IL-35 plays a key role in regulatory T (Treg) and regulatory B (Breg) cell functions in mammals. CD25 has been demonstrated as one of the markers of Treg cells, and CD19+CD25hiCD71hi cells have been verified as a type of Breg cells in humans. These results indicate that there is a close relationship between IL-35 and CD25+ cells. In mammals, CD25 (alias IL-2Rα) has been identified as having high affinity and specificity for IL-2 binding, and is closely linked and structurally related to IL-15Rα, which having high affinity for IL-15 binding. In teleost, IL-15Rα can bind to both IL-2 and IL-15, with higher affinity to IL-15 than IL-2, and has been termed a CD25-like molecule in some research studies. To date, no studies of IL-35 and IL-15Rα have been documented in fish. In this work, five isoforms of IL-15Rα were cloned from grass carp, and a monoclonal antibody to the protein was developed. The results of flow cytometry and quantitative real-time PCR analyses demonstrated that grass carp IL-35 subunit genes EBI3a and IL-12p35 were mainly expressed in IL-15Rα+ cells, while the expression levels of IL-10 and TGF-β in IL-15Rα+ and IL-15Rα− cells were insignificant. Recombinant grass carp IL-35 (rgcIL-35) could increase the proportion of IL-15Rα+ cells in leukocytes, and a certain proportion of IL-15Rα+ cells also appeared in myeloid cell subset II after stimulation with rgcIL-35. Meanwhile, the migration, phagocytic ability, and bactericidal ability of grass carp neutrophils were significantly decreased after stimulation with certain concentrations of rgcIL-35. Moreover, neutrophil apoptosis could be significantly inhibited by rgcIL-35.

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.

Pneumonia caused by Pasteurella (Mannheimia) haemolytica was induced in weaned beef heifer calves, approximately 6 months of age. Calves were treated at 20 h after challenge with therapeutic doses of danofloxacin or tilmicosin. Peripheral blood neutrophils were collected at 3, 24 and 48 h after treatment. The ex vivo effects on neutrophil function, neutrophil apoptosis, and hematological parameters were examined, as was the effect on percentage lung consolidation. Neutrophil function assays included random migration under agarose, cytochrome C reduction, iodination, Staphylococcus aureus ingestion, chemotaxis, and antibody-dependent and antibody-independent cell-mediated cytotoxicity. Apoptosis was determined using a cell death detection kit. Killing was performed at 72 h after treatment. Statistical comparisons were made among the three groups of challenged-treated animals: saline, danofloxacin, and tilmicosin. Comparisons were also made between nonchallenged nontreated animals (NCH) and challenged saline-treated animals. There were no significant differences for any of the neutrophil function assays or neutrophil apoptosis among the challenged-treated groups. This suggests that danofloxacin and tilmicosin have no clinically significant effects on neutrophil function or apoptosis. There were also no significant differences in percentage lung consolidation among the challenged-treated groups. Significant differences were found between the NCH calves and the challenged saline-treated calves in several neutrophil assays, which were attributed to effects of P. haemolytica infection.

Four different experimental models for Streptococcus suis-induced disease were compared to find a model that closely mimics naturally occurring disease in conventional pigs. Fourteen, 2-week old pigs free of S. suis type 2 were used in 2 experiments. In experiment 1, 3 pigs were inoculated intravenously (IV) and 3 pigs intranasally (IN) with S. suis. Two out of 3 of the IV-inoculated pigs exhibited signs of severe central nervous system disease (CNS) and were euthanized. Streptococcus suis type 2 was isolated from whole blood, joints, and serosal surfaces of both pigs. No clinical signs and no growth of S. suis were detected in the IN-inoculated pigs. In experiment 2, 4 pigs were inoculated IV and another 4 were inoculated IN with the same isolate as in experiment 1. One hour before inoculation the IN-inoculated pigs were given 5 mL of 1% acetic acid intranasally (IN-AA). All the IV-inoculated pigs showed CNS disease and lameness, and 2 of the pigs became severely affected and were euthanized. All the IN-AA inoculated pigs exhibited roughened hair coats and 2 pigs developed severe CNS disease and were euthanized. Streptococcus suis was isolated from the joints and blood of 3 pigs in the IV-inoculated group. Streptococcus suis was isolated from blood of 2 pigs, meninges of 3 pigs, and joints of 1 pig in the IN-AA inoculated group. Natural exposure to S. suis most likely occurs by the intranasal route. The IN-AA model should serve as a good model for S. suis-induced disease, because the natural route of exposure is intranasal and the IN-AA model was effective in inducing disease that mimics what is observed in the field.

