Distribution of immunoglobulin-bearing leukocytes in bovine mammary tissue infected chronically with Staphylococcus aureus

Mammary parenchymal and test end tissues from cows with chronic Staphylococcus aureus mastitis were examined to determine the distribution of immunoglobulin (Ig) G1- and IgG2-bearing leukocytes. Leukocytes bearing IgG2 predominated in S. aureus infected quarters, with highest numbers observed at the Furstenberg's rosette followed by streak canal and parenchymal tissue areas. Significantly more IgG1- and IgG2-bearing leukocytes were observed at the Furstenberg's rosette and significantly more IgG2-bearing leukocytes were observed at the streak canal of S. aureus infected quarters compared to uninfected quarters. Receptors for cytophilic IgG on neutrophils and macrophages may increase efficiency of phagocytosis and improve the antimicrobial effectiveness of these cells in treat end tissues.     

Analysis of surface antigen expression and host defense function in leukocytes from calves heterozygous or homozygous for bovine leukocyte adhesion deficiency

OBJECTIVE: To analyze surface antigen expression and functional responses of leukocytes from calves heterozygous and homozygous for bovine leukocyte adhesion deficiency (BLAD). ANIMALS: 8 clinically normal calves, 4 calves heterozygous for BLAD, and 4 calves homozygous for BLAD. PROCEDURE: Surface antigen expression was examined by flow cytometric analysis of leukocytes stained with monoclonal antibodies. Neutrophil function analyses included phagocytosis and killing of Candida albicans and measurement of respiratory burst activity using cytochrome c and dihydrorhodamine 123 assays. Differential leukocyte counts also were performed. RESULTS: Leukocytes from heterozygous calves were similar to those of clinically normal calves with respect to surface antigen expression, C albicans phagocytosis and killing, and respiratory burst activity. In contrast, neutrophils from calves homozygous for BLAD had significantly reduced phagocytic and yeast-killing capacity but had higher respiratory burst activity than cells from clinically normal or heterozygous calves. Homozygous calves also had extreme neutrophilia and significantly more immature neutrophils. CONCLUSIONS: The heterozygous BLAD genotype does not cause detectable functional differences in leukocytes, compared with those of clinically normal calves. In contrast, leukocytes from homozygous calves seem to upregulate alternative host defense capabilities (eg, respiratory burst activity) to partially compensate for the lack of typical adherence-dependent host defense functions.     

Determination of the immunotoxic potential of heavy metals on the functional activity of bottlenose dolphin leukocytes in vitro

Heavy metals may affect the immune system of cetaceans. But no information exists on their effects on the bottlenose dolphin (Tursiops truncatus) immune system, although this species is a coastal top predator which can bioaccumulate high concentrations of them. This work studies the effects of Hg (1, 5 and 10mg/L), Al (2,5, 25 and 50mg/L), Cd (1, 10, 20 and 40mg/L), Pb (1, 10, 20 and 50mg/L) and Cr (1 and 10mg/L), on the function of phagocytes and lymphocytes isolated from the peripheral blood of bottlenose dolphins under in vitro conditions. Cell viability, apoptosis, lymphocyte proliferation and phagocytosis were evaluated. Viability and lymphoproliferation were measured with Alamar Blue assay, and apoptosis and phagocytosis were evaluated with flow cytometry. Apoptosis was detected as mechanism of cell death after cadmium and mercury exposure. A significant reduction in the lymphoproliferative response was registered by exposure to 1mg/L of mercury, 10mg/L of cadmium and 50mg/L of lead. Decreased phagocytosis was also observed at 5mg/L of mercury, 50mg/L of aluminium and 10mg/L of cadmium. Chromium did not present any effects on any immune assay at the concentrations tested. The concentrations of heavy metals that were found to affect the functional activity of bottlenose dolphin leukocytes are within the environmental ranges reported in the tissues of bottlenose dolphins. These results support the hypothesis that exposure to these contaminants, particularly mercury and cadmium could lead to a reduction in host resistance to disease in these animals.     

