Optimum conditions for the turkey lymphocyte transformation test.

Optimum conditions for turkey lymphocyte transformation tests were determined. Thrice-washed turkey buffy-coat cells obtained after slow centrifugation (40 x g, 10 minutes) responded well to mitogenic stimulation. Turkey lymphocytes isolated on Ficoll-containing separation media largely lost their ability to respond to mitogens. Maximum responses were obtained with 2 x 10(7) lymphoid cells/ml. Responses to the mitogens were greatest when bovine fetal serum was used at a 2.5% concentration or pooled turkey serum and autologous plasma were used at a 1.25% concentration. Higher concentrations of turkey serum or plasma decreased the responses when sub-optimum doses of concanavalin-A (Con A) or phytohemagglutinin-P (PHA-P) were used. Serum-free cultures gave higher stimulation indices than cultures with serum only when sub-optimum doses of Con A or PHA-P were used. Optimum mitogen concentrations varied with individual birds, timing of the culture, temperature of incubation, and serum concentration in the cultures. Responses were usually greatest with final concentrations of 5 micrograms Con A/ml, 10 micrograms PHA-P/ml, and 20 micrograms pokeweed mitogen (PWM)/ml and when the cultures were incubated in 96-well microplates at 40 C in humidified air with 5% CO2 for 40-42 hours with pulsing with 3H-thymidine during the final 16 hours of incubation.     

Duck lymphocytes--III. Transformation responses to some common mitogens

The lymphocyte transformation (LT) test was performed using duck blood lymphocytes stimulated with phytohaemagglutinin (PHA), concanavalin A (Con A), lentil lectin (LC), Roman snail lectin (HP), peanut agglutinin (PNA), Bandeiraea simplicifolia seed lectin (BSS), wheat germ agglutinin (WGA), horseshoe crab lectin (HSC), pokeweed mitogen (PWM) and E. coli lipopolysaccharide (LPS). Cells were cultured in microtitre trays, at 41.6 degrees C, 8 x 10(5) cells in 200 microliters medium (= 4 x 10(6) cells/ml) supplemented with 10% pooled duck serum. Mitogens were added at final concentrations of 0.1-100 micrograms/ml and triplicate cultures at each concentration were harvested daily for scintillation counting 6 hr after addition of 1 microCi [3H]thymidine. Three patterns of response were observed. The responses to Con A, LC, HP and HSC were greatest at high mitogen concentrations (40-100 micrograms/ml) throughout the 7 days of culture. With PHA, PNA, WGA and LPS maximum stimulation was obtained at 3-5 days, at which time the cells were responding to lower concentrations of mitogen than were required at other times during the experiment. The response to BSS and PWM showed increasing sensitivity to lower concentrations of mitogen during the first 3 days of culture and then stimulated most strongly at 2-10 micrograms/ml in cultures harvested after 4-7 days. Cells from two ducks were cultured for 3 and 5 days with selected concentrations of these mitogens; the results confirmed the variation in response to different mitogens. It is possible that these patterns of response are the outcome of stimulating different populations of duck lymphocytes.     

Changes in lymphocyte blastogenic response of mares during the perinatal period

A fluorometric assay was applied to evaluate blastogenesis of equine lymphocytes. Optimal culture conditions were as follows; concentrations of phytohaemagglutinin-P (PHA), concanavalin A (Con A) and pokeweed mitogen (PWM) were 1 microgram/ml, 40 micrograms/ml and 10 micrograms/ml, respectively, when 5 X 10(5) lymphocytes were incubated with culture medium containing 20% pooled horse serum (PHS) for 120 hours. The relative mean stimulation index of healthy non-pregnant mares were 5.107 +/- 0.323 (M +/- SE) with PHA, 4.019 +/- 0.183 with Con A and 3.610 +/- 0.131 with PWM. Sequentially the blastogenic responses of lymphocytes from twenty mares were observed during various stages of the perinatal period. Response decreased gradually before parturition was lowest at the time of parturition (PHA: 1.923 +/- 0.174, Con A: 1.698 +/- 0.206 and PWM: 1.706 +/- 0.177), and then increased gradually after parturition towards non-pregnant levels.     

