Cytologic examination of bronchoalveolar lavage fluid (BALF), including phenotypic analysis of lymphocytes, was performed on 32 Standardbreds with poor race performance and endoscopic examination findings characteristic of inflammatory airway disease (IAD). Nucleated cell counts in BALF from IAD-affected horses were higher than those in control horses; the cytologic profile of BALF in affected horses included mixed inflammation, characterized by mild neutrophilia, lymphocytosis, and monocytosis. Eosinophil and mast cell counts were not higher in the IAD-affected group, compared with those in the control group; however, 4 IAD-affected horses had marked eosinophilia (24.7 +/- 4.8% SEM) in BALF. Phenotypic analysis of lymphocytes in BALF obtained from IAD-affected horses revealed a low proportion of CD4-positive cells and B cells, compared with those in the control group; these findings may have been representative of a greater proportion of non-B, non-T cells (null cells) in horses with IAD. The cytologic profile of BALF obtained from horses with IAD differed from that in horses affected with chronic obstructive pulmonary disease, suggesting that the pathogenesis of inflammation in horses with IAD may differ from that of chronic obstructive pulmonary disease.

Equine neutrophil antibody was raised in rabbits inoculated with equine neutrophils isolated to purity > 99.0%, using Percoll density-gradient sedimentation. Neutrophil antibody was detected by use of agar gel diffusion, leukoagglutination, indirect immunofluorescence, staphylococcal protein A and streptococcal protein G binding, and phagocytic inhibition techniques. Precipitin lines and leukoagglutination were seen in antiserum dilutions of 1:4 and 1:64, respectively. The specific nature of leukoagglutination was characterized by the formation of rosette-like clumps of neutrophils. Specific bright membranous fluorescence was seen in neutrophils treated with the antiserum and exposed to fluorescein-conjugated goat anti-rabbit immunoglobulin, and staphylococcal protein A and streptococcal protein G. Whereas the indirect immunofluorescence and protein G-binding tests were equally sensitive and resulted in titer of 1:256, the protein A-binding test was less sensitive and resulted in titer of only 1:32. Nonspecific binding of protein A and protein G was noticed as uniform or patchy cellular fluorescence in a small number of neutrophils. Treatment of neutrophils with antiserum up to dilution of 1:8 resulted in a significant (P < 0.05) suppression of phagocytosis of opsonized zymosan particles. Thus, protein G-binding and indirect immunofluorescence tests are highly sensitive to detect neutrophil antibody and may be used to diagnose immune-mediated neutropenias in horses and, possibly, in other animal species.

A study was conducted to develop valid estimates of lymphocyte count (LC; cells per microliter) of individual, clinically normal dairy cattle. Estimated weighted regression was used on repeated measures of individual LC to examine 6 models predicting LC as a function of age in cattle not infected with bovine leukemia virus. The generalized growth curve model of analysis of variance was used to estimate intercepts, slopes, and prediction limits for the models and to compare the LC-to-age relationship between Holstein and Guernsey breeds. The best-fitting model (P = 0.0001) with the narrowest prediction interval was LC = 4,414.4 - 84.6X, where X = (age - 48) if age less than or equal to 48 months, and X = 0 if age > 48 months, and 163.6 and 8.1 are the SE of the estimates, respectively. Upper one-sided 95%-predicted normal LC tended to be higher than estimates derived from traditional hematologic keys that use confidence limits of mean LC. Difference was not found in the LC-to-age relationship between the Holstein and Guernsey cattle (P = 0.67). Results of this study provided estimates of normal LC that are more specific in diagnosing lymphocytosis in individual cattle.

The cytotoxic effect of Moraxella bovis 118F on bovine neutrophils was evaluated and characterized by use of a 51Cr release assay. Neutrophils harvested from healthy adult cattle were labeled with 51Cr. The leukocidic activity produced by M bovis 118F, a hemolytic strain of M bovis, was heat-labile. A live culture of strain 118F, at a ratio of 100 bacteria/neutrophil, released 97.7% of the 51Cr from labeled neutrophils. Neither a heat-killed preparation of M bovis 118F nor a live or heat-killed preparation of M bovis IBH63 (a nonhemolytic and nonpathogenic strain) induced significant (P > 0.05) release of 51Cr. Moraxella bovis 118F broth culture filtrates prepared for evaluation of leukocidic activity also were evaluated for hemolytic activity. These 2 toxic activities had several characteristics in common. Both were filterable, heat-labile, produced by a hemolytic strain, and were released during early logarithmic phase growth from broth cultures. Leukocidic and hemolytic activities were protected from degradation by phenylmethyl sulfonyl fluoride, a serine protease inhibitor. Leukocidic and hemolytic activities were dependent on calcium ions. Filtrate resulted in 54.1% 51Cr release from labeled neutrophils and contained 646.7 hemolytic U/ml, respectively, when saline (0.85% NaCl) + 10 mM CaCl2 solution was used as diluent. Neither saline solution nor saline + 10 mM MgCl2 solution supported leukocidic or hemolytic activity. Serum, obtained from several calves 10 to 38 days after M bovis inoculation, substantially neutralized leukocidic and hemolytic activities, compared with paired preinoculation serum samples. In addition, no significant difference (P > 0.05) was detected when the ability of each calf's postinfection serum to neutralize leukocidic activity was compared with the ability of the serum to neutralize hemolytic activity.

