Haemophilus somnus-induced interference with bovine neutrophil functions

The effect of Haemophilus somnus on bovine polymorphonuclear leukocyte (PMN) function was examined in vitro with whole cells and fractions extracted from the surface of this bacterium. The ability of PMNs to iodinate protein and ingest Staphylococcus aureus was significantly inhibited in the presence of live cells, heat-killed whole cells or supernatant fluid from heat-killed cells, but not in the presence of washed, heat-killed cells. None of the fractions inhibited nitroblue tetrazolium (NBT) reduction by PMNs. The PMN inhibitory factors were further characterized. The material that inhibited S. aureus ingestion was found to be a heat-stable cell surface material of greater than 300 000 MW. The fraction inhibiting iodination of protein was found to be less than 10 000 MW.     

Pasteurella haemolytica leukotoxin: chemiluminescent responses of peripheral blood leukocytes from several different mammalian species to leukotoxin- and opsonin-treated living and killed Pasteurella haemolytica and Staphylococcus aureus

A luminol-dependent chemiluminescence (LDCL) assay was used to assess the response of polymorphonuclear leukocyte (PMN) preparations from 4 species of ruminants (ie, cattle, sheep, goats, and antelopes) and 6 species of nonruminants (ie, swine, dogs, cats, rabbits, horses, and persons) to both opsonized and nonopsonized preparations of living and heat-killed Pasteurella haemolytica and Staphylococcus aureus and to opsonized and nonopsonized heat-killed strains of each bacterium in the presence of sterile culture supernatant (leukotoxin) from P haemolytica. The LDCL responses of PMN preparations from each of the species studied were greater for living than for heat-killed S aureus. The most efficient LDCL emission was observed with reaction mixtures containing opsonized living S aureus. Regardless whether they contained killed or living bacteria, the opsonized S aureus preparations elicited LDCL emissions more efficiently than did the corresponding nonopsonized preparations. Living P haemolytica cells and their sterile culture supernatant inhibited the LDCL emissions of phagocytically stimulated PMN preparations from ruminants, but not those from nonruminants. The LDCL response of ruminant PMN to nonopsonized living P haemolytica was characterized by the development of a peak response at 10 minutes of incubation followed by a precipitous decrease and a subsequent complete cessation of chemiluminescence. The peak LDCL response was higher for opsonized living P haemolytica than for nonopsonized living bacteria, and the increased response lasted longer. However, opsonization of living P haemolytica with the serum samples tested only temporarily spared the ruminant PMN preparations from the detrimental effects of leukotoxin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Chemotactic response of equine polymorphonuclear leucocytes to Streptococcus equi

Streptococcus equi infection in horses is characterised by intense infiltration of lymph nodes by polymorphonuclear leucocytes (PMNs) suggesting a potent chemotactic response to the organism or its products. Equine PMNs were separated using Ficoll-Hypaque medium and used in an assay of chemotaxis under agarose to study the components of S equi involved in this response. Results showed that complement-derived chemotactic factors generated by activation of the alternative complement pathway were important in chemotactic responses to S equi. Both whole bacteria and peptidoglycan preparations were potent complement activators, whereas purified M protein was less active. In contrast, S equi culture supernatant protein did not activate complement; instead it directly inhibited migration of PMNs. Moreover, PMNs, when incubated with culture supernatant of a non-haemolytic strain, showed signs of cellular degeneration suggesting the presence of a cytotoxin distinct from haemolysin.     

Depression of polymorphonuclear leukocyte function associated with experimentally induced Escherichia coli mastitis in sows

In a study of susceptibilities of sows from 2 herds to experimentally induced Escherichia coli mastitis, a marked difference was seen. The "susceptible" sows were from a conventional herd and "resistant" sows were from a specific-pathogen-free herd. The purpose of the study was to determine whether deficient neutrophil function was associated with increased susceptibility to E coli-induced mastitis. Four in vitro procedures were used to evaluate polymorphonuclear leukocyte (PMN) function: (i) random migration under agarose, (ii) ingestion of 125I-iododeoxyuridine-labeled Staphylococcus aureus, (iii) quantitative nitroblue tetrazolium reduction, and (iv) iodination. After parturition and intramammary inoculation with E coli, sows from the susceptible herd were neutropenic and the neutrophils which were present in the peripheral blood had reduced function. Specifically, there were depressed random migration under agarose, S aureus ingestion, and iodination when compared with PMN function in resistant sows. These data indicate that susceptibility to E coli mastitis was associated with deficiencies in PMN numbers and function. Potential causes of the neutrophil dysfunction are discussed and include possible systemic hormonal aberrations or the presence of an inapparent viral or bacterial infection.     