Optimal conditions for in vitro mitogen-induced proliferation of peripheral blood lymphocytes in breeding foxes

The proliferative response of fox peripheral blood lymphocytes to nonspecific mitogens: leucoagglutinin (LA), concanavalin A (Con A) and pokeweed mitogen (PWM) was studied. Microcultures were kept at 39 degrees C in a humidified atmosphere containing 5% CO2. The highest 3H-thymidine incorporation was observed, when Con A was used, while LA and PWM showed weaker but significant stimulatory action. Optimal doses of mitogens were: 5 micrograms/ml for Con A, 5 micrograms/ml for LA and a dilution of 1:100 for PWM. The maximal stimulation index for Con A was about 240 and up to 100 for LA or PWM. The maximal lymphocyte proliferation was observed when culture media were supplemented with 10% serum. When proliferation kinetics were studied, the peak response was observed on Day 2.     

Mitogen stimulation of canine normal and myasthenia gravis lymphocytes

Responses of canine lymphoid tissues to mitogens were studied in five normal dogs and in two dogs with acquired myasthenia gravis (MG). In the normal dogs, lymph-node-derived lymphocytes gave the most consistent proliferative responses to concanavalin A (Con A), phytohemagglutinin (PHA), and pokeweed mitogen (PWM), as determined by thymidine incorporation; and in most cases PHA, lipopolysaccharide (LPS), and PWM stimulated total IgG production, as determined by ELISA. Splenic lymphocytes had the greatest capacity for increased total IgG production. In the myasthenic dogs total IgG production by unstimulated lymph-node-derived lymphocytes was 88 micrograms/ml and 153 micrograms/ml, much higher than that of unstimulated normal dog lymphocytes (mean less than 1.0 microgram/ml). All mitogens resulted in suppression rather than stimulation of IgG production by lymphocytes from dogs with MG. Production of antibodies to acetylcholine receptors (AChRs) was detected in the supernatants of lymphocyte cultures from one of the dogs with MG at a rate of 78 fmol/5 x 10(5) cells per week and was not detected in culture supernatants of control dogs. This study demonstrates that lymph nodes may be an important site of antibody production in myasthenic dogs and provides the necessary groundwork for future studies of the cellular immunology of canine MG.     

Comparative studies on in vitro mitogen-induced proliferation of peripheral blood lymphocytes in dog and breeding fox.

In vitro blastogenesis of dog and fox lymphocytes was compared by a microculture technique. The highest 3H-thymidine incorporation in cultures of dog lymphocytes was observed at day 3, while in those of fox at day 2, incubated either at 37 degrees C or at 39 degrees C. Lymphocytes cultured at 39 degrees C incorporated more tritiated thymidine than did cells cultured at 37 degrees C. The stimulation index (SI) of dog peripheral blood lymphocytes to both mitogens concanavalin A (Con A) and leucoagglutinin (LA) was in a similar range, while pokeweed mitogen (PWM) showed a weaker but significant stimulatory action. The blastogenesis of fox lymphocytes was the greatest in Con A stimulated cultures. The mitogenic potency of LA and PWM was about half of that of Con A, with no essential difference between them. Maximum lymphocyte proliferation of dog and fox was observed when culture media were supplemented with 10% fetal calf serum (FCS).     

