Effect of transportation on lower respiratory tract contamination and peripheral blood neutrophil function

Objective To evaluate the effect of transportation on lower respiratory tract contamination and peripheral blood neutrophil function in horses and to compare results from transported horses with those obtained in earlier experiments from horses confined with heads elevated. Design A prospective study. Procedure Six horses were transported by road for 12 h. Clinical and haematological examination, transtracheal aspiration and cell function studies were conducted before and after transportation. Results obtained after transportation were compared to pre-transportation values. Results After transportation, peripheral blood leucocyte and neutrophil numbers were increased and rectal temperatures were elevated. Transtracheal aspirates showed an accumulation of purulent respiratory tract secretions with increased numbers of bacteria, particularly β-haemolytic Streptococcus spp and members of the Pasteurellaceae family. Three horses also had increased numbers of bacteria from the Enterobacteriaceae family relative to corresponding samples from earlier studies. Phagocytosis by peripheral blood neutrophils was significantly reduced, while the oxidative burst activity of peripheral blood leucocytes was either unchanged or enhanced. Clinical Implications Bacterial contamination of the lower respiratory tract occurs as a routine consequence of transportation of horses and is likely to be an important determinant in the development of transport-associated respiratory disease. Inflammatory airway secretions and increased numbers of bacteria were rapidly cleared, without clinical evidence of significant pulmonary disease and without additional treatment, in normal horses that were allowed to lower their heads after transportation. Peripheral blood neutrophilia and a reduction in neutrophil phagocytic function were evident for at least 36 h after transportation, suggesting that horses may require a number of days to recover from the stress of transportation. As the potential role of bacteria from the Enterobacteriaceae family in the development of transport-associated respiratory disease has not been elucidated, horses which develop clinical disease following transportation should undergo thorough bacteriological investigation to ensure appropriate treatment.     

The effect of high intensity exercise on the functional capacity of equine pulmonary alveolar macrophages and BAL-derived lymphocytes

The effect of strenuous exercise on the functional capacity of pulmonary alveolar macrophages (PAM) and bronchoalveolar lavage-derived lymphocytes was determined in eight horses prior to and after 7 weeks of training. Strenuous exercise had no effect on the total cell count or the percentage of live cells in bronchoalveolar lavage (BAL) samples prior to or following training. However, training was associated with a significant increase in the total cell count of pre-exercise BAL samples and a significant reduction in the percentage of live cells in post-exercise samples. Strenuous exercise was associated with impaired phagocytosis by PAM after 7 weeks of training but had no effect on similar samples obtained from untrained horses. The oxidative burst activity of PAM was significantly increased following strenuous exercise for both untrained and trained horses. BAL -derived lymphocyte oxidative burst was similarly affected following training. These results suggest that strenuous exercise and training may influence pulmonary immune cell function.     

Effects of pentoxifylline on equine neutrophil function and flow properties.

Pentoxifylline has been reported to improve peripheral vascular circulation by altering the flow properties of blood. To determine if the hemorrheological effects of pentoxifylline were mediated by alterations in neutrophil function and/or flow properties, we evaluated the drug's effects on equine neutrophils in vitro. Pentoxifylline, at a concentration of 1 x 10(-1) M, but not at concentrations of 1 x 10(-6) M to 1 x 10(-2) M, markedly suppressed neutrophil superoxide production, zymosan phagocytosis and adherence to nylon wool. Pentoxifylline failed to improve neutrophil filterability through 3 mu polycarbonate filters at any concentration tested. We conclude that equine neutrophil function and flow properties are unlikely to be affected by pentoxifylline concentrations achievable in vivo.     

