The acute phase response in calves experimentally infected with bovine viral diarrhoea virus and/or Mannheimia haemolytica.

The aim of this investigation was to study differences and similarities in the acute phase response of calves experimentally infected in the respiratory tract with either bovine viral diarrhoea virus (BVDV) or Mannheima haemolytica (Mh), or with a combination of both (BVDV/Mh). A non-inoculated control group was also included. The acute phase response was measured by serum or plasma concentrations of the acute phase proteins (APPs) haptoglobin, serum amyloid A (SAA) and fibrinogen, and of cortisol, prostaglandin F2alpha-metabolite and interferon-alpha (IFN-alpha) activity. Clinical symptoms were also recorded and were most severe in the BVDV/Mh group. The symptoms were mild to moderate in the BVDV group, while none, or very mild symptoms were observed in the Mh group. In all inoculated groups, a significant acute phase response was observed, with elevated values of haptoglobin, SAA and fibrinogen, while the control group remained unaffected throughout the study. In general, the magnitude of the response was similar, but the duration of elevated concentrations of APPs was significantly longer in the BVDV/Mh group than in the BVDV group, reflecting the duration of the clinical symptoms. However, in the single infection groups, the APP response and the clinical symptoms were not correlated. The IFN-alpha activity increased in all BVDV-inoculated animals, but no response in cortisol and PGF2alpha-metabolite concentrations was observed after infection. Basal levels of serum concentrations of haptoglobin, SAA and fibrinogen were established and may be used for evaluating calf health in herds. The duration of elevated haptoglobin, SAA and fibrinogen values did not differ significantly within groups indicating that their value as indicator of disease is equal.

Effect of subcutaneous injection of ginseng on cows with subclinical Staphylococcus aureus mastitis.

Cows with subclinical mastitis caused by Staphylococcus aureus were subjected to subcutaneous injections with either an extract from the root of Panax ginseng CA Meyer at a dose of 8 mg/kg body weight per day for 6 days, or with saline as a control. The injection areas were checked for adverse reactions. The daily milk production was measured before and after treatment. Blood was collected for total and differential leucocyte counts, identification of lymphocyte subpopulations using flow cytometry, lymphocyte proliferation test, and neutrophil phagocytosis and oxidative burst assay. Quarter milk samples were collected for bacteriological analysis and somatic cell counts (SCC). After the end of treatment, the numbers of S. aureus-infected quarters and milk SCC tended to decrease in ginseng-treated cows. Phagocytosis and oxidative burst activity of blood neutrophils were significantly increased 1 week after ginseng treatment, but the proliferative response of blood lymphocytes did not change significantly. The number of monocytes in ginseng-injected cows was significantly higher 1 week post-treatment than pre-treatment, and the number of lymphocytes was significantly higher than pre-infusion at 2 and 3 weeks after ginseng treatment. Similar changes were not observed in the control group. The present findings indicate that ginseng treatment can activate the innate immunity of cows and may contribute to the cow's recovery from mastitis. It is therefore suggested that ginseng has a potential as a stimulator of the immune system of dairy cows.

Changes in some blood micronutrients, leukocytes and neutrophil expression of adhesion molecules in periparturient dairy cows.

Dairy cows are highly susceptible to infectious diseases, like mastitis, during the period around calving. Although factors contributing to increased susceptibility to infection have not been fully elucidated, impaired neutrophil recruitment to the site of infection and changes in the concentrations of some micronutrients related with the function of the immune defence has been implicated. Most of the current information is based on studies outside the Nordic countries where the conditions for dairy cows are different. Therefore, the aim of the study was to evaluate changes in blood concentrations of the vitamins A and E, the minerals calcium (Ca), phosphorous (P), and magnesium (Mg), the electrolytes potassium (K) and sodium (Na) and the trace elements selenium (Se), copper (Cu) and zinc (Zn), as well as changes in total and differential white blood cell counts (WBC) and expression of the adhesion molecules CD62L and CD18 on blood neutrophils in Swedish dairy cows during the period around calving. Blood samples were taken from 10 cows one month before expected calving, at calving and one month after calving. The results were mainly in line with reports from other countries. The concentrations of vitamins A and E, and of Zn, Ca and P decreased significantly at calving, while Se, Cu, and Na increased. Leukocytosis was detected at calving, mainly explained by neutrophilia, but also by monocytosis. The numbers of lymphocytes tended to decrease at the same time. The mean fluorescent intensity (MFI) of CD62L and CD18 molecules on blood neutrophils remained constant over time. The proportion of CD62L+ neutrophils decreased significantly at calving. The animals were fed according to, or above, their requirements. Therefore, changes in blood levels of vitamins, minerals and trace elements were mainly in response to colostrum formation, changes in dry matter intake, and ruminal metabolism around calving. Decreased levels of vitamins A and E, and of Zn at calving might have negative implications for the functions of the immune defence. The lower proportion of CD62L+ neutrophils at calving may result in less migration of blood neutrophils into the tissues, and might contribute to the increased susceptibility to infections at this time.

