The metabolic responses of equine articular cartilage to incubation with bacterial lipopolysaccharide (LPS) were studied, using explant cultures of articular cartilage obtained from the metatarsophalangeal joints of 15 horses, age of which ranged from 3 months to 20 years. For comparison, explants were also established from the metatarsophalangeal joints of 3 calves. Explants were cultured for 3 days in medium containing various concentrations of LPS from 0 (control) to 100 microgram/ml. Glycosaminoglycan (GAG) released during the 3-day incubation was determined by a spectrophotometric assay, using the dye 1,9-dimethylmethylene blue. Newly synthesized GAG content was assayed by measuring [35S]sulfate incorporation during a 3-hour pulse labeling period. In addition, prostaglandin E2 (PGE2) synthesis was quantified, using a [3H]PGE2 radioimmunoassay kit and magnetic separation. Finally, explants from 3 animals were used to evaluate the effect of supplementing culture medium with 5% serum on the response of explants to LPS, and explants from 1 horse were used to compare responses to stimulation with LPS derived from 2 bacterial sources. Equine explants cultured with bacterial LPS had a dose-dependent decrease in synthesis and increase in release of GAG, and these responses were significantly (P < 0.0001) greater in explants from younger horses. In addition, equine explants had a significant (P = 0.0001) dose-dependent increase in concentration of PGE2 released into the culture medium in response to incubation with LPS. Comparison of data for GAG synthesis from equine and bovine explants revealed a significant (P = 0.025) difference in responsiveness to LPS between the 2 species. Equine explants tended to have a greater suppression of GAG synthesis in response to incubation with increasing concentrations of LPS than did age-corrected bovine samples.

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.

The nucleotide sequence of a chromosomally encoded antigen-expressing gene of Borrelia coriaceae was determined and used as a target for the polymerase chain reaction (PCR). Two primer sets were designed specifying the amplification of 269- and 701-bp DNA fragments. Primer set I, producing the short amplicon, was tenfold more sensitive than primer set II. As little as 10 fg of purified B coriaceae DNA could consistently be detected. The PCR assays, containing controlled numbers of whole spirochetes, allowed detectable amplification of 2 to 10 organisms. An internal, nonradioactively labeled gene-specific probe verified specificity of the PCR amplicons. Neither primer set cross-reacted with other related spirochetes. This PCR assay was adapted and found suitable for identification of B. coriaceae in biological samples, such as blood and thymus. Evidence for presence of B. coriaceae in biological samples was not found in tissue samples obtained from experimentally infected cows and their fetuses. These data failed to establish a definite association between B. coriaceae and epizootic bovine abortion.

We used monoclonal antibodies and immunohistologic examination of lymph nodes, to elucidate the pathogenesis of lymphosarcoma induced by, infection with bovine leukemia virus (BLV). The superficial cervical lymph nodes from 3 BLV-infected but apparently healthy sheep and 5 sheep with full-blown lymphosarcoma were examined. We also investigated the integration of bovine leukemia provirus by use of Southern blotting. In lymph nodes from sheep lacking clinical signs of infection, in which the provirus had been integrated at multiple sites in the genome, many large hypertrophic follicles were observed in the cortex. These follicles had germinal centers consisting of CD4+T cells and B cells that expressed surface IgM (sIgM) and major histocompatibility complex (MHC) class-II antigens, but not B cell-specific B2 molecule. The percentage of CD4+T cells in the cortex was significantly (P < 0.05) higher than that of the controls and sheep with lymphosarcoma. In all sheep with lymphosarcoma, the lymph nodes were completely destroyed by proliferating neoplastic cells, and in addition, small atrophic follicles, which consisted of normal B-cell marker-positive cells, were seen near the trabecula and the subcapsule. In these instances, neoplastic cells appeared to be a monoclonal population derived from a single CD5- B-cell lineage and to be classified as 2 types, CD5-CD4-CD8-B2+MHC class-II+sIgM+ and CD5-CD4-CD8-B2+MHC class-II+sIgM-. Moreover, CD8+T cells infiltrated diffusely throughout the tumorous lymph nodes apart from the atrophic follicles, and CD4+T cells were observed around atrophic follicles. Both types of T cells were small-size, normal lymphocytes with round and noncleaved nuclei, and were apparently non-neoplastic cells. In fact, after separation by use of a panning method, it seems that, in blood mononuclear cells from BLV-infected sheep without clinical signs of infection, but in lymphosarcomatous stages, the proviral genome was integrated only in B cells and not in T cells. Thus, we conclude that the host's immune response may be still maintained at a lymphosarcomatous stage.

