Functional variation in endogenous and exogenous immunoglobulin binding to bovine neutrophils relative to parturition.

Ten healthy first- and second-lactation Holstein cows were observed from 1 week before to 1 week after calving and at postpartum day 30 to determine polymorphonuclear neutrophil (PMN) functional variation and immunoglobulin binding profiles. Blood and mammary PMN were obtained 3 times weekly and within 24 hours of calving. Functional traits measured included phagocytosis of Staphylococcus aureus and in vitro chemotaxis through micropore filters in a Boyden chamber. Additionally, PMN were evaluated for endogenous binding of IgG1, IgG2, IgA, and IgM before and after in vitro chemotaxis. Exogenous binding of the same isotypes was determined after incubation in pooled colostrum, purified immunoglobulin, and pooled sera. Phagocytosis results indicated a significant and transient increase in percentage of milk PMN with associated, rather than phagocytosed, bacteria for 1 week after calving. Blood PMN phagocytosis was not significantly different during this period. Though total chemotaxis was essentially unchanged, the percentage of PMN that were unable to complete migration increased substantially on the day of calving, an effect that disappeared by postpartum day 4. A significant (P < 0.01) positive correlation (r = 0.29) between percentage of PMN migrating completely through the micropore filter and percentage of blood PMN with associated bacteria was observed. Changes were not observed in endogenous immunoglobulin binding, with the exception of a peak in relative fluorescence intensity for IgG1 on the day of calving; this disappeared within 2 days after calving. Correlations between relative intensities of IgG2 and IgM, and percentage of mammary neutrophils phagocytosing were 0.37 and 0.70.(ABSTRACT TRUNCATED AT 250 WORDS)

Opsonization of Staphylococcus aureus by bovine immunoglobulin isotypes.

The ability of specific bovine Ig isotypes to enhance phagocytosis of Staphylococcus aureus by polymorphonuclear neutrophils was studied. Polymorphonuclear neutrophils were isolated from the blood of 14 lactating Holstein cows. Antibodies against S. aureus M10 were produced by two Holstein cows immunized via intramuscular injections and injections in the area of the supramammary lymph node with M10 emulsified in dextran sulfate. The IgG1, IgG2, and IgM were prepared from immune sera. Fluorescein-labeled, formalin-killed S. aureus M10 were opsonized with the respective isotypes prior to incubation with polymorphonuclear neutrophils. Percentage of polymorphonuclear neutrophils phagocytosing averaged 37.4, 1.1, 15.9, and 9.4% for immune sera, IgG1, IgG2, and IgM, using a M10: polymorphonuclear neutrophils ratio of 10:1; and 77.1, 1.8, 32.1, and 57.9 using a 40:1 ratio. When IgG1 was incubated with either IgG2 or IgM, phagocytosis was reduced to 10.0 and 5.0%, respectively, using the 10:1 ratio and 24.2 and 44.7%, respectively, using the 40:1 ratio. Significant variation occurred among cows in the ability of polymorphonuclear neutrophils to undergo phagocytosis independent of isotype and S. aureus M10: polymorphonuclear neutrophil ratio. These data show that IgG2 and IgM are opsonic for bovine polymorphonuclear neutrophils and that IgG1 inhibits the activity of both. These results will be helpful to determine immunization protocols to solicit synthesis of bovine IgM and IgG2 specific for S. aureus.

Prediction of mastitis using milk somatic cell count, N-acetyl-beta-D-glucosaminidase, and lactose.

The objectives of this work were 1) to examine the responsiveness of SCC, lactose concentration, and NAGase activity in milk to changes in bacteriological status and 2) to develop models for predicting bacteriological status of mammary glands. Data included 550 cows in 10 commercial herds. Natural logarithm NAGase and log cell count were most responsive to changes in bacterial status. The log NAGase was relatively more effective in identifying major from minor pathogen infections, whereas log SCC was better able to differentiate between infected and uninfected classes. Non-transformed NAGase, SCC, and lactose were considerably less responsive to infection status. Logistic regression of bacterial status on herd, lactation number, milk, log SCC, log NAGase, and stage of lactation was performed. The least significant variables were removed in a stepwise process. Final predictors of infection status were herd, log SCC, and log NAGase. The role of log SCC was to discriminate infection from no infection, whereas log NAGase discriminated major from minor pathogens. The log NAGase, alone or in combination with log SCC, added substantially to the detection power of the model. Chi-square goodness of fit tests found no significant differences between observed and predicted infection probabilities. Substitution of herd averages for log SCC and log NAGase for the herd variables resulted in significant differences between predicted and observed herd infection probabilities.

