Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure, This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

Objective-To determine the effect of caprine arthritis-encephalitis virus (CAEV) infection on expression of interleukin-16 (IL-16). Animals-6 goats experimentally infected with CAEV and 6 age-matched healthy uninfected control goats. Procedure-Peripheral blood mononuclear cells (PBMCs) and synovial membrane cells from infected and control goats cultured with or without phytohemagglutinin were analyzed for IL-16 mRNA by use of a reverse transcriptase-polymerase chain reaction assay with goat-specific primers, after cloning and sequencing of a 384-bp fragment of the goat IL-16 gene. Synovial fluid, serum, and culture supernatants of PBMCs and synovial cells of control and CAEV-infected goats were analyzed for IL-16 by use of an ELISA. Results-The 384-bp product was 86% homologous to the corresponding human IL-16 nucleotide sequence. Higher expression of IL-16 mRNA in PBMCs (unstimulated or stimulated with phytohemagglutinin) was detected in samples from CAEV-infected goats, compared with control goats, but the difference was not significant. Synovial membrane cells infected in vitro had higher expression than uninfected control cells. Higher IL-16 concentration was detected in synovial fluid, serum, and culture supernatants of PBMCs of infected goats than in samples from control goats. Conclusions and Clinical Relevance-Infection with CAEV increases expression of IL-16, a proinflammatory and chemotactic cytokine. This cytokine appears to be constitutively expressed at low concentrations in normal uninfected PBMCs and synovial membrane cells. Increased production of IL-16 in CAEV infection may partly be responsible for increased lymphoid cell infiltrations observed in arthritic joints and other tissues of CAEV-infected goats.

Marked differences in bone marrow cellularity were observed between cattle affected with leukocyte adhesion deficiency (LAD) and control cattle. The number of nucleated cells in bone marrow was 2.9 to 8.8 times higher in cattle affected with LAD, compared with controls. The myeloid-to-erythroid ratio of bone marrow from 3 cattle affected with LAD ranged from 2.4 to 12. Deficient CD18 expression on neutrophils isolated from bone marrow of cattle with LAD was clearly detected by flow cytometric analysis. Neutrophils from bone marrow of cattle affected with LAD appeared round and not flat, after adherence to plastic wells under agarose, whereas neutrophils from bone marrow of clinically normal cattle were firmly spread on the surface of plastic wells. In the chemotaxis under-agarose assay, many pseudopodia were detected on bone marrow neutrophils from clinically normal cattle, but were not detected on bone marrow neutrophils from cattle with LAD. Activities of chemotactic movements and phagocytosis of neutrophils isolated from bone marrow of cattle affected with LAD were documented to be severely impaired.

Binding of endogenous and exogenous homologous IgG, and IgM to bovine neutrophils before and after in vitro migration through micropore filters, and in vivo migration through mammary tissues after intramammary injection of endotoxin was evaluated by use of flow cytometry. Immunoglobulin binding to neutrophils at 4 and 37 C was also evaluated. Before and after in vitro migration, neutrophils with endogenously bound IgG, and IgM averaged 1 and 2% and 23 and 7%, respectively. Before and after in vivo migration, IgG2 and IgM binding averaged 1 and 7% and 26 and 15%, respectively. Before and after in vitro migration, binding of purified IgG2 and IgM averaged 75 and 67% and 8 and 24%, respectively. Before and after in vivo migration, percentage of neutrophils binding purified IgG2 and IgM averaged 92 and 98% and 54 and 70%, respectively. When serum was used as a source of exogenous immunoglobulins, binding of total Igg after in vitro migration increased from 5% to 28% and of IgM from 4% to 20%. After in vivo migration, binding increased from 21% to 47% and from 24% to 56%, respectively. Exogenous binding of IgG2 at 4 and 37 C averaged 75 and 84%, and binding of IgM averaged 8% at either temperature. Endogenous IgG2 was unaffected by temperature; however, binding of IgM decreased from 23% at 4 C to 2% at 37 C. These data indicate that endogenous binding was higher for IgM before migration than after migration, in vitro and in vivo. Furthermore, migration in vivo through cellular matrices induced receptor upregulation for IgG and IgM. Source and concentration of ligand and serum components, other than immunoglobulins, appeared to contribute to receptor expression and availability. Neutrophils that were exposed to endotoxin and migrated into milk expressed more receptors than did unstimulated and nonmigrating neutrophils. The association of IgM with its receptor was temperature-dependent.

