We investigated the ability of an antibody-specific, O antigen-based ELISA to document Salmonella typhimurium herd infections by screening of milk samples. Three cattle populations, 20 herds with no history of salmonellosis, 8 herds with history of S. typhimurium episodes within the previous 7 months, and 220 herds of unknown disease status, were tested. A herd was considered ELISA positive if at least 5% of the cows had OD values > 0.3. Among the 20 herds without history of salmonellosis, only 2 herds were ELISA positive, whereas all 8 herds with a known history of salmonellosis were ELISA positive (herd specificity, 0.9 and herd sensitivity, 1.0). A significant correlation (P < 0.001) was found between the OD values of serum and milk samples from cows in the herds with a history of salmonellosis. It was concluded that ELISA testing of individual milk samples can be used for surveillance of herds for S. typhimurium infections, but further modifications are needed to test bulk tank milk samples.

A monoclonal antibody (MAB) against equine tumor necrosis factor-alpha (Eq TNF) was used to investigate the role of TNF in cytokine, eicosanoid, and metabolic responses of Miniature Horses given endotoxin. Plasma concentrations of interleukin 6 (IL-6), lactate, thromboxane A2 metabolite, and prostacyclin metabolite (6-keto-PGF(1 alpha)) were measured in 10 Miniature Horses given 0.25 micrograms of lipopolysaccharide (LPS; Escherichia coli O55:B5)/kg of body weight. Five horses were given Eq TNF MAB and 5 were given isotype-matched MAB as control. All horses were given 1.86 mg of antibody/kg by IV infusion, 5 minutes before LPS was given IV. Blood samples were taken 20 minutes before and at multiple intervals for 24 hours after LPS was given. Interleukin 6 bioactivity in plasma was measured, using IL-6-dependent cell line (B9). Eicosanoid activities were assessed by enzyme immunoassay, and plasma lactate concentration was determined enzymatically. Data were analyzed by ANOVA and Tukey's honest significant difference test for significant (P < 0.05) effect of treatment. Horses given Eq TNF MAB had significantly (P < 0.050) lower peak mean +/- SEM IL-6 (59 +/- 29 U/ml), lactate (16 +/- 2.00 mg/dl), and 6-keto-PGF(1 alpha) (254 +/- 79 pg/ml) values then did horses given control MAB (880 +/- 375 U/ml for IL-6; 26 +/- 0.04 mg/dl for lactate; and 985 +/- 290 pg/ml for 6-keto-PGF(1 alpha)). There was no effect of anti-TNF treatment on LPS-induced thromboxane A2 metabolite production. Tumor necrosis factor mediated IL-6, lactate, and prostacyclin responses, without affecting thromboxane production in horses given LPS.

Receptors for opsonins, such as complement component C3b (CR1) and immunoglobulins, Fc receptors, interact with adhesion glycoproteins in mediating immune functions. Defects in expression of the adhesion glycoproteins CD11/CD18 results in severely hampered in vitro and in vivo adherence-related functions of leukocytes. Little is known regarding the effect of leukocyte adhesion deficiency (LAD) on ligand binding and receptor expression. We investigated the binding and expression of CR1 and Fc receptors by bovine neutrophils isolated from dairy calves suffering from LAD, compared with clinically normal (hereafter referred to as normal) age-matched calves. Neutrophils were also assayed for endogenously bound IgG and IgM and for exogenous binding of C3b, IgG1, IgG2, IgM, and aggregated IgG (aIgG), using flow cytometry. Luminol-enhanced chemiluminescence (CL) production in response to IgG2 opsonized zymosan was studied, and specific inhibition of CL was used to determine the specificity of IgG2 binding. Activation of protein kinase C with phorbol myristate acetate was used to determine the effect of cellular activation on expression of CR1. A greater percentage of neutrophils from normal calves bound C3b than did neutrophils from LAD-affected calves. Receptor expression was similar. Activation with phorbol myristate acetate resulted in increased expression of CR1 on neutrophils from normal and LAD-affected calves, but expression was almost twofold greater on neutrophils from normal calves. There was no difference between LAD-affected and normal calves in percentage of neutrophils that bound endogenous IgG and IgM. A greater percentage of neutrophils from normal calves bound exogenous IgM than did neutrophils from LAD-affected calves. Receptor expression for aIgG was greater on neutrophils from LAD-affected calves than on those from normal calves. Luminol-enhanced CL of neutrophils in response to IgG2 opsonized zymosan was not different between LAD-affected and normal calves. Results indicate increased binding and expression of Fc receptors for aIgG and decreased binding and expression for C3b and IgM on neutrophils from calves with LAD. Leukocyte adhesion deficiency may be compounded by added defects in the expression and binding of receptors for opsonins, such as C3b and IgM.

