Immunological response to long-term transport stress in mature horses and effects of adaptogenic dietary supplementation as an immunomodulator

REASONS FOR PERFORMING STUDY: Little information exists on the immunological effects of transport or the use of supplements to minimise transport stress. OBJECTIVES: To establish baseline ranges and evaluate immunophenotypic and functional changes associated with transport and a nutritional 'adaptogen' supplement. METHODS: Horses received either supplement (n = 10) or placebos (n = 9) during the 30 day study. After 28 days in stalls, 12 horses (6 supplement; 6 placebo) were transported for 24 h, then unloaded and recovered. Venous blood samples were collected on Days 1, 14 and 28 to establish baselines, and on Days 28, 29 and 30 to examine changes during transport and recovery. RESULTS: Transport prompted elevations (P<0.05) in cortisol concentration, neutrophil count and white blood cell counts, while lymphocyte subpopulation counts (CD3+, CD4+, CD8+, CD21+) decreased (P<0.05). Normal phenotypic lymphocyte profiles returned within 24 h of recovery. Supplement effects on immunophenotype (CD21+ and CD8+) were observed in stabled horses (P<0.05), but not in transported horses. CONCLUSIONS: These results provide insights into the immunological mechanisms associated with long-term transport. POTENTIAL RELEVANCE: The existence of a small window of immunological uncertainty follows long-term transportation, enhancing the potential risk of infectious disease in susceptible individuals.     

Changes in peripheral blood leucocyte counts and subpopulations after experimental infection with BVDV and/or Mannheimia haemolytica

Leucocyte counts and subpopulations were studied in peripheral blood from calves experimentally infected in the respiratory tract with either bovine virus diarrhoea virus (BVDV) or Mannheimia haemolytica (Mh), or with a combination of both agents (BVDV/Mh). A non-inoculated control group was included. Peripheral blood samples were obtained for total leucocyte counts, and for neutrophil, lymphocyte and monocyte counts. The numbers of blood lymphocytes expressing the surface antigens CD4, CD8, WC1, B and IL-2R were analysed using flow cytometry. The results showed that BVDV inoculation induced a significant decrease in total leucocyte counts and in neutrophil and lymphocyte numbers, while Mh inoculation induced significant increases in total leucocyte counts and neutrophils, while the lymphocyte count decreased. In the BVDV/Mh group, the total leucocyte count and the lymphocyte numbers decreased significantly. In this group, the lymphocyte numbers remained on a very low level throughout the rest of the study. The numbers of CD4+, CD8+ and WC1+ lymphocytes decreased significantly compared with before inoculations mainly in the BVDV and BVDV/Mh groups. The drops were most pronounced in the BVDV/Mh group. The numbers of B+ lymphocytes and IL-2R+ cells did not change significantly.     

Evaluation of leukapheresis and thrombocytapheresis in the horse

Continuous-flow centrifugation leukapheresis techniques were used to collect 300-ml volumes of leukocyte-rich plasma from 5 nonmedicated horses and from 5 corticosteroid-stimulated horses. White blood cell counts and differential counts were performed on the horses before (base line) and up to 48 hours after leukapheresis. Systemic administration of hydrocortisone increased numbers of total WBC and neutrophils and improved harvest of these cells. Nonmedicated horses had a mean yield of 3.38 X 10(10) leukocytes in the 300-ml volume. Stimulated horses yielded a mean of 6.88 X 10(10) leukocytes. After leukapheresis, WBC counts decreased a mean of 38% in nonstimulated horses and decreased a mean of 30% in stimulated horses. By 6 hours after leukapheresis, circulating WBC counts of horses in both groups had returned to preleukapheresis values. The relationship between neutrophil yield and the 4 variables (preleukapheresis WBC count, preleukapheresis neutrophil count, preleukapheresis lymphocyte count, and the PCV of the leukocyte-rich plasma) were examined, using simple (pair-wise) correlation and multiple linear regression. A significant positive correlation was found between neutrophil yield and preleukapheresis WBC and neutrophil counts. Because sodium citrate was used in the collection system to prevent extracorporeal blood coagulation, ionized and total serum calcium concentrations were monitored before and after leukapheresis. Although total serum calcium concentrations remained unchanged, ionized calcium concentrations decreased approximately 33% from base-line values during the 2-hour leukapheresis procedures. Occasional mild muscle fasciculations were the only adverse clinical signs of citrate toxicity exhibited by the horses.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of cross-tying horses during 24 h of road transport

