Peripheral Blood Neutrophil Function and Lymphocyte Subpopulations in Cycling Mares

The purpose of this study was to evaluate different parameters of the immune status in the mare, during the follicular and the luteal phases of the oestrous cycle, in two consecutive years. Functional competence of peripheral blood neutrophils, such as chemotaxis, phagocytosis and oxidative burst was assessed under physiological cyclic conditions (Exp. I). In the second year of this study (Exp. II), besides peripheral blood neutrophil phagocytosis and oxidative burst analysis, circulating lymphocyte subsets were also characterized. The reproductive status in a total of 17 adult cycling mares was evaluated by ultrasonography and further confirmed by plasma progesterone levels. Chemotaxis tests were performed using porous membranes inserted in transwell chambers. Lipopolysaccharide (LPS) from Escherichia coli and N‐formyl‐Met‐Leu‐Phe (fMLP) were used as chemoattractants. Measurement of phagocytosis and oxidative burst in blood neutrophils were assessed by flow cytometry using commercially available kits. Quantification of T‐lymphocyte subsets was assessed by indirect immunofluorescence staining after incubation with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers by flow cytometry. Natural killer cells and B cells were estimated mathematically. No significant difference was found in migration, phagocytosis and oxidative burst at either phase of the oestrous cycle. Statistical analysis of total white blood cell counts also showed no significant difference between either phase of the oestrous cycle, although there was a tendency for blood neutrophils to increase in number under the progesterone influence (p = 0.09). Lymphocytic subpopulations did not differ throughout the oestrous cycle. Overall, our results suggest that luteal and follicular phases in cycling mares may not influence the immune status of the mare.     

Characterization of peripheral blood and pulmonary leukocyte function in healthy foals

Studies in infants and foals indicate an age-dependent maturation of peripheral lymphocyte subsets. The age-dependent relationship for maturation of cellular immune responses, such as phagocytosis and lymphocyte responses of the peripheral and pulmonary-derived leukocytes, has not been characterized in foals. Lymphocyte subpopulations, mitogen stimulation response of lymphocytes, lymphokine-activated killing cell activity, phagocytosis and oxidative burst activity, and serum immunoglobulin (Ig) classes G and M concentrations were determined in developing foals. This study illustrates age-dependent changes in immunoglobulin class concentrations, lymphocyte subsets, and EqMHC Class II expression in cells of the peripheral blood and lungs of developing neonatal-to-weanling foals. The increase in peripheral blood and BAL B-lymphocytes and serum immunoglobulins in developing foals suggests expansion of immune cell populations during a time in which environmental pathogen exposure is great. General immune function, mitogenic responses, LAK cell activity, opsonized phagocytosis, and oxidative burst activity of newborns was similar to the adult horse. Total immune-cell numbers, rather than function, seemed to be the limiting factor in the development of the equine neonatal immune system. There was an age-related percent increase in the appearance of pulmonary lymphocytes, but a percent decrease in macrophages. Although development of the respiratory immune system follows changes in the peripheral blood, cellular expansion, activation, and migration may occur at a slower pace, making the respiratory environment susceptible to pathogens prior to optimal immune system maturity.     

Serum Leptin Concentrations in Lipizzan Fillies

To determine seasonal changes in circulating leptin concentrations, as well as leptin fluctuations in various patterns of seasonal ovarian activity, blood samples were taken in 107 Lipizzan fillies aged 1–4 years over the whole year from January to December. Serum concentrations were measured in samples obtained once a month (leptin) or twice a month (progesterone), and evaluated with respect to the age of animals (yearlings, 2-, 3- and 4-year olds) and the pattern of ovarian activity (anoestrous, seasonal cycle, prolonged cyclicity, continuous cyclicity). The mean and monthly leptin concentrations in 3- and 4-year olds were significantly higher than in yearlings and 2-year olds (p < 0.001). Circulating leptin levels in 3- and 4-year olds increased significantly in summer, to peak in August and then decline significantly (p < 0.001) in autumn. The fluctuations in younger fillies were less evident. The highest mean serum leptin concentrations were observed in fillies exhibiting continuous cyclicity and the lowest in those, exhibiting seasonal cyclicity (p < 0.001). In anoestrous fillies, leptin concentrations were lower than in fillies with prolonged or continuous cyclicity but higher than in fillies with seasonal cyclic activity (p > 0.05).     