Lymphocyte transformation test in veterinary clinical immunology

Lymphocyte transformation test is a powerful tool in laboratory testing of immunologic competence of animals. The impaired function of the lymphocytes or presence of mitogenesis suppressing factors in the patient serum were detected by comparing lymphocyte transformation (expressed as thymidine incorporation) obtained in media containing either autologous, homologous, or fetal calf serum additions. Most valuable results were obtained by using at least two, preferably three, different phytomitogens: concanavlin A (Con A), pokeweed mitogen (PWM), and pl ytohemagglutinin (PHA) at optimal concentrations (Con A, 15 μg/ml, PWM and PHA, 5 μg/ml) and decreased concentrations (Con A, 5 μg/ml, PWM and PHA, 1 μg/ml). Mitogenesis induced by lipopolysaccharide was considerably smaller and not used routinely. With 2 × 10⁵ lymphocytes/well, the background count of unstimulated lymphocytes in autologous serum in healthy dogs was usually between 100 and 400 counts/min (CPM), in clinically healthy cattle and horses from 200 to over 2000 CPM. Higher CPM were rarely detected without clinical disease. Increased background counts were often associated with viral infections, leukemias and lymphoreticular hyperplasias, decreased background counts were associated with various diseases. The stimulation indexes (SI) of healthy animals in autologous serum with Con A, (5 μg/ml) or PWM or PHA (1 μg/ml) were in the range from 100 to 1000 in the dogs, in the tens for Con A and in hundreds for PWM and PHA in horses and cattle. Increased SI were present during the incubation period of various diseases. Decreased SI were associated with numerous infectious and lymphoreticular diseases and were caused by any of the following: (1) the presence of serum immunosuppressive factor(s) in the patient serum, (2) the decreased response of lymphocytes to mitogens, or (3) increased mitogenicity of lymphocytes due to unidentified serum factors in absence of phytomitogens.     

Transformation of chicken lymphocytes stimulated by concanavalin A: response parameters.

The culture conditions for obtaining the maximum transformation of chicken lymphocytes stimulated by the mitogen Concanavalin A are described in this paper. Lymphocytes were cultured in round bottom microtiter tissue culture plates and the incorporation of 3H-methyl-thymidine (3H-TdR) into lymphocyte deoxyribonucleic acid was used to quantitatively evaluate transformation. Maximum incorporation of 3H-methyl-thymidine occurred when 2 x 10(6) lymphocytes and 4 micrograms of Concanavalin A were added to each culture. Deoxyribonucleic acid synthesis was greatest during the period of 18 to 36 hours after addition of mitogen. Differences between chicken and mammalian lymphocyte cultures are discussed.     

An evaluation of the mitogenic reactivity of intestinal intraepithelial lymphocytes of chickens.

A colorimetric assay employing MTT (3-[4,5-dimethylthiazole-2-yl], 2-5-diphenyltetrazolium bromide) was used to determine the mitogenic response of intestinal intraepithelial lymphocytes (i-IELs) of chickens to T- and B-cell mitogens. Comparisons between mitogenic responses of i-IELs and peripheral blood lymphocytes (PBLs) were made to examine potential relationships. The results from this study indicated that T-cell mitogens, concanavalin A (Con A), and phytohemagglutinin-P (PHA-P) induced mitogenic stimulation in i-IELs. Although stimulation indexes of both i-IELs and PBLs were similar, the optical densities (ODs) of i-IEL cultures containing Con A or PHA-P were 20- to 50-fold lower than the ODs of PBL cultures containing the same mitogen. The lower conversion of MTT to formazan resulting in lower ODs in i-IEL cultures indicated a lower level of cellular activity in the i-IELs than in the PBLs. The mitogenic responses of both i-IELs and PBLs to Con A and PHA-P were dose dependent. The responsive concentration of Con A for i-IELs was within the range of 25-50 micrograms/ml, whereas the responsive concentration of PHA-P for i-IELs was 50 micrograms/ml. Three days of incubation was found to be adequate to induce a significant (P < 0.05) mitogenic response for both T-cell mitogens. Lipopolysaccharide was unable to induce a mitogenic response in i-IELs, which was attributed to the lack of B cells in the i-IEL population. This technique may prove useful in evaluating and studying the role of i-IELs in local cell-mediated immune responses of the gastrointestinal tract.     

