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)-α production; and mRNA expression of TNF-α, interleukin (IL)-1β, 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-α, and cell lysates were assayed to determine PCA. Expression of mRNA for TNF-α, IL-1β, 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-α production in a concentration-dependent manner. Furthermore, E5564 significantly inhibited LPS-induced mRNA expression of TNF-α, IL-1β, 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.
Objective—To determine the prevalence of detectable serum IgG concentrations in calves prior to ingestion of colostrum and to assess whether a detectable IgG concentration was related to dam parity, calf birth weight, calf sex, season of calving, or infectious agents that can be transmitted transplacentally.
Animals—170 Holstein dairy calves.
Procedures—Serum samples were obtained from calves prior to ingestion of colostrum, and serologic testing for bovine viral diarrhea virus (BVDV) and Neospora caninum was performed. Relative risk, attributable risk, population attributable risk, and population attributable fraction for calves with a detectable serum IgG concentration attributable to positive results for N caninum and BVDV serologic testing were calculated. Logistic regression analysis was used to determine whether dam parity, calf sex, season of calving, and calf weight were associated with precolostral IgG concentration.
Results—90 (52.9%) calves had a detectable total serum IgG concentration (IgG ≥ 16 mg/dL). Relative risk, attributable risk, population attributable risk, and population attributable fraction for calves with a detectable serum IgG concentration attributable to positive results for N caninum serologic testing were 1.66, 0.34, 0.014, and 0.03, respectively. Calf sex, calf birth weight, and season of calving were not significant predictors for detection of serum IgG in precolostral samples.
Conclusions and Clinical Relevance—Prevalence of IgG concentrations in precolostral serum samples was higher than reported elsewhere. There was no apparent link between serum antibodies against common infectious agents that can be transmitted transplacentally and detection of measurable serum IgG concentrations.
Objective—To evaluate the effect of diets enriched with eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) on in vivo production of interleukin (IL)-1, IL-6, tumor necrosis factor (TNF)-α, prostaglandin E2 (PGE2), and platelet-activating factor (PAF) in dogs.
Animals—15 young healthy dogs.
Procedures—Dogs were randomly allocated to receive an isocaloric ration supplemented with sunflower oil (n = 5), fish oil (5), or fish oil plus vitamin E (5) for 12 weeks. At week 12, in vivo production of inflammatory mediators was evaluated in serum at multiple time points for 6 hours following stimulation with IV administration of lipopolysaccharide (LPS).
Results—Serum activity or concentration (area under the curve) of IL-1, IL-6, and PGE2 significantly increased after LPS injection in all groups but to a lesser extent in dogs receiving the fish oil diet, compared with results for dogs receiving the sunflower oil diet. Serum activity of TNF-α and PAF concentration also increased significantly after LPS injection in all groups but did not differ significantly among groups.
Conclusions and Clinical Relevance—A fish oil–enriched diet consisting of 1.75 g of EPA/kg of diet and 2.2 g of DHA/kg of diet (dry-matter basis) with an n-6:n-3 fatty acid ratio of 3.4:1 was associated with significant reductions in serum PGE2 concentrations and IL-1 and IL-6 activities. Results supported the use of EPA- and DHA-enriched diets as part of antiinflammatory treatments for dogs with chronic inflammatory diseases. Additional studies in affected dogs are warranted to further evaluate beneficial anti-inflammatory effects of EPA- and DHA-enriched diets.
Objective—To determine the effects of ketamine hydrochloride on hemodynamic and immunologic alterations associated with experimentally induced endotoxemia in dogs.
Animals—9 mixed-breed dogs.
Procedures—In a crossover study, dogs were randomly allocated to receive ketamine (0.5 mg/kg, IV, followed by IV infusion at a rate of 0.12 mg/kg/h for 2.5 hours) or control solution (saline [0.9% NaCl] solution, 0.25 mL, IV, followed by IV infusion at a rate of 0.5 mL/h for 2.5 hours). Onset of infusion was time 0. At 30 minutes, lipopolysaccharide (LPS; 1 μg/kg, IV) was administered. Heart rate (HR), systolic arterial blood pressure (SAP), plasma tumor necrosis factor (TNF)-α activity, and a CBC were evaluated.