Six primiparous Holstein cows were fed a Se-deficient diet, beginning at least 90 days before their first calving, and 6 other primiparous cows were given the same diet plus a supplement of 2 mg of Se/cow/d as sodium selenite. All cows were fed their diets for the duration of the experimental period. One uninfected quarter of each cow was injected with 25 microgram of Escherichia coli endotoxin at postpartum week 5. Leukocytes were isolated by centrifugation from milk collected at postinjection hour 16. Isolated cells were 92 +/- 3% neutrophils and were incubated with Staphylococcus aureus or E coli in a 1:300 ratio. Phagocytosis and intracellular killing by neutrophils were assessed after 0, 30, 60, and 90 minutes by a fluorochrome assay, using acridine orange. Viability of neutrophils was assessed by use of trypan blue. Superoxide anion production and hydrogen peroxide production by neutrophils also were determined. Cows fed Se-deficient diets had significantly (P < 0.05) lower blood Se concentration and blood glutathione peroxidase activity than cows fed Se-supplemented diets. Selenium status had no effect on the phagocytic capacity of neutrophils. Neutrophils obtained from cows fed Se-supplemented diets killed a significantly (P < 0.05) higher percentage of ingested bacteria than did neutrophils from cows fed the Se-deficient diet. Viability was significantly (P < 0.05) reduced by incubation with S aureus in neutrophils from both groups of cows, with neutrophils from Se-deficient cows having lower viability. Superoxide anion production did not differ significantly between neutrophils from the 2 groups, but extracellular hydrogen peroxide concentration was significantly (P < 0.05) higher in neutrophils harvested from milk of cows fed the Se-deficient diet.

A study was conducted to examine sources of variation introduced into a phagocytosis assay as a result of the isolation of neutrophils from bovine blood, including variation attributable to isolation of neutrophils from blood, variation between duplicate determinations of percentage phagocytosis, and the variation in the ability of neutrophils isolated from blood (over repeated collections from the jugular vein) to phagocytose. For the phagocytosis assay, jugular venous blood from each of 4 cows was divided into 2 equal portions. The neutrophils were isolated by lysis of red blood cells with 0.2% sodium chloride. The neutrophils (2 X 10(7)) were incubated in duplicate with 32P-labeled Staphylococcus aureus ([32P]SA; 2 X 10(8)) inskimmed milk samples (2.5% final concentration) prepared from 4 cows. This process was repeated thrice on neutrophils isolated from 4 cows at 2-week intervals. The proportions of variation in percentage of 32P-labeled S aureus phagocytosed between duplicate neutrophil isolations and between duplicate assay determinations were 0 and 1%. Differences among skimmed milk sources and among runs, using blood neutrophils taken at different times from the same donor cow, accounted for 62 and 36% of the total variation. The results indicated that variation arising from blood neutrophil isolation introduced into a phagocytosis assay within a single-day trial is of no concern. The large variation among skimmed milk sample sources indicated differences among cows in the ability of their milk to support phagocytosis. The variation in neutrophil isolations over time for any cow was considered too large to allow for evaluation of physiologic and environmental effects on phagocytosis of neutrophils isolated from blood.

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.

Canine circulating neutrophils, isolated by a blood lysing technique, were incubated with 7-S nerve growth factor (NGF), at final concentrations between 12.5 and 800 ng/ml, for 30 minutes at 37 C. Neutrophil cytosolic H2O2 production, measured by flow cytometry, after 7-S NGF incubation was not significantly different from that produced at 37 C (baseline temperature controls) alone. Phorbol myristate acetate (PMA; 100 ng/ml) stimulation of neutrophils produced cytosolic H2O2 concentrations almost 13 times that of baseline temperature control neutrophils. Preincubation of neutrophils with 7-S NGF (100 to 800 ng/ml, 30 minutes, 37 C) and subsequent stimulation by PMA resulted in augmented H2O2 production in excess of twice that of neutrophils treated with PMA alone, and almost 30 times that of baseline temperature controls.

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.