Bovine pneumonic pasteurellosis: chemiluminescent response of bovine peripheral blood leukocytes to living and killed Pasteurella haemolytica, Pasteurella multocida, and Escherichia coli

A luminol-dependent chemiluminescence (LDCL) assay was used to evaluate the response of bovine polymorphonuclear leukocytes; (neutrophils [PMN]) to living and heat-killed Escherichia coli, Pasteurella multocida (type A, serotype 3), and P haemolytica (biotype A, serotype 1), and to heat-killed P haemolytica and sterile culture supernatant from living P haemolytica. Control cultures containing PMN that had not been phagocytically stimulated with bacteria had a modest increase in LDCL during the initial 10 minutes of incubation, followed by a gradual decline throughout the 120-minute incubation period. Bovine PMN emitted LDCL more efficiently when the cells were exposed to living E coli or P multocida than when they were exposed to the same bacteria killed by heat. The mean LDCL values for reaction mixtures containing living E coli or P multocida peaked at 30 minutes of incubation and remained above values for mixtures containing the same heat-killed bacteria. Kinetics of the LDCL response of bovine PMN to heat-killed P haemolytica were similar (although reduced in amplitude) to that observed with killed E coli or P multocida. The LDCL response of bovine PMN to living P haemolytica was not like that for E coli or P multocida, and was characterized by the development of a peak response at 10 minutes followed by a precipitous decrease in responsiveness and a subsequent complete cessation of LDCL. Addition of sterile culture supernatant from living P haemolytica to test samples containing heat-killed P haemolytica induced a response similar to that obtained with the living microorganism.(ABSTRACT TRUNCATED AT 250 WORDS)     

Association of increased estradiol and progesterone blood values with altered bovine polymorphonuclear leukocyte function

Polymorphonuclear leukocyte (PMN) function and serum concentrations of estradiol, progesterone, and cortisol (hydrocortisone) were monitored concurrently in clinically normal cows during the estrous cycle. Five parameters were used to evaluate PMN function: (i) random migration under agarose, (ii) ingestion of 125I-labeled Staphylococcus aureus, (iii) nitroblue tetrazolium (NBT) reduction, (iv) iodination, and (v) antibody-dependent cell-mediated cytotoxicity. Increased serum estradiol concentrations were associated with enhanced random migration, but had no apparent effect on NBT reduction, iodination, or ingestion of S aureus by bovine PMN. Increased serum estradiol was also associated with increased serum cortisol. Increased serum progesterone values were associated with a depression of NBT reduction and iodination by PMN, but with enhanced random migration and antibody-dependent cell-mediated cytotoxicity. These results indicate that physiologic changes in steroid hormone values during the normal estrous cycle of the cow are associated with alterations in PMN function.     

Effects of ACTH administration on bovine polymorphonuclear leukocyte function and lymphocyte blastogenesis

Yearling steers were treated with ACTH to determine the effect of increased plasma cortisol concentration on bovine lymphocyte and polymorphonuclear leukocyte (PMN) function. The administration of ACTH caused a significant (P less than 0.01) increase in serum cortisol concentration and depression of lymphocyte blastogenesis in response to phytohemagglutinin and concanavalin A. The response to pokeweed mitogen was also depressed, but not significantly. Random migration by PMN was significantly enhanced by ACTH treatment, but there was no effect on ingestion of Staphylococcus aureus, nitroblue tetrazolium reduction, or antibody-dependent cell-mediated cytotoxicity by PMN. The iodination reaction, which evaluates the activity of the myeloperoxidase-hydrogen peroxide-halide antibacterial system of the PMN, was significantly impaired after ACTH treatment. These data indicate that specific parameters of lymphocyte and neutrophil function were impaired directly or indirectly by elevated in vivo concentrations of plasma cortisol.     

Evaluation of bovine polymorphonuclear leukocyte function

Bovine polymorphonuclear leukocytes (PMNs) were isolated from the peripheral blood of cattle. Five in vitro procedures were utilized to evaluate PMN function: 1) Random migration under agarose, 2) Ingestion of ¹²⁵I-iododeoxyuridine labeled $\text{Staphylococcus aureus}$, 3) Quantitative nitroblue tetrazolium reduction, 4) Chemiluminescence and 5) Iodination. Normal values for bovine PMNs are reported and interpretation of results is discussed. The PMN function tests were designed so that all 5 procedures may be performed in a short period of time on the same cell preparation. This allows for the detection and partial characterization of a potential PMN dysfunction.     