Effect of estrogen (E2) and progesterone (P4) on uterine antibacterial activity and immunoglobulin concentrations in mares was studied. In 2 in vitro experiments, 6 mixed-breed mares were ovariectomized, and uterine fluid and blood serum were analyzed. Antibacterial assay methods were used to determine inhibitory effects on Streptococcus zooepidemicus of uterine fluid samples collected on days 3, 5, and 8, and serum obtained on day 8 of treatment. Single radial immunodiffusion methods were used to quantify amounts of IgA and IgG in uterine fluid and serum on days 3, 5, 8, and 14 of treatment. Neither E2 nor P4 increased activity of serum and uterine fluid against S zooepidemicus. Numbers of colony-forming units per milliliter of bacteria were significantly (P < 0.01) lower in control Hanks' balanced salt solution with 1.0% gelatin (HBSSG) than in uterine fluids. Bacterial numbers were significantly (50%) greater in uterine fluids and serum than in HBSSG controls for both treatments. Both fluids, especially serum, supported significantly (P < 0.01) more growth of S zooepidemicus than did HBSSG when incubated for 0, 2, and 4 hours. These findings are in contrast to previous reports of antibacterial activity in the uterus of sexually intact mares undergoing an estrous cycle: great reduction of bacterial count in uterine fluid from mares in diestrus, and significant increases in bacterial numbers in uterine fluid or serum from mares in estrus. Treatment comparisons between serum and uterine fluid IgA and IgG concentrations were not significantly different, although overall IgA concentration in the uterus was higher than concentration in serum. The IgG concentration in uterine fluid was higher in P4- than E2-treated mares. However, IgG concentration was significantly (P < 0.01) higher in uterine fluid on day 8 in P4-treated mares than on day 3 or 5. Results of this study indicate that neither immunoglobulin concentration nor hormone treatment has a direct effect on streptocidal activity.

Growth of Pasteurella haemolytica A1 in RPMI 1640 medium containing 0.5% bovine serum albumin (BSA) for 2.5 hours enhanced culture supernatant leukotoxic activity [30,700 +/- 12,900 toxic units/ml, compared with leukotoxic activity of culture supernatants produced in RPMI 1640 medium alone (120 +/- 40 toxic units/ml)]. Gel filtration chromatography of the leukotoxic activity from RPMI 1640 medium supernatants in buffer containing 50 mM NaCl indicated a single leukotoxic activity peak (peak I) eluting near the gel resin molecular mass exclusion limit (estimated molecular mass of approx 8,000 kd). In contrast, culture supernatants produced in RPMI 1640 plus bovine serum albumin medium (RPMI + BSA) had peak I and 2 additional leukotoxic activity peaks (peaks II and III) with estimated molecular mass of approximately 80 and < 30 kd, respectively. All leukotoxic activity peaks were composed of approximately 100-kd molecular mass leukotoxin protomer, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and immunoblotting with a monoclonal antibody against leukotoxin. Subjecting culture supernatant leukotoxic activity produced in RPMI + BSA to gel filtration chromatography in buffer containing 500 mM NaCl or 6M urea resulted in detection of only a single leukotoxic activity peak with estimated approximate molecular mass of 250 and 800 kd, respectively. These findings suggest that P haemolytica exists as a high molecular mass aggregate with low leukotoxic activity which, in the presence of BSA, partially disaggregates to multiple toxin forms with enhanced leukotoxic activity. Some of these leukotoxin forms interact with dextran-based gel resins at low ionic strength.

Poly(methacrylic acid) hydrogels were tested for oral delivery of a vaccine against Pasteurella haemolytica infection in cattle. Culture supernatants of P haemolytica, the most common bacterium associated with pneumonia in cattle, were used as the antigens in the vaccine. Hydrogels containing culture supernatants were administered orally to calves. Calves were then challenge-exposed with virulent P haemolytica. Calves were euthanatized 3 days after challenge exposure. The lungs of each calf were scored for severity and size of pneumonic lesions. Results indicated that vaccinated calves had smaller, less severe pneumonic lesions and lived longer than nonvaccinated calves. These results indicated that hydrogels can be used to deliver vaccines orally to calves to enhance resistance to pneumonia caused by P haemolytica.

The effect of interleukin 1 (IL-1) on equine articular cartilage was investigated, using a cartilage explant culture system. Measurement of [35S]O4 incorporation revealed synthesis of matrix proteoglycan by cartilage to be decreased 45, 59.7, and 37.5% after 1, 3, and 5 days, respectively, in culture in the presence of 5 U of IL-1/ml. There was no change in proteoglycan degradation as determined by measurement of [35S]O4 release into the culture medium. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cartilage-conditioned medium indicated that exposure of cartilage to IL-1 caused a decrease in total protein synthesis by 45, 68, and 87% after 1, 3, and 5 days, respectively, in culture while selectively inducing synthesis of the 57-kd neutral metalloproteinase stromelysin (matrix metalloproteinase-3) in young and adult horses. Identification of stromelysin was confirmed by functional characterization and immunoprecipitation. Baseline total protein synthesis, as well as specific synthesis of stromelysin in cartilage from adult and aged horses, was markedly less than that of young horses. The IL-1-induced induced reduction in total protein synthesis may not be a characteristic of equine articular cartilage from affected joints of horses with naturally acquired osteoarthritis as indicated by an overall increase in protein synthesis by osteoarthritic explants. Introduction of IL-1 into an equine articular cartilage explant culture system resulted in decrease of matrix component synthesis and increase in specific degradative enzyme synthesis and activity. Articular cartilage from aged horses had markedly less overall metabolic activity, compared with cartilage from young horses. Articular cartilage from affected joints of horses with naturally acquired osteoarthritis did not have metabolic alterations identical to those of IL-1-stimulated normal articular cartilage from the same individual, necessitating reevaluation of the validity of the IL-1-induced model of osteoarthritis. Osteoarthritis is a common, naturally acquired disease of horses, and tissue from animals of all ages and stages of osteoarthritis is available. The equine model of osteoarthritis may afford an important means of studying the alterations in articular cartilage metabolism as a function of age and disease severity.