Canine lymphocyte cultures in vitro: Evaluation of peripheral blood lymphocyte response to mitogens

Lymphocytes from dog peripheral blood have been stimulated in vitro with 3 different mitogens (Con A, PHA and PWM). Culture medium was RPMI 1640 enriched with either autologous plasma, fetal calf serum or a newly described defined serum substitute. In such cultures the number of surviving and activated cells was measured by cytofluorometry and the proliferation was assessed by thymidine incorporation. In unstimulated cultures, up to 70% of all cells had disappeared (died) during the first 42 hours of incubation, whereas the number of viable cells was reduced to 50–60% in mitogen stimulated cultures. Of the surviving lymphocytes, between 25–40% of the cells appeared to have an elevated RNA-content (activated or G₁ cells). By comparison between thymidine incorporation and number of mitogen induced G₁ cells, a very high correlation was found (r=0.92). However, the slope of the regression line was much lower than expected. The low thymidine incorporation per activated cell was primarily related to the high cell death and a resulting dilution of tritiated thymidine. Indeed, preliminary results suggested that the same thymidine incorporation per G_1b cells could be obtained if peripheral blood lymphocytes were washed immediately before pulsing as could be obtained with lymphnode cells without washing.     

Neonatal calf serum immunoregulation of lymphocyte blastogenic responses

The ability of lymphocytes from newborn calves to undergo blastogenic responses to the mitogens Concanavalin A (Con A), phytohemagglutinin (PHA), or Pokeweed Mitogen (PWM), and immunomodulation of these responses by neonatal calf serum was assessed as a function of age. Lymphocytes were obtained from thymus, spleen, and mesenteric lymph nodes of 1-, 2- to 3-, 5- to 7-, and 9- to 10-d-old calves, aliquoted and incubated (+/- mitogens) in sera from 1-, 2-, 3-, or 7- to 10-d-old calves. Lymph-node lymphocytes responded least when cultured in sera from 1-d-old calves, regardless of mitogen or age of cell donor; the response increased as age of serum donor increased (P less than .05). Splenic lymphocytes responded similarly (P less than .005). However, when cultured in sera from older calves, splenic lymphocytes from older calves responded greater to PWM than did those from younger calves. Thymic lymphocytes responded minimally to PWM and PHA. Their response to Con A increased (P less than .005) with age of serum donor calf, but the effect was greatest on lymphocytes from 5- to 7-d-old calves. Mixing experiments with varying ratios of 1-d-old calf serum: 10-d-old calf serum suggested that serum from 1-d-old calves contained suppressive activity. Serum cortisol level (measured by radioimmunoassay) was 30 +/- 4.6 ng/ml in calves at 6 h of age and declined to 5.5 +/- 1.1 ng/ml by 10 d. Charcoal treatment to remove steroids did not enhance blastogenesis. Addition of cortisol (50 ng/ml) to charcoal-treated sera resulted in inhibition of response to PHA, but no change in response to Con A or PWM. Further investigation is indicated to characterize this immunosuppressive activity and to establish its relationship to disease susceptibility.     

Development of optimal conditions for lymphokine production by chicken lymphocytes

Chicken thymus, spleen, and bursa lymphocytes were isolated by different methods and incubated under differing conditions in order to obtain and characterize avian lymphokines. The biological activity of lymphokine-containing cell culture supernatants was measured by their antiviral activity (interferon(IFN)-units) and by their capacity to induce cytostatic effects in bone-marrow-derived macrophages (50% cytostasis-inducing dose, CID). Lymphokine production by thymus lymphocytes required concanavalin A (ConA)-stimulation, while spleen cells, when cultured at high density, released CID and IFN activities into the culture medium even without mitogen-stimulation. By way of comparison, the highest lymphokine content was found in the supernatant of lymphocyte cultures, which were incubated for 72 hours at 41 degrees C after stimulation with an optimal ConA dose. For stimulation of thymus lymphocytes 30 micrograms ConA/ml were found to be optimal, independent of serum content and cell density in the cultures. In contrast, the optimal ConA dose for spleen lymphocytes not only depended on the serum content but also on the cell density in the cultures and varied within a range of 2.5 micrograms and 45 micrograms ConA/ml.     

