disease), EEE, and tetanus given as multivalent preparations or as multiple vaccine courses. Trial II compared vaccines from two manufacturers licensed to protect against EHV-1 abortion, and measured EHV-1-specific interferon-gamma (IFN-gamma) mRNA production in addition to serological evidence of antigenicity. In Trial I significant differences were found between the antigenicity of different commercial vaccines that should be considered in product selection. It was difficult to identify vaccines that generate significant immune responses to respiratory viruses. The most dramatic differences in vaccine performance occurred in the case of the tetanus antigen. In Trial II both vaccines generated significant antibody responses and showed evidence of EHV-1-specific IFN-gamma mRNA responses. Overall there were wide variations in vaccine response, and the vaccines with the best responses were not produced by a single manufacturer. Differences in vaccine performance may have resulted from differences in antigen load and adjuvant formulation.     

Vascular responses in the equine digit.

The digital circulation was isolated in 12 ponies under pentobarbital anesthesia. Blood flow was either controlled by a pump or measured under natural perfusion. The responses to rapid changes and stoppages of blood flow indicated no evidence of autoregulation or reactive hyperemia. Local administration of acetylcholine, histamine, and prostaglandins E1 and E2 decreased prevenous resistance, whereas epinephrine and serotonin caused prevenous constriction. Large doses of epinephrine and serotonin decreased venous caliber. The effects of prostaglandins A1 and F2alpha were variable. The equine digital vasculature responds to changes in flow and to vasoactive agents like the canine forelimb skin vasculature.     

In vitro responses of equine digital vessels to dopamine and fenoldopam.

The in vitro responses of isolated vascular preparations of digital arteries and veins obtained from healthy anaesthetised horses were determined for dopamine and fenoldopam. The digital vessels were harvested, cut into 4 mm vascular segments, suspended in tissue baths and attached to force-displacement transducers. Dose-response studies between 10(-8) and 10(-4)M concentrations were performed for all drugs. The change in tension of each vascular ring was measured in grams of force. The reactivity between palmar and plantar digital vessels and baseline vascular responses were determined for dopamine. The vascular responses of dopamine were compared to in vitro data for other known vasoconstrictor agents. The mechanism of vasoconstriction induced by dopamine was further defined using prazosin, a specific competitive alpha-1 adrenoceptor antagonist. The vasodilating ability of fenoldopam, a dopamine-1 (DA-1) receptor agonist, was also determined using noradrenaline- preconstricted vascular segments from palmar digital vessels. The effective concentration to produce 50 per cent of the maximal response (EC50) and the maximal contraction in grams of force per milligram of the vascular ring (g/mg) were calculated. There were no differences in the reactivity between the palmar and plantar digital vessels. Dopamine produced intense constriction in arteries and veins but only at very high molar concentrations. Prazosin decreased significantly the sensitivity of the veins to dopamine (increased the mean EC50 values) but not the arteries. Prazosin had no effect on the maximal contractions of the vessels. Fenoldopam produced very little relaxation of either the arteries or veins. These results suggest that dopamine produces constriction in equine digital arteries and veins and that the constriction is only partially mediated by alpha-1 adrenoceptors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of the second-generation synthetic lipid A analogue E5564 on responses to endotoxin in [corrected] equine whole blood and monocytes

OBJECTIVE: To evaluate proinflammatory effects of the second-generation synthetic lipid A analogue E5564 on equine whole blood and isolated monocytes and to determine the ability of E5564 to prevent LPS (lipopolysaccharide)-induced procoagulant activity (PCA); tumor necrosis factor (TNF)-alpha production; and mRNA expression of TNF-alpha, interleukin (IL)-1beta, IL-6, and IL-10 by equine monocytes. SAMPLE POPULATION: Venous blood samples obtained from 19 healthy horses. PROCEDURES: Whole blood and monocytes were incubated with Escherichia coli O111:B4 LPS, E5564, or E5564 plus E coli O111:B4 LPS. Whole blood and cell supernatants were assayed for TNF-alpha, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-alpha, IL-1beta, IL-6, and IL-10 by monocytes was determined by use of real-time quantitative PCR assay. RESULTS: Minimal proinflammatory effects were detected in whole blood and monocytes. In addition, E5564 inhibited LPS-induced PCA and TNF-alpha production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-alpha, IL-1beta, and IL-10 and decreased LPS-induced expression of IL-6. CONCLUSIONS AND CLINICAL RELEVANCE: The second-generation synthetic lipid A analogue E5564 lacked agonist activity in equine whole blood and monocytes and was a potent antagonist of enteric LPS. Therefore, E5564 appeared to be the first lipid A analogue that has potential as an effective therapeutic agent in horses with endotoxemia.     