Transportation stress has been implicated as a predisposing factor to respiratory disease in horses. Cross-tying horses individually in stalls is common practice for transporting show and racehorses, but horses also travel in small groups or individually without being restricted by tying. The objective of this study was to compare physiological responses of horses travelling cross-tied or loose during 24 h of road transport. Ten horses were used in a cross-over design consisting of two 4 day trials. In the first trial, 6 horses were cross-tied, while 2 pairs of horses were loose in enclosed compartments. Treatments were reversed in the second trial. Baseline samples were collected on Day 1, horses transported on Day 2, and recovery data collected on Days 3 and 4. Blood samples were collected daily at 0800, 1100 and 2000 h. The mean responses in all horses of serum cortisol, lactate, glucose, alpha1-acid glycoprotein, and total protein concentrations, packed cell volume (PCV), white blood cell (WBC) counts and aminotransferase and creatine kinase were was elevated significantly from baseline during the 4 day study. The response of white blood cell counts, neutrophil to lymphocyte ratios and glucose and cortisol concentrations was significantly elevated in the cross-tied compared to the loose group during transport and recovery. This study supports the recommendation of allowing horses during long-term transportation to travel loose in small compartments, without elevating their head by cross-tying.     

The effects of prednisone and azathioprine on circulating immunoglobulin levels and lymphocyte subpopulations in normal dogs.

This study investigates serum immunoglobulin (SIg) levels and lymphocyte subpopulations in normal dogs in response to putative immunosuppressive doses of prednisone and/or azathioprine. The objectives were to quantify SIg levels and lymphocyte subpopulations, including Thy-1+, CD4+, CD8+ and B cells, in normal dogs both before and after the administration of prednisone and/or azathioprine at 2 mg/kg, PO, each. Eighteen beagles were divided into 3 groups of 6 dogs each. Blood samples for radial immunodiffusion assay of IgG, IgM and IgA, complete blood count (CBC)and flow cytometry were collected prior to the administration of any drugs and again after 14 d of azathioprine, prednisone or azathioprine and prednisone. Peripheral blood mononuclear cells were isolated using density centrifugation and were incubated with monoclonal antibodies reacting with CD4+, CD8+, Thy-1+ and membrane immunoglobulin. Lymphocyte subsets were quantified using flow cytometry. Azathioprine-treated dogs had no significant changes in SIg levels or lymphocyte subpopulations. Prednisone-treated dogs had significant (P < 0.05) decreases in all SIg levels, all lymphocyte subpopulations and erythrocyte numbers, and had an increase in neutrophil counts. Prednisone and azathioprine-treated dogs had significant (P < 0.05) decreases in serum IgG levels and Thy-1+ and CD8+ lymphocyte subpopulations, with an increase in the CD4:CD8. These dogs also had a significant decrease in erythrocyte number and a significant increase in the monocyte count. These findings suggest that azathioprine and prednisone in combination or prednisone alone may be useful for the treatment of T cell-mediated diseases since decreased circulating T cell levels were demonstrated following treatment. The combination of drugs or azathioprine alone may not be appropriate for treatment of acute or autoantibody-mediated immune disease, because SIg levels were minimally affected by treatment.     

Myeloid and Megakaryocytic Hypoplasia in Related Standardbreds

Myeloid and megakaryocytic bone marrow hypoplasia in association with moderate to profound neutropenia was observed in 8 young Standardbred horses sired by the same stallion; 7 horses were intermittently thrombocytopenic. Evaluation of serial neutrophil counts in 2 horses suggested that a cyclic variation in neutrophil numbers was present, that lymphocyte numbers increased when neutrophil counts decreased, and that platelet counts decreased when neutrophil counts decreased. Preliminary bone marrow cultures indicated that myeloid progenitor cells were present and that these cells were able to respond to exogenous growth factors by differentiating. A bone marrow microenvironment or growth factor defect is suspected. Seven of 8 horses died or were euthanized. One horse with moderate neutropenia and a normal platelet count has been racing for 3 years. Necropsies in 4 horses did not reveal a cause for the myeloid hypoplasia. A familial basis for the disease is suspected.     