Mitogen-induced transformation of sheep and goat peripheral blood lymphocytes in vitro: The effects of varying culture conditions and the choice of an optimum technique

Peripheral blood lymphocytes (PBL) prepared by centrifugation of heparinized sheep or goat jugular venous blood on Ficoll-Triosil were shown to incorporate methyl-[H³]-thymidine ([H³]-Tdr) in vitro in response to lymphocyte mitogens.Optimal conditions for transformation included the culture of 2.5 × 10⁵ viable cells per round bottomed culture well in 250μl medium RPMI-1640 supplemented with fetal calf serum (FCS) at 10% for goat or 15% for sheep lymphocytes. Optimum incorporation of [H³]-Tdr by sheep PBL was recorded after 3–5 days and was achieved in response to 100μg/ml phytohaemagglutinin (PHA), 20μl/ml pokeweed mitogen (PWM), 10μg/ml Concanavalin-A (Con-A) and 50μg/ml bacterial lipopolysaccharide (LPS). For goat PBL the optimum mitogen concentrations were 50μg/ml PHA, 20μl/ml PWM, 5μg/ml Con-A and 50μg/ml LPS. Optimum PHA concentrations were influenced by the level of FCS supplementation, higher concentrations of PHA being required for optimum response when the concentration of FCS was increased.While variability within preparations was small there was considerable variation in the magnitude of the response between preparations, which was sufficient to confound comparisons between different experiments and between animals. The variability between preparations could not be attributed to changes in sensitivity of PBL to mitogens or to the influence of erythrocyte contamination of the PBL preparations. While these results are in general agreement with previous reports of optimal conditions for the measurement of ruminant PBL to mitogens, there are some important differences which are discussed in the context of the available literature.     

Serum and tissue cage fluid concentrations of ciprofloxacin after oral administration of the drug to healthy dogs

Ciprofloxacin, a fluoroquinolone antimicrobial agent, was administered orally to 4 healthy dogs at dosage of approximately 11 and 23 mg/kg of body weight, every 12 hours for 4 days, with a 4-week interval between dosing regimens. Serum and tissue cage fluid (TCF) concentrations of ciprofloxacin were measured after the first and seventh dose of each dosing regimen. The peak concentration was greatest in the serum after multiple doses of 23 mg/kg (mean +/- SEM; 5.68 +/- 0.54 micrograms/ml) and least in the TCF after a single dose of 11 mg/kg (0.43 +/- 0.54 micrograms/ml). The time to peak concentration was not influenced by multiple dosing or drug dose, but was longer for TCF (6.41 +/- 0.52 hour) than for serum (1.53 +/- 0.52 hour). Accumulation of ciprofloxacin was reflected by the area under the concentration curve from 0 to 12 hours after administration (AUC0----12). The AUC0----12 was greatest in the serum after multiple doses of 23 mg/kg (31.95 +/- 1.90 micrograms.h/ml) and least in the TCF after a single dose of 11 mg/kg (3.87 +/- 1.90 micrograms.h/ml). The elimination half-life was not influenced by multiple dosing or dose concentration, but was greater for TCF (14.59 +/- 1.91 hours) than for serum (5.14 +/- 1.91 hours). The percentage of TCF penetration (AUCTCF/AUCserum) was greater after multiple doses (95.76 +/- 6.79%) than after a single dose (55.55 +/- 6.79%) and was not different between doses of 11 and 23 mg/kg. Both dosing regimens of ciprofloxacin resulted in continuous serum and TCF concentrations greater than 90% of the minimal inhibitory concentration for the aerobic and facultative anaerobic clinical isolates tested, including Pseudomonas aeruginosa.     

Serum and synovial fluid concentrations of ampicillin trihydrate in calves with suppurative arthritis

Eight calves with suppurative arthritis were each given a single intramuscular injection of ampicillin trihydrate at a dose of 10 mg/kg. Ampicillin concentrations were measured serially in serum and in suppurative and normal synovial fluid over a 24-hour period. The mean peak serum concentration was 2.5 +/- 0.54 micrograms/ml 2 hours after injection. The highest concentration in normal synovial fluid was 3.5 +/- 0.40 micrograms/ml at 4 hours and the highest concentration in suppurative synovial fluid was 2.7 +/- 0.58 micrograms/ml at 2 hours. Overall mean ampicillin concentration in normal synovial fluid for the first 8 h (2.9 +/- 0.32 micrograms/ml) was significantly different from that in suppurative synovial fluid (2.1 +/- 0.33 micrograms/ml) and serum (1.9 +/- 0.30 micrograms/ml; p less than 0.05).     