Results—Mean SAP was significantly reduced in dogs administered ketamine or saline solution at 2 and 2.5 hours, compared with values at time 0. However, there was no significant difference between treatments. At 1, 2, and 2.5 hours, dogs administered ketamine had a significantly lower HR than dogs administered saline solution. Although plasma TNF-α activity significantly increased, compared with values at time 0 for both groups, ketamine-treated dogs had significantly lower peak plasma TNF-α activity 1.5 hours after LPS administration. All dogs had significant leukopenia and neutropenia after LPS administration, with no differences detected between ketamine and saline solution treatments.
Conclusions and Clinical Relevance—Administration of a subanesthetic dose of ketamine had immunomodulating effects in dogs with experimentally induced endotoxemia (namely, blunting of plasma TNF-α activity). However, it had little effect on hemodynamic stability and no effect on WBC counts.
Objective—To determine the phosphodiesterase (PDE) isoenzymes in equine platelets and evaluate their influence on platelet adhesion.
Sample Population—Platelets obtained from healthy New Forest Pony geldings that ranged from 12 to 20 years of age (mean ± SEM, 17.3 ± 1.1 years).
Procedures—PDE isoenzyme activity in equine platelets was determined by use of a 2-step radioactive assay. Functional importance of PDE isoenzymes was established by use of selective inhibitors in a colorimetric adhesion assay.
Results—PDE1, PDE2, PDE3, and PDE5 and small amounts of PDE4 were found in equine platelets. Inhibition of PDE3 abolished platelet adhesion almost completely, whereas inhibition of PDE4 and PDE5 had little effect.
Conclusions and Clinical Relevance—Function of equine platelets can be influenced by inhibition of PDE3. Selective PDE3 inhibitors may be clinically useful to regulate platelet function. They offer the advantage of increased potency with fewer adverse effects, compared with those for nonselective PDE inhibitors.
Objective—To evaluate the effects of a standardized exercise test to exhaustion in horses on leukocyte function ex vivo.
Animals—6 Thoroughbred geldings.
Procedures—Blood samples were obtained from each horse before exercise; at exhaustion (termed failure); and at 2, 6, 24, 48, and 72 hours after exercise to evaluate hematologic changes, rate of leukocyte apoptosis, and leukocyte production of reactive oxygen species (ROS) ex vivo. To assess leukocyte function, leukocyte ROS production in response to stimulation with lipopolysaccharide, peptidoglycan, zymosan, and phorbol myristate acetate was evaluated. Apoptosis was evaluated via assessment of caspase activity in leukocyte lysates.
Results—In response to lipopolysaccharide, production of ROS by leukocytes was significantly increased at 2 hours and remained increased (albeit not significantly) at 6 hours after exercise, compared with the preexercise value. In the absence of any stimulus, leukocyte ROS production was significantly increased at 6 and 24 hours after exercise. In contrast, ROS production in response to phorbol myristate acetate was significantly decreased at 6, 24, and 72 hours after exercise. Leukocyte ROS production induced by zymosan or peptidoglycan was not altered by exercise. Leukocytosis was evident for 24 hours after exercise, and neutrophilia was detected during the first 6 hours. A significant increase in the rate of leukocyte apoptosis was detected at failure and 72 hours after exercise.
Conclusions and Clinical Relevance—Results indicated that strenuous exercise undertaken by horses causes alterations in innate immune system functions, some of which persist for as long as 72 hours after exercise.
Objective—To determine cell membrane receptors involved in phagocytosis of Mycobacterium avium subsp paratuberculosis (MAP) organisms.
Sample Population—Monocytes were obtained from healthy adult Holstein dairy cows that were test negative for MAP infection on the basis of bacteriologic culture of feces and serologic test results.
Procedures—Monocytes or bovine macrophage cell line (BoMac) cells were incubated with MAP organisms for 30, 60, or 120 minutes with or without inhibitors of integrins, CD14, or mannose receptors. Phagocytosis was evaluated by light microscopy or by flow cytometry. CD11a/CD18, CD11b, and CD14 expression on monocytes and BoMac cells was evaluated by use of flow cytometry.
Results—Monocytes and BoMac cells rapidly phagocytized MAP organisms. However, compared with BoMac cells, monocytes had a greater total capacity to phagocytize MAP organisms. Addition of neutralizing anti-integrin antibodies (anti-CD11a/CD18 and anti-CD11b) substantially inhibited phagocytosis by monocytes during the first 60 minutes of incubation with MAP organisms, but were less effective at 120 minutes of incubation. Anti-CD11a/CD18 and anti-CD11b antibodies were less effective in inhibiting phagocytosis by BoMac cells. Addition of inhibitors of CD14 or mannose receptors also inhibited phagocytosis of MAP by monocytes. Addition of a combination of integrin and mannose inhibitors had an additive effect in reducing phagocytosis, but addition of integrin and CD14 inhibitors did not have an additive effect.