Histomorphologic/morphometric evaluation, leukocyte scintigraphy, and myeloperoxidase activity were used to determine whether neutrophils accumulate in the large colon of horses during low-flow ischemia and reperfusion. Twenty-four adult horses were assigned to 1 of 3 groups: group 1, sham-operated (n = 6); group 2, 6 hours of ischemia (n = 9); and group 3, 3 hours of ischemia and 3 hours of reperfusion (n = 9). Low-flow ischemia of the large colon was induced in horses of groups 2 and 3 by reducing colonic arterial blood flow to 20% of baseline. Radiolabeled (99mTc) autogenous neutrophils were injected at 175 minutes, which corresponded to 5 minutes prior to reperfusion in group-3 horses. Full-thickness biopsy specimens of the left ventral colon were collected at baseline and at 30-minute intervals for 6 hours; a portion of the biopsy specimen was placed in formalin for histologic examination, and the remainder was used to measure mucosal radioactivity and myeloperoxidase activity. There were no differences in baseline mucosal neutrophil index, mucosal neutrophil numbers, submucosal venular neutrophil numbers, mucosal radioactivity, or mucosal myeloperoxidase activity among groups, or over time in group-1 horses. Neutrophils accumulated in the colonic mucosa during ischemia and further increased at reperfusion, as indicated by neutrophil index (morphology) and mucosal neutrophil numbers (morphometry); mucosal neutrophil index was significantly (P < 0.05) greater in group-3 horses during reperfusion than at the corresponding periods of ischemia in group-2 horses. Neutrophil numbers were significantly (P < 0.05) increased in submucosal venules at 10 minutes of reperfusion in group-3 horses and were significantly (P < 0.05) greater in group-3 than in group-2 horses during the interval from 3 to 6 hours. Mucosal radioactivity significantly (P < 0.05) increased at reperfusion in group-3 horses; there was a trend (P = 0.076) toward greater mucosal radioactivity in group-3, compared with group-2 horses, throughout the 3- to 6-hour interval. There were no differences in mucosal myeloperoxidase activity among or within any of the 3 groups over time. Neutrophils accumulated in the large colon of horses during low-flow ischemia and reperfusion. Neutrophil infiltration was detected by histologic examination and leukocyte scintigraphy, but not by measurement of myeloperoxidase activity. The accumulation of neutrophils during ischemia and the further neutrophil infiltration during reperfusion indicate that neutrophils may contribute to reperfusion injury of the large colon.

Alterations in the size and functions of porcine alveolar macrophages exposed to sublytic amounts of heat-labile, hemolytic cytotoxin produced by Actinobacillus pleuropneumoniae (App) serotype 1, strain HS54 into the culture medium were studied in vitro. Alveolar macrophages were sensitive to the cytotoxin; treatment of the macrophages with low concentrations of cytotoxin (0.016 hemolytic unit) resulted in severe, irreversible cell swelling. However, high doses of cytotoxin (2.0 hemolytic units) were required to cause substantial cell death, as indicated by the influx of propidium iodide into and release of lactate dehydrogenase from cells. Macrophages exposed to low, sublytic doses of cytotoxin failed to migrate toward chemoattractant, were unable to attach to glass, and failed to phagocytize optimally opsonized erythrocytes. Macrophages already attached to glass surfaces detached when exposed to sublytic doses of cytotoxin. The swelling and impairment of functions of alveolar macrophages observed in this study could not be attributed to endotoxic effects, because heat treatment of the cytotoxin preparation for 60 minutes at 60 C resulted in complete loss of cytotoxicity. We conclude that sublytic doses of heat-labile, hemolytic cytotoxic substances produced by App depress alveolar macrophage function at concentrations likely to develop in association with acute pulmonary infection with App. The Apx (A pleuropneumoniae Rtx toxins) exotoxins secreted by the bacteria into culture medium were considered responsible for the toxic activity of the cytotoxin preparation. The Apx of the App field strain used in this study were likely to be similar to those of serotype-1 reference strain (S4707). Analysis by use of DNA-DNA hybridization indicated that genomic DNA of the field strain contained sequences similar to those encoding structural protein of ApxI (apxIA) and ApxII (apxIIA) of the serotype-1 reference strain. Therefore, Apx produced by the field strain of App used in this study are likely to be of similar pathogenic importance worldwide.