Effects of canine distemper virus on natural killer cell activity in dogs

An in vitro 51Cr-release assay was developed to detect the cytotoxicity of natural killer cells (NK) of canine peripheral blood mononuclear leukocytes to canine distemper virus (CDV) target cell membrane-bound antigens. Leukocytes from 23 young (greater than or equal to 1 week of age), CDV-naive gnotobiotic dogs could discriminate between noninfected control and CDV-infected Vero target cells. However, the amount of preinfection NK activity did not positively correlate with the ultimate outcome of the disease process when these same dogs were given virulent R252-CDV. Evaluation of preinfection and postinfection CDV-specific NK activity indicated that infection-associated increases in cytolysis of CDV-infected or noninfected Vero targets did not occur. In vitro infection of peripheral blood leukocytes with CDV did not change the kinetics or magnitude of NK-mediated cytolysis of homologous virus-infected or other NK-susceptible target cells.     

Effects of cold treatment and ketosis induced by starvation on interferon production in leukocytes of lactating cows.

The production of interferons in blood and milk leukocytes of three groups of cows was measured to determine the effect of 6-days cold treatment (-2 degrees to -8 degrees C) and/or starving. The first group (cold) was treated with low ambient temperature (-2 degrees C to -8 degrees C) 11 hours every day for 6 days, the second (cold and starved) was treated with low temperature and starved for 6 days. The third group (controls) was fed normally and kept in a barn at room temperature (18 degrees to 20 degrees C). The leukocytes of the control and the cold treated cows responded normally to interferon induction with Newcastle Disease Virus (NDV) and mitogens: phytohemagglutinin (PHA) and concanavalin A (ConA). The cows treated with low temperature and starved for 6 days developed biochemical blood changes of ketosis. Leukocytes of these cows with ketosis produced less interferon (p less than 0.05) than before starvation and less than leukocytes of the control cows and the cold treated cows. It can be assumed that ketosis caused by starving decreases the ability of a cow's leukocytes to produce interferons.     

In vitro effects of prepartum concentrations of oestradiol-17 beta on cell-mediated immunity and phagocytosis by porcine leukocytes

The in vitro effect of oestradiol-17 beta (Oe2) on cell-mediated immunity (CMI) and phagocytosis by leukocytes obtained from ten and eight sows, respectively, was studied. The concentrations of Oe2 used were 300 pmol/l and 3000 pmol/l, to reflect a low and a high prepartum concentration of the hormone. The CMI was measured by lymphocyte proliferation (LP) tests performed on whole blood and purified mononuclear cells (MNC) and by estimation of the production of interleukin-2 (IL-2) in the latter type of culture. In the whole blood LP test, the mitotic response to concanavalin A (Con A) was increased by 29% (P less than or equal to 0.05) when the Oe2 concentration was 300 pmol/l. The higher concentration of Oe2 (3000 pmol/l) increased the amount of IL-2 in supernatants from Con A-stimulated MNC by 17% (P less than or equal to 0.05). The phagocytic function of polymorphonuclear leukocytes was determined in a chemiluminescence assay. In this assay, the maximal rate of phagocytosis was reduced by 12% (P less than or equal to 0.01) and the area under the curve by 10% (P less than or equal to 0.05) at an Oe2 concentration of 3000 pmol/l. The results show that prepartum concentrations of Oe2 can promote CMI in vitro, but weaken phagocytosis by porcine leukocytes.     