Whole blood re-calcification time in equine colic

Whole blood re-calcification times were evaluated as a measure of endotoxin-associated coagulopathy in horses. First, the effects of endotoxin concentration and duration of in vitro incubation of citrated whole blood with endotoxin on the whole blood re-calcification time of blood collected from healthy horses were determined. Increasing concentrations or incubation times of endotoxin accelerated the whole blood re-calcification time. This effect was attributed mainly to increased monocyte thromboplastin activity. Second, whole blood re-calcification time, a clotting profile, plasma factor VII activity and plasma endotoxin concentration on blood samples obtained from 35 equine colic patients and 10 healthy horses were determined. Compared with healthy horses, colic patients had a longer mean whole blood re-calcification and prothrombin time, lower per cent factor VII activity and higher mean fibrin degradation products concentration. Within the colic patient group, horses that did not survive had detectable endotoxin in plasma, longer whole blood re-calcification and prothrombin times, and lower plasma factor VII activity, compared with colic patients that survived. These data indicate that colic patients with endotoxaemia experience hypercoagulable states, followed by consumptive coagulopathy. Although the cause of endotoxin-associated coagulopathy is likely multi-factorial, increased expression of monocyte thromboplastin activity may be involved in the pathogenesis of coagulopathy. The whole blood recalcification time is a simple, fast and inexpensive way to detect coagulopathy during endotoxaemia and determine the prognosis for survival.     

Streptococcus equi but not Streptococcus zooepidemicus produces potent mitogenic responses from equine peripheral blood mononuclear cells

Streptococcus equi causes equine strangles. The acute disease has many of the hallmarks of an acute response including high fever, elevated plasma fibrinogen and neutrophilia, affects known to be mediated by proinflammatory cytokines. The objective of this study was to screen-culture supernatants from equine clinical isolates of S. equi and S. zooepidemicus for stimulation of mitogenic responses by horse peripheral blood mononuclear cells. Mitogenicity comparable to that of concanavalin A was detected in culture supernatants of S. equi strains but not in those of S. zooepidemicus. Mitogenicity was neutralised by Proteinase K and a post-strangles convalescent serum, and evidence for the presence of both thermo-resistant and thermo-labile mitogenic factors was obtained. Release of proteinaceous immunogenic mitogens in combination with the antiphagocytic protein SeM unique to S. equi may therefore contribute to some of the severe clinical manifestations of acute strangles in the horse.     

Hemodynamic responses of the equine digit to intravenous and digital arterial infusion of dopamine

In 6 adult horses anesthetized with pentobarbital, the hemodynamic responses of the equine digit to infusion of dopamine were evaluated by use of an isolated extra corporeal pump perfused digital preparation. Digital blood flow was maintained at a constant rate that was independent of systemic hemodynamic changes. Three sequential experiments were performed on each horse. In the first experiment (n = 6), dopamine was infused IV at rates of 1.0, 2.5, and 5.0 micrograms/kg/min. For the second experiment (n = 5), dopamine (400 micrograms/ml) was infused into the digital artery at the rates of 0.07, 0.7, and 1.2 ml/min. The third experiment (n = 5) consisted of a 5-minute intra-arterial infusion of phentolamine followed by the intra-arterial infusion of dopamine while continuing the infusion of phentolamine. Digital venous, arterial, and capillary pressures, total digital vascular resistance, and precapillary to postcapillary resistance ratios were determined in each experiment. Systemic infusion of dopamine did not induce changes in the hemodynamics of the digital vasculature. Digital arterial infusion of dopamine alone resulted in a dose-dependent increase in arterial pressure, total digital vascular resistance, and an increase in the precapillary to postcapillary resistance ratio. Phentolamine attenuated the vasoconstrictive response elicited by intra-arterial infusion of dopamine.     