Mammary gland immunology around parturition. Influence of stress, nutrition and genetics.

Adequate immune functions are essential for the defence against udder infections. Detailed knowledge about the immune response and important defence factors is essential in order to find new ways for the prevention and treatment of udder infections leading to mastitis. Work should be concentrated on ways of minimising the negative influence on immune functions and/or ways of stimulating these functions, especially during periods of immune suppression. A depression of important immune functions has been reported around parturition and there is a higher prevalence of clinical mastitis and other diseases during this period. Immunosuppression is often associated with high levels of glucocorticoids in blood, a common finding around parturition and during stressful conditions. A number of stressors are present around calving, e.g. parturition, onset of lactation and changes in feeding and management regimes. Adequate management including feeding strategies and routines are important for the immune functions. Metabolic stress as well as deficiencies in vitamins and minerals around parturition and during the first month of lactation can have a negative influence on the immune functions and thereby increase the risks for udder infections and mastitis. There seem to be a genetic variability in certain immune functions among periparturient cows. This might indicate a possibility to find markers for genetic selection of individuals with a well-developed immune system without negative effects on milk productivity.

Studies on the modulation of leucocyte subpopulations and immunoglobulins following intramammary infusion of beta 1,3-glucan into the bovine udder during the dry period.

Inflammatory and immunological reactions after intramammary infusion of beta 1,3-glucan were studied during the steady dry period and involution phase of the bovine udder. The effects of a single intramammary infusion of two different doses (100 and 200 mg) of beta 1,3-glucan were evaluated during the steady dry period. In a second study, the effects of beta 1,3-glucan at drying off were studied by using two treatment regimens; a single infusion at drying off, compared with two infusions of the compound, at drying off and again 2 weeks later. Total and differential leucocyte counts were measured in both blood and udder secretions. Additionally, the expression of receptors for CD14 and MHC class II on leucocytes, and the expression of receptors for CD4, CD8, WC1, IL2R and B-cells on lymphocytes was measured in mammary secretions by flow cytometric analyses. The concentrations of immunoglobulins in udder secretions were measured by radial immunodiffusion. The results showed that a single intramammary infusion of beta 1,3-glucan during the steady dry period causes transient enhancement of some aspects of the inflammatory and immune responses. The increases in somatic cell counts, numbers of monocytes/macrophages, and in proportions of CD14+ and MHC class II+ leucocytes in udder secretions were dose-dependent. Infusion of beta 1,3-glucan also slightly increased the proportion of CD4+ lymphocytes and the concentrations of IgG1 and IgG2 in dry secretions. Infusion of beta 1,3-glucan at drying off seemed to accelerate the involution process through an increase in somatic cells, particularly in the numbers of macrophages, in mammary secretions. The numbers of lymphocytes and polymorphonuclear leucocytes, the proportions of IL2R+ lymphocytes, the proportions of CD14+ or MHC class II+ leucocytes and the concentrations of IgG1 and IgG2 also increased in comparison with untreated controls. Moreover, a second infusion of beta 1,3-glucan tended to prolong this response, indicating that this might be an effective means of enhancing the mammary defence against udder infections closer to calving. In conclusion, the results indicate the intramammary infusion of beta 1,3-glucan could be used to enhance the defence mechanisms of the bovine udder against infections, especially during early involution.

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.

Effect of intramammary infusion of beta-1,3-glucan or interleukin-2 on leukocyte subpopulations in mammary glands of sheep.

OBJECTIVE: To investigate effects of intramammary infusion of beta-1,3-glucan or recombinant ovine interleukin-2 (rOvIL-2) on blood and mammary leukocyte subpopulations and their expression of various surface antigens in sheep. ANIMALS: 12 healthy multiparous, nonpregnant, nonlactating fine-wool Merino ewes. PROCEDURE: Beta-1,3-glucan in pyrogen-free saline solution (PFSS; n = 6), rOvIL-2 in PFSS (3), or PFSS (3) was infused on days 0 and 7 in 1 udder half of each ewe. Jugular vein blood and mammary secretion samples were taken before infusion and on days 2, 7, and 21 after infusion. Total and differential leukocyte counts were obtained, and blood and mammary cells were labeled for flow cytometry. RESULTS: Slight swelling of the mammary glands was observed on day 2 after rOvIL-2 but not after beta-1,3-glucan infusion. Both substances induced significant increase in mammary secretion leukocyte numbers, compared with controls. Beta-1,3-Glucan induced an influx of monocyte/macrophages, whereas neutrophils were the predominating cell population after rOvIL-2 infusion. Beta-1,3-glucan induced selection for CD4+, B-cell+, WC1+, and L-selectin+ lymphocytes on day 14 after infusion. By comparison, rOvIL-2 induced selection for B-cell+ and L-selectin+ lymphocytes on days 14 and 21 and depletion of CD8 and, to some degree, of IL-2R+ lymphocytes. Beta-1,3-Glucan induced an increase in the proportion of CD14+ leukocytes, indicating selective migration of monocyte/ macrophages to the nonlactating udder. CONCLUSION: Beta-1,3-Glucan and rOvIL-2 can modulate nonspecific immunity in the udder of sheep but may exert their effects by differing mechanisms. Clinical Relevance-Stimulation of the nonspecific defense against udder infections may improve control of mastitis.