Effects of racing on lymphocyte proliferation in horses

OBJECTIVE: To measure the lymphocyte proliferation response in horses 12 to 16 hours after completion of a race. ANIMALS: 8 Thoroughbreds that competed in 14 races and 3 control Thoroughbreds that did not race. PROCEDURE: Horses participated in races during the late afternoon or evening. Venous blood samples were collected on a morning before a race (1 or 2 days before the race or on the day of the race), on the afternoon of a race (40 to 60 minutes after the race), and on the morning of the day after a race (12 to 16 hours after the race). Lymphocyte proliferation responses and WBC count were measured in samples obtained in the mornings. Plasma cortisol was measured in all samples. RESULTS: Lymphocyte proliferation responses were significantly reduced and WBC counts significantly increased 12 to 16 hours after a race. Plasma cortisol concentrations were significantly increased 40 to 60 minutes after a race. In samples from the control horses, lymphocyte proliferation responses, WBC counts, or plasma cortisol concentrations did not differ significantly among time periods. CONCLUSIONS AND CLINICAL RELEVANCE: A decrease in proliferative responses of circulating lymphocytes can be found as late as 12 to 16 hours after a horse participates in a race. Although the clinical consequences of these exercise-related alterations of the immune response are not yet known, managers of horses should take into account that the immune system of a horse may be affected by racing.     

Effects of enterocentesis on peritoneal fluid constituents in the horse

Peritoneal fluid was collected from 15 clinically normal horses and was analyzed for nucleated cell (NC) counts and specific gravity. Six horses (controls, group 1) were subjected to abdominocentesis only, with a teat cannula, every 24 hours for 5 days. There were no marked changes in the peritoneal fluid of these horses over the 5-day period. Peritoneal fluid was collected from 6 other horses (group 2) with an 8.89-cm 18-gauge needle. The needle was then advanced until intestinal fluid was obtained. Peritoneal fluid was then collected with teat cannulas at 24-hour intervals for an additional 4 days. Peritoneal fluid NC counts from group 2 horses were significantly increased (P less than 0.05) at peak values 2 days after enterocentesis. Specific gravities of peritoneal fluid from group 2 horses were increased on days 1 and 2 after enterocentesis (P greater than 0.05). Peritoneal fluid from 3 other horses (group 3) was collected before enterocentesis (base line) and again at 4-hour intervals after enterocentesis. Peritoneal fluid NC counts of group 3 horses were markedly increased above base-line values 4 hours after enterocentesis and continued to increase for up to 12 hours after enterocentesis when the experiment was terminated. All horses that underwent enterocentesis remained clinically normal except 1 group 3 horse that had a fever (39.6 C) 24 hours after enterocentesis.     

Evaluation of iatrogenic hemarthrosis of the metacarpophalangeal joint as a method of induction of temporary reversible lameness in horses

OBJECTIVE: To determine whether iatrogenic hemarthrosis of the metacarpophalangeal joint could be used as a model for temporary reversible joint pain in horses. ANIMALS: 8 adult horses. PROCEDURE: Each horse was evaluated on a treadmill before and after injection of 1 metacarpophalangeal joint with 10 mL of autogenous blood. Horses were evaluated subjectively and objectively by use of a computerized force measurement system at intervals until lameness abated. The mean force difference between injected and noninjected limbs at all time periods after injection was compared with the difference between limbs at baseline. From each horse, synovial fluid samples collected before and 24 hours and 30 days after injection were analyzed for total protein concentration and cell type and number. Venous blood samples were collected before and 6 and 24 hours after injection for assessment of plasma cortisol concentration. RESULTS: For 24 hours after injection, the mean force difference between injected and noninjected limbs was significantly increased over baseline. The greatest force difference was detected after 2 and 4 hours. Baseline and 24-hour force data were not significantly different. Compared with baseline values, synovial fluid protein concentration and nucleated cell and RBC counts were increased significantly at 24 hours after injection but were not different at 30 days after injection. No significant changes in plasma cortisol concentration were detected at any time point. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, iatrogenic hemarthrosis of the metacarpophalangeal joint appears to induce temporary reversible lameness with a mild to moderate degree of synovitis.     

Effects of exercise stress on various immune functions in horses.

