E.N. Noordhuizen-Stassen

β-hydroxybutyrate levels in peripheral blood and ketone bodies supplemented in culture media affect the in vitro chemotaxis of bovine leukocytes

The role of ketone bodies on chemotactic capacities of leukocytes was characterized in two experiments. Experiment I was performed to investigate the association between serum beta-hydroxybutyrate concentrations (BHB) and in vitro chemotaxis of leukocytes. Cows were divided into low-BHB, medium-BHB, and high-BHB ones and classified according to their BHB. Leukocytes from high-BHB cows had a significantly lower chemotactic differential than leukocytes from low-BHB cows (p < 0.01). The effect of adding ketone bodies into in vitro chemotaxis cultures on leukocytes chemotaxis was studied in Experiment II. Either individual or a combination of commercial ketone bodies - sodium salts of BHB (BHBA), lithium salt of acetoacetate (ACAC), and acetone (Acetone) - were diluted in culture media and divided into eight concentrations corresponding to concentrations of bovine subclinical and clinical ketosis. For leukocytes from medium- and high-BHB cow, the chemotactic indexes of leukocytes were reduced by ACAC and Acetone. Chemotactic differentials of cultures with ACAC and acetone supplementation from both sources of leukocytes were significantly lower than that of the control culture (p < 0.05). For leukocytes from high-BHB cows, chemotactic indexes were suppressed in a ketone-body environment. In conclusion, leukocytes from naturally-occurring ketotic cows have lower chemotactic differentials than those from non-ketotic cows, and a chemotactic capacity indicated by a chemotactic differential is impaired when leukocytes migrate in an environment with ketone bodies in vitro.

A Cox proportional-hazards model with time-dependent covariates to evaluate the relationship between body-condition score and the risks of first insemination and pregnancy in a high-producing dairy herd

The objective of this study was to determine the relationship between body-condition score and postpartum reproductive efficiency in dairy cattle. Data on body-condition score, reproduction, diseases, and production from 1404 lactations of 639 cows, calving between January 1984 and November 1996 on a commercial farm, were used. The Cox proportional-hazards model with time-dependent covariates was used to qualify the relationship between body-condition score on a scale of 1 to 5 and risks of first insemination and pregnancy. Cows with body condition at calving < 3 had lower first-insemination risks. Loss of body-condition score between calving and 45 days after calving was associated with increased days open and days-to-first insemination. Cows with body-condition scores < 2 after Day 45 and before first insemination were less likely to be inseminated and become pregnant compared with cows that had higher body condition. Milk yield, mastitis, lameness, milk fever and genital infection were associated with a reduction in reproductive efficiency. The results indicated that loss of body-condition and actual body-condition scores before conception or first insemination or both can be used as a prognosis for days open and days-to-first insemination in dairy herds.

Increased surface expression of CD11b receptors on polymorphonuclear leukocytes is not sufficient to sustain phagocytosis during Escherichia coli mastitis in early postpartum dairy cows.

Phagocytosis, CD11a and CD11b adhesion receptor expression, O2-production and maturity of circulating polymorphonuclear leukocytes (PMN) were studied during acute coliform mastitis in early postpartum dairy cows to obtain a better insight in the role of neutrophils in the pathology of this disease. The mammary gland of twelve newly calved high-yielding dairy cows was experimentally infected with Escherichia coli. Variability in clinical signs of mastitis and inhibition of milk production among cows was very high. There was a significant negative correlation between the number of circulating neutrophils immediately before infection and severity of mastitis represented by the decrease in milk production of non-infected quarters two days after infection. Pre-infection phagocytosis of E. coli, CD11a and CD11b receptor expression, phorbol myristate acetate (PMA)-induced O2-production and maturity of neutrophils on a per cell basis were not related to severity of mastitis. However, significant correlations between severity of mastitis and the total number of phagocytic PMN and mature PMN in blood immediately before infection were found. PMN characteristics responded differently to mastitis depending on the severity of the disease. Neutrophil functions from cows classified as severe (S) and moderate (M) responders to infection of the mammary gland were compared. Surface expression of CD11a receptors on PMN was decreased in all cows 24 h after infection, and this decrease was long-continued in S responders. A biphasic upregulation of the number of CD11b receptors on PMN was observed with a more pronounced response in S cows. PMN phagocytosis was decreased 12 h after infection in S cows and 18 h after infection in S and M cows and was normalized 24 h post-infection. The decrease of phagocytosis coincided with the first peak of CD11b receptor expression. Phorbol myristate acetate (PMA)-induced production of O2-by PMN was decreased for three days after infection in S responders compared to only one day in M responders and was followed by an upregulation. These data demonstrate a complexity in alterations of PMN functions during mastitis and suggest the involvement of differences in systemic factors dependent on severity of mastitis.