Modulation of endotoxin-induced inflammation in the bovine teat using antagonists/inhibitors to leukotrienes, platelet activating factor and interleukin 1 beta.

A leukotriene biosynthesis inhibitor (MK886), a platelet-activating factor (PAF)-receptor antagonist (WEB 2086), a recombinant human interleukin-1 receptor antagonist (IL-1ra) and a polyclonal antibody to recombinant bovine IL-1 beta (anti-rBoIL-1 beta) was used to investigate the involvement of leukotrienes, PAF and IL-1 beta during endotoxin-induced inflammation in the bovine teat cistern. Endotoxin alone was infused into one teat cistern and endotoxin in combination with an inhibitor/antagonist was infused into another teat cistern of the same animal. Teat cistern samples were taken before infusion and at 3.5 and 7 h after infusion, and the numbers of neutrophils were counted. Saline infusion was used as control. The inhibitors/antagonists were also tested in combination with leukotriene B4 (LTB4), PAF and rBoIL-1 beta, respectively. MK886 or WEB 2086 significantly reduced the accumulation of neutrophils mainly between 3.5 and 7 h after infusion, indicating roles for leukotrienes, probably LTB4, and PAF in neutrophil accumulation during endotoxin-induced inflammation. As WEB 2086 also reduced cell accumulation between 0 and 3.5 h, PAF was implicated also in the early influx of neutrophils. WEB 2086 almost completely inhibited PAF-induced cell accumulation between 0 and 3.5 h. LTB4 did not induce significant cell accumulation in the teat cistern. IL-1ra did not affect endotoxin-induced neutrophil accumulation whereas anti-rBoIL-1 beta reduced total cell accumulation and, to some degree, accumulation between 0 and 3.5 h after infusion. Infusion of IL-1ra significantly inhibited cell accumulation induced by rBoIL-1 beta. Anti-rBoIL-1 beta also significantly reduced neutrophil accumulation induced by rBoIL-1 beta, but to a lesser degree. The results suggest roles for leukotrienes, most likely LTB4, and PAF, and to a lesser extent IL-1 beta, during endotoxin-induced neutrophil migration into the bovine teat cistern. The potential of the inhibitors/antagonists as therapeutic agents for bovine mastitis should be investigated.

Leukocyte and cytokine accumulation in the ovine teat and udder during endotoxin-induced inflammation.

The accumulation of leukocytes, ovine serum albumin and the cytokines interleukin-1 beta(IL-1 beta), tumour necrosis factor-alpha(TNF-alpha), interleukin-8 (IL-8), granulocyte-macrophage colony-stimulating factor (GM-CSF) and interferon-gamma(IFN-gamma) was studied during endotoxin-induced inflammation in lactating and dry ovine udders, and in the teat cisterns of dry ewes after surgical closure of the passage between the teat and udder cisterns. Samples were taken before infusion and hourly up to 10 h after infusion of 0.1, 1 or 10 micrograms of endotoxin, or infusion of pyrogen-free saline (PFS) as a control. Rectal temperatures were measured. A significant dose- and time-dependent accumulation of leukocytes, mainly neutrophils, was observed in the lactating udders and in the teat cisterns. In the dry udders, the leukocyte accumulation was significant for time but not for dose. Peak numbers of cells were reached at 3-4 h in the dry udders and in the teat cisterns, but not until 10 h after infusion in the lactating udders. The changes in the ovine serum albumin concentrations mostly paralleled changes in leukocyte numbers. A role was indicated for TNF-alpha, IL-8 and GM-CSF, but not for IL-1 beta and IFN-gamma, during endotoxin-induced inflammation in the ovine udder. Release of TNF-alpha, IL-8 and GM-CSF was most prominent in lactating udders, peaking at 2 or 3 h after infusion, but was also detected in dry udders and teat cisterns. Detectable levels of IL-1 beta and IFN-gamma were occasionally found in all three groups.