Repeatability of flow cytometric and classical measurement of phagocytosis and respiratory burst in bovine polymorphonuclear leukocytes

Five methods for measurement of phagocytosis and respiratory burst activity of bovine blood polymorphonuclear leukocytes (PMNs) were evaluated. Eight cows were repeatedly sampled over a two week period and parallel samples tested in all five assays to assess the repeatability and stability of the methods. In the flow cytometric phagocytosis assay, ingestion of fluorescein labeled bacteria was measured, and in the flow cytometric assay for respiratory burst, oxidation of a dye by reactive oxygen species was recorded. In the classical assays, bactericidal effect on opsonized, live bacteria was quantified by the conversion of an indicator substance, superoxide anion production was assayed by the reduction of cytochrome c, whereas myeloperoxidase activity was determined with a radioactive iodination assay. The results showed that the Phagotest, Bursttest, cytochrome c and iodination assays gave repeatable results when samples were run in the same setup on the same day. Although day-to-day variability was significant in all assays, the described methods comprise a panel of useful tests for the evaluation of phagocytosis and respiratory burst activity in bovine PMNs. The flow cytometric methods represent a convenient alternative to the classical methods for measurement of phagocytosis and respiratory burst in bovine blood PMNs.     

Determination of phagocytosis of 32P-labeled Staphylococcus aureus by bovine polymorphonuclear leukocytes

A procedure for the measurement of phagocytosis by bovine polymorphonuclear leukocytes (PMN) of 32P-labeled Staphylococcus aureus was modified so that a larger number of samples could be compared in a single run, and smaller volumes of sample, PMN, and 32P-labeled S aureus could be used. Results were highly reproducible, with a coefficient of variation between duplicate determinations of less than or equal to 2%. Lysostaphin was prepared from the supernatant of S staphylolyticus and was compared with a commercially available preparation. Effects of lysostaphin on PMN and influence of incubation media on release of 32P from 32P-labeled S aureus by lysostaphin were examined.     

In vitro bactericidal efficacy of equine polymorphonuclear leukocytes against Corynebacterium equi

Polymorphonuclear leukocytes from adult horses were separated from whole blood, using a 2-step Percoll gradient, and were tested for bactericidal function against Corynebacterium equi. Staphylococcus aureus, an organism against which equine neutrophils have proved efficacy, was a positive control. The percentage of uptake after a 15-minute preincubation of the neutrophils and bacteria in the presence of normal horse serum was also calculated. The results indicated that equine neutrophils effectively phagocytosed and killed C equi and S aureus. The percentage of uptake for S aureus (95% +/- 3%) was greater than that for C equi (85% +/- 6%) (P less than 0.001), but the bactericidal efficacy was equivalent. More than 90% of the ingested or attached bacteria were destroyed during the 3-hour incubation period (mean percentage of C equi killed = 96 +/- 2%; mean percentage of S aureus killed = 91 +/- 8%). These results indicated that a failure of bacterial killing by neutrophils is unlikely to be important in the pathogenesis of C equi pneumonia in the horse.     

Effects of bovine mammary secretion during the early nonlactating period and antibiotics on polymorphonuclear neutrophil function and morphology.

The effect of bovine mammary secretion during the early nonlactating period and of antibiotic preparations on bovine polymorphonuclear neutrophil (PMN) phagocytic function and morphology were evaluated in a series of in vitro multifactorial experiments. Benzathine cloxacillin (CL), benzathine cephapirin (CE), sodium novobiocin (NO), and a combination of dihydrostreptomycin with procaine penicillin G (DP) were prepared in the presence and absence of a peanut oil aluminum monostearate vehicle. The PMN were isolated from bovine blood, and the effect of each antibiotic preparation on PMN function and morphology was evaluated in a buffer, fat, skin, and a combination of fat with skim from bovine mammary secretion during the nonlactating period. The fat and skim were diluted with buffer to approximate their concentration in mammary secretion. Phagocytic functions of PMN were monitored by fluorescent microscopy, which made it possible to estimate both ingestion and intracellular killing of bacteria by PMN. Changes in PMN morphology were monitored by transmission electron microscopy. The ability of PMN to ingest and kill Staphylococcus aureus ATCC 25923 was significantly decreased by fat, skim, CL, CE, NO, and DP. Effects of some antibiotics on ingestion and killing of bacteria by PMN were influenced by the addition of vehicle and by interactions with mammary secretion. Neutrophil morphology was altered by fat, skim, CL, CE, NO, and DP. The detrimental effects of CL, CE, NO, and DP on PMN morphology were influenced (some significantly) by the presence of vehicle and interactions with mammary secretion. There were significant correlations among secretion- and antibiotic-induced changes in PMN ingestion of bacteria, PMN killing of bacteria, and PMN morphology.     