Neutrophil function in septic dogs

BACKGROUND: Leukocytes appear to enter a hypo-inflammatory state in human patients with a severe bacterial infection, whereas, in swine, intra-abdominal sepsis produces an initial increase and subsequent decrease in neutrophil Fc receptor mediated phagocytosis. HYPOTHESIS: Impaired neutrophil function (hypo-inflammatory state) develops in dogs with sepsis. ANIMALS: Thirteen adult client-owned dogs that developed clinical signs consistent with sepsis were assessed for evidence of neutrophil dysfunction. These results were compared with those of 12 healthy dogs. METHODS: Flow cytometry combined with the appropriate fluorescent markers allowed for quantification of the phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis of immune complexes, neutrophil phagocytosis of opsonized Escherichia coli, and the intracellular concentration of reduced glutathione as a measure of oxidative stress. RESULTS: The phagolysosomal oxidative burst after Fc receptor-mediated phagocytosis was significantly lower in neutrophils from septic dogs (mean fluorescence intensity +/- standard deviation; 118 +/- 13 and 165 +/- 27 for septic and control dogs, respectively, p = 0.001), although the phagocytosis of opsonized E. coli was significantly increased (155 +/- 74 and 77 +/- 44 for septic and control dogs, respectively, p = 0.004). Intracellular reduced glutathione was not significantly different in neutrophils from septic and healthy control dogs. CONCLUSIONS: An important component of neutrophil function is decreased in septic dogs. The diminished oxidative burst after Fc receptor-mediated phagocytosis in neutrophils from septic dogs could hinder the ability of the innate immune system to clear bacterial infections or it might help protect these patients from the systemic consequences of infection.     

Onset of puberty and duration of seasonal cyclicity in Lipizzan fillies

REASONS FOR PERFORMING STUDY: The onset of puberty and duration of seasonal cyclicity in Lipizzan fillies and young mares have been determined mostly by empirical data, but very little is known about the hormonal changes at puberty and during the seasonal cyclicity in this breed. OBJECTIVES: To determine the onset of puberty and the course of the cyclicity in young Lipizzan mares from age 1-4 years. METHODS: Blood samples were collected twice a month for 2 years from 46 Lipizzan fillies age 1-4 years. Progesterone concentrations, indicating ovarian activity, were measured using an EIA kit. Values above 6.4 nmol/l were taken to indicate luteal phase and those below 3.4 nmol/l, oestrus. RESULTS: Fifty percent of Lipizzan fillies entered puberty during the first spring after their birth at mean +/- s.e. age 15.25 +/- 0.31 months and 50% during the second spring, at age 25.2 +/- 0.57 months. For several fillies of all ages, manifesting anoestrus during the winter months, the oestrus started in March and reached a peak in June to September. The duration of the oestrus activity was age-dependent, at 2.37 +/- 0.62 months in yearlings and 8.56 +/- 0.46 months in fillies age 4 years. In December, >50% of fillies age 2-4 years still exhibited oestrus activity and some entered anoestrus in January the following year. A small proportion of fillies age 3-4 years remained cyclic during the whole year, indicating that factors other than photoperiod can be involved in regulating seasonal cyclicity. CONCLUSIONS AND POTENTIAL RELEVANCE: Our results demonstrate the influence of seasonal factors on the onset of puberty and on the course of oestrus activity in the majority of Lipizzan fillies. However, certain exceptions were also demonstrated but reasons for the prolonged periods of oestrus activity in some Lipizzan fillies were not identified, and the role of the hormone leptin should be studied in future investigations.     