Cytokine expression by pulmonary leukocytes from calves challenged with wild-type and leukotoxin-deficient Mannheimia haemolytica

The objective of this study was to assess the role of leukotoxin (LKT) in modulating the pulmonary cytokine response of calves challenged with Mannheimia haemolytica. Thirty-six calves, seronegative to LKT and M. haemolytica whole cell antigen were divided into three groups (I, II and III). Calves in groups I and II were challenged by the intra-bronchial route with 25 mL of phosphate buffered saline (PBS) containing 0.44×10(9) cfu/mL of LKT deficient (lkt(-)) and 25 mL of PBS containing 0.31×10(9) cfu/mL of wild-type (wt) M. haemolytica serotype 1, respectively. Group III calves were challenged intra-bronchially with 25 mL of sterile PBS. Leukocytes were collected from broncho-alveolar lavage fluid (BALF) 4 days before and at 1, 3, and 6 days post-inoculation (p.i.). Expression of the following cytokines in the recovered leukocytes was measured using real-time PCR: interleukin (IL)-1β, -8, -10, -12 (p40) and TNF-α. The amount of TNF-α produced was also quantified by ELISA. Although a statistically significant difference in the expression of these cytokines was not observed between groups challenged with the wt and lkt(-) strains, the wt infected group (II) did exhibit higher mean clinical scores. Overall, there was considerable variation in the composition of the BALF between the groups and by day 7 p.i., both lkt(-)- and wt-challenged calves had seroconverted to M. haemolytica.     

Superoxide production by stimulated equine polymorphonuclear leukocytes-inhibition by anti-inflammatory drugs

Polymorphonuclear leukocytes (PMNLs) were isolated from an inflammatory exudate induced in the intercarpal joints of horses by an administration of carrageenin. Their superoxide production at rest and following stimulation with either serum-treated zymosan (STZ) or phorbol myristate acetate (PMA) was measured by cytochrome-c reduction. Stimulation of the cells increased the cytochrome-c reduction 10-15 times that of resting cells. The maxima were 20 nmol of reduced cytochrome-c per 10(6) cells per ml at 120 min (STZ) and 35 nmol of reduced cytochrome-c per 10(6) cells per ml at 60 min (PMA). The maximum inhibition of the cytochrome-c reduction by superoxide dismutase (Palosein) was 83.6% (STZ stimulation) and 72.1% (PMA stimulation). The non-steroidal anti-inflammatory drugs, phenylbutazone, salicylic acid, aspirin, sodium salicylate in addition to D-penicillamine and dimethylsulfoxide caused dose-dependent inhibition of the cytochrome-c reduction when the cells were stimulated by PMA. The maximum inhibitions were 64% and 36% for aspirin (10(-2) M), 32% and 17% for phenylbutazone (10(-3) M), 15% and 31% for dimethylsulfoxide (6.4 x 10(-1) M), 32% and 19% for salicylic acid (10(-2) M), 0% and 17% for sodium salicylate (10(-2) M) and 2.2% and 2.5% for D-penicillamine (10(-4) M) when the cells were stimulated by STZ and PMA, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Basis of melengestrol acetate action as a progestin

To provide insight into potential mechanisms contributing to the various biological responses of cattle to treatment with progesterone, norgestomet, and melengestrol acetate (MGA), MCF-7 cells were utilized to determine the relative binding affinity of the progesterone receptor for MGA, norgestomet, progesterone, and a progesterone agonist (R5020), and to determine if progesterone, MGA, or norgestomet have estrogenic and/or anti-estrogenic activities. The progesterone receptor had greater affinity (P<0.05) for MGA, R5020, and norgestomet than for progesterone; and the affinity for norgestomet exceeded (P<0.05) that of MGA and R5020. Estrogen stimulates proliferation of MCF-7 cells; therefore these cells have been utilized as a bioassay to detect estrogenic and anti-estrogenic activity. Progesterone (10(-11) to 10(-5)M) did not promote cellular proliferation. However, MGA (10(-8), 10(-7), and 10(-6)M) increased (P<0.05) cell proliferation compared to the control group (10(-11) to 10(-9) and 10(-5)M MGA did not stimulate cell proliferation), and MGA-induced cell proliferation (10(-8)M) was reduced (P=0.095) by an estradiol antagonist (ICI 182,780; ICI). Cellular proliferation increased (P<0.05) with norgestomet (10(-5)M) compared to the control group (10(-11) to 10(-6)M norgestomet did not stimulate cell proliferation) and the increased proliferation was decreased (P<0.05) by ICI. Neither progesterone nor MGA demonstrated anti-estrogenic activity. Norgestomet (10(-10) to 10(-6)M) did reduce (P<0.05) maximal estrogen-stimulated cell proliferation, but not to basal levels. In summary, the affinities of the progesterone receptor for norgestomet, MGA, and progesterone are consistent with their effective dose to inhibit ovulation in vivo, but their progestin and their estrogenic/anti-estrogenic activities cannot fully explain why progesterone and norgestomet are more capable of reprogramming the reproductive axis in anestrous postpartum cows compared to MGA.     

