Immune response of sheep to bluetongue virus: in vitro induced lymphocyte blastogenesis

Sheep were inoculated with 2 ml of 10(7) plaque forming units per ml of purified prototypes of the four United States serotypes (10, 11, 13 and 17) of bluetongue virus. Nine weeks following the initial inoculation, a challenge inoculation with homologous virus was done. Animals were followed for virus isolation and evidence of cell-mediated immunity by weekly lymphocyte stimulation tests (LST). Two dilutions (10 micrograms/ml and 1 microgram/ml) of pure virus from each of the purified serotypes were used as antigen as were the phytomitogens phytohemagglutinin, Concanavalin A, and pokeweed mitogen. LST data were analyzed by the analysis of variance method and reported as counts per minute and stimulation index (SI). Significant SI were observed following primary and secondary challenge with both homologous and heterologous virus. There was evidence of lymphocyte perturbations characterized by a sharp decrease in response to mitogens following primary and secondary challenge lasting for one week followed by a significant increase in blastogenesis three to four weeks after inoculation of virus. These results provide evidence that cell-mediated immunity is evident in bluetongue infection, that there is cross reactivity between viral serotypes and that BTV infection leads to perturbations in lymphocyte function including suppression of responses. An increase in the blastogenic response to phytomitogens correlated with viral clearance.     

Lymphocyte subset proliferative responses of Mycobacterium bovis-infected cattle to purified protein derivative

Despite highly successful eradication efforts in several countries, Mycobacterium bovis infection of cattle remains a significant health concern worldwide. Immune mechanisms of resistance to and/or clearance of M. bovis infection of cattle, however, are unclear. Recent studies have provided evidence supporting a role for CD4(+), CD8(+), and gammadelta TCR(+) T cells in the response of cattle to M. bovis. In the present study, we utilized a flow cytometric-based proliferation assay to determine the relative contribution of individual lymphocyte subsets in the response to M. bovis infection and/or sensitization with mycobacterial purified protein derivative (PPD). Peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle proliferated in response to in vitro stimulation with M. bovis PPD. CD4(+) T cells and gammadelta TCR(+) cells were the predominate subsets of lymphocytes responding to PPD. gammadelta TCR(+) cells also proliferated in non-stimulated cultures; however, the gammadelta TCR(+) cell proliferative response of infected cattle was significantly (p<0.05) greater in PPD-stimulated cultures as compared to non-stimulated cultures. Intradermal injection of PPD for comparative cervical testing (CCT) induced a boost in the in vitro proliferative response of CD4(+) but not gammadelta TCR(+) cells of infected cattle. Administration of PPD for CCT also boosted interferon-gamma (IFN-gamma) production by PBMC of infected cattle following in vitro stimulation with M. bovis PPD. Injection of PPD for CCT did not, however, elicit a proliferative or IFN-gamma response in cells isolated from non-infected cattle. These data indicate that CD4(+) and gammadelta TCR(+) cells of M. bovis-infected cattle proliferate in a recall response to M. bovis PPD and that the CD4(+) cell response is boosted by intradermal injection with PPD for CCT.     

Effect of recombinant human interferon-alpha in vitro and in vivo on mitogen-induced lymphocyte blastogenesis in cats