In vitro growth of mastitis-inducing Escherichia coli in milk and milk fractions of dairy cows

The outcome of E. coli mastitis in cows ranges from mild to severe in individual animals. This study explored the hypothesis that milk from individual cows differs in its growth medium properties for E. coli, and whether possible variation could be related to specific milk constituents. To mimic the early phase of intramammary E. coli infection, a low inoculum size and a short incubation period were used. Cell-reduced, cell- and fat-free (skim) and cell- and fat-free and protein-reduced (whey) fractions were prepared from whole milk samples (n=18). Ten ml of whole milk, milk fractions and brain heart infusion broth (BHI) were inoculated with approximately 100cfu E. coli. After 6h of incubation, bacterial counts were assessed by dilution plating in triplicate. Bacterial counts in whole milk differed up to a 100-fold between cows, which was not associated with SCC. Bacterial counts were significantly higher in whey fractions than in whole milk, cell-reduced and skim fractions and variation in whey was smaller, indicating that the acid-precipitable protein fraction contains the milk constituents of major relevance for inhibition of and variation in bacterial growth. The presence of fat and cells added to bacterial growth inhibition to a lesser extent. In conclusion, in vitro growth of E. coli in milk differs substantially between individual cows within an incubation period comparable with the early phase of intramammary infection. This suggests that the growth medium properties of milk could be of importance in the pathogenesis of E. coli mastitis and subsequent outcome of disease.

A procedure for parallel isolation of white blood cells, granulocyte and purified neutrophil suspensions from the peripheral blood of cattle

A rapid method is described for parallel isolation of white blood cells, granulocytes and purified neutrophils from peripheral blood of normal cattle. The mean recovery (+/- S.D.) of white blood cells, granulocytes and purified neutrophils isolated from peripheral blood of 13 cows was 66.4 +/- 12.6%, 68.7 +/- 20.0% and 38.0 +/- 20.9%, respectively. The mean purity of the isolated granulocyte and neutrophil suspensions was 94.0 +/- 3.8% and 95.0 +/- 6.0%, respectively. Viability of isolated cells was more than 97%.

Flow cytometric measurement of neutrophil alkaline phosphatase before and during initiation of an induced Escherichia coli mastitis in cattle

In 12 healthy cows, neutrophil alkaline phosphatase (NAP) activity was measured by flow cytometer before and during an experimentally induced Escherichia coli mastitis, to study the role and increase of NAP in Gram-negative bacterial infections. Percentage of neutrophils containing alkaline phosphatase and intensity of NAP activity were measured. Preinfection percentage of neutrophils with enzyme activity varied between 64.0% and 84.4% and the intensity of enzyme activity was low in all cows. After induction of infection, percentage of neutrophils with enzyme activity showed a significant decrease on day 1 followed by an significant increase on day 3. NAP intensity increased significantly on the second and third day after infection. This increase of intensity was significantly, positively correlated with the severity of infection. From this study we may conclude that variation in susceptibility to E. coli mastitis could not be explained by preinfection NAP levels. The post-infection increase of NAP activity, that was found following an induced infection was more a result of increased enzyme intensity per neutrophil, then from an increase of percentage neutrophils with enzyme activity. Furthermore, a strong correlation was found between NAP intensity and severity of inflammation. There was evidence that the more severely diseased animals showed stronger NAP intensity increase.

WITHIN DAY AND BETWEEN DAY VARIATION OF THE IN VITRO UNDER AGAROSE CHEMOTAXIS ASSAY IN BOVINE

The present study was conducted to determine within day variation (experiment I) and between day variation (experiment II) of the in vitro under agarose chemotaxis assay. Further, results from experiment II were used to estimate a more stable immunological parameter for the chemotactic activity. In experiment I, blood samples of eight cows were taken every 4 h starting at 0800 during a 24 h period. This procedure was replicated on three different days with peripheral white blood cells of lactating bovine. Chemotactic differential showed variation within a day. The differences between samplings were not constant over the days, but varied randomly from day to day. In experiment II, 12 cows were followed for 8 consecutive days and blood samples for chemotaxis assay were taken once a day at 0730. Differences between the days were significant. With a conditional auto regression model of the first order adjusted least squares means of each cow were estimated over the 8 consecutive days. The chemotactic value of a day was used to estimate the value of the next day. Expanding the model with more previous days did not improve the model. The results of this study indicate that blood samples for chemotaxis should be taken at the same time of the day to control for within day variation. If a sequence of chemotactic values is available we strongly suggest working with adjusted least square means of chemotactic differentials. These adjusted means show less random variation and are a more stable parameter for chemotactic activity.