Concanavalin A-induced suppressor cell activity in intestinal mucosal leukocytes obtained from healthy cows

Leukocytes isolated from the intraepithelium, lamina propria, and aggregated lymphoid follicles (ALF) of the bovine small intestinal mucosa were activated by concanavalin A (conA) to generate suppressor activity against the proliferative response of autologous and allogeneic leukocytes to conA, phytohemagglutinin, and pokeweed mitogen. Freshly obtained intraepithelium, lamina propria, and ALF leukocytes, preincubated with 25 to 50 micrograms/ml of conA for 24 to 48 hours, were able to inhibit the mitogenic responses of autologous and allogeneic lymphocytes when cocultured at a ratio greater than or equal to 1:1 (suppressor cells to responder cells). Depletion of adherent cells (monocytes/macrophages) by sequential plastic and gel adherence completely abrogated the conA-induced suppressor activity of all 3 leukocytes populations. However, reconstitution with autologous or allogeneic monocytes/macrophages during the induction phase restored the inducible suppressor activity. Addition of recombinant human interleukin-2 at a concentration as low as 5 U/ml during the responder phase enabled the responder population to recover the response apparently impaired by the conA-treated ALF leukocytes. At a concentration of 10 U/ml, human interleukin-2 was not only able to restore the responder population's response to phytohemagglutinin stimulation, but highly enhanced its proliferative ability. Although quantitative variations were observed between different cell populations and cells obtained from individual cows, the extent and general pattern of the inducible suppressor activity were similar in cells from cows studied.     

Effects of tramadol and o‐desmethyltramadol on canine innate immune system function

ObjectiveTramadol is a commonly used opioid analgesic in dogs, particularly in dogs with a compromised immune system. An opioid may be selected for its immunomodulatory effects. Consequently, the objective of this study was to investigate the effects of tramadol on immune system function by evaluating the effect of tramadol and o-desmethyltramadol (M1) on the function of canine leukocytes in vitro. The hypothesis was that tramadol and M1 would not alter polymorphonuclear leukocyte (PMN) phagocytosis, PMN oxidative burst, or stimulated leukocyte cytokine production capacity of tumor necrosis factor (TNF)-a, interleukin (IL)-6, and IL-10.Study designIn vitro pharmacodynamic study.AnimalsSix healthy dogs.MethodsBlood from six dogs was obtained and incubated with various concentrations of tramadol and M1. Phagocytosis and oxidative burst were assessed using flow cytometry, and lipopolysaccharide (LPS), lipoteichoic acid (LTA) and peptidoglycan (PG)-stimulated leukocyte production of TNF, IL-6, and IL-10 were measured using a canine specific multiplex assay.ResultsNo differences were detected in phagocytosis or oxidative burst with any drug concentration. Tramadol did not alter leukocyte cytokine production, however, M1 significantly blunted IL-10 production.ConclusionsTramadol and its metabolite M1 were sparing to PMN phagocytosis and oxidative burst in dogs in vitro. Tramadol did not alter leukocyte cytokine production, however, M1 blunted IL-10 production at clinically achievable concentrations suggesting that M1 may promote a proinflammatory shift.Clinical relevanceThese data suggest that tramadol has minimal effect on phagocytosis and oxidative burst, and may promote a proinflammatory shift. Therefore, tramadol may be an ideal opioid analgesic in dogs at high risk of infection. Further investigation in vivo is warranted.     