Evaluation of peripheral blood neutrophil function in tumor‐bearing dogs

Background: Peripheral blood neutrophils of untreated human cancer patients have been shown to have normal, increased, and decreased phagocytic activity, killing capacity, and/or oxidative burst activities. Objectives: The objectives of this study were to evaluate oxidative burst and phagocytic activities of peripheral blood neutrophils from tumor‐bearing dogs before therapy and compare them with neutrophil function of healthy control dogs. Methods: Heparinized whole blood was obtained from dogs with high‐grade lymphoma (n=23), sarcoma (n=13), or carcinoma (n=11), and healthy control dogs (n=11) for flow cytometric evaluation of oxidative burst and phagocytic activities. Percentage of bursting cells and amount of oxidative burst activity were determined after stimulation with phorbol 12‐myristate 13‐acetate (PMA) or Escherichia coli. Percentage of phagocytic cells and amount of phagocytic activity were determined after incubation with fluorescent E. coli. Results: Compared with control dogs, dogs with sarcoma (P=.004) and carcinoma (P=.05) had a lower percentage of neutrophils exhibiting oxidative burst activity after stimulation with PMA. Phagocytic activity was significantly lower in dogs with sarcomas compared with control dogs (P<.0001) and dogs with lymphoma (P=.01). Conclusions: Untreated carcinomas and sarcomas in dogs may suppress the percentage of neutrophils capable of oxidative burst when stimulated by PMA. Furthermore, sarcomas also may suppress the amount of phagocytic activity per neutrophil. Until further studies can be performed, the clinical significance of these findings is unknown.     

Effects of training on resting peripheral blood and BAL-derived leucocyte function in horses

In this study, the effects of prolonged, high intensity training on aspects of peripheral blood and bronchoalveolar lavage (BAL)-derived leucocyte function were evaluated in 8 horses. All horses undertook a 7 week endurance training programme, followed by 5 weeks of high intensity training (HIT). Thereafter, horses were divided into control (C) and overtraining (OT) groups. The frequency and intensity of training were increased more substantially for horses in the OT group. Training was terminated in week 32 when horses in the OT group demonstrated a significant performance reduction. Peripheral blood and BAL samples were collected from 4 horses in C and OT groups in training weeks 7, 11, 14, 18, 22, 28 and 32. Flow cytometric techniques were used to assess phagocytosis by peripheral blood neutrophils and pulmonary alveolar macrophages (PAM), and oxidative burst activity of neutrophils, PAM, peripheral blood and BAL-derived lymphocytes. Peripheral blood neutrophil phagocytosis (internalisation) increased during the initial HIT period and decreased from week 16 when the training workload was increased for both groups. The oxidative burst activity of peripheral blood neutrophils and lymphocytes similarly increased and then decreased in response to training. The oxidative burst activity of PAM was reduced towards the end of the overtraining phase of the programme. Pulmonary alveolar macrophage phagocytosis and oxidative burst activity of BAL-derived lymphocytes demonstrated no change throughout the course of the study. There was no difference in results obtained from C or OT group horses, suggesting that protracted HIT, rather than overtraining, was associated with impaired cell function. The detrimental effects observed in peripheral blood neitrophil and PAM function may indicate impaired nonspecific immunity which may adversely affect the health and performance of horses undergoing protracted periods of intense training.     

Differential inhibition of equine neutrophil function by phosphodiesterase inhibitors

Neutrophils are recruited to the lungs of horses with chronic obstructive pulmonary disease (COPD) and exhibit increased activity after antigen challenge, which may contribute to inflammation and lung damage. Inhibition of phosphodiesterase isoenzymes (PDEs) has been shown to attenuate human neutrophil functions including superoxide production, leukotriene (LT)B4 biosynthesis, enzyme and chemokine release. As equine neutrophils contain predominantly the isoenzyme, PDE4, the present study was undertaken to investigate the effects of rolipram, a PDE4 inhibitor, on equine neutrophil function. For comparison, the effects of the nonselective PDE inhibitor, theophylline, were examined. Cells from both normal horses and COPD horses in remission were used. Superoxide production was significantly inhibited by both rolipram [32.2 +/- 2.6 vs. 10.1 +/- 1.1 nmol/10(6) cells and 49.8 +/- 6.8 vs. 22.7 +/- 2.2 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M human recombinant (hr) C5a] and theophylline (19.0 +/- 0.6 vs. 10.2 +/- 0.6 nmol/10(6) cells and 24.3 +/- 2.1 vs. 10.7 +/- 0.9 nmol/10(6) cells for normal and COPD susceptible horses, respectively, in response to 10(-7) M C5a). However, superoxide production induced by serum treated zymosan was inhibited only by theophylline (10(-3) M). Neither hrC5a- nor platelet activating factor (PAF)-induced neutrophil adherence to fibronectin coated plastic was reduced by rolipram (10(-5) M). These results demonstrate that the effects of PDE inhibitors on equine neutrophils are both stimulus and function dependent. The PDE4 inhibitors may reduce neutrophil activation in vivo in horses with COPD.     