Innate immune traits differ between Meishan and Large White pigs

A panel of innate immune traits were compared between Meishan and Large White pigs. These pigs were of similar age and kept under the same environmental conditions to reduce non-genetically derived variation in immune traits. The animals were all apparently healthy and were not experimentally challenged with any pathogen during the study. The measures only required a small blood sample. Total white cell counts were similar between the pig breeds. However, the numbers of lymphocytes, neutrophils and monocytes differed significantly, with Meishans having higher neutrophil and monocyte counts and lower lymphocyte counts. Flow cytometric methods were used to determine quantitatively the characteristics and function of neutrophils and monocytes. Meishan neutrophils were smaller and less complex than Large White neutrophils, and phagocytosis of Escherichia coli and the ensuing oxidative burst was lower in Meishan neutrophils compared to Large White neutrophils. Monocyte phagocytosis of E. coli was significantly less than that of neutrophils in both breeds but the function of Meishan monocytes as measured by phagocytosis and tumour necrosis factor alpha (TNFalpha) release did not differ from that of Large White monocytes. Levels of acute phase proteins also differed between the breeds with a significantly higher proportion of Meishans having elevated serum amyloid A levels. However, Meishans had lower alpha(1)-acid glycoprotein levels than Large Whites and haptoglobin levels were similar. Such differences in innate immune traits may have implications in the resistance to infection by a broad range of pathogens and subsequent disease effects in these breeds. Further studies are warranted to investigate the genes underlying these traits.     

Effects of midazolam on equine innate immune response: a flow cytometric study

Benzodiazepines (BDZ) are among the most frequently used class of psychotropic drugs employed in veterinary medicine in Brazil and worldwide due to their anxiolytic, muscle relaxant and anticonvulsant effects [J. Clin. Pharmacol. 33 (1993) 124]. Peripheral benzodiazepine receptor (PBR) sites were described in peripheral organs, endocrine steroidogenic tissues and immune organs and cells. Midazolam is a mixed-type agonist of PBRs. The present study is focused on the effects of midazolam on equine peripheral blood neutrophils, peritoneal macrophages and cortisol levels in plasma. Adult horses were treated with a single dose of midazolam (0.06 or 0.1 mg/kg) or with 0.9% NaCl. Immune cells were collected 24 h after treatment for flow cytometry analysis of Staphylococcus aureus-induced phagocytosis and oxidative burst. Plasma cortisol concentration was measured 30, 90, 180 and 360 min after midazolam treatment. Midazolam induced a dose-dependent reduction on: (1) peripheral blood neutrophil and peritoneal macrophage oxidative burst; (2) the capacity of both peripheral blood neutrophils and peritoneal macrophages to phagocyte S. aureus. Increments on plasma cortisol concentration were not found after 0.06 and 0.1 mg/kg of midazolam. The effects on oxidative burst of neutrophils and macrophages from horses treated with midazolam were interpreted as a consequence of an impairment of S. aureus-induced phagocytosis. The present data suggest that midazolam, most probably acting on PBRs present on equine macrophage and neutrophil membranes, might have changed some mechanisms related to both phagocytosis and oxidative burst. These results support the use of flow cytometry to identify functional properties and dysfunction of equine immune cells. They also confirm the notion that changes in the functional capacity of the immune system may represent an important hazard of exposure to drugs or chemicals.     

Modulation of leukocyte populations and immune responses in sheep experimentally infected with Anaplasma (formerly Ehrlichia) phagocytophilum