In vitro responses of equine colonic arterial and venous rings to adenosine triphosphate.

OBJECTIVE: To evaluate the in vitro effects of adenosine tryphosphate (ATP) on vasomotor tone of equine colonic vasculature. SAMPLE POPULATION: Arteries and veins from the left ventral colon of 14 mixed-breed horses euthanatized for reasons unrelated to cardiovascular or gastrointestinal tract disease. PROCEDURES: Endothelium-intact and -denuded arterial and venous rings were precontracted with 10(-7) and 1.8 x 10(-8) M endothelin-1, respectively. In 1 trial, endothelium-intact rings were also incubated with 10(-4) M N omega-nitro-L-arginine methyl ester (L-NAME) to inhibit nitric oxide (NO) production. Adenosine triphosphate (10(-8) to 10(-3) M) was added in a noncumulative manner, and relaxation percentage versus time curves were generated. Areas under the curves (ie, percentage of relaxation time) were calculated. RESULTS: Relaxation response of arterial and venous rings to ATP was dose-dependent. Percentage of relaxation time in response to 10(-4) and 10(-3) MATP was significantly greater, compared with that for rings not treated with ATP Removal of endothelium attenuated but did not eliminate the relaxation response. Addition of L-NAME did not attenuate the relaxation response in arteries. At higher concentrations, the vascular response to ATP was biphasic. CONCLUSIONS AND CLINICAL RELEVANCE: ATP applied to equine colonic arterial and venous rings with and without intact endothelium induced a biphasic response characterized by transient contraction followed by slow, substantial, and sustained relaxation. This ATP-induced response is possibly mediated by a mechanism other than NO. Adenosine triphosphate may be a useful treatment to modulate colonic vasomotor tone in horses with strangulating volvulus of the ascending colon.     

Characterization and comparison of the responses of equine digital arteries and veins to endothelin-1

OBJECTIVE: To compare the responses of equine digital arteries (EDAs) and equine digital veins (EDVs) to endothelin-1 (ET-1) and determine the role of the endothelium and type of receptors involved in the modulation and mediation of those responses, respectively. SAMPLE POPULATION: 5 to 9 palmar digital vessels/experiment from 28 healthy horses. PROCEDURE: Rings of dissected vessels were mounted under tension between force transducer wires in organ baths containing Krebs-Henseleit solution at 30 degrees C. Responses of EDAs and EDVs (with intact [+e] or denuded [-e] endothelium) to cumulative concentrations of ET-1 (10(-10) to 3 X 10(-7) M) were compared. For (+e)EDAs and (+e)EDVs precontracted with a thromboxane-mimetic (U44069; 10(-8) M) and (-e)EDAs and (-e)EDVs, responses to an ETB receptor agonist (S6c; 10(-10) to 3 X 10(-7) M) were evaluated. Responses to ET-1 (10(-7) M) in (-e)EDAs and (-e)EDVs were evaluated after incubation with an ETA receptor antagonist (BQ-123; 3 X 10(-7) M), an ETB receptor antagonist (BQ-788; 3 X 10(-7) M), or vehicle solution. RESULTS: Endothelin-1 induced a concentration-dependent contraction of endothelium-intact and -denuded EDAs and EDVs; EDVs were more sensitive. Neither vessel type relaxed in response to S6c, although 2 of the (-e)EDAs contracted mildly. Whereas BQ-123 inhibited the (-e)EDA and (-e)EDV responses to ET-1, BQ-788 had no effect. CONCLUSIONS AND CLINICAL RELEVANCE: Endothelin-1 induced digital vasoconstriction (marked constriction in veins). This action was unaffected by endothelium and mediated predominantly by ETA receptors. These findings suggest ET-1 can induce selective digital venoconstriction.     