Chemotactic locomotion and luminol-dependent chemiluminescence of neutrophils, mitogen-induced lymphocyte blastogenesis, serum cortisol concentration, immunoglobulin quantification, and leukocyte counts were determined to evaluate the effect of a single strenuous exercise in horses. Increased serum cortisol concentration (P less than 0.01) and an increased neutrophil-to-lymphocyte ratio (P less than 0.05) indicated that horses had been stressed. The chemotactic index and peak chemiluminescence production decreased significantly (P less than 0.05 and P less than 0.01, respectively) 1 day after exercise. Mitogen-induced blastogenesis of lymphocytes and serum immunoglobulin values remained unchanged in response to exercise. Results of this study indicated that a single bout of exercise may transiently impair neutrophil antimicrobial functions and nonspecific defense mechanisms, but not specific immunity in horses.     

The effect of three different doses of sodium pentosan polysulphate on haematological and haemostatic variables in adult horses

OBJECTIVE: To evaluate the effects of three different doses of sodium pentosan polysulphate (PPS) on haematological and haemostatic variables in adult horses. DESIGN: Eight adult standardbred horses were used. All horses received a single injection of 0, 3, 6, and 10 mg/kg of PPS at the beginning of each treatment week for 4 weeks so that by the end of the study all horses had received all four doses of PPS. Blood samples were collected at 0, 1, 2, 3, 4, 6, 8, 12, 24, 48, and 168 h after each weekly injection of PPS. Variables measured were packed cell volume, haemoglobin, red blood cell count, mean corpuscular volume, mean corpuscular haemoglobin, mean corpuscular haemoglobin concentration, platelet count, white cell count, neutrophil count, lymphocyte count, eosinophil count, monocyte count, serum protein, fibrinogen, prothrombin time, and activated partial thromboplastin time (PTT). Data were analysed using an ANOVA. Significance was set at P < 0.05. RESULTS: There was a dose-dependent increase in PTT. A significant increase in PTT occurrred in all treatment groups when compared to horses receiving 0 mg/kg in which there was no change over time. The PTT values all returned to baseline by 48 h after treatment. The mean neutrophil count was higher 3 h after treatment when compared to time 0. Horses receiving 3 mg/kg of PPS had a higher lymphocyte count 4 h after injection, and those receiving 6 and 10 mg/kg had higher counts at 3,4,6 and 8 h after injection when compared to time 0. At 8 h after injection horses receiving 6 and 10 mg PPS had higher lymphocyte counts than horses not receiving PPS. CONCLUSIONS: PPS causes a dose-dependent prolongation of PTT in horses. At the dose rates currently recommended for treatment of joint problems in horses this increase was small and remained elevated from baseline for up to 24 h. Based on these findings doses of PPS up to 3 mg/kg should not be administered to horses within 24 h of high stress activities or where physical injury may occur.     

The effect of experimental infectious mastitis on leukocyte subpopulations and cytokine production in non-lactating ewes.

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichia coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin + lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1 beta (IL-1 beta), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1 beta, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Glucocorticoid receptor down-regulation in neutrophils of periparturient cows

OBJECTIVE: To determine effects of parturition on glucocorticoid receptor (GR) expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts in periparturient dairy cows. ANIMALS: 23 Holstein cows. PROCEDURE: Blood samples were collected from 8 multiparous and 5 primiparous periparturient cows at various times from 28 days before parturition until 14 days after parturition. Glucocorticoid receptor expression in neutrophils, serum cortisol concentration, and total blood leukocyte and neutrophil counts were determined. Results were compared with results from control samples obtained from 5 multiparous and 5 primiparous Holstein cows in midpregnancy. RESULTS: Neutrophils from periparturient cows had 49% reduction in GR expression at calving, compared with GR expression 2 to 4 weeks before calving, and 39% reduction, compared with neutrophils from cows in midpregnancy. Reduction in neutrophil GR expression began 1 week before calving and was most severe at calving and 24 hours after calving; a significant difference in GR expression was detected between primiparous and multiparous cows. Serum cortisol concentrations and total leukocyte and neutrophil counts were significantly increased at calving and returned to baseline values by 24 hours after calving. Significant negative correlations were detected between neutrophil GR expression and serum cortisol concentration, total leukocyte count, and neutrophil count. CONCLUSIONS AND CLINICAL RELEVANCE: Reduced GR expression in blood neutrophils of periparturient dairy cows was associated with increased serum cortisol concentrations, leukocytosis, and neutrophilia. Thus, GR down-regulation in neutrophils may be involved in periparturient neutrophil dysregulation and may cause increased susceptibility to mastitis.     