Anaplasma phagocytophilum infection in sheep is characterized by an immune suppression as indicated by impaired antibody response, reduced lymphocyte response and reduced oxidative burst. The effect of A. phagocytophilum infection on leucocyte populations, especially lymphocytes, was therefore investigated in six sheep experimentally infected with A. phagocytophilum, and compared with leucocyte populations from control animals.To investigate the ability of the infection to interfere with the cellular and humoral responses to specific antigens, the animals were vaccinated with commercial vaccines at the time of experimental infection, and monitored for 56 days.There were reduced percentages of gammadelta T-cells and CD4+ T-cells in peripheral blood of infected animals throughout the study period, and these cell populations showed a down-regulation of CD25 expression; while there was a relative increase in CD8+ T-cells. The reduction in CD25+ gammadelta T-cells involved a subpopulation of WC1+ gammadelta T-cells. During the first 2 weeks of the study there were reduced percentages of B-cells and leukocytes expressing MHC II and CD11b, though this decrease changed to a relative increase later in the study. The relative reductions in leucocyte populations corresponded with the observed leucopenia during the first 3 weeks post-infection, which involved lymphocyte, neutrophil and eosinophil subsets [Vet. Immunol. Immunopathol. 86 (2002) 183]. There was a reduced expression of CD11b and CD14 on granulocytes during the first 2 weeks of the study, which corresponded with the previously reported leucopenia involving neutrophils and eosinophils. Antibody responses to vaccines, lymphocyte in vitro proliferative responses to antigens and mitogens, and in vitro IFN-gamma responses to antigens were reduced up to 4 weeks after infection.     

Primary and anamnestic responses of bovine bronchoalveolar and peripheral blood lymphocyte subsets to aerosolized Pasteurella haemolytica A1

Site-specific responses of bronchoalveolar and peripheral blood lymphocyte subsets were compared during primary and anamnestic immune responses against live Pasteurella haemolytica A1 (Ph1). Eight 1-year old calves were sequentially exposed intrabronchially with aerosolized Ph1 on days 0, 14, and 21, and two calves were sham exposed. Bronchoalveolar and peripheral blood lymphocytes were analyzed before each Ph1 exposure, and on days 3 and 7 post exposure using single and two-color flow cytometry to identify CD2+, CD4+, CD8+, CD21+, CD45R+, CD25+ and gammadelta lymphocyte subsets. Significant differences (p < 0.05) in bronchoalveolar and peripheral blood lymphocyte subsets were observed before Ph1 exposure. Subsequent aerosol exposures, resulted in significant (p < 0.05) changes in bronchoalveolar lymphocyte subsets and the CD4:CD8 bronchoalveolar lymphocyte ratio, but concomitant changes were not observed in peripheral blood lymphocytes. Expression of CD2, CD4 and CD8 lymphocyte differentiation antigens was consistently lower and more heterogeneous on bronchoalveolar lymphocytes. Differential analysis of bronchoalveolar leukocytes revealed a significant increase in bronchoalveolar lymphocytes and neutrophils during anamnestic responses.     

Blood Lymphocyte Subpopulations, Neutrophil Phagocytosis and Proteinogram During Late Pregnancy and Postpartum in Mares

The aim of this study was to evaluate peripheral blood lymphocyte subpopulations, neutrophil phagocytic capacity and proteinogram characteristics in mares, during the last trimester of pregnancy and in postpartum. Measurement of phagocytosis and quantification of T‐lymphocyte subsets were done by flow cytometry. Quantification of T‐lymphocyte subsets was performed with monoclonal antibodies specific for CD2, CD3, CD4 and CD8 cell markers. Natural killer and B‐cell counts were estimated mathematically. Serum proteinogram was obtained by electrophoresis. No significant differences were observed between gestation and postpartum on CD4⁺, CD8⁺ and NK⁺ lymphocyte subsets, CD4 : CD8 ratio and phagocytosis. The percentage of cells expressing CD3 (64.2 ± 1.8) and CD2 (68.4 ± 1.7) (Mean ± SEM) was reduced during gestation vs postpartum (69.7 ± 1.5 and 73.8 ± 1.4 respectively) (p < 0.05). During pregnancy, CD19⁺ (31.6 ± 1.7) was higher than in postpartum (26.2 ± 1.4) (p < 0.05). Total T cells (2911 ± 227 cells/μl), T helper cells (2144 ± 169 cells/μl) and T‐cytotoxic cells (767 ± 68 cells/μl) were depressed in pregnancy, when compared with postpartum (4093 ± 337 cells/μl; 3004 ± 276 cells/μl; 1089 ± 94 cells/μl respectively) (p < 0.01). Total white blood cell count was reduced during pregnancy (8815 ± 427 cells/μl) with respect to postpartum (10742 ± 446 cells/μl) (p < 0.01), while neutrophil count did not change. Total proteins, albumin, α₁,α₂,β₁, β₂, γ globulins and albumin : globulin did not differ. Our results suggest that the physiological immune depression occurring in mares, during gestation might be due to T‐helper and T‐cytotoxic lymphocytes reduction.     