A suction blister technique was used in 10 healthy dogs to remove the epidermis from the dermis in a standardized way. Collection chambers were attached to these skin windows and filled with autologous serum to attract exudative neutrophils. The chambers were emptied by fine-needle aspiration at 4-hour intervals and were refilled with serum for 24 hours after the Int aspiration. The collected cells were counted, differentiated, and stained, using the trypan blue dye-exclusion method to determine cell viability. Multiple skin biopsy specimens obtained during the procedure were examined histologically. The chamber fluid collected after 24 hours was cultured for bacteria. Increasing numbers of viable neutrophils were collected during the 24-hour period from the induced skin windows. In all but 1 dog, sufficient viable neutrophils could be collected to perform further functional tests in vitro. Our conclusion is that this technique might be useful to study chemotaxis in vivo and to perform functional tests on exudative neutrophils.

Corynebacterium pseudotuberculosis phospholipase D (PLD) significantly affected viability of ovine neutrophils after 24 hours' exposure, This effect was more marked in cells that ingested PLD emulsified in oil. Treatment of neutrophils with PLD significantly (P < 0.05) reduced the ability of these cells to migrate toward activated sheep serum. The PLD was not chemotactic, but it activated normal sheep serum, producing factors that were chemotactic for neutrophils.

Cultures of bovine alveolar macrophages were inoculated with type-1 and type-8 adenoviruses, initially isolated from calves with respiratory tract disease, and functional properties of the cells were observed over a period of 10 to 11 days. Both viruses replicated in macrophages; viral titers were low (< 3.75 log10 TCID50/0.1 ml), and intranuclear inclusions were detected by indirect immunofluorescence in 5 to 10% of the cells from 3 days after inoculation. Highest titers were induced by type-1 adenovirus, which also induced the greatest functional changes. Expression of Fc and complement receptors was reduced by both viruses, although the greatest effects were seen with type 1. Phagocytosis of Candida krusei cells was reduced following type 1 infection, whereas phagocytosis in type-8-infected cells was not different from that of noninfected macrophages. Ability to kill ingested Candida cells also was reduced following type-1 infection, whereas type-8-infected macrophages had lower killing ability only at 2 to 4 days after inoculation. Neither virus had substantial effects on the production of neutrophil chemotactic factors by the macrophages.

Chemotaxis under agarose was evaluated to establish an assay system and to characterize chemotacticresponses of canine neutrophils. A method for the measurement of canine neutrophil chemotaxis was established, with optimal responses obtained with agarose containing 10% pooled canine serum, a concentration of 5 X 10(5) cells/well, zymosan-activated serum (ZAS), or autologous serum or plasma as the chemoattractants, and a 120-minute incubation period. Canine neutrophils responded well to ZAS, heat-inactivated ZAS, autologous serum and plasma, and heat-inactivated pooled serum. Chemotactic activity was proportional to the concentration of serum used as the chemoattractant. Mean (+/- SD) random migration, chemotaxis, chemotactic index, and chemotactic differential of neutrophils from 9 healthy Greyhounds were 1.09 (+/- 0.23), 1.95 (+/- 0.38), 1.82 (+/- 0.31), and 0.86 (+/- 0.32) mm,respectively.

Ovulation has been likened to an inflammatory process. Inflammatory cells accumulate in the ovulating follicle, presumably because of chemotactic factors. Chemotactic activity was measured in fluid aspirated from follicles of estrous mares 0, 12, 24, and 36 hours after ultrasonographic detection of a 35-mm follicle and IV treatment with 2,500 IU of human chorionic gonadotropin. Chemotaxis was assessed by measuring directional migration of equine neutrophils under agarose. Follicular fluid acted as a chemoattractant for neutrophils, but there was no significant difference in chemotactic activity among different time intervals after administration of human chorionic gonadotropin. On the basis of results of various treatments, chemotactic properties of serum and follicular fluid were similar. Chemotactic activity was significantly reduced by heating (56 C for 30 minutes) and by trypsinization and was virtually removed by charcoal treatment. Dialyzing the follicular fluid (3,500 and 8,000 molecular weight cut-off) significantly reduced the chemotactic activity of follicular fluid and serum. The importance of chemotactic factors in the process of ovulation in the mare is yet to be established.

Important procedural factors in the under-agarose assay for porcine neutrophil migration were identified, and optimal conditions were established. Three factors were tested: the concentration of zymosan-activated serum inoculated into the outer well; the number of neutrophils inoculated into the center well; and the time of incubation of the agarose plates. All factors had a significant (P < 0.0001, 0.0001, and 0.01, respectively) effect on the chemotactic index of porcine neutrophils. The optimal combination of these 3 factors was undiluted zymosan-activated serum as the chemoattractant, 8 x 10(5) neutrophils inoculated into the center well, and 5 hours of incubation. The assay was validated, using standard conditions, and the data were used to predict the number of pigs and/or repetitive assays needed to identify differences among experimental groups.