The effect of recombinant human interferon-alpha (rHuIFN-alpha) in vitro and in vivo on mitogen-induced lymphocyte blastogenesis was evaluated in specific-pathogen-free cats. Pre-incubation of isolated feline peripheral blood lymphocytes (PBL) in vitro with either 10(4) or 10(3) International Units (U) of rHuIFN-alpha for 24 h significantly suppressed (P less than 0.001 and 0.01, respectively) blastogenic responses to the phytomitogens concanavalin A (Con A) and pokeweed mitogen (PWM). Lower doses of IFN (range, 10-10(-3) U/ml) neither suppressed nor enhanced mitogenesis. In the absence of phytomitogens, incubation of PBL with 10(4) - 10(2) U (P less than 0.001) or 10 U (P less than 0.05) of rHuIFN-alpha/ml resulted in a significant decrease in incorporation of [methyl-3H] thymidine into newly synthesized cellular DNA. Cultures of PBL exposed continuously for 4 days to rHuIFN-alpha doses of 10(4) U/ml or less did not demonstrate specific reductions in cell viability, indicating that the observed antiproliferative actions of IFN apparently were independent of any direct cytotoxic effects. To investigate the dose-response effects of rHuIFN-alpha in vivo on lymphocyte blastogenesis, individual groups of cats were evaluated on 3 consecutive days before and then 24 h after each cat was inoculated intramuscularly with either a high dose (10(6) U/kg), moderate dose (10(4) U/kg), or a relatively low dose (10(2) U/kg) of rHuIFN-alpha. Cats inoculated with 10(6) U of rHuIFN-alpha/kg had significantly reduced (P = 0.037) blastogenic responses to Con a at 24 h postinoculation compared to preinoculation values; mean PWM responses were also decreased, but this effect was not statistically significant. In contrast, inoculation of cats with either 10(4) or 10(2) U of rHuIFN-alpha/kg significantly enhanced (P = 0.05 or 0.008, respectively) Con A-induced blastogenesis and had no discernible effect on PWM responses. These findings suggest that very high doses of rHuIFN-alpha given parenterally may be associated with suppression of certain T-cell responses in cats; conversely, much lower doses may be immunoenhancing.     

In vitro depression of bovine lymphocyte function by treatment of cultured bovine lymphocytes with physiologic concentrations of hydrocortisone

The effect of hydrocortisone (hydrocortisone sodium succinate) on bovine lymphocyte blastogenesis in response to Staphylococcus aureus antigens and phytohemagglutinin was measured in vitro. Lymphocytes isolated from the blood of cows were treated for 6 to 8 days with physiologic hydrocortisone concentrations known to be inducible by environmental stress (10 ng/ml), acute clinical mastitis (25 ng/ml), or adrenocorticotropin treatment (45 ng/ml). All 3 concentrations of hydrocortisone caused a depression (P less than 0.01) in lymphocyte blastogenesis in response to phytohemagglutinin and S aureus antigen extract. Hydrocortisone concentrations as low as 10 pg/ml caused a depression in the lymphocyte blastogenic response to phytohemagglutinin. Marked variation existed among cows in the normal response of their nontreated lymphocytes and in the degree of depression of lymphocyte function after the in vitro treatment with hydrocortisone. Macrophage depletion experiments showed that the suppressive effect of hydrocortisone was not mediated by induction of suppressor macrophages. The data suggest that T-cell function was impaired directly by hydrocortisone treatment.     

Bovine leukosis virus in sheep,lymphocyte modification and surface-immunoglobulin-bearing cell numbers

Lymphocytes from sheep experimentally infected with bovine leukosis virus (BLV) and from non-infected normal sheep were examined for the presence of surface Ig by an immunofluorescence test. Surface Ig-bearing lymphocytes in blood from BLV-infected sheep increased when lymphocyte counts of blood were elevated in comparison with normal animals. The mitogen stimulation of cultured lymphocytes from BLV-infected sheep and from non-infected normal sheep was determined by measuring ³H-thymidine incorporation. Peripheral blood lymphocytes (PBL) from BLV-infected leukemic sheep with elevated PBL counts responded poorly to phytohemagglutinin M and concanavalin A but responded well to lipopolysaccharide compared with lymphocytes from uninfected animals. In BLV-infected preleukemic sheep with low PBL counts, stimulation indices of mitogen responses of lymphocytes with phytohemagglutinin M, concanavalin A, and pokeweed mitogen were low compared with those of lymphocytes from uninfected animals. The results indicated that B cells were affected by BLV infection in sheep as suggested by the increased number of surface Ig-bearing lymphocytes and that significant alteration of mitogen stimulation of lymphocytes occured in sheep with BLV infection.     