The objective of this work was to evaluate a proprietary ultrafiltered bovine whey product for its in vitro influence on the function of neutrophils from normal and dexamethasone-treated cattle, and for its in vivo influence on neutrophil function in periparturient dairy cows. The ultrafiltered bovine whey was produced by hyperimmunizing cows to various bacterial pathogens by intramammary injection, collecting and pooling the colostrum and milk for the first 3 wk after parturition, then separating and processing the whey through a filter with a nominal molecular mass cut off of 10,000 Da. In vitro treatment of neutrophils from normal calves with ultrafiltered bovine whey significantly increased neutrophil random migration, cytochrome C reduction, iodination activity, antibody-dependent cell-mediated cytotoxicity, and antibody-independent cell-mediated cytotoxicity. Cytochrome C reduction was the only neutrophil function parameter significantly enhanced by the in vitro treatment of neutrophils from dexamethasone-treated cattle with ultrafiltered bovine whey. In vivo treatment of periparturient cows with ultrafiltered bovine whey did not alter the total or differential leukocyte counts in the animals but did significantly increase the total erythrocyte counts. In vivo treatment with ultrafiltered bovine whey also significantly increased neutrophil iodination activity in the periparturient cows. Neutrophil iodination activity (a measure of the myeloperoxidase/hydrogen peroxide/halide antibacterial system) is a very potent bactericidal mechanism of neutrophils and has previously been shown to be suppressed in periparturient cows.

The effects of single or dual infection with bovine immunodeficiency-like virus (BIV) and/or, bovine leukemia virus (BLV) on bovine immune function were examined over a 4 year period. Holstein calves were infected with BIV (four calves), BLV (five calves), BIV and BLV (five calves), or sham inoculated (three calves). Lymphocyte blastogenesis to mitogens, seven tests of neutrophil function, and mononuclear cell subset analysis by flow cytometry (BoCD4, BoCD8, BoCD2, BoWC1, sIgM+, and monocytes) were performed at regular intervals to 49 months post-infection. These data were analyzed for main effects of each virus and interaction as a 2 x 2 factorial. BIV infected cattle had lower neutrophil antibody-dependent cell-mediated cytotoxicity and iodination responses during 2 of the 4 years post-infection (P < 0.05). BIV infection was not associated with any long-term significant changes in lymphocyte blastogenesis to mitogens or changes in mononuclear cell subset numbers in blood. There was a tendency for animals infected with BIV alone to have decreased lymphocyte blastogenic responses to mitogens, but this was not statistically significant. BLV infection caused an increase in total mononuclear cells with no dramatic shift in the relative proportions of the various subsets. Co-infection with BIV and BLV did not consistently cause a different response than either virus did individually. One BIV infected animal died of non-BLV lymphosarcoma 7 months after infection. All other animals had no unusual clinical signs. In summary, infection with BIV caused a significant, temporary decrease in neutrophil function with no consistent statistically significant alteration in lymphocyte blastogenesis or mononuclear cell numbers during the first 4 years after infection. BLV infection caused an increase in lymphocyte numbers, and there appeared to be no synergism between the viruses.

OBJECTIVE To develop a model to study the kinetics and relative amounts of cytokines produced by liver cells during enteric infection. DESIGN Salmonella enteriditis lipopolysaccharide (LPS)- or live S choleraesuis-stimulated isolated livers from clinically normal pigs and pigs with active acute phase response. ANIMALS 7- to 14-day-old salmonellosis-free pigs, 4 to 12/group. PROCEDURE Livers were removed and perfused with oxygenated Krebs-Henseleit solution for 30 minutes and with S choleraesuis or LPS added for 7 minutes. Livers were then perfused with 500 ml of fresh solution in a closed loop procedure for 180 minutes. Perfusate samples were collected for tumor necrosis factor-alpha (TNF alpha) and interleukin 6 (IL-6) bioassays. RESULTS Tumor necrosis factor-alpha values remained constant during perfusion of normal livers and increased in those exposed to LPS. Interleukin 6 values increased in perfusate from normal livers from 30 to 150 minutes, then decreased. In livers from pigs with an active acute phase response, TNF alpha values were reduced; IL-6 appeared by 2 minutes and decreased after 25 minutes. CONCLUSIONS Isolated livers could be kept viable for 3 hours, and IL-6 and TNF alpha could be measured by the bioassays used. CLINICAL RELEVANCE Model can be used for studying and modifying the response of liver cells to infective agents.