Effect of parenteral l-alanyl-l-glutamine administration on phagocytic responses of polymorphonuclear neutrophilic leukocytes in dogs undergoing high-dose methylprednisolone sodium succinate treatment

Objective-To determine whether parenteral l-alanyl-l-glutamine (Ala-Gln) administration modulated phagocytic responses of polymorphonuclear neutrophilic leukocytes (PMNs) from dogs undergoing high-dose methylprednisolone sodium succinate (MPSS) treatment. Animals-15 healthy Beagles. Procedures-Dogs were randomly assigned to 3 treatment groups (n = 5/group): 38-hour IV infusion of saline (0.9% NaCl) solution (control group), saline solution with 8.5% amino acids (2.3 g/kg/d), or saline solution with 8.5% amino acids (1.8 g/kg/d) and 20% l-alanyl-l-glutamine (Ala-Gln; 0.5 g/kg/d). High-dose MPSS treatment was initiated at the same time that IV infusions began, such that a total dose of 85 mg of MPSS/kg was administered through multiple IV injections over a 26-hour period. The infusions were maintained until 12 hours after the last MPSS injection. Blood samples collected before MPSS injections began and 2, 12, and 24 hours after injections ceased were used to evaluate PMN function. Results-MPSS injections resulted in an increase in the total number of circulating leukocytes and increases in neutrophil and monocyte counts but did not affect lymphocyte, eosinophil, or basophil counts. Lymphocyte counts in the Ala-Gln group were higher than in the control group 12 hours after MPSS injections finished. Relative to preinfusion values, phagocytic capacity, oxidative burst activity, and filamentous actin polymerization of PMNs were suppressed in all dogs except those that received Ala-Gln. Conclusions and Clinical Relevance-Parenteral Ala-Gln administration in dogs resulted in an increase in PMN phagocytic responses that were suppressed by high-dose MPSS treatment.     

Prolonged suppression of the innate immune system in the horse following an 80 km endurance race

REASONS FOR PERFORMING STUDY: An increased susceptibility to bacterial and viral infections of the respiratory tract, which results in a loss of performance, has been reported in racehorses. Much research has focused on the influence of high-intensity exercise of a short duration on immune system function in horses, but scant attention has been given to prolonged endurance exercise as an immune modulator. OBJECTIVES: The objective of this study was to evaluate the effect of an 80 km endurance race on the monocyte and neutrophil oxidative burst, serum cortisol, glutamine and plasma glucose concentrations in 8 endurance-trained horses (mean +/- s.d. age 9.4 +/- 2.2 years). METHODS: Blood samples were drawn from the horses prior to and following an 80 km ride. RESULTS: Mean time for completion of the 80 km race was 306 +/- 40 mins. Immediately post race mean serum cortisol concentration, blood monocyte and neutrophil counts were higher and blood lymphocyte counts and plasma glucose concentration were lower compared with prerace values (P < 0.05). Neutrophil and monocyte oxidative burst activity decreased following the race and had not regained prerace values after 3 days of rest (P < 0.05). CONCLUSIONS: The present study indicates that long duration exercise in horses has a negative impact on the function of the innate immune system that lasts several days post race. Precise mechanisms instigating the fall in innate immune system function are unclear and multifactorial, but may be attributed, at least in part, to a high serum cortisol response during very prolonged exercise. POTENTIAL CLINICAL RELEVANCE: A prolonged bout of exercise results in a long-term suppression of the innate immune system function in horses which may, in part, account for the observed increase of infectious episodes in horses during training.     

Separation of equine bronchopulmonary lavage cells by density gradient centrifugation and expression of procoagulant activity in unpurified cells and cell subpopulations

Bronchopulmonary lavage was performed in 10 healthy horses and in 39 horses with chronic pulmonary disease. The predominant cell types were macrophages in healthy horses and neutrophils in severely diseased horses. Procoagulant activity (PCA) was detected in all 32 cell-free supernatants examined and in all 49 unpurified cell suspensions. Cells were separated by centrifugation on discontinuous gradients prepared either with Percoll or with Metrizamide. Macrophages were enriched in subpopulations of low density. Neutrophils could not be purified by density gradient centrifugation using either gradient medium. PCAs of cell subpopulations were plotted against their respective macrophage, neutrophil, and lymphocyte content. PCA was positively correlated with macrophage content (P less than 0.001) and negatively correlated with neutrophil (P less than 0.02) and lymphocyte (P less than 0.001) content. Therefore, PCA of equine lung cells most likely originates from macrophages as shown in other species. The density shift of lung neutrophils requires further investigation.     