Conclusions and Clinical Relevance—Multiple receptors are involved in phagocytosis of MAP organisms. Although CD11/CD18 receptors appear to be the major receptors used by MAP at early time points, mannose receptors and CD14 also contribute substantially to phagocytosis.
Objective—To assess the effect of maternal cells or cellular components on neonatal immune responses to intracellular pathogens in calves.
Animals—15 Holstein calves.
Procedures—Calves were fed whole colostrum, frozen colostrum, or cell-free colostrum within 4 hours after birth. Leukocytes were obtained from calves before feeding colostrum and 1, 2, 7, 14, 21, and 28 days after ingestion. Proliferative responses against bovine viral diarrhea virus (BVDV) and mycobacterial purified protein derivatives were evaluated. Dams received a vaccine containing inactivated BVDV, but were not vaccinated against mycobacterial antigens.
Results—All calves had essentially no IgG in circulation at birth, but comparable and substantial concentrations by day 1. Calves that received whole colostrum had enhanced responses to BVDV antigen 1 and 2 days after ingestion of colostrum. In contrast, calves that received frozen colostrum or cell-free colostrum did not respond to BVDV. No differences were identified among the 3 groups in response to mycobacterial antigens.
Conclusions and Clinical Relevance—Results indicated that transfer of live maternal cells from colostrum to neonatal calves enhanced responses to antigens against which the dams had previously responded (BVDV), but not to antigens to which the dams were naïve (mycobacterial purified protein derivatives). Results suggested that cell-mediated immune transfer to neonates can be enhanced by maternal vaccination.
Objective—To determine serum concentrations of the selected acute-phase proteins (APPs) haptoglobin, serum amyloid A (SAA), and C-reactive protein (CRP) in pigs experimentally inoculated with classical swine fever (CSF) and African swine fever (ASF) viruses.
Animals—8 crossbred (Large White × Landrace) 10-week-old pigs.
Procedures—Pigs were allocated to 2 groups (4 pigs/group). One group was inoculated with the CSF virus Alfort 187 strain, whereas the other groupwas inoculated with the ASF virus Spain 70 isolate. Blood samples were collected at various time points. At the end of the study, pigs were euthanized and a complete necropsy was performed, including histologic and immunohistochemical analyses.
Results—Serum concentrations of APPs increased in pigs inoculated with CSF and ASF viruses, which suggested an acute-phase response in the course of both diseases. The most noticeable increase in concentration was recorded for SAA in both groups (up to a 300-fold increase for CSF virus and an approx 40-fold increase for ASF virus), followed by CRP and then haptoglobin, which each had only 3- to 4-fold increases.
Conclusions and Clinical Relevance—Serum concentrations of APPs increased significantly in pigs inoculated with CSF and ASF viruses. However, differences were evident in serum concentrations of the proteins evaluated in this study.
Objective—To evaluate the role of the mitogen-activated protein kinase extracellular signal-regulated kinase (MAPKERK) pathway in the interaction between Mycobacterium avium subsp paratuberculosis (MAP) organisms and bovine monocytes.
Sample Population—Monocytes obtained from healthy adult Holstein dairy cows that were not infected with MAP organisms.
Procedures—Monocytes and MAP organisms were incubated together with or without a specific inhibitor of the MAPKERK pathway (PD98059), and the capacity of monocytes to express tumor necrosis factor alpha (TNF)-α and interleukin (IL)-10 and -12, produce nitric oxide, acidify phagosomes, kill MAP organisms, and undergo apoptosis was evaluated.
Results—The MAPKERK pathway was activated within 10 minutes after addition of MAP organisms to monocytes. Addition of PD98059 to monocyte-MAP mixtures decreased monocyte TNF-α and IL-12 mRNA expression but had no effect on IL-10 mRNA expression. Treatment with PD98059 failed to induce significant alterations in phagosome acidification, organism killing, nitric oxide production, or apoptosis of MAP-exposed monocytes.
Conclusions and Clinical Relevance—Results indicated that the MAPKERK pathway was activated during the interaction of MAP organisms with monocytes, which initiated TNF-α and IL-12 mRNA expression but failed to initiate antimicrobial activity. The MAPKERK pathway may be involved in initiating proinflammatory and proimmune responses in MAP infection in cattle.