An 11-month-old Holstein calf experimentally infected with bovine immunodeficiency-like virus (BIV) developed T-cell lymphosarcoma 5 months postinoculation, concurrent with progressive monocytosis. Tumors were found in the thymus, multiple lymph nodes, and brain. Tumor cells were CD2+, CD4-, CD8-T cells. Infectious BIV could be recovered from splenic tissue and blood mononuclear cells. Bovine leukemia virus was not present. Because this calf was part of an ongoing experiment on the pathogenesis of BIV infection, immune function data were also available both before and after lymphosarcoma developed. Neutrophil and monocyte function were normal, but lymphocyte blastogenesis was enhanced before the development of lymphosarcoma. Follicular hyperplasia in lymphoid tissues was also seen. This case raises the possibility that BIV infection may cause or be associated with some cases of atypical T-cell lymphosarcoma, without evidence of immune suppression at the time of tumor onset.

The activity of four recombinant human cytokines on porcine neutrophils was evaluated. Porcine neutrophils were treated with varying doses of recombinant human tumour necrosis factor-alpha (rHu-TNF), interferon-gamma (rHu-IFN), interleukin-8 (rHu-lL-8), or granulocyte-macrophage colony-stimulating factor (rHu-GM-CSF). The function of treated neutrophils was compared with that of non-treated controls in the following assays: antibody-independent neutrophil cytotoxicity (AINC), antibody-dependent cell-mediated cytotoxicity (ADCC), iodination, Staphylococcus aureus ingestion, cytochrome C reduction, random migration, and chemotaxis. Treatment with rHu-TNF produced significant (P < 0.05) depression of neutrophil random migration (2.5, 25, and 250 ng ml-1 rHu-TNF) and iodination (250 ng ml-1) and a near significant (P = 0.08) depression in ADCC (250 ng ml-1). Treatment with 25,000 U ml-1 of rHu-IFN caused a significant increase in AINC. At lower doses of rHu-IFN, there was a trend (0.05 < P < or = 0.08) toward depression of AINC (250 U ml-1) and ADCC (25 U ml-1) and enhancement of iodination (250 U ml-1). Treatment with 50 ng ml-1 of rHu-IL-8 caused a near significant increase (P = 0.06) in AINC. There were no significant differences noted when porcine neutrophils were treated with rHu-GM-CSF (2.5-2500 U ml-1). No synergism was noted between rHu-TNF and rHu-IFN.

Salmonella typhimurium infection in swine causes an enterocolitis followed by a persistent carrier state, but little is known about the mechanisms that allow this organism to colonize and persist in host tissues. Neutrophils provide a first line of defense against invading pathogens such as Salmonella typhimurium. The purpose of this study was to evaluate porcine neutrophil function after in vivo exposure to Salmonella and to determine if the immunomodulator, bacillus Calmette Guerin (BCG), exerts any effect on neutrophil function or on the colonization and persistence of S. typhimurium in the pig. Compared to negative controls, neutrophils from pigs exposed to S. typhimurium exhibited significantly decreased iodination, cytochrome-C reduction, antibody-dependent cell-mediated cytotoxicity, random migration, and chemotaxis (P less than or equal to 0.05). Neutrophil bactericidal activity against S. typhimurium was significantly enhanced. Most of the significant differences were noted in the first two days after exposure to Salmonella. Often the functional alterations were biphasic, peaking again 7-10 days after exposure. BCG alone significantly depressed random migration and cytochrome-C reduction in unstimulated neutrophils. The clinical course, colonization pattern, and persistence of Salmonella were similar between pigs receiving BCG and untreated pigs. These data suggest that S. typhimurium infection causes a depression in oxidative metabolism and motility, yet an increase in overall bactericidal activity against S. typhimurium in circulating porcine neutrophils. It also appears that BCG treatment, as reported here, does not enhance resistance of pigs to S. typhimurium colonization or reduce the number of persistent organisms in the porcine ileum.