Effects of 17beta-estradiol,bisphenol A and tributyltin chloride on germ cells of Caenorhabditis elegans

Effects of a one-generation exposure to a natural estrogen, 17beta-estradiol (E2), and environmental pollutants such as bisphenol A (BPA) and tributyltin chloride (TBTCL) on the number of germ cells were investigated in the hermaphrodite Caenorhabditis elegans. The eggs of gravid adult worms isolated by alkaline hypochlorite treatment were seeded on a test chemical-containing NGM (nematode growth medium) agar plate without cholesterol. After incubation for 6 days at 16 degrees C, the germ cells of adult worms were stained with 4', 6-diamino-2-phenylindole dihydrochloride (DAPI). The staining procedure was completed within one hour and the stained germ cells were counted under a fluorescence microscope without dissection. The number of germ cells in the worms treated with E2 (10(-10)-10(-6) M) and BPA (10(-9)-10 (-5) M) was significantly increased. Maximal increases were observed at 10(-8) M E2 (156 +/- 15.3% of control) and 10(-5) M BPA (168 +/- 20.0 % of control). TBTCL (10(-9)-10(-6) M) significantly decreased the number of germ cells. The minimal decrease was observed at 10(-6) M TBTCL (30.2 +/- 3.51% of control). These results indicate that changes in the number of germ cells are a sensitive indicator of the effects of chemicals on the reproductive system. Since the method described in this paper is a novel, simple, time- and money-saving bioassay, C. elegans is an excellent model with which to determine the reproductive toxicity of chemicals including environmental pollutants.     

Conflict of estrogenic activity by various phthalates between in vitro and in vivo models related to the expression of Calbindin-D9k

Phthalates are suspected to disrupt the endocrine system, especially through estrogenic effects. In the present study, we investigated the effects of various phthalates and compared them with those of estrogenic compounds that disrupt the female reproductive system. To assess the effects of these phthalates, alteration of the Calbindin-D9k (CaBP-9k) gene was measured as a biomarker because rat CaBP-9k gene carries an estrogen response element (ERE) which is involved in estrogen responsiveness of the gene during the estrous cycle. In this study, phthalates were tested for estrogenic properties in in vitro and in vivo models. First, the E-Screen assay was used to measure the proliferation of MCF-7 cells, a human breast cancer cell line. Treatments with 17beta-estradiol (E2; 9-fold) and 17alpha-estradiol (EE; 9-fold) induced MCF-7 cell proliferation at concentrations of 10(-9) M. Phthalates induced an increase in MCF-7 proliferation at concentration of 10(-6) M up to 10(-4) M. Nbutyl benzyl phthalate (BBP; 6-fold vs. vehicle), dicyclohexyl phthalate (DCHP; 8-fold), 2-ethylhexyl phthalate (DEHP; 6-fold) and di-n-butyl phthalate (DBP; 7-fold) at the concentration of 10(-4) M induced in an increase in MCF-7 proliferation after 6 d of treatment compared to vehicle. However, significant increase in MCF-7 proliferation was induced by diethyl phthalate (DEP). Second, we investigated the expression of CaBP-9k in the uterus of immature rats after oral treatment with BBP, DCHP, DEHP, DBP or DBP (600 mg/kg per day) in this in vivo model, because the immature rat model is highly sensitive to exposure to estrogenic chemicals. None of the phthalates induced the expression of CaBP-9k mRNA and its protein in the neonatal uterus as analysed by Northern and Western blot analyses, respectively. Although phthalates induced an increase in MCF-7 cell proliferation by an estrogenic effect, they could not induce CaBP-9k expression in the in vivo system, suggesting that the assays of estrogenic effects of various phthalates conducted in vitro and in vivo expression of CaBP-9k may produce conflicting results.     