A comparison of foal and adult horse neutrophil function using flow cytometric techniques

Flow cytometric assays were used to compare phagocytic and oxidative burst activity of neutrophils from healthy foals less than 7 days of age with the activity of cells from healthy adult horses. The phagocytosis of Staphylococcus aureus by foal neutrophils was less than that observed for adult neutrophils when autologous serum was used as the source of opsonins in the assay. The use of adult serum did not significantly improve the ability of foal neutrophils to attach bacteria. The oxidative burst activity of foal neutrophils was equivalent to that of adult cells. However, when serum or plasma was incorporated into the oxidative burst assay, foal neutrophils demonstrated greatly reduced autofluorescence and a suppressed response to phorbol myristate acetate (PMA), relative to that demonstrated by adult cells. These results suggest that peripheral blood neutrophils from foals have a reduced ability to phagocytose bacteria relative to that exhibited by adult horse neutrophils and that the oxidative burst activity of foal neutrophils is down-regulated in response to an unidentified serum factor(s). Such changes may contribute to the increased susceptibility of foals to septic disease.     

High versus low body condition in mares: interactions with responses to somatotropin,GnRH analog,and dexamethasone

Mares that had previously been fed to attain body condition scores (BCS) of 7.5 to 8.5 (high) or 3.0 to 3.5 (low) were used to determine the interaction of BCS with the responses to 1) administration of equine somatotropin (eST) daily for 14 d beginning January 20 followed by administration of GnRH analog (GnRHa) daily for 21 d and 2) 4-d treatment with dexamethasone later in the spring when mares in low BCS had begun to ovulate. The majority of mares with high BCS continued to cycle throughout the winter, as evidenced by larger ovaries (P < 0.002), more corpora lutea (P < 0.05), greater progesterone concentrations during eST treatment (P < 0.04), and more (P < 0.05) large- and medium-sized follicles. Treatment with eST alone or in combination with GnRHa had no effect (P > 0.05) on ovarian activity or ovulation. Plasma leptin concentrations were greater (P < 0.002) in mares with high BCS; however, there was no effect (P > 0.10) of eST treatment. Plasma IGF-I concentrations were greater (P < 0.0001) in mares treated with eST compared with mares given vehicle, and mares with high BCS had greater IGF-I (P < 0.02) and LH concentrations (P < 0.02) than mares with low BCS. Plasma leptin concentrations in mares with high BCS were increased (P < 0.001) within 12 h of dexamethasone treatment; the leptin response (P < 0.001) in mares with low BCS was greatly reduced (P < 0.001) and transient. Glucose and insulin concentrations also increased (P < 0.0001) after dexamethasone treatment in both groups, and the magnitude of the response was greater (P < 0.0001) in mares with high BCS than in mares with low BCS. In summary,low BCS in mares was associated with a consistent seasonal anovulatory state that was affected little by eST and GnRHa administration. In contrast, all but one mare with high BCS continued to experience estrous cycles and(or) have abundant follicular activity on their ovaries. The IGF-I response to eST treatment was also reduced in mares with low BCS, as was the basal leptin concentration and leptin response to dexamethasone. Although low BCS and leptin concentrations were associated with inactive ovaries during winter and early spring, mares with low BCS eventually ovulated in April and May while leptin concentrations remained low.     