Cattle persistently infected with bovine viral diarrhea (BVD) virus have decreased neutrophil and lymphocyte functions. We reevaluated these functions and further characterized the inhibition of persistent BVD virus infection in neutrophils, using sensitive kinetic assays. In addition, the influence of in vitro incubation of neutrophils with recombinant bovine interferon gamma (rBoIFN gamma) and in vitro incubation of lymphocytes with recombinant bovine interleukin-2 was evaluated. Significant (P less than 0.05) decrease in random migration under agarose, Staphylococcus aureus ingestion, cytochrome-C reduction, iodination, antibody-independent cell-mediated cytotoxicity, oxidant production, and cytoplasmic calcium flux were observed in neutrophils from cattle persistently infected with BVD virus, compared with noninfected control cattle. Incubation of neutrophils from noninfected controls with rBoIFN gamma significantly (P less than 0.05) decreased random migration under agarose, cytochrome-C reduction, and cytoplasmic calcium flux. Neutrophils from cattle persistently infected with BVD virus also had decreased random migration under agarose after incubation with rBoIFN gamma; in addition, antibody-independent cell-mediated cytotoxicity, elastase release, and cytoplasmic calcium flux were significantly enhanced. The rBoIFN gamma induced significantly (P less than 0.05) different effects on chemotaxis, cytochrome-C reduction, iodination, and cytoplasmic calcium flux of neutrophils from infected and control cattle. The rBoIFN gamma was more effective at improving the function of neutrophils from cattle persistently infected with BVD virus, compared with neutrophils from controls. Lymphocytes from infected cattle had decreased blastogenesis in response to phytohemagglutinin, concanavalin A, and pokeweed mitogen.(ABSTRACT TRUNCATED AT 250 WORDS)

The present study was designed to determine the effect of recombinant porcine somatotropin (rpST; 5, 15, or 25 mg.pig-1.d-1 for 57 d) on a variety of immune function variables. We observed no significant effect of rpST treatment on the gross pathology of the pigs, histopathology of the immune system organs, total and differential white blood cell counts, lymphocyte blastogenic response to mitogens, or the neutrophil functions of chemotaxis, ingestion, reduction of cytochrome C, iodination, and antibody-dependent cell-mediated cytotoxicity. Those variables that were significantly affected by rpST treatment include a decreased hemoglobin and packed red blood cell volume (at some time points for all three rpST dosages), a decrease in plasma protein level at the 25-mg dose, a small increase in neutrophil random migration (at all three rpST dosages), and a decrease in IgG antibody response to tetanus toxoid at 15 d after immunization (but not at d 8, 22, or 29). Additionally, rpST treatment was associated with a decreased rate of BW gain (at 15-mg dose), increased liver and kidney weights (at all three dosage levels), and an increased incidence of renal tubular cytoplasmic vacuolation of minor severity. There were no observed differences in the overall health of the pigs due to rpST treatment, based on clinical observations as well as determination of antibody titer to, and isolation of, common swine pathogens. Therefore, there was no evidence that the observed influence of rpST treatment on immune function would be clinically relevant.

SUMMARY Recombinant human interleukin-2 (rhil-2) was evaluated for its influence on total and differential wbc counts, lymphocyte blastogenic responsiveness to mitogens, and several measurements of neutrophil function in clinically normal and in dexamethasone-treated cattle. A single dose of rhil-2 (2.5 × 107 U) given sc had no influence on the total or differential wbc count; however, it did cause an inhibition of neutrophil random migration. The other measurements of neutrophil function (Staphylococcus aureus ingestion, cytochrome C reduction, iodination, and antibody-dependent and antibody-independent cell-mediated cytotoxicity) evaluated were not significantly altered. The rhil-2 treatment was associated with a significant (P < 0.01) decrease in uptake of [3H]thymidine in unstimulated lymphocytes and a tendency toward enhanced blastogenesis of lymphocytes stimulated with phytohemagglutinin. This enhancement was significant (P < 0.05) only when the results were expressed as a stimulation index. Lymphocyte responsiveness to concanavalin A and pokeweed mitogen was not significantly influenced by rhil-2 administration. Dexamethasone (0.04 mg/kg) administered every 24 hours for 3 consecutive days altered the wbc count and several measurements of lymphocyte and neutrophil function. The administration of a single dose of rhil-2 (2.5 × 107 U) 8 hours after the first dose of dexamethasone did not alter the influence of dexamethasone on any of the measurements. These results indicated that rhil-2 has some biologic activity in cattle, but when used as administered here, did not overcome the influence of dexamethasone on the in vitro measurements of lymphocyte and neutrophil function that were evaluated.