Modulation of the cytokine responses in equine macrophages following TACE-inhibition

The detrimental effects of the pro-inflammatory cytokine TNF-alpha during equine acute abdominal disease are well known. Its pivotal role in many human diseases has led to various in-depth studies regarding its release mechanism, in particular by TNF-alpha converting enzyme (TACE). In this study we investigated the inhibitory effect of a TACE-inhibitor on cytokine release (TNF-alpha, IL-1alpha and IL-6) in three different cell models, including U937 cells, a recently established equine macrophage cell line, known as eCAS, and primary equine PBMC. The aim was to show the similarity of TNF-alpha release through the TACE mechanism in human and equine cells after stimulation with LPS. Results indicate that, by using a TACE-inhibitor, TNF-alpha, IL-1alpha and IL-6 release can be reduced in both equine cell models and achieved comparable results in the human U937 cells. These results suggest that equine TNF-alpha, like its human homologue, is released from its membrane-bound position by TACE.     

Modified in vitro method to label equine red blood cells with technetium 99m in concentrated whole blood

An in vitro method to label equine RBC with technetium 99m was modified to achieve quantitative labeling of cells in concentrated whole blood. After a blood sample was incubated with a reducing agent (stannous citrate), an oxidizing reagent (NaOCl) and a chelating agent (EDTA) were added to inactivate residual Sn2+ in the plasma. This step prevented premature reduction of pertechnetate in plasma. Labeling of RBC from 9 healthy horses, using a standard whole blood protocol, resulted in only moderate labeling efficiency (44 to 85%) and indicated a linear relationship between labeling efficiency and PCV. Effects of increased incubation time, increased incubation temperature, prelabeling sedimentation, and double addition of NaOCl/EDTA were investigated in whole blood from 10 healthy horses. Labeling efficiency was improved by each independent factor and by combination of factors. Highest labeling efficiencies (96 to 97%) were achieved when blood samples were sedimented for 20 minutes before being labeled, regardless of incubation time or incubation temperature. Morphologic features of RBC were unaffected by labeling procedures. In vivo whole blood clearance time for labeled cells was determined in 5 healthy horses. Sedimented blood samples were labeled, using a standard 15-minute incubation time at 20 to 22 C. Mean clearance half-time for 5 horses was approximately 20 hours. More than 95% of 99mTc activity was associated with the cells during the 24 hours after reinjection.     

Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.     

Serum antibody responses to equine herpesvirus 1 glycoprotein D in horses, pregnant mares and young foals

The envelope glycoprotein D of equine herpesvirus 1 (EHV-1 gD) has been shown in laboratory animal models to elicit protective immune responses against EHV-1 challenge, and hence is a potential vaccine antigen. Here we report that intramuscular inoculation of EHV-1 gD produced by a recombinant baculovirus and formulated with the adjuvant Iscomatrix elicited virus-neutralizing antibody and gD-specific ELISA antibody in the serum of over 90% of adult mixed breed horses. The virus-neutralizing antibody responses to EHV-1 gD were similar to those observed after inoculation with a commercially available killed EHV-1/4 whole virus vaccine. Intramuscular inoculation of EHV-1 gD DNA encoded in a mammalian expression vector was less effective in inducing antibody responses when administered as the sole immunogen, but inoculation with EHV-1 gD DNA followed by recombinant EHV-1 gD induced increased gD ELISA and virus-neutralizing antibody titres in six out of seven horses. However, these titres were not higher than those induced by either EHV-1 gD or the whole virus vaccine. Isotype analysis revealed elevated gD-specific equine IgGa and IgGb relative to IgGc, IgG(T) and IgA in horses inoculated with EHV-1 gD or with the whole virus vaccine. Following inoculation of pregnant mares with EHV-1 gD, their foals had significantly higher levels of colostrally derived anti-gD antibody than foals out of uninoculated mares. The EHV-1 gD preparation did not induce a significant mean antibody response in neonatal foals following inoculation at 12 h post-partum and at 30 days of age, irrespective of the antibody status of the mare. The ability of EHV-1 gD to evoke comparable neutralizing antibody responses in horses to those of a whole virus vaccine confirms EHV-1 gD as a promising candidate for inclusion in subunit vaccines against EHV-1.     

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.