Effect of plasma transfusion on neutrophil function in healthy and septic foals

OBJECTIVE: To evaluate the effect of plasma transfusion on phagocytosis and oxidative burst activity of peripheral blood neutrophils from healthy and septic equine neonates with sub-optimal passive transfer of maternal immunity. ANIMALS: Nine healthy and seven septic foals with suboptimal passive transfer of maternal immunity (serum IgG < 8 g/L) presented to participating veterinary hospitals for plasma transfusion, and seven healthy foals less than 7 days of age and with circulating IgG concentrations > or = 8 g/L. PROCEDURE: Foals with serum IgG concentrations < 8 g/L were assessed as healthy or septic. Sepsis was recognised by positive bacterial cultures and/or sepsis scores of > or = 11. All foals received between 1 and 3 L of plasma to boost circulating IgG concentrations to > or = 8 g/L. Serum IgG concentrations were determined before and following transfusion by glutaraldehyde coagulation test and confirmed by single radial immunodiffusion assays. Neutrophil phagocytosis and oxidative burst activity were determined before plasma transfusion and at 0 h, 12 h, 24 h, 48 h and 5 d following treatment. Neutrophil function from seven healthy foals less than 7 d of age and with circulating IgG concentrations of > or = 8 g/L was similarly evaluated on a single occasion. RESULTS: Plasma treatment significantly increased circulating IgG concentrations for healthy and septic foals. Oxidative burst activity of neutrophils from septic foals was significantly increased 5 days following treatment, relative to 0 h post treatment. Other differences were not significant but suggested a transient decrease in phagocytosis by neutrophils from healthy foals and increased phagocytosis by neutrophils from septic foals immediately following transfusion. Oxidative burst activity of neutrophils from septic foals tended to be less than that of healthy foals at all sampling times. Serum IgG concentrations were not correlated with neutrophil phagocytosis, but were correlated with oxidative burst activity. CONCLUSIONS: Plasma transfusion did not improve neutrophil function of healthy foals, suggesting that such treatment may be of equivocal benefit for healthy neonates. Conversely, improved neutrophil function was observed following treatment of septic foals, suggesting that plasma transfusion was beneficial for these foals. Oxidative burst activity of neutrophils from septic foals was lower than that of neutrophils from healthy foals and was significantly improved 5 days post treatment, when compared with values obtained immediately following treatment.     

Cellular and humoral defence mechanisms in mares susceptible and resistant to persistent endometritis

Both random and directional migration of blood neutrophils from 9 mares susceptible to persistent endometritis were significantly less (p less than 0.05) than neutrophils from 8 resistant mares. Serum from susceptible mares had significantly more (p less than 0.01) chemotactic activity than serum from resistant mares. Although phagocytosis of yeast blastospores by blood neutrophils from 4 resistant and 3 susceptible mares was similar, uterine neutrophils from susceptible mares were significantly worse (p less than 0.01) at phagocytosis than uterine neutrophils from resistant mares. Uterine washings from 17 susceptible mares were significantly better at opsonising yeast blastospores than washings from 14 resistant mares; however, washings from both groups had a similar ability to promote killing of S. zooepidemicus by neutrophils. When an immunologically non-specific endometritis was induced, washings from 3 susceptible mares were significantly worse at promoting bactericidal activity by 144 h than washings from 4 resistant mares (p less than 0.01). Haemolytic complement activity was significantly greater (p less than 0.001) in washings from 17 susceptible mares than from 14 resistant mares. Induction of acute endometritis resulted in high levels of haemolytic complement activity in 2 of 3 susceptible mares at 24 and 144 h, but only in small increases in 4 resistant mares. Thus, some abnormalities in neutrophil function were detected and a possible defect in promotion of neutrophil bactericidal activity by uterine secretions from susceptible mares but there was no evidence for any deficiency in haemolytic complement activity.     

Effects of bovine leukemia virus infection on milk neutrophil function and the milk lymphocyte profile