精氨酸在体内和体外试验中对鲤鱼免疫力的影响

本试验采用体外和体内2种方法来研究精氨酸(Arg)对鲤鱼免疫力的影响。体外试验中,在培养基中分别添加0、0.5、1.0、1.5和2.0mmol/L的Arg,将肾脏白细胞培养不同的时间段后测定增殖指数、呼吸爆发活力、吞噬活力和杀菌率。体内试验中,将平均体重37g的鲤鱼随机分为5组,每组3个重复,每个重复10条,分别体内注射0、25、50、100和200 mg/kg鱼体重的Arg,商业饲料投喂2周,进行肾脏白细胞增殖指数、呼吸爆发活力和吞噬活力的测定,以及血清和肝胰脏一氧化氮(NO)含量、一氧化氮合成酶(NOS)活性及血清白蛋白(ALB)含量的测定。体外试验结果表明:添加Arg能够提高肾脏白细胞的增殖指数、呼吸爆发活力、吞噬活力和杀菌率。培养12和24h后,1.0、1.5和2.0mmol/L Arg组肾脏白细胞增殖指数显著高于0mmol/L Arg组(P0.05);培养6、12和24h后,1.0mmol/L Arg组肾脏白细胞呼吸爆发活力显著高于0mmol/L Arg组(P0.05);培养12和24h后,1.0、1.5和2.0mmol/L Arg组肾脏白细胞吞噬活力显著高于0mmol/L Arg组(P0.05);培养18h后,1.0、1.5和2.0 mmol/L Arg组肾脏白细胞杀菌率显著高于0 mmol/L Arg组(P0.05)。体内试验结果表明:50、100和200 mg/kg Arg组肾脏白细胞呼吸爆发活力、吞噬活力和增殖指数显著高于0mg/kg Arg组(P0.05);100和200mg/kg Arg组血清ALB和NO含量显著高于0 mg/kg Arg组(P0.05),50、100和200mg/kg Arg组血清NOS活性显著高于0mg/kg Arg组(P0.05);50、100和200mg/kg Arg组肝胰脏NO含量显著高于0mg/kg Arg组(P0.05),25和50mg/kg Arg组肝胰脏NOS活性显著高于0mg/kg Arg组(P0.05)。由此可见,Arg提高了鲤鱼肾脏白细胞的免疫力,体外试验表明Arg的适宜浓度为1.0mmol/L。正常摄食情况下,Arg在鲤鱼体内注射适宜浓度为50~100mg/kg。     

Sequential response of milk leukocytes, albumin, immunoglobulins, monovalent ions, citrate, and lactose in cows given infusions of Escherichia coli endotoxin into the mammary gland

Changes in concentrations of both the cellular and the humoral components of milk are known to occur during mastitis. This study was conducted to determine temporal changes in the concentrations of leukocytes, albumin, immunoglobulins (Ig), monovalent ions, lactose, and citrate in milk during the initial phases of simulated mastitis. Ten cows whose udders were pathogen free and had milk leukocyte counts of less than 0.5 X 10(6)/ml were used. Two dosages of Escherichia coli endotoxin were administered to simulate various degrees of mastitis. Two quarters in each cow were infused with the endotoxin and the other 2 served as controls. Quarter milk samples were collected frequently before and after infusion. Within 2 hours after infusion of a 100-micrograms dose of endotoxin, clinical mastitis was observed in most of the infused quarters. Leukocytes, albumin, IgG1, and conductivity showed significant increases. Values before infusion and at postinfusion (PI) hour 2 were as follows: leukocytes, 0.33 and 3.65 X 10(6)/ml, respectively; albumin, 0.38 and 4.49 mg/ml; IgG1, 0.34 and 0.79 mg/ml; and conductivity, 6.0 and 6.9 mmho. Average of the peak values and their average relative time of appearance after infusion were as follows: leukocytes, 28.82 X 10(6)/ml at 16 hours; albumin, 9.37 mg/ml at 4 hours; IgG1, 1.35 mg/ml at 4 hours; and conductivity, 95.5 mmho at 10 hours. The IgG1 values tended to remain high in the presence of rapidly declining albumin concentrations, indicating the possibility of an active, rather than a passive, transfer of IgG1 from the circulation. The response to the 10-micrograms dose of endotoxin ranged from subclinical to clinically mild mastitis with lesser cellular and humoral responses.     

