No indication of Coxiella burnetii infection in Norwegian farmed ruminants.

BACKGROUND: Infection with Coxiella burnetii, the cause of Q-fever, has never been detected in Norwegian animals. Recognising the increasing prevalence of the infection in neighbouring countries, the aim of the study was to perform a survey of Norwegian farmed ruminants for the prevalence of C. burnetii infection. RESULTS: Milk and blood samples from more than 3450 Norwegian dairy cattle herds, 55 beef cattle herds, 348 dairy goat herds and 118 sheep flocks were serologically examined for antibodies against C. burnetii. All samples were negative for antibodies against C. burnetii. The estimated prevalences of infected herds were 0 (95% confidence interval: 0% - 0.12%), 0 (0% - 12%), 0 (0% - 1.2%) and 0 (0% - 10%) for dairy cattle herds, beef cattle herds, goat herds and sheep flocks, respectively. CONCLUSIONS: The study indicates that the prevalence of C. burnetii infection in farmed Norwegian ruminants is low, and it cannot be excluded that Norway is free of the infection. It would be beneficial if Norway was able to maintain the current situation. Therefore, preventive measures should be continued.

Lymphocyte subpopulations and neutrophil function in calves during the first 6 months of life.

Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and thereafter every month until the age of 6 months. The percentages and absolute values of CD4+, CD8+ gammadelta TCR+ and WC1+ T cells, CD21+ B cells and NKp46+ NK cells were determined by flow cytometry, and the expression of the interleukin-2 receptor alpha chain (CD25) was measured to assess the level of activation of the lymphocyte subpopulations. Neutrophil phagocytosis, respiratory burst and bactericidal activity were measured in five different neutrophil function assays. Most of the parameters examined reached a stable level during the first 6 months of life. The proportions of CD4+ and CD8+ lymphocytes remained relatively stable during the study period, while there was a moderate decrease in the relative percentage of gammadelta T cells from birth to approximately 5 months of age. However, the absolute numbers of gammadelta T cells per millilitre of blood remained stable throughout the study period and did not display significant variation with age. The percentage of cells expressing the B-cell maturation marker CD21 increased significantly over the first 5 months of life. The proportion of NK cells showed substantial variation during the study. Marked differences in the relative proportions of the lymphocyte subpopulations were noted between the individual calves, and the individual ranking of the animals was largely maintained over time. CD25 expression was detected on a mean of 6.6% of the CD4+ cells, while a lower percentage of the other lymphocyte subpopulations expressed this receptor. Phagocytic activity was demonstrated in approximately 90% of the neutrophils, and this proportion remained stable during the entire study period, while respiratory burst activity showed a moderate decrease during the first 2 months of life. The present study shows that the T-cell subpopulations are present in peripheral blood of calves at levels comparable with adult values, while the B-cell population increases significantly with age. The decrease in the relative percentage of gammadelta T cells appears to be attributable to an increase in the absolute numbers of CD4+ and CD21+ cells, rather than a change in absolute gammadelta T-cell numbers. Furthermore, the results indicate that the neutrophilic granulocytes are functional and able to mount an effective response in young calves from the first week of life.

Hematologic values in calves during the first 6 months of life.

BACKGROUND: Age-related changes in hematologic values are known to occur in many species. Few published studies include repeated measurements of hematologic parameters in calves during the first months of life. OBJECTIVE: The aim of the present study was to monitor hematologic values by sequential measurements from birth to 6 months of age in 15 healthy calves of the Norwegian Red breed, and compare the results to reference intervals for adult, lactating dairy cows. METHODS: Fifteen clinically healthy calves were sampled every week during the first 5 weeks of life and every month thereafter until 6 months of age. Hematologic values were measured using the ADVIA 120 hematology system. Reference intervals were determined for 75 healthy adult cows of the same breed. RESULTS: Compared with adult reference intervals, the MCV was lower and the RBC count was higher in calves throughout the investigation period. Hemoglobin concentration stayed largely within the adult reference interval. Mean MCHC was lower than adult values for 5 weeks, then increased and reached adult values by weeks 10-12. The mean lymphocyte count for calves reached adult reference values at weeks 6-8, and the mean monocyte count increased steadily until weeks 14-16. For most leukocytes, interindividual variation was larger during the first 5-8 weeks of life. The mean platelet count for calves was higher than the adult reference interval until weeks 19-21 of age. CONCLUSIONS: Age-specific reference intervals for calves from birth to 6 month of age are needed for RBC count, MCV, MCHC, red cell distribution width, and platelet and lymphocyte counts.