Effect of anticapsular antibodies on neutrophil phagocytosis of Staphylococcus aureus.

One of the major virulence factors of Staphylococcus aureus is development of an exopolysaccharide capsule in vivo, which inhibits recognition of antibodies to highly antigenic cell wall by neutrophils. To circumvent this inhibition, an attempt was made to produce anticapsular antibodies. Three cows per group were immunized in midlactation by injections in the area of the supramammary lymph node and intramuscularly and were boosted on d 14, 42, and 70 with three variants of Smith S. aureus: compact, unencapsulated; diffuse, rigid capsule; and diffuse large clearing, exceptionally large flaccid capsule using dextran sulfate as adjuvant. Serum agglutination and ELISA titers of cows immunized with diffuse and diffuse large clearing increased after immunization and after each boost and remained elevated to the end of the experiment at 112 d. Phagocytosis of diffuse and diffuse large clearing, measured by flow cytometry, was enhanced by immunization with either organism. No antibody response to capsule or enhanced phagocytosis of diffuse developed in cows immunized with compact. However, anticompact antibodies were opsonic for diffuse large clearing. These data show that bovine antibodies to S. aureus capsule are opsonic for bovine neutrophils and that capsule plays a role in inhibition of cell-wall opsonization of S. aureus.

N-acetyl-beta-D-glucosaminidase activities, milk somatic cell counts, and blood leukocyte and erythrocyte counts in cows after heat-induced stress or after intravenous administration of adrenocorticotropic hormone.

Six midlactation, nonpregnant cows were subjected to heat-induced stress or to repeated injections of adrenocorticotropic hormone (ACTH). The heat stress (experiment 1) consisted of subjecting the cows to 7 days of fluctuating temperatures (21 C during the day, 32 C during the night). The ACTH-induced stress (experiment 2) was accomplished by giving each of the cows 100 IU of ACTH twice daily for 4 consecutive days. The cows' milk somatic cell counts (MSCC), milk N-acetyl-beta-D-glucosaminidase (NAGase) activity, quarter milk production (ie, milk production per mammary quarter), blood leukocyte counts, and plasma concentrations of NAGase were monitored in both experiments. Although heat stress did not result in significant differences in NAGase, heat stress did significantly (P less than 0.05) decrease milk production. The ACTH administrations resulted in increased milk NAGase activity, MSCC, and blood leukocyte counts, and in decreased plasma NAGase activity and quarter milk production. Therefore, milk NAGase activity and MSCC were not affected by short-term heat stress, but were increased by ACTH-induced stress. Blood erythrocyte concentrations were not affected by heat stress or by ACTH-induced stress.

Effects of estradiol benzoate and estrus on N-acetyl-beta-D-glucosaminidase activity and somatic cell concentration in milk.

Quarter composite milk samples were obtained from five cows free of intramammary infection throughout an estrous cycle and after injection of corn oil and of estradiol benzoate (1 microgram/kg body weight) in corn oil. Samples were assayed for NAGase activity and somatic cell concentration. Least squares means for the enzyme were 1.46, 1.47, 1.43, and 1.41 natural logarithm nanomoles per minute per milliliter for control, estrus, corn oil, and estradiol benzoate, respectively. Milk somatic cell count means were 2.77, 2.71, 2.92, and 3.10 loge X 10(3) cells/ml. Treatment had no significant effect on enzyme activity or cell counts. Results suggest no adjustment of NAGase or cell count is necessary for stage of estrous cycle. When administered at physiological dosages, exogenous estrogen did not appear to influence either measure.

Variation in N-acetyl-beta-D-glucosaminidase activity and somatic cell count among various milk fractions.

Six Holstein cows with uninfected quarters were quarter machine milked for four consecutive morning milkings. Foremilk, bucket, and stripping fractions were collected for all milkings. For the first three milkings, an additional milk sample was collected 1 h after milking. After the final milking, oxytocin (20 IU) was injected in the tail vein 20 min after milking, and residual milk was collected. All samples were assayed for NAGase activity and somatic cell concentration. Mean NAGase activity for foremilk, bucket, and stripping samples were 1.65, 1.55, and 1.84 loge nmol/min/ml. Hour after milking and residual samples averaged 2.07 and 1.78. Cell counts (loge thousand cells/ml) for foremilk, bucket, and strippings averaged 2.52, 2.91, and 4.30. Hour after and residual samples averaged 4.56 and 5.53. Results suggest that among uninfected cows, foremilk approximates bucket levels of NAGase and cell count.