Broad spectrum antimicrobial activity of leukocyte extracts from the American alligator (Alligator mississippiensis)

Leukocytes were isolated from whole blood of wild alligators by differential sedimentation. The leukocytes were disrupted in 5% AcOH and the crude extracts processed by ultrafiltration. The extracts were subjected to solvent exchange (0.1% AcOH) and the fraction that contained macromolecules between 1 and 10 kDa were subjected to further analyses. The acid extracts of the alligator leukocytes exhibited substantial antimycotic activities against six of eight species of Candida yeast tested. In addition, the alligator leukocyte extracts were effective as antimicrobial agents against 10 of 12 bacterial species, and displayed moderate activity against two enveloped viruses (human immunodeficiency virus-1 and herpes simplex virus-1_HF). Kinetic analyses revealed that the antimycotic effects of the leukocyte extract occurred rapidly, with 64% fungal growth inhibition within 3 min of exposure. The molecule(s) responsible for the antimicrobial activities were sensitive to proteases, heat-stable, acid soluble, and in the 1–10 kDa range. These data suggest that alligator leukocytes express cationic peptides that are responsible for their antimicrobial properties.     

Superoxide production in phagocytes obtained from Mycobacterium marinum-stimulated goldfish (Carassius auratus) that were exposed to copper.

OBJECTIVE: To investigate the effects of copper exposure and recovery from copper toxicosis on the nonspecific immune response in Mycobacterium marinum-inoculated goldfish. ANIMALS: Goldfish (Carassius auratus) with a mean weight of 33.5 g. PROCEDURE: Superoxide (O2-) production was measured in fish 2 to 6 weeks after injection with phosphate-buffered saline (PBS) solution or M marinum (10(2) to 10(7) colony-forming units [CFU]/fish). Then, paired groups of fish were injected with PBS solution or 10(4) CFU of M marinum and exposed to copper (100 microg/L) for 7 days or for 4 days with 3 days of recovery. One paired group not exposed 14 days later to copper served as control fish. Phagocyte production of O2-was measured by use of the nitroblue tetrazolium reduction assay. Inflammation and bacterial colony counts were determined by use of routine histologic and microbiologic procedures. RESULTS: Superoxide production achieved a maximal response 2 to 4 weeks after M marinum inoculation. Compared with control fish, O2- production increased in the groups exposed to copper but then decreased in the exposed groups that were allowed to recover. Superoxide response and peritoneal inflammation were greater in M marinum-inoculated groups than in non-inoculated groups. CONCLUSIONS: Copper exposure and inoculation with M marinum increased O2- production, whereas recovery after exposure decreased O2- production, even in fish that were immunostimulated by M marinum. CLINICAL RELEVANCE: When the antimicrobial oxidative response is suppressed after copper exposure, steps should be taken to avoid imposing additional stress and minimize the possibility of resurgent or secondary pathogenic infections.     

Effect of Vitamin D 3 on Cadmium Retention by Laying Hens

The effect of cadmium (Cd) and vitamin D 3 on Cd retention in the organism of laying hens was observed. Hens ( n =48) were divided into eight groups with six animals per group as follows: experimental groups 1, 3 and 5 were administered Cd (0.3, 0.6 and 6.0 mg CdCl 2 kg -1 body weight, respectively) daily in water and groups 2, 4 and 6 were administered the same CdCl 2 concentrations supplemented with vitamin D 3 (100 IU hen -1 ). C1 was a control group without any supplements and C2 a control group supplemented only with vitamin D 3 . Samples of inner organs were analysed by atomic absorption spectrophotometry after 6 months of Cd exposure. A significant elevation in Cd levels, mainly in the liver and kidneys, was found. A significant decrease ( P h 0.01) in Cd levels in the liver was recorded after the addition of 6.0 mg CdCl 2 kg -1 body weight and vitamin D 3 , in comparison with the group without vitamin D 3 (18.76 vs. 10.33 mg kg -1 ). A similar decrease in Cd levels in the kidney ( P h 0.001) at the same supplementation dose of Cd was obtained in comparison with the group without vitamin D 3 (145.32 vs. 60.37 mg kg -1 ). The results confirmed that vitamin D 3 is able to reduce the Cd content in the organism of laying hens. The main effect of vitamin D 3 was demonstrated by decreasing Cd retention in the liver and especially in the kidneys.     