Staphylococcus aureus enterotoxin D is secreted in milk and stimulates specific antibody responses in cows in the course of experimental intramammary infection.

An enterotoxin D (SED)-producing strain of Staphylococcus aureus was used to infect one mammary gland of each of 17 lactating dairy cows. All glands became infected and shed bacteria over a sampling period of 3 weeks. Serum and milk antibodies specific for SED were monitored by an enzyme-linked immunosorbent assay for 12 weeks. Elevated anti-SED antibodies were detected in all cows after infection, and immunoglobulin of the G2 subclass comprised most of the specific serum response. SED was detected in mastitic milk samples from two cows at levels of 5 to 10 ng/ml. An in vitro lymphocyte proliferation assay showed that SED at levels below 10 pg/ml induced proliferation of bovine lymphocytes and that sheep antiserum specific for SED neutralized this proliferative response. Sera obtained from the cows pre- and postinfection inhibited lymphocyte proliferation at SED concentrations of 10 and 50 ng/ml, respectively. The addition of SED to whole blood or to isolated neutrophils had no significant effect on neutrophil function in vitro. The results show that SED is secreted during mammary gland infection, is mitogenic for bovine lymphocytes, and stimulates the production of specific antibodies.

Staphylococcus aureus capsular polysaccharide types 5 and 8 reduce killing by bovine neutrophils in vitro.

Isogenic variants of Staphylococcus aureus strain Reynolds expressing either no capsule or capsular polysaccharide (CP) type 5 (CP5) or type 8 (CP8) were used to assess the effect of CP on bacterial killing and the respiratory burst of bovine neutrophils. The effects of antisera specific for CP5 and CP8 were also evaluated. The killing of live bacteria by isolated neutrophils was quantified in a bactericidal assay, while the respiratory burst after stimulation with live bacteria in whole blood was measured by flow cytometry. The expression of a CP5 or CP8 capsule protected the bacteria from being killed by bovine neutrophils in vitro (P <0.001), and the capsule-expressing variants did not stimulate respiratory burst activity in calf whole blood. The addition of serotype-specific antisera increased the killing of the capsule-expressing bacteria and enhanced their stimulating effect in the respiratory burst assay (P <0.01). When the S. aureus variants were grown under conditions known not to promote capsule expression, there were no significant differences between them. The present study demonstrates that the expression of S. aureus CP5 or CP8 confers resistance to opsonophagocytic killing and prevents the bacteria from inducing respiratory burst of bovine neutrophils in vitro and that these effects can be reversed by the addition of serotype-specific antisera.

Flow cytometric measurement of neutrophil respiratory burst in whole bovine blood using live Staphylococcus aureus.

A rapid and simple method for measurement of respiratory burst in neutrophil granulocytes in whole bovine blood is described. The respiratory burst was stimulated by live Staphylococcus aureus, and the production of reactive oxygen species quantified by the conversion of intracellular dihydrorhodamine 123 to the green fluorescent rhodamine 123, measured by flow cytometry. Assay conditions, including bacterial and dihydrorhodamine 123 concentrations and incubation time, were determined. Repeatability and precision of the method were assessed by testing parallel samples from clinically healthy dairy cows. In vitro and in vivo inhibition of respiratory burst was investigated, and labelling with a granulocyte marker antibody was performed. Stimulation with live S. aureus induced green fluorescence in the neutrophil granulocytes in a whole blood preparation. The fluorescence intensity increased with increasing bacterial concentration and increasing incubation time. Agreement analysis showed that the method gave repeatable results, and the intra-assay variability of the method was relatively low. The method is considered a useful technique for measurement of neutrophil respiratory burst in whole bovine blood.

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes.

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.