The effects of bovine leukemia virus (BLV) on the immune response have been extensively investigated; however, its effects on mammary gland immunity are only speculative. Although BLV has a tropism for B cells, it can affect both adaptive and innate immunities because these systems share many effector mechanisms. This scenario is the basis of this investigation of the effects of BLV on mammary gland immunity, which is largely dependent upon neutrophilic functions. Thus, the present study sought to examine neutrophilic functions and the lymphocyte profile in the milk of naturally BLV-infected cows. The viability of the milk neutrophils and the percentage of milk neutrophils that produced reactive oxygen species (ROS) or phagocytosed Staphylococcus aureus were similar between BLV-infected and BLV-uninfected dairy cows. Furthermore, the expression of CD62L and CD11b by the milk neutrophils and the percentage of milk neutrophils (CH138⁺ cells) that were obtained from the udder quarters of the BLV-infected cows were not altered. Conversely, the median fluorescence intensity (MFI) representing intracellular ROS production and the phagocytosis of S. aureus, the expression of CD44 by the milk neutrophils and the percentage of apoptotic B cells were lower in the milk cells from BLV-infected dairy cows, particularly those from animals with persistent lymphocytosis (PL). The lymphocyte subsets were not different among the groups, with the exception of the percentage of CD5⁻/CD11b⁻ B cells, which was higher in the milk cells from BLV-infected cows, particularly those with PL. Thus, the present study provides novel insight into the implications of BLV infection for mammary gland immunity.     

Immunologic Profiles of Peripheral Blood Leukocytes and Serum Immunoglobulin G Concentrations in Perinatal Mares and Neonatal Foals (Heavy Draft Horse)

The purpose of this study was to examine sequential changes in the immunologic parameters of perinatal mares and neonatal foals of the heavy draft horse. Blood samples were collected from clinically healthy pregnant mares and their newborn foals every week from 1 month before the expected foaling date, and 1 hour, 1 day (24-48 hours), and 1, 2, 3, and 4 weeks after foaling. Peripheral blood samples were used to examine total leukocyte counts (n = 20), differential leukocyte counts (n = 20), lymphocyte subpopulations (n = 13), lymphocyte responses to mitogens (n = 10), neutrophil phagocytic function (n = 12), and serum immunoglobulin G (IgG) concentrations (n = 10). In perinatal mares, remarkable changes observed included increased neutrophils, decreased lymphocytes, decreased CD4⁺ T lymphocytes, and decreased lymphocyte responses to mitogens at delivery. These changes were speculated to be the result of physical stress associated with delivery. In neonatal foals, increase in the phagocytic function of neutrophils, and increase in serum IgG concentration after suckling colostrum and increase of lymphocytes accompanied by physiologic growth were observed. Compared to dams, foals showed lower phagocytic function of neutrophils before suckling and fewer lymphocytes and lower lymphocyte responses to mitogens within 1 day after birth. This study revealed immunologic dynamics in perinatal mares and neonatal foals. Immunologic functions are suppressed in foaling mares and are immature in neonatal foals, especially before colostral intake. We expect these data will be useful for further studies in the field of clinical immunology, and preventive medicine.     

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.     

Non-immune control of trypanosomosis: in vitro oxidative burst of PMA- and trypanosome-stimulated neutrophils of Boran and N'Dama cattle

An in vitro assay that measures the generation of superoxide anions (O2-) was used to assess the level of oxidative burst of phorbol myristate acetate (PMA)- and trypanosome-stimulated neutrophils isolated from healthy Boran and N'Dama cattle, and those infected with Trypanosoma congolense. PMA stimulation of healthy bovine neutrophils resulted in between 300-400 % increase in O2- generation. Neutrophils of Boran cattle exhibited slightly higher but insignificant O2- generation capacity than those of the N'Dama breed. In vitro stimulation by trypanosomes of neutrophils isolated from Trypanosoma congolense-infected cattle caused significant increases in O2- generation, especially on days 14, 28 and 42 post-infection, of both breeds of cattle. No significant differences were observed in O2- generation capacity of the neutrophils of both breeds of infected cattle throughout the period of assay. The results of this study have shown that PMA and trypanosomes do cause an enhanced in vitro oxidative burst, hence trypanosome phagocytosis and killing activity of neutrophils. Neutrophils have been shown to play very significant roles in parasite clearance, hence reduction of trypanosome parasitaemia. The rates of both in vitro generation of O2- and trypanosome phagocytosis over time did not differ significantly between Boran and N'Dama breeds of cattle, even during T congolense infection in this study. Hence, it may be inferred that sustained and higher parasitaemia, more pronounced neutropenia, inadequate bone marrow response and less effective trypanosome-specific immune response, rather than defective neutrophil trypanosome destruction, may be the problem of trypanosusceptible cattle breeds.     