Effect of chronic clenbuterol administration and exercise training on immune function in horses

Effects of longitudinal exercise training and acute intensive exercise (simulated race test) on immune function have not been reported in horses. Clenbuterol, a beta2-adrenergic agonist, is used to manage inflammatory airway disease in horses. This study investigated the interaction of 8 wk of exercise training with or without 12 wk of clenbuterol administration in horses. Twenty-three untrained standardbred mares (10 +/- 3 yr, Mean +/- SE) were used and divided into four experimental groups. Horses given clenbuterol plus exercise (CLENEX; n = 6) and clenbuterol alone (CLEN; n = 6) received 2.4 microg/kg BW of clenbuterol twice daily (in an average volume of 20 mL) on a schedule of 5 d on and 2 d off for 12 wk. The CLENEX group was also aerobically trained 3 d/wk. Mares given exercise alone (EX; n = 5) were aerobically trained for 3 d/wk, and the control group (CON; n = 6) remained sedentary. Both EX and CON horses were administered similar volumes (approximately 20 mL) of molasses twice daily. A simulated race test (SRT) resulted in an elevation in lymphocyte number postexercise (P < 0.05). There was no significant difference after acute exercise in either monocyte or granulocyte number. Acute exercise resulted in a decrease (P < 0.05) in the percentage of CD4+ and an increase (P < 0.05) in the percentage of CD8+ cells. The SRT resulted in a decreased lymphoproliferative response to pokeweed mitogen (P < 0.05). A SRT had no effect on antibody production in response to equine influenza vaccine. The EX group demonstrated greater cortisol concentrations at rest and at all other time points postexercise after completing the training regimen compared with CLENEX horses (P < 0.05). Preexercise (SRT) peripheral blood monocyte number was lower in CLENEX horses than in other treatment groups (P < 0.05). Clenbuterol and exercise training did not significantly affect post-SRT changes in leukocyte numbers. Exercise training resulted in a decrease (P < 0.05) in the percentage of CD8+ cells post-SRT compared with other groups, but the percentage of CD4+ cells was not altered by either clenbuterol or exercise conditioning. Lymphocyte proliferative response was not affected by clenbuterol or exercise treatment. Horses demonstrated responses to bouts of acute exercise as noted with other species, namely humans and rodents.     

The effect of exercise and conditioning on equine red blood cell characteristics

The effect of exercise and conditioning on 2,3-diphosphoglycerate levels was studied in nine mature horses. During a 12 minute exercise bout producing heart rates of 165 bpm, 2,3-DPG was significantly increased (p<.05). In addition, exercising levels of 2,3-DPG were increased (p<.05) approximately 8% after a six-week submaximal conditioning program. These increases could not be entirely attributed to changes in erythrocyte number. Mean corpuscular volume was also increased during exercise (p<.05) but was not altered by conditioning.     

Effects of training and strenuous exercise on hematologic values and peripheral blood leukocyte subsets in racing sled dogs

OBJECTIVE: To determine the effects of training and sustained submaximal exercise on hematologic values in racing sled dogs. DESIGN: Cohort study. ANIMALS: 39 Alaskan sled dogs bred for endurance racing. Procedures-Blood samples were collected prior to initiation of a 7-month training regimen (n=39), after completion of the training regimen (19), and after completion of an 1,100-mile race (9), and a CBC, differential cell count, and flow cytometry for leukocyte surface antigens were performed. RESULTS: Both training and exercise caused significant decreases in PCV and hemoglobin concentration and significant increases in total WBC count. In contrast, training and exercise were not found to have significant effects on absolute numbers or fractions of CD4+ or CD8+ lymphocytes, other than a significant increase in the fraction of CD8+ lymphocytes associated with training. CONCLUSIONS AND CLINICAL RELEVANCE: Results suggested that training and exercise induced changes in several hematologic values in racing sled dogs. Extracellular fluid volume expansion was the likely explanation for the training-induced decrease in PCV, and acute blood loss secondary to gastrointestinal tract bleeding was likely responsible for the decrease in PCV associated with acute exercise.     