A method was developed to evaluate frequent milking as a means of controlling intramammary infection. An artificial intramammary environment was used to determine growth responses of Escherichia coli (P4) to natural changes in the mammary gland resulting from bacterial invasion. Physical conditions manipulated in this model were growth medium, temperature, and oxygen tension. Mathematical modeling was then incorporated to generate predictions concerning growth dynamics of the organism when milking frequency was changed. To test accuracy of the model, initial predictions were derived from bacterial growth data in which E coli was incubated in tryptose soy broth for 12 hours at 37 C and PO2 equal to 23.3 mm of Hg. These predictions matched closely with experimental data in which 12-, 4-, and 2-hour milking intervals were simulated in the artificial intramammary environment. The mathematical model was then used to characterize growth rate data from in vitro experiments in ultra-high temperature-treated milk and in vivo experimental infection data generated with E coli (P4). Predictions generated from this model suggested that increasing milking frequency to 4 or 6 times daily controls growth of E coli for a prolonged period and that 12 times daily milking may lead to elimination of the bacterium.

Cardiopulmonary responses were evaluated in 12 dogs undergoing endoscopy (gastroscopy and enteroscopy). Constant endoscopic insufflation was used to distend the stomach and small intestine for 30 minutes in groups of small (< 10 kg n = 4), medium (10 to 20 kg n = 4), and large (> 20 kg n = 4) dogs. Cardiopulmonary measurements within groups prior to gastric distention (preendoscopy) were compared with postendoscopy measurements and with those made during endoscopy. After distending the stomach and small intestine, increased luminal pressure within the body of the stomach and in the descending duodenum (P < 0.05) and increased abdominal girth (P < 0.05) were observed, with the greatest changes in small dogs. Caudal vena cava pressures and mean arterial and pulmonary artery pressures increased (P < 0.05) during endoscopy. Cardiac index varied, with small dogs having greater cardiac index (P < 0.05) during endoscopy, compared with that in medium and large dogs. Minute volume remained unchanged during insufflation, despite a decrease in tidal volume (P < 0.05), because of an increase in respiratory rate (P < 0.05). Arterial blood gas analysis revealed a mild, mixed metabolic/respiratory acidosis in all groups. Although cardiopulmonary changes associated with gastrointestinal tract endoscopy were common, the changes were often small and of little clinical significance.

The role of interleukin 1 (IL-1) as an inflammatory mediator during mastitis and the therapeutic effect of recombinant human IL-1 receptor antagonist (IL-1ra) for bovine mastitis was studied. Cows were intramammarily infused with lipopolysaccharide (25 g) in 1 mammary gland. Half the cows also received infusions of 5 mg of IL-1ra into the same mammary gland just prior to endotoxin infusion and 4, 8, and 12 hours later. After endotoxin infusion, tumor necrosis factor and high IL-1 bioactivity were detected in whey from infused glands. Vascular permeability changes and neutrophil accumulation in milk paralleled the appearance of cytokines. A systemic reaction, characterized by pyrexia and an increase in blood cortisol concentration, also were observed. Milk yield was inhibited and milk composition was altered in infused and noninfused glands. The increase in IL-1 bioactivity in milk after endotoxin infusion was almost completely prevented in glands receiving IL-1ra. However, IL-1ra had no effect on local inflammation, systemic reaction, or impairment in productive performance. These results indicate that IL-1 does not mediate its effect within the milk compartment, and suggest either that IL-1 is not critical to the mastitic response or that intramammary infusion of IL-1ra does not place the antagonist where IL-1 interacts with its receptor.

A series of experiments was conducted to document tumor necrosis factor-alpha (TNF) activity in serum of swine after inoculation with Salmonella spp endotoxin and after oral or respiratory tract challenge exposure with live Salmonella spp. For experiment 1, a potentially lethal dose of S typhimurium endotoxin (25 microgram/kg of body weight) was administered IV, and serum TNF activity was measured. High TNF (approx 700 IU/ml) activity at 1 to 2 hours after administration of the inoculum was associated with death, whereas lower TNF (approx 30 IU/ml) activity was associated with a general prolonged state of shock. For experiment 2, pigs were administered a nonlethal dose (5 microgram/kg, IV) of either S typhimurium or S choleraesuis endotoxin. Difference in the ability to induce porcine serum TNF activity was not observed between strains. During experiment 3, pigs were inoculated with 104 colony-forming units of S typhimurium chi4232 either orally by gelatin capsule (GC) or by intranasal (IN) instillation. A late serum TNF response (17 IU/ml) was measured at 6 weeks after IN inoculation. A serum TNF response was not detected in GC-inoculated pigs. All tissues and feces were test-negative for S typhimurium prior to the 6-week TNF response. Serum TNF activity may be related to clearance of S typhimurium after respiratory tract exposure, but it is not important to or indicative of clearance of orally presented S typhimurium in swine. During experiment 4, pigs were inoculated with 106 colony-forming units of S typhimurium chi4232 similarly as for experiment 3. Challenge exposure with this medium-size dose of inoculum induced a prolonged peak serum TNF response (37 IU/ml) between 2 and 4 weeks after IN inoculation. Again, serum TNF activity was not detected in GC-inoculated pigs. Data suggest that clearance of a medium-size dose (106) of inoculum may be influenced by the prolonged higher serum TNF activity. For experiments 5 and 6, pigs were inoculated IN with 103, 106, 108, or 109 S choleraesuis chi3246. A measurable, yet statistically nonsignificant, serum TNF response was observed for all doses. Pigs inoculated by GC with 108 S choleraesuis chi3246 had similar results. High does (> 106) of live S choleraesuis were associated with clinical signs of endotoxic shock. Clearance of S choleraesuis, or lack thereof, did not correlate with serum TNF activity.