The Effect of Experimental Infectious Mastitis on Leukocyte Subpopulations and Cytokine Production in Non-lactating Ewes

The interactions between leukocytes and cytokines during the acute response to intramammary infections in the dry mammary gland of sheep were studied. Dry ewes were experimentally infected in one udder half with either Staphylococcus aureus or Escherichta coli, or infused with saline as control. Udder secretion samples, blood samples and udder tissue samples were collected before and 4, 8 and 24 h after infections/infusions. Total and differential leukocyte counts were calculated in both blood and mammary secretions, and flow cytometry was used to detect the presence of CD4+, CD8+, WC1+, IL-2R+, CD18+ or L-selectin+ lymphocytes, CD18+ or L-selectin + neutrophils, and CD14+ leukocytes. Moreover, the concentrations of interleukin-1β+ (IL-1β), interleukin-8 (IL-8) and granulocyte-macrophage colony stimulating factor (GM-CSF) in udder secretions were measured using ELISA, and RT-PCR was used to detect the presence of corresponding cytokine mRNA in udder tissue biopsies. The results suggest an association between the concentrations of IL-1β, IL-8 and the intensity of neutrophil infiltration of the infected gland. Immunologically relevant changes in proportions of lymphocyte subpopulations might also occur in the acute phase of the inflammatory reaction of the udder. Greater cellular and cytokine responses to E. coli infection may have contributed to the milder clinical picture and more rapid resolution of infection than that seen for S. aureus. Enhancing the production of pro-inflammatory cytokines may improve defence against bacterial mastitis.     

Continuous-flow centrifugation hemapheresis in the horse

In a continuous-flow centrifugation apheresis technique adapted for blood-component separation and collection in horses, hydroxyethyl starch was not required for erythrocyte sedimentation. The efficacy and separation characteristics of whole blood from 10 horses were evaluated at various gravitational forces (700 to 1,500 rpm), using a constant withdrawal rate (100 ml/min). Maximum leukocyte collection occurred at 700 rpm (P less than 0.01), and optimal neutrophil collection occurred at 700 to 750 rpm (P less than 0.01). Although neutrophil counts decreased and lymphocyte counts remained constant at higher rpm settings, an optimal rpm range could not be determined for lymphocyte collection. Peak platelet collection occurred at 1,500 rpm. The response of whole blood from 5 horses was evaluated at lower (500 to 700 rpm) and higher (1,400 to 2,200 rpm) centrifuge speeds. A constant whole blood withdrawal rate of 100 ml/min and a leukocyte collection rate of 3 ml/min were maintained. The optimal rpm settings were determined and compared with values obtained from the 10 horses. Significant differences (P less than 0.01) did not exist between maximum numbers of leukocytes collected at low and high speeds. There was a significant (P less than 0.01) decrease in neutrophils and a concomitant increase in the numbers of lymphocytes recovered at the higher rpm. Platelet yields increased as rpm was increased to 2,000.     

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity)

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.     

Age-related quantitative alterations in lymphocyte subsets and immunoglobulin isotypes in healthy horses

OBJECTIVE: To characterize age-associated changes in lymphocyte population subsets and immunoglobulin isotypes. ANIMALS: 30 healthy young light-breed horses (5 to 12 years old) and 30 healthy aged light-breed horses (> 20 years old). PROCEDURE: Lymphocyte subset populations were identified, using monoclonal antibodies to cell surface markers CD5, CD4, CD8, and IgG. Subset populations were quantitated by use of flow cytometric analysis of antibody-stained cells. Serum immunoglobulin concentration was determined using single radial immunodiffusion. RESULTS: Absolute cell counts of total lymphocytes, T cells, CD4+ and CD8+ T cells, and B cells were decreased in aged horses, compared with young horses. There was a significant decrease in the percentage of CD8+ cells and an increase in the CD4+-to-CD8+ cell ratio in the aged population, compared with young horses. However, serum concentration of IgG, IgG(T), IgM, or IgA did not differ with age. CONCLUSIONS AND CLINICAL RELEVANCE: In horses, total lymphocyte count and lymphocyte subset cell counts decrease with age. Age-matched control values are necessary for optimal evaluation of hematologic variables in aged horses. The decrease in lymphocyte subset cell counts in healthy aged horses mimics that seen in other species and may contribute to an age-associated decrease in immunocompetency.