Effects of antibiotics on phagocyte recruitment, function, and morphology in the bovine mammary gland during the early nonlactating period

The effects of 2 antibiotic preparations administered intramammarily on phagocyte recruitment, function, and morphology were evaluated at the beginning of the nonlactating period. Twelve cows with no clinical or microbiologic evidence of mastitis were assigned to 1 of 2 treatment groups. At the end of lactation, 1 of the antibiotic preparations was infused in a fore- and hind quarter of each cow; the remaining quarters were untreated controls. One group was given benzathine cephapirin; the second group was given sodium novobiocin. Secretion samples were collected from 1 treated and 1 control quarter at 16 hours, and from the remaining 2 quarters at 64 hours after treatment. Total and differential somatic cell counts were determined, and morphology of mammary polymorphonuclear neutrophils (PMN) and macrophages was observed by transmission electron microscopy. In vitro ingestion and killing of Staphylococcus aureus by mammary PMN and macrophages were assessed by fluorescent microscopy, using acridine orange stain. Cells resident in a fixed volume of secretion were incubated with a known concentration of S aureus. Total cell and PMN concentrations were higher in treated than in control quarters. Neutrophils were the predominant cell type in both treated and control quarters over the sampling period. As measured in this study, in vitro ingestion and killing of S aureus by individual PMN from treated quarters was reduced. Antibiotic treatment also increased the proportion of morphologically abnormal phagocytes. There were significant correlations among PMN ingestion, killing, and morphology. However, increased PMN concentrations tended to compensate for the reduced phagocytic function of individual cells. Therefore, efficacy of antibiotic treatment of nonlactating cows may depend, at least in part, on increased PMN concentration, which may tend to compensate for reduced phagocytic function. Compared with PMN, macrophages appeared to have only a minor role in phagocytosis of bacteria.     

Measurement of phagocytosis of 32P-labeled Staphylococcus aureus by bovine leukocytes: lysostaphin digestion and inhibitory effect of cream.

A procedure to measure phagocytosis by blood and milk neutrophils was developed. One milliliter of heat-killed 32P-labeled Staphylococcus aureus ([32P]SA) (180-200 X 10(6) CFU), 1 ml of phosphate-buffered saline solution (PBSS), and 2 ml of serum, whole milk, skimmed milk, whey, or PBSS were incubated in duplicate for 60 minutes at 37 C. Isolated blood or milk nuetrophils (polymorphonuclear leukocytes (PMN), 25 X 10(6) cells/ml; 1 ml) were added and incubated at 37 C for 30 minutes. Unphagocytosed [32P]SA organisms were lysed by incubation with 5 ml of lysostaphin (10 U) at 37 C for 30 minutes, and the PMN and phagocytosed T2P]SA were removed by centrifugation. Radioactivity of the supernatant was determined in a scintillation spectrometer and was used in estimate the percentage of [32P]SA phogocytosed. With this procedure, 25 assays in duplicate could be conducted each day with an expected coefficient of variation between duplicates of 5.6%. Blood PMN phagocytosed 80, 44, 74, 72, and 11% of the [32P]SA when incubated in serum, whole milk, skimmed milk, whey, and PBSS, respectively. Mik PMN phagocytosed 78, 44, 72, 74, and 22%, respectively. The addition of cream to either skimmed milk or serum reduced phagocytosis of [32P]SA by both blood and milk PMN. The inhibitory effect of cream was verified by the microscopic observation that PMN containing large quantities of ingested fat contained fewer S aureus. Seemingly, PMN upon entering the alveoli of the mammary gland become less efficiently phagocytic for bacteria, because of the presence of milk fat globules. This phef intramammary infection by invading mastitic pathogens.     