Method for evaluation of Mycobacterium bovis purified protein derivative tuberculin in experimentally infected cattle.

A method for the evaluation of Mycobacterium bovis purified protein derivative (PPD) tuberculin in experimentally infected cattle is presented. The development of skin test responses in M bovis-infected cattle was determined for International Standard PPD-S, M bovis PPD-2, and M bovis PPD-5 at 24, 48, and 72 hours. Significantly larger reactions (dermal thickness) were observed at 48 and 72 hours than at 24 hours (P = 0.001). Statistically significant differences were not detected in the responses obtained with M bovis PPD-2, M bovis PPD-5, and International Standard PPD-S if comparisons were made at approximately the same concentrations in M bovis-infected cattle (P greater than 0.25). In Mycobacterium avium-infected cattle, M bovis PPD-2 produced skin test responses that were significantly smaller than responses obtained using M avium PPD-2 (P = 0.001). Significant variation was not observed in the PPD-S responses in 2 groups of M bovis-infected cattle (P greater than 0.1).     

The in vitro response of turkey lymphocytes to steroid hormones

The in vitro mitogen response of whole blood turkey lymphocytes to various concentrations of steroid hormones was evaluated. Corticosterone (COS) at concentrations between 1 and 80 ng/ml significantly suppressed the proliferative response (3H-thymidine incorporation) to phytohemagglutinin (PHA) and concanavalin A (ConA). Non-mitogen-stimulated (NMS) cells were suppressed at concentrations of COS above 5 ng/ml. Progesterone significantly suppressed NMS cells at concentrations of 80 ng/ml, PHA-stimulated cells at concentrations of 500 ng/ml, and ConA-stimulated cells at concentrations of 1000 ng/ml. beta-Estradiol enhanced the response of NMS cells at concentrations of 500 ng/ml, had no effect on PHA-stimulated cells, and suppressed the response of ConA-stimulated cells at concentrations greater than 500 ng/ml. Testosterone affected only the ConA response, causing suppression at concentrations above 2000 ng/ml. Corticosterone and progesterone caused 80 and 95% suppression, respectively, of the proliferative response to ConA when compared with non-hormone-treated cells. The possible implications of steroid hormone-induced immunosuppression in the pathogenesis of aspergillosis is discussed.     

The canine lymphocyte: effect of streptolysin O on the proliferative response of canine lymphocytes

The effects of Streptolysin O (SLO) on canine peripheral blood lymphocytes were studied using the lymphocyte blastogenesis test (LBT). Canine lymphocytes stimulated with SLO produced a response that was similar to the response obtained with the commonly used phytomitogens: phytohemagglutinin (PHA), concanavalin A (CON A) and pokeweed mitogen (PWM). When SLO was added to any of the phytomitogens and incubated with canine lymphocytes, an additive or stimulatory response was obtained. However, mixing the phytomitogens in any combination did not produce a similar additive or stimulatory response. The results were interpreted to mean that in the canine system, the binding of SLO to lymphocytes does not sterically interfere with receptors for PHA, CON A, or PWM. Additionally, SLO may stimulate a population of lymphocytes that is distinct from that stimulated by the phytomitogens. Moreover, it would appear that the phytomitogens probably stimulate the same or an overlapping population of lymphocytes. Experiments using enriched lymphoid cell populations were used to characterize the lymphocytes stimulated by phytomitogens and SLO. Lipopolysaccharides from two different bacteria were unable to cause a significant lymphoproliferative response.     