Variation in mitogen-induced ovine lymphocyte blastogenesis: Adherent cells or 2-mercaptoethanol restore randomly depressed responses

Peripheral blood leucocytes (PBL) isolated 23 times over a 6-week period frommm four normal sheep showed considerable variation in serially tested responses to predetermined optimal concentrations of concanavalin A (Con A), phytohaemagglutinin (PHA), and pokeweed mitogen (PWM). Con A responses, in particular, varied widely and were often randomly depressed (21 of 91 times compared to 15 of 91 times for PHA or PWM). The addition of as few as 1% adherent cells (AC) to depressed cultures fully restored the PBL proliferative response to normal levels. Addition of greater numbers of AC (5 or 10%) had little further enhancing effect on depressed cultures. The addition of 1, 5, or 10% AC to cultures that were responding at normal levels increased responses only slightly. Autologous or allogeneic AC were equally effective. Addition of 2-mercaptoethanol (2-ME) to depressed cultures only partially restored the blastogenic response to Con A and had little effect on normal cultures.     

Serum and skin concentrations after multiple-dose oral administration of trimethoprim-sulfadiazine in dogs.

Six healthy adult mixed breed dogs were each given 5 oral doses of trimethoprim (TMP)/sulfadiazine (SDZ) at 2 dosage regimens: 5 mg of TMP/kg of body weight and 25 mg of SDZ/kg every 24 hours (experiment 1) and every 12 hours (experiment 2). Serum and skin concentrations of each drug were measured serially throughout each experiment and mean serum concentrations of TMP and SDZ were determined for each drug for 24 hours (experiment 1) and 12 hours (experiment 2) after the last dose was given. In experiment 1, mean serum TMP concentration was 0.67 +/- 0.02 micrograms/ml, and mean skin TMP concentration was 1.54 +/- 0.40 micrograms/g. Mean serum SDZ concentration was 51.1 +/- 12.2 micrograms/ml and mean skin SDZ concentration was 59.3 +/- 9.8 micrograms/g. In experiment 2, mean serum TMP concentration was 1.24 +/- 0.35 micrograms/ml and mean skin TMP concentration was 3.03 +/- 0.54 micrograms/g. Mean serum SDZ concentration was 51.6 +/- 9.3 micrograms/ml and mean skin SDZ concentration was 71.1 +/- 8.2 micrograms/g. After the 5th oral dose in both experiments, mean concentration of TMP and SDZ in serum and skin exceeded reported minimal inhibitory concentrations of TMP/SDZ (less than or equal to 0.25/4.75 micrograms/ml) for coagulase-positive Staphylococcus sp. It was concluded that therapeutically effective concentrations in serum and skin were achieved and maintained when using the manufacturer's recommended dosage of 30 mg of TMP/SDZ/kg (5 mg of TMP/kg and 25 mg of SDZ/kg) every 24 hours.     

Serum and tissue fluid norfloxacin concentrations after oral administration of the drug to healthy dogs

Norfloxacin, a 4-quinolone antibiotic, was administered orally to 4 healthy dogs at dosages of 11 and 22 mg/kg of body weight, every 12 hours for 4 days, with a 4-week interval between dosing regimens. Serum and tissue cage fluid (TCF) norfloxacin concentrations were measured at 0, 0.5, 1, 1.5, 2, 3, 4, 5, 6, 8, 10, and 12 hours after the first and seventh dose of each dosing regimen. When administered at a dosage of 11 mg/kg, the mean peak serum concentration (Cmax) was 1.0 microgram/ml at 1 hour, the time of mean peak concentration (Tmax) after the first dose. After the seventh dose, the Cmax was 1.4 micrograms/ml at Tmax of 1.5 hours. The Tmax for the TCF concentration was 5 hours, with Cmax of 0.3 microgram/ml and 0.7 microgram/ml after the first and seventh dose, respectively. When administered at a dosage of 22 mg/kg, the serum Tmax was 2 hours after the first dose, with Cmax of 2.8 micrograms/ml. After the seventh dose, the serum Tmax was 1.5 hours, with Cmax of 2.8 micrograms/ml. The Tmax for the TCF concentration was 5 hours after the first and seventh doses, with Cmax of 1.2 micrograms/ml and 1.6 micrograms/ml, respectively. After the seventh dose, the serum elimination half-life was 6.3 hours for a dosage of 11 mg/kg and was 6.7 hours for a dosage of 22 mg/kg. For serum concentration, the area under the curve from 0 to 12 hours (AUC0----12) was 8.77 micrograms.h/ml and 18.27 micrograms.h/ml for dosages of 11 mg/kg and 22 mg/kg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunosuppression of in vivo and in vitro lymphocyte responses in swine induced by Trichinella spiralis or excretory-secretory antigens of the parasite.