Effects of dexamethasone, levamisole, and dexamethasone-levamisole combination on neutrophil function in female goats

Effects of dexamethasone, levamisole, or combined dexamethasone-levamisole administration on polymorphonuclear neutrophil (PMN) function in healthy, adult female goats were studied. Goats were assigned to treated (n = 6) and control (n = 6) groups. In experiment 1, treated goats were given levamisole (6 mg/kg of body weight, IM). In experiment 2, treated goats were given 0.1 mg of dexamethasone/kg, IV, for 3 consecutive days, 1 mg of dexamethasone/kg, IV, for 6 consecutive days, and 6 mg of levamisole/kg, IM, with a 4th injection of 1 mg of dexamethasone/kg. All injections were administered 12 hours before blood collection. The PMN were evaluated for random migration and chemotaxis under agarose, ingestion of Staphylococcus aureus, cytochrome C reduction, iodination, and antibody-dependent cell-mediated cytotoxicity. Levamisole alone did not alter the function of caprine PMN. Both doses of dexamethasone caused increased random migration and decreased cytochrome C reduction and iodination. Dexamethasone resulted in no changes in chemotaxis, S aureus ingestion, and antibody-dependent cell-mediated cytotoxicity. Random migration and cytochrome C reduction returned toward base line in cells from dexamethasone and levamisole-treated goats. Although iodination activity in cells from dexamethasone-treated goats remained significantly (P less than 0.05) lower than those of controls after levamisole administration, a rebound toward base-line activity occurred.     

Porcine neutrophil function in the presence of virulent and avirulent Salmonella choleraesuis

Porcine polymorphonuclear leukocytes (PMNLs) may be activated by bacteria to begin phagocytosis followed by oxidative and non-oxidative mechanisms of killing. The purpose of this study was to identify differences between virulent and avirulent Salmonella choleraesuis (S. choleraesuis) strains, 38 and 9 respectively, in their interactions with porcine PMNLs using five different assays. (1) Staphylococcus aureus (S. aureus) ingestion was determined by exposure of porcine PMNLs to a mixture of S. choleraesuis and 125I labeled S. aureus. There was a 2.98% and 22.20% decrease in S. aureus ingestion by mouse-avirulent S. choleraesuis 9 and mouse-virulent S. choleraesuis 38 respectively. (2) Iodination of proteins was done by exposing zymosan stimulated porcine PMNLs to S. choleraesuis in the presence of 125I and measuring its incorporation into porcine PMNL proteins. This assay indicated a 73.7% and 74.7% decrease in iodination by S. choleraesuis 9 and S. choleraesuis 38, respectively. (3) Cytochrome c reduction was performed by using porcine PMNLs, zymosan, and S. choleraesuis to determine the bacterial effect on superoxide anion production. S. choleraesuis 9 and S. choleraesuis 38 inhibited superoxide anion production by 78.0% and 92.6%, respectively. (4) Lactoferrin release from porcine PMNLs was measured by an ELISA using the supernatant from the cytochrome c assay. Results indicate a 52.0% and 61.0% increase in lactoferrin release by S. choleraesuis 9 and 38 respectively. (5) The bactericidal assay was performed by counting cfus of S. choleraesuis after preliminary incubation with porcine PMNLs, followed by killing of extracellular S. choleraesuis and lysis of porcine PMNLs. Survival of S. choleraesuis 9 and E. coli (control) were 7.50% and 1.37%, respectively, in contrast to 52.62% survival of the virulent S. choleraesuis 38. These results indicate that both strains inhibited protein iodination and caused a slight increase in lactoferrin release, but the virulent S. choleraesuis 38 inhibited S. aureus ingestion, cytochrome c reduction, and survived porcine PMNL killing more effectively than the avirulent S. choleraesuis 9.     

Enhancement of lymphocyte blastogenesis and neutrophil function by avridine in dexamethasone-treated and nontreated cattle