Blood culture and stimulation conditions for the diagnosis of tuberculosis in cervids by the Cervigam assay

Mitogen- and antigen-induced interferon-gamma (IFN-gamma) responses of peripheral blood leucocytes from cervids were evaluated by a commercial whole-blood assay. The assay was applied to Mycobacterium bovis-infected white-tailed deer and reindeer, M bovis BCG-vaccinated white-tailed deer and elk, and unvaccinated, uninfected white-tailed deer, fallow deer, elk and reindeer. The responses of the M bovis-infected white-tailed deer to pokeweed mitogen (PWM) varied with time and between individuals. The responses of the M bovis-infected reindeer to PWM and M bovis purified protein derivative (PPD) were positively associated. Samples from tuberculosis-free captive herds in various parts of the USA were also evaluated. Four per cent of fallow deer, 20 per cent of elk, 44 per cent of white-tailed deer, and 91 per cent of reindeer had responses to PWM exceeding 0.25 Delta optical density, that is, PWM stimulation minus no stimulation. The specificity of the responses to M bovis PPD and a Mycobacterium tuberculosis complex-specific antigen rESAT-6:CFP-10, excluding animals not responding to PWM, ranged from 78 per cent to 100 per cent and was dependent upon the species and the positive response cut-off value. The results show that the commercial assay is valid for the detection of TB in reindeer; however, further development of the assay will be required before it is used in surveillance programmes for white-tailed deer, fallow deer, and elk.     

In vitro lymphocyte stimulation by concanavalin A and with histamine as a co-mitogen in dogs with atopic dermatitis.

Cell-mediated immune function was assessed in a group of dogs with atopic dermatitis by measuring the responses of peripheral-blood lymphocytes (PBL) to various concentrations of Concanavalin A (Con A) and comparing them to those of normal dogs. No difference from normal was found in any of the stimulation indices neither was spontaneous tritium uptake of unstimulated cells different between the groups. We also measured the response to Con A stimulation in vitro of PBL preincubated for 24 h, either in cell-culture medium at 37 degrees C, or in whole blood containing EDTA at room temperature, as an indirect measure of function of a subgroup of suppressor cells. Preincubation caused enhancement of mitogenesis for normal dog lymphocytes but not for the atopic dog cells, particularly for suboptimal concentrations of Con A. No differences were found in the responsiveness following incubation in cell-culture medium between normal and atopic dog cells but for both groups the cells preincubated in whole blood were generally more responsive. Histamine, which is one of the mediators of type 1 hypersensitivities such as atopy, can modulate lymphocyte function. At 10(-4) and 10(-8) M histamine, when added simultaneously with Con A, enhanced mitogenesis of normal dog PBL but suppressed mitogenesis of atopic dog PBL. By using histamine H1 and H2 antagonists, we concluded that histamine enhanced mitogenesis via H1, receptors and suppressed it via H2 receptors. Our results suggest that there are abnormalities in lymphocyte function in dogs with atopic dermatitis which may be important in the pathogenesis of the disease.     

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.     

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.     

Effect of corticosteroid on porcine leukocytes: age-related effects of corticosteroid inhibition on porcine lymphocyte responses to mitogens

The effects of prednisolone sodium succinate on the responses of porcine lymphocytes to phytohemagglutinin (PHA) and pokeweed mitogen (PWM) were investigated. Sensitivity of peripheral blood lymphocytes (PBL) to the synthetic glucocorticoid, prednisolone, was related to age of the lymphocyte donor. The greatest sensitivity was found in PBL from animals less than one week old; PBL from animals between 2 to 4 months retained some glucocorticoid sensitive cells; whereas, PBL from animals older than 6 months were exceptionally resistant to steroid. Similar age-associated sensitivities were found for lymphocytes from lymph node, spleen and thymus. Significant differential sensitivities among the various lymphoid organs were found with the thymic lymphocyte possessing the greatest sensitivity to steroid and the PBL lymph node and splenic lymphocytes possessing the highest resistance to the suppressive effects of steroid. The age related differences in sensitivity to steroid did not appear to be caused by differences in the number of steroid receptors because lymphocytes from susceptible and resistant animals had similar numbers of receptors. The results suggest that the age related sensitivity may be associated with a higher percentage of sensitive thymic-derived lymphocyte in the PBL, lymph node and spleen of the younger animals. Results of this study also suggest that the adult pig (6 months) should be classified as a steroid resistant species.     