Effects of recombinant granulocyte-macrophage colony-stimulating factor on bovine peripheral blood and mammary gland neutrophil function in vitro.

Modulation of peripheral blood and mammary gland neutrophil function following in vitro exposure to recombinant bovine granulocyte-macrophage colony-stimulating factor (rBoGM-CSF) was studied. Bovine blood and mammary gland neutrophils were cultured for 9 h in media containing 0.005, 0.05 or 0.5 microgram/mL rBoGM-CSF. Neutrophils treated with rBoGM-CSF exhibited significantly more chemotactic and bactericidal activities and tended to produce more superoxide anion than control cells. The effects of rBoGM-CSF on bovine neutrophil populations appeared to be dose-dependent. The production of superoxide anion and the bactericidal activity of mammary gland neutrophils were consistently higher than blood neutrophils. Only moderate increases in lipopolysaccharide-induced mammary gland neutrophil functions were observed following incubation with rBoGM-CSF which suggests that there may be a threshold of immunomodulation for these prestimulated cells. It may be possible to augment the functional capacity of bovine neutrophil populations in vivo through the therapeutic application of rBoGM-CSF and consequently enhance resistance of dairy cattle to bacterial infections.     

Culture and characterisation of equine peripheral blood mesenchymal stromal cells

Although the use of mesenchymal stromal cells (MSCs) for the treatment of orthopaedic injuries in horses has been reported, no official guidelines exist that classify a particular cell as an equine MSC. Given the limited characterisation of peripheral blood (PB)-derived equine MSCs in particular, this study aimed to provide more detailed information in relation to this cell type. Mesenchymal stromal cells were isolated from equine PB samples and colony forming unit (CFU) assays as well as population doubling times (PDTs) (from P₀ to P₁₀) were performed.Two types of colonies, ‘fingerprint’ and dispersed, could be observed based on macroscopic and microscopic features. Moreover, after an initial lag phase (as indicated by a negative PDT at P₀ to P₁) the MSCs divided rapidly as indicated by a positive PDT at all further passages. Immunophenotyping was carried out with trypsin- as well as with accutase-detached MSC to evaluate potential trypsin-sensitive epitope destruction on particular antigens. Isolated MSC were positive for CD29, CD44, CD90 and CD105, and negative for CD45, CD79α, MHC II and a monocyte/macrophage marker, irrespective of the cell detaching agent used. Trilineage differentiation of the MSCs towards osteoblasts, chondroblasts and adipocytes was confirmed using a range of histochemical stains.     

Effect of exercise on the partitioning of equine respiratory resistance

Pleural, tracheal, pharyngeal and mask pressures as well as airflow and tidal volume of five ponies on a treadmill (incline 8.3 degrees) were recorded simultaneously while resting, walking, trotting slowly, trotting fast, after standing for 30 secs and 5 mins after the end of the exercise. The curves obtained were used to calculate total pulmonary resistance (RL), lower airway resistance (RLA) and upper airway resistance (RUA). The latter was also divided into nasal resistance (Rnas) and laryngeal plus extrathoracic tracheal resistance (Retr + lar). Furthermore, the inspiratory and expiratory components of each of these R values were estimated. Levels of RL, RLA and RUA were increased significantly during exercise but, during the recovery period, the values were significantly lower than those pre-exercise. RUA represented 82 per cent of RL at rest and this percentage did not change significantly during and after exercise. The nasal resistance to RUA ratio was always higher than 0.5. The fact that RL increased with exercise intensity was due to the increase of RUA during inspiration, and mainly a result of the increase of RLA during expiration. At fast trot, RLA represented 5 and 50 per cent of RL during inspiration and expiration respectively. It was concluded that heavy exercise induces in ponies an increase of RL, one reason for which could be the partial collapse of the extrathoracic and intrathoracic airways during inspiration and expiration respectively.     