Characterization of the in vitro responses of equine cecal longitudinal smooth muscle to endothelin-1

OBJECTIVE: To characterize the in vitro response of equine cecal longitudinal smooth muscle (CLSM) to endothelin (ET)-1 and assess the role of ETA and ETB receptors in those ET-1-induced responses. ANIMALS: 36 horses without gastrointestinal tract disease. PROCEDURE: To determine cumulative concentration-response relationships, CLSM strips were suspended in tissue baths containing graded concentrations of ET-1 (10(-9) to 10(-6)M) with or without BQ-123 (ETA receptor antagonist); with or without IRL-1038 (ETB receptor antagonist); or with both antagonists at concentrations of 10(-9), 10(-7), and 10(-5)M. To determine the percentage change in baseline tension of CLSM, the areas under the curve during the 3-minute periods before and after addition of each dose were compared. Also, the effects of ET-1 and a combination of selective ETA and ETB receptor antagonists on electrically evoked contractions were studied. RESULTS: ET-1 caused sustained increases in CLSM tension in a concentration-dependent manner. Contractile responses to ET-1 were not significantly inhibited by either BQ-123 or IRL-1038 alone at any concentration; however, responses were significantly inhibited by exposure to the antagonists together at a concentration of 10(-5)M. Electrical field stimulation did not change the spontaneous contractile activity of CLSM and did not significantly alter the tissue response to ET-1, BQ-123, or IRL-1038. CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that ET-1 has a contractile effect on equine CLSM that is mediated via ETA and ETB receptors. In vitro spontaneous contractions of equine CLSM apparently originate in the smooth muscle and not the enteric nervous system.     

Interleukin-2 production by mitogen-stimulated intestinal mucosal leukocytes from cattle

Leukocytes isolated from intraepithelium, lamina propria, and aggregated lymphatic follicles of the small intestine of healthy adult cattle were tested for their ability to produce interleukin 2 (IL-2) by in vitro stimulation of cells with mitogens. Supernatants from interepithelial leukocytes, lamina propria leukocytes, and cultures stimulated with concanavalin A (conA), phytohemagglutinin, and pokeweed mitogen contained growth factors with the capacity to maintain proliferation of a bovine IL-2-dependent lymphoblastoid cell line. Interleukin-2 activity was demonstrated in supernatants of all 3 conA-stimulated leukocyte populations as early as 20 hours after initiation of culture, reached peak values at 30 to 50 hours, and decreased by 72 hours. Although quantitative variations of IL-2 production were observed between various cell types and among cattle, conA was the most potent in inducing IL-2 activity in all 3 leukocyte populations. Supplementation of culture medium with 2-mercaptoethanol or phorbol myristrate acetate neither induced IL-2 production nor enhanced mitogen-induced IL-2 production. Addition of indomethacin to conA-stimulated cultures enhanced IL-2 production. Although depletion of adherent cells did not affect IL-2 production, total elimination of Ia-positive accessory cells inhibited its production by all 3 cell populations. Lymphocytes responsible for IL-2 production in aggregated lymphatic follicle population were presumptive T cells because they were nylon wool-nonadherent, B26A positive (monoclonal antibody directed against pan T cells), pIg45A negative (antibody directed against pan B cells), and considered peanut agglutination-positive.