Some immune responses in cattle exposed to Mycobacterium paratuberculosis after injection with modified-live bovine viral diarrhea virus vaccine

Delayed-type hypersensitivity responses to Mycobacterium paratuberculosis purified protein derivative (PPD) were decreased in cows experimentally exposed to M. paratuberculosis 7 days after exposure to a modified-live bovine viral diarrhea virus (ML-BVDV) vaccine. In vitro lymphocyte blastogenic responses to phytohemagglutinin were decreased in each of 3 cows 7 days after exposure to ML-BVDV vaccine. Also, decreased lymphocyte blastogenic responses to M. paratuberculosis PPD were observed in cultures of 2 of 3 cows 7 days after exposure to ML-BVDV vaccine. No significant differences in enzyme-linked immunosorbent assay reactions were detected in sera of M. paratuberculosis-infected cattle collected before and at 4 and 12 weeks after exposure to ML-BVDV vaccine.     

Effect of cortisol in vitro and in vivo on production of bovine interleukin 2

The influence of cortisol in vitro and in vivo on lymphocyte proliferative responses and interleukin 2 (IL2) production was evaluated in Hereford feeder calves. Cortisol, added to bovine mononuclear cell cultures, reduced (P less than 0.05) mitogen-stimulated lymphocyte proliferative responses and IL2 production. Lower IL2 activity from cortisol-treated cell cultures was not caused by a cortisol-mediated cytotoxicity or a residual cortisol effect on the IL2-indicator cell line. Calves given ACTH (1.0 IU/kg of body weight, IM) twice daily for 2 days had increased (P less than 0.001) plasma cortisol concentrations when compared with those of saline-treated controls. Leukocytosis (P less than 0.002), characterized mainly by a neutrophilia (P less than 0.007), was evident in ACTH-treated calves. Lymphocyte proliferative responses to the phytomitogens, concanavalin A, phytohemagglutinin, and pokeweed mitogen were decreased (P less than 0.05) in calves with increased plasma cortisol concentrations. Interleukin 2 production was lower (P less than 0.05) in concanavalin A-stimulated lymphocyte cultures from ACTH-treated calves. Seemingly, lower lymphocyte proliferative responses in cortisol-treated mononuclear cell cultures and in ACTH-treated calves were caused partly by lower IL2 production.     

B and T lymphocytes in the bovine mammary gland: Rosette formation and mitogen response

Characterization of lymphocyte subpopulations in the bovine mammary gland was accomplished using cells obtained from dry secretions. Correlation of cell surface properties with functional capacity was attempted by assaying the ability to form erythrocyte-antibody (EA) rosettes, erythrocyte-antibody-complement (EAC) rosettes, and sheep erythrocyte (E) rosettes and the ability to respond to phytohemagglutinin (PHA), Concanavalin A (Con A), and pokeweed mitogen (PWM) in the lymphocyte stimulation test. Results were compared with those obtained for peripheral blood lymphocytes (PBL) from the same animals. Mammary gland lymphocytes (MGL) formed significantly fewer (p < .01) EA and EAC rosettes, but significantly greater (p < .01) E rosettes compared to PBL. MGL were significantly less responsive (p < .05) to mitogens than were PBL. MGL contained a large proportion of T lymphocytes, which do not respond to T lymphocyte mitogens in culture.     

Ascaris suum infection in pigs sensitizes lymphocytes but suppresses their responsiveness to phytomitogens

The effect of Ascaris suum infection and treatment with fenbendazole on the blastogenic response of peripheral blood lymphocytes to A. suum antigens and to three phytomitogens was assayed by the lymphocyte transformation technique. Repeated infections with A. suum led to the development of sensitized lymphocytes primarily responding to egg hatching fluid antigen. Treatment with fenbendazole decreased the number of specific sensitized lymphocytes, but favorably increased the resistance of pigs to reinfection. Immunity to reinfection did not correlate with the strength of the blastogenic response to A. suum antigens. Repeated infection with A. suum negatively affected the development of the blastogenic response to phytomitogens in the pigs, leading to a partial depression of the responsiveness of lymphocytes and to the partial suppression by serum. Responses to pokeweed mitogen were affected more than the responses to concanavalin A and phytohemagglutinin.     