Effects of feeding intensity during the dry period on leukocyte and lymphocyte sub-populations, neutrophil function and health in periparturient dairy cows

The objective of this investigation was to study (1) the numbers of leukocytes, (2) the proportions of lymphocytes expressing CD4, CD8, WC1, B or IL2R and (3) neutrophil phagocytosis and oxidative burst activity in blood around parturition in three groups of dairy cows fed different levels of a total mixed ration during the last eight weeks before calving. All cows were fed ad libitum during the first eight weeks of lactation. Serum concentration of the acute phase protein serum amyloid A (SAA), the milk somatic cell count (SCC) and disease incidence were also recorded. Special emphasis was given to the weeks just before and just after calving as dairy cows are known to be immune suppressed during this period. Dry period diet had only minor effects on leukocyte numbers, and did not influence neutrophil phagocytosis and oxidative burst. In addition, no effect was observed on disease incidence or SAA concentrations. However, an increase in the proportion of B-lymphocytes and a decrease in the proportion of WC1+ T lymphocytes were observed after calving in cows fed high or low energy rations during the dry period, but not in cows fed a medium energy ration. The weeks just before and after parturition were characterised by neutrophilia, eosinopenia, lymphopenia and monocytosis, but time had no effect on neutrophil phagocytosis and oxidative burst. The proportions of CD4+, CD8+, B+ and IL-2R+ lymphocytes increased in early lactation relative to the mid dry period. In addition, the concentration of SAA increased dramatically at calving. The results emphasise the need for more studies to clarify the complex interactions between nutrition and immunity during the peripartum period in dairy cattle.     

Hormonal patterns in normal and hyperleptinemic mares in response to three common feeding-housing regimens

We previously reported that a rise in plasma leptin concentrations followed the rise in insulin and glucose in meal-fed horses, whereas horses maintained on pasture had little fluctuations in hormonal patterns. We have also described a hyperleptinemic-hyperinsulinemic condition that occurs in about 30% of our light horse mares of high body condition maintained on pasture. The present experiment was designed to 1) study the effect of 3 common feeding-housing regimens on leptin and other metabolic hormones in mares and 2) determine whether the hyperleptinemic condition interacted with these regimens. Six light horse mares with high body condition (average score = 7) were assigned to 2 simultaneous 3 x 3 Latin squares, 1 with normal mares (leptin = 0.1 to 6 ng/mL) and 1 with mares displaying hyperleptinemia (>10 ng/mL). Three feeding-housing regimens were compared: ad libitum pasture, ad libitum native grass hay in an outdoor paddock, and single morning feedings of a pelleted concentrate and hay at 0700 in a barn. Five days of acclimation to the feeding regimens were followed by a 36-h period of hourly blood collection to characterize the hormonal characteristics. Leptin concentrations were elevated (P < 0.001) in mares predetermined to be hyperleptinemic compared with normal mares, regardless of the feeding regimen. Leptin was greatest (P < 0.01) in mares on pasture and least in mares fed hay. Variations over time (P < 0.01) were present for all hormones and metabolites studied. Glucose and insulin concentrations were greatest (P < 0.01) in mares on pasture, with meal-fed mares exhibiting an immediate rise in plasma concentrations of both after feeding. Mares on hay had low and constant concentrations of glucose, insulin, and leptin, with no apparent fluctuations. Cortisol, prolactin, and IGF-I did not differ with leptin status, whereas GH differed due to feeding-housing regimen (P < 0.02); there was also an interaction of leptin status and feeding-housing regimen for GH concentrations (P = 0.094). It was concluded that 1) estimates of hormonal secretion in horses based on frequent sampling, depending upon the hormone in question, can be profoundly affected by the feeding-housing regimens, and 2) the hyperleptinemic condition persists under differing conditions of feeding-housing.