The lipoidal amine, N,N-dioctadecyl-N',N'-bis (2-hydroxyethyl) propanediamine (avridine or CP 20,961), formulated in liposomes, was evaluated for its effect on leukocyte kinetics, lymphocyte blastogenesis, and polymorphonuclear leukocyte (PMN) function in dexamethasone-treated and nontreated cattle. In the 1st experiment, cattle were given avridine in a single IM injection of 0.1, 1.0, or 10 mg/kg of body weight. All doses induced swelling at the injection site, a febrile response, and a leukocytosis due to a neutrophilia. Mononuclear cell numbers were normal. All 3 groups of avridine-treated animals had a higher mean lymphocyte blastogenic response to mitogens on the 4 days after administration than did the control nontreated animals. Avridine administration was associated with an enhanced ability of PMN to ingest Staphylococcus aureus and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The highest dose (10 mg/kg) was associated with a depression of the ability of PMN to iodinate protein. An effect of avridine on PMN random migration under agarose or nitroblue tetrazolium (NBT) reduction was not observed. In a 2nd experiment, cattle were given no treatment, 0.04 mg of dexamethasone/kg IM, or 10 mg of avridine/kg IM followed 24 hours later by 0.04 mg of dexamethasone/kg. Dexamethasone administration caused a leukocytosis due to a neutrophilia with normal mononuclear cell numbers, an enhancement of PMN random migration under agarose, and an inhibition of NBT reduction, iodination, and ADCC activity of PMN. Dexamethasone did not have a detectable effect on lymphocyte blastogenesis or on ingestion of S aureus by PMN.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effects of Pasteurella haemolytica lipopolysaccharide on selected functions of bovine leukocytes

Lipopolysaccharide (LPS) was obtained from Pasteurella haemolytica serotype 1, using phenol-water extraction, and was evaluated for its ability to modify the biological activity of bovine leukocytes. The LPS was not toxic to polymorphonuclear leukocytes (PMN). The LPS preparation had little effect on random migration of PMN or mononuclear leukocytes (MNL), but caused a 2.5- to 3- fold increase in migration of cells within a whole peripheral blood leukocyte preparation. Phagocytosis of [125I]-labeled Staphylococcus aureus by PMN decreased with low (2.5 micrograms/10(6) cells) and high (65 micrograms/10(6) cells) concentrations of LPS and increased with moderate concentrations of LPS (5 to 25 micrograms/10(6) cells). The LPS enhanced nitroblue tetrazolium reduction by PMN. Moderate LPS concentrations (25 to 50 micrograms/10(6) cells) were mitogenic for MNL, whereas high LPS concentrations (300 micrograms/10(6) inhibited [3H]thymidine incorporation by MNL.     

The interaction of Rhodococcus equi and foal neutrophils in vitro

Polymorphonuclear neutrophil leukocytes (PMNL) from 8 healthy foals (2-14 weeks of age) and 2 foals with bacterial pneumonia were separated from whole blood using a 2 step Percoll gradient. Purified PMNL were tested for bactericidal function against Rhodococcus equi and Staphylococcus aureus in the presence of normal horse serum. The percentage uptake after a 15-min pre-incubation of PMNL and bacteria was also calculated. Ultrastructural examination of the interaction of R. equi and normal foal PMNL was performed after 15 min incubation. Results indicated that foal PMNL effectively phagocytose and destroy R. equi and S. aureus in the presence of normal horse serum. The mean percent uptake for R. equi was 99.3 +/- 0.4% and for S. aureus 99.9 +/- 0.1%. Further, 97.8 +/- 0.1% ingested R. equi and 98.4 +/- 0.1% ingested S. aureus were destroyed in the 15-min incubation period. Over the 3-h incubation, 91.9% of remaining R. equi were killed, but only 49.2 +/- 31.9% of S. aureus (P less than 0.01). Total bactericidal effect of foal PMNL, however, was 99.3 +/- 0.4% against R. equi and 99.9 +/- 0.1% against S. aureus. The percentage uptake and total bactericidal efficacy of neutrophils from sick foals was greater than 95%. Ultrastructural examination of the PMNL-R. equi interaction after 15 min incubation revealed phagocytosis of the bacteria and morphologic changes consistent with neutrophil degranulation. This study suggests that a defect in PMNL bactericidal capability is not likely to be a contributing factor in the pathogenesis of R. equi pneumonia in foals.     

Evaluation of the polymorphonuclear cell functions of bottlenose dolphins

The functions of polymorphonuclear cells (PMN) are the important non-specific defense mechanisms in the immune system. Especially marine mammals are protected by these mechanisms from the aquatic environment with a large variety of microorganisms. Therefore, we examined the PMN functions of bottlenose dolphins in order to obtain the normal ranges and to standardize the techniques. PMNs were isolated by using lymphocyte isolate solution whose density was 1.077; superoxide production was assessed by nitroblue tetrazolium reduction test (NBT) and phagocytosis was tested by using polystyrene latex beads. We showed that the optimal incubation time was 30 min in NBT assay and 12 hr in phagocytosis assay for dolphin PMNs.