Blood ionized calcium (Ca2+) and pH; plasma lactate concentrations; and total protein, total calcium (CaT), albumin, and phosphorus concentrations in serum were determined in 40 healthy horses before (T1), at the finish line (T2), and 10 minutes after the finish (T3) of the cross-country phase of a 3-dayevent competition. Mean (+/- SEM) Ca2+ concentrations decreased from 6.22 +/- 0.04 mg/dl at T1 to 5.04 +/- 0.07 mg/dl at T2 (P less than or equal to 0.05). This decrease was accompanied by a nonsignificant increase in CaT between T1 and T2. The mean (+/- SEM) percent ionization of calcium decreased significantly (P less than or equal to 0.05), from 50.9 +/- 2.75% at T1 to 40.3 +/- 3.58% at T2. Significant increases in mean albumin, total protein, phosphorus, and lactate concentrations and a significant decrease in mean pH were observed at T2 (P less than or equal to 0.05). At T3, mean Ca2+ and percent ionization had increased, but remained significantly less than resting values. Mean CaT was significantly decreased at T3, compared with values at T1 and T2. Correlation of mean Ca2+ concentration with all other measured variables at each time was evaluated; correlation coefficients between mean Ca2+ and all other variables were low (r2 less than or equal to 0.38), indicating low biological significance.

Uptake of ferritin by M cells in follicle-associated epithelium at various sites in the small and large intestines was examined in 4 healthy 5-week-old pigs by use of electron microscopy. A 2.5% solution of ferritin in saline was injected into ligated loops of the jejunum and ileum containing aggregations of lymphoid follicles (Peyer's patches), as well as into intestinal loops containing lymphoglandular complexes at the ileocecal junction, in the central colonic flexure, and in the rectum. As negative control, saline solution was injected into loops at identical localizations. After an exposure period of 2 hours, uptake of ferritin by M cells, but not by enteroabsorptive cells of the small and large intestines, was observed. Numbers of M cells with ferritin and total M cells were counted and the percentage was calculated. Total number of M cells was highest in lymphoglandular complexes in the rectum and lowest on domes of the ileal Peyer's patch. High numbers of M cells with ferritin were found on domes of the jejunal Peyer's patch, and in lymphoglandular complexes at the ileoceral entrance and in the rectum. Only a few M cells on domes of the ileal Peyer's patch and in lymphoglandular complexes in the central colonic flexure contained ferritin. The percentage of M cells with internalized ferritin was similar on domes of the ileal Peyer's patch, and in lymphoglandular complexes at the ileocecal junction and in the rectum. It was higher on domes of the jejunal Peyer's patches and lower in lymphoglandular complexes of the central colonic flexure. Ferritin was found in the apical tubulovesicular system, multivesicular bodies, and a few vacuoles in the central area of M cells. Ferritin was exocytosed into the lateral intercellular spaces next to M cells. Uptake of ferritin by intraepithelial cells in the follicle-associated epithelium could not be documented, but ferritin was present in vesicles of subepithelial macrophages.

Release of enzymes from cytoplasmic granules has been postulated to have a major role in neutrophil-mediated tissue injury. Secretion or release of primary granules, specific granules, and cytosolic enzymes by bovine neutrophils was examined by quantifying the release of beta-glucuronidase, B12-binding protein, and lactate dehydrogenase, respectively, in response to predetermined amounts of phorbol myristate acetate, calcium ionophore, and opsonized zymosan. These responses were compared with the enzyme release induced by exposure to live or dead, unopsonized or opsonized Pasteurella haemolytica. The greatest release of beta-glucuronidase, B12-binding protein, and lactate dehydrogenase was observed in neutrophils exposed to live organisms partially because of neutrophil lysis. Bovine neutrophils respond markedly to particulate agonists, live or dead, pathogenic or nonpathogenic, by a selective release of specific granules, an effect enhanced by opsonization. Particulate agonists induce minimal primary granule release other than that induced by cell death. Because bovine neutrophils contain quantitatively high numbers of specific granules, the high rate of secretion/ release in response to P haemolytica organisms could have a major role in the tissue responses that characterize the lesions of pneumonic pasteurellosis.