Effect of high intensity training on anaerobic capacity of middle gluteal muscle in Thoroughbred horses

We hypothesize that high intensity training for Thoroughbred horses that have been subjected to conventional training could further improve the metabolic properties of the middle gluteal muscle. Nine well-trained horses were subjected to high intensity (80-100% Vdot;O(2)max, 5 minx2) training for 12 weeks. Biopsy samples were obtained from the muscle before and after 4 and 12 weeks of training. Three of the 9 horses did not complete the training programme. In the remaining 6 horses, activities of succinic dehydrogenase (SDH), phosphofructokinase (PFK) and 3-hydroxy acyl CoA dehydrogenase (HAD), and the composition of myosin heavy chain isoforms were analyzed by biochemical techniques. After 12 weeks of training, a significant increase was found in PFK activity but not in the SDH and HAD activities. There were no significant changes in the composition of myosin heavy chain isoforms. The high intensity training in this study was effective at increasing glycolytic enzyme activity, indicating the possibility to improve anaerobic capacity, which potentially could contribute greatly to performance in Thoroughbred horses. This study also highlighted a fact that high intensity training should be given with the great care to prevent the skeletal muscle injuries.     

Effect of Cysticercus cellulosae on neutrophil function and death

Neutrophils, eosinophils and macrophages interact with invading parasites and naive hosts. The initial reaction of leukocytes is the generation of reactive oxygen species (ROS). The cytotoxic effects of extracts derived from intact Cysticercus cellulosae and from the scolex or membrane fractions on neutrophils were examined. DNA fragmentation of neutrophils was observed when cells were incubated with an extract from the intact metacestode; however, the addition of antioxidant enzymes to the incubation medium had a protective effect. The scolex and membrane extracts did not affect DNA fragmentation of neutrophils. Hydrogen peroxide production of neutrophils incubated with metacestode fractions from C. cellulosae increased by 190% (total extract), 120% (scolex) or 44% (membrane). An increase in antioxidant catalase activity (28%) concomitant with the increased production of ROS was observed in neutrophils incubated with metacestode fractions, which could be an attempt at self-protection. ROS production by neutrophils in the presence of the intact cysticerci extract did not alter phagocytosis. In contrast, the scolex and membrane fractions increased the phagocytic capacity of neutrophils by 44 and 28%, respectively. The results showed that the extract from intact C. cellulosae was toxic for neutrophils via ROS production, leading to DNA fragmentation and inhibition of phagocytic capacity, but neutrophils are able to protect themselves against oxidative stress by via catalase activity.     

The effect of strenuous exercise on mRNA concentrations of interleukin-12, interferon-gamma and interleukin-4 in equine pulmonary and peripheral blood mononuclear cells

The effect of strenuous exercise on the mRNA concentrations of interleukin-12p35 subunit (IL-12p35), interferon-gamma (IFN-gamma) and interleukin-4 (IL-4) in equine pulmonary and peripheral blood mononuclear cells (PBMCs) was investigated. We hypothesized that strenuous exercise would suppress the expression of IL-12p35, IFN-gamma and augment the expression of IL-4. Eleven horses were randomly divided into two groups, a stall-confined control group (n=5) and an exercise-conditioned treatment group (n=6). Bronchoalveolar and PBMCs were obtained from horses in the treatment group prior to the commencement of a 9-week conditioning program and 24h after the completion of a maximum exercise test conducted in week 12. Samples were obtained simultaneously from control horses. Differential counts were performed on the bronchoalveolar lavage cells. Real-time PCR was performed on the pulmonary and PBMCs to quantitate cytokine expression using equine-specific primers and Taqman probes. Target gene expression was normalized to 18s rRNA expression. With the exception of IL-4 in the BALF cells, mRNA for the three cytokines was detected in the mononuclear cells from all horses at both sampling times. There were no significant differences in the cytokine mRNA concentrations between the two groups of horses at either of the sampling times. These findings demonstrate that strenuous treadmill exercise does not exert a deleterious effect on gene expression for IL-12p35, IFN-gamma or IL-4 when assessed in horses 24h following the intense physical activity.