Effects of aflatoxin M1 intake at physiologic levels on newborn dairy calves

When aflatoxin-contaminated grain is consumed by dairy cows, aflatoxin M1 is excreted in the milk. Sixteen neonatal male Holstein calves were given milk which had been collected from cows given 5 to 6 mg of aflatoxin B1 each day. The calves were examined for possible detrimental effects of the mycotoxin at pseudophysiologic concentrations. Calves were allotted to 1 of 4 groups given different milk dietary aflatoxin M1 concentrations: group 1--given 0 microgram of aflatoxin M1/L (undetectable); group 2--given 0.5 microgram/L; group 3--given 1 microgram/L; and group 4--given 2 micrograms/L. Whole milk equal to 8% of body weight was fed daily and adjusted each week to maintain this ratio. Water and a 15% crude protein complete calf starter ration were offered ad libitum for the 6-week feeding study. Weekly blood samples were collected via jugular venipuncture and analyzed for serum alkaline phosphatase and aspartate aminotransferase activities. Daily means for milk dry matter intake (in kg) and complete ration intake (in kg) for the calf groups were as follows: 0.46 and 0.36 for group 1; 0.46 and 0.25 for group 2; 0.42 and 0.18 for group 3; and 0.49 and 0.40 for group 4. Significant differences in complete ration and total dry matter intake were noted. The average daily gains (in kg) and gains in height at withers (in cm) were 0.39 and 4.1 for group 1; 0.36 and 4.0 for group 2; 0.29 and 5.7 for group 3; and 0.42 and 5.1 for group 4.(ABSTRACT TRUNCATED AT 250 WORDS)     

MHC class II-restricted, CD4(+) T-cell proliferative responses of peripheral blood mononuclear cells from Mycobacterium bovis-infected white-tailed deer

White-tailed deer are significant wildlife reservoirs of Mycobacterium bovis for cattle, predators, and, potentially, humans. Infection of cattle with M. bovis stimulates an antigen-specific T-cell response, with both CD4(+) and CD8(+) cells implicated in protective immunity. Few studies, however, have examined lymphocyte subset responses to experimental M. bovis infection of white-tailed deer. In this study, a flow cytometric proliferation assay was used to determine the relative contribution of individual peripheral blood mononuclear cell subsets of M. bovis-infected white-tailed deer in the recall response to M. bovis antigen. Naive deer were challenged with M. bovis by cohabitation with infected deer. These M. bovis-challenged deer developed significant in vivo (delayed-type hypersensitivity) and in vitro (proliferative) responses to M. bovis purified protein derivative (PPD). At necropsy, typical tuberculous lesions containing M. bovis were detected within lungs and lung-associated lymph nodes of infected deer. The predominant subset of lymphocytes that proliferated in response to in vitro stimulation with PPD was the CD4(+) subset. Minimal proliferative responses were detected from CD8(+), gamma delta TCR(+), and B-cells. Addition of monoclonal antibodies specific for MHC II antigens, but not MHC I or CD1 antigens, abrogated the proliferative response. Together, these findings indicate that while CD4(+) cells from infected deer proliferate in the recall response to M. bovis antigens, this response is not sufficient to clear M. bovis and immunologic intervention may require stimulation of alternate subsets of lymphocytes.     

Specific in vitro lymphocyte response of chickens injected with killed Mycobacterium tuberculosis.

An in vitro microassay for lymphocyte transformation, using 3H-thymidine incorporated into avian peripheral blood leukocytes, is described. The transformation responses of 1 X 10(6) leukocytes stimulated by the nonspecific mitogen, phytohemagglutinin-M (PHA-M), were equivalent with 68- and 92-