Twenty Holstein steers subclinically infected with coccidia were allotted to 2 groups of 10 steers each. One group received a diet containing 0.5 mg of decoquinate/kg of body weight. After 25 days on the diet, there was no difference between the groups in lymphocyte blastogenic responsiveness to mitogens; however, there were differences in neutrophil function. Lymphocytes from steers of the decoquinate-fed group had decreased random migration under agarose, enhanced cytochrome C reduction, and enhanced iodination activity. Other measures of neutrophil function evaluated (chemotactic index, Staphylococcus aureus ingestion, and antibody-dependent and -independent cell-mediated cytotoxicity) were not affected. After 30 days of decoquinate feeding, half of the cattle in each group received 5 daily IM injections of dexamethasone (0.04 mg/kg of body weight). The dexamethasone-treated steers from the group that did not have decoquinate in the diet developed clinical coccidiosis, whereas the decoquinate-treated steers remained clinically normal. Lymphocyte and neutrophil function were again evaluated for a 3-day period beginning 4 days after dexamethasone treatment was halted. Neutrophils from the steers that developed clinical coccidiosis after dexamethasone administration had significantly (P less than 0.05) inhibited random migration under agarose, cytochrome C reduction, and iodination activity, but significantly (P less than 0.01) enhanced S aureus ingestion. The feeding of decoquinate prevented the inhibition of neutrophil cytochrome C reduction and lessened the inhibition of neutrophil iodination in the dexamethasone-treated group. Dexamethasone treatment was associated with an inhibition of lymphocyte blastogenic responsiveness to phytohemagglutinin in principals as well as controls.

Three dosages (0.1, 0.5, and 2.5 mg/animal, subcutaneously), of recombinant bovine interferon-gamma (rBoIFN-gamma) were evaluated for their in vivo influence on neutrophil function and lymphocyte blastogenesis in cattle. The optimal of the three dosages tested (0.5 mg/animal or 1.1 x 10(6) U/animal) was then evaluated for its influence on neutrophils and lymphocytes in both normal and dexamethasone-treated cattle. One animal, which received 2.5 mg of rBoIFN-gamma, died by 24 h after administration due to acute diffuse interstitial pneumonia with interlobular edema and emphysema. The two highest dosages used caused fever at 24 h and the highest dosage caused a decrease in lymphocyte blastogenesis at 24 h after administration. The influence of rBoIFN-gamma on neutrophil function was dose dependent and depended on the baseline values for neutrophil function. Random migration by neutrophils was consistently inhibited in animals that received 0.5 mg or more of rBoIFN-gamma. Staphylococcus aureus ingestion and antibody-dependent cell-mediated cytotoxicity by neutrophils was enhanced by rBoIFN-gamma treatment in both dexamethasone-treated cattle and in nondexamethasone-treated cattle, which had relatively low values for these parameters before treatment. Iodination by neutrophils was also enhanced by rBoIFN-gamma when either a suboptimal concentration of neutrophil stimulant was used or when the cattle were treated with dexamethasone. In summary, the rBoIFN-gamma had greater immunomodulator activity in immunosuppressed than in normal cattle. The in vivo influence of rBoIFN-gamma therefore depends on the physiologic status of the animal.

Compared with neutrophils from healthy dogs, neutrophils from 2 dogs with primary ciliary dyskinesia had increased distance of random migration, but fewer of the neutrophils migrated. The affected dogs had an increase in the numbers of Staphylococcus aureus phagocytized. Lymphocyte blastogenesis in the affected dogs in response to standard mitogens was considered to be normal.