The in vivo and in vitro effects of Trichinella spiralis excretory-secretory (ES) antigens on porcine peripheral blood lymphocyte (PBL) responses induced with mitogens (phytohemagglutinin, PHA; concanavalin A, Con A; pokeweed mitogen, PWM) or unrelated antigen (Protein A) were studied to determine whether ES antigens depress lymphocyte responses in experimental swine trichinosis, and/or if this response was manifested after lymphocytes from infected pigs had been pretreated with ES antigens. Additionally, the range of inhibition of lymphocyte responses was tested in parasite-free pigs using different doses of ES antigens and compared with the responsiveness of control cultures from the same animals. The responses of lymphocytes from pigs inoculated with 4 x 10(3) muscle larvae (ML) were strongly depressed (P < 0.05) at post-inoculation days (PID) 7 (after stimulation with PHA), 14, 35 (Con A or PWM), and 49 (PWM). At PID 56 and 63 the lymphocytes from T. spiralis-infected pigs responded better (P < 0.05) to all three mitogens than those from non-infected controls. After 7 weeks post-inoculation, PBL which were pretreated with 10 or 250 micrograms ml-1 of ES antigens showed significantly weaker (P < 0.05, P < 0.001) responses to PWM or PHA, respectively, than those from non-infected animals. The responsiveness of lymphocytes from both groups of pigs to Protein A was not affected by the pretreatment with ES antigens in vitro. The responses of lymphocytes from the parasite-free pigs induced by PHA, PWM or Protein A were strongly depressed (P < 0.01) after in vitro pretreatment regardless of the dose of ES antigens (5, 10, 15, or 20 micrograms ml-1) applied.     

Culture conditions for blastogenic responses of bovine mammary mononuclear cells

Several experimental parameters were examined to determine optimal conditions for proliferative responses of mammary mononuclear cells (MMC) obtained from six nonlactating dairy cows. These parameters were: pre-incubation of cells in medium prior to assay, mitogen concentration, assay incubation time, and type of culture medium. Response variables included viability of cells and the rate of proliferation as assessed by tritiated thymidine incorporation. Pre-incubation of cells in medium had no effect on the proliferative response of MMC. Whereas Concanavalin A (ConA; 3.3 or 6.6 micrograms/ml) and phytohemagglutinin (PHA; 1, 5, 10 micrograms/ml) did stimulate proliferation of MMC, the higher doses did not stimulate greater proliferation than the lower doses of mitogens. The greatest mitogenic response was obtained on days 2 and 3 of incubation. Proliferative responses were significantly higher at all mitogen levels tested in a 50-50 mixture of Rosewell Park Memorial Institute medium 1640 and Liebovitz-15 medium (RPMI/L-15) than in RPMI alone. Viability of MMC was also significantly higher in the RPMI/L-15 medium. To test whether the significant effect of media on blastogenesis was specific for mononuclear cells from the bovine mammary gland, peripheral blood lymphocytes (PBL) from four dairy cows were cultured with ConA and PHA in a mitogen assay in both RPMI and RPMI/L-15. Viability was measured on day of collection and on all culture days. PBL were stimulated equally in both media. PBL viability decreased significantly on day 1 in both RPMI and RPMI/L-15. These results suggest that the optimal culture conditions for blastogenic responses of mammary mononuclear cells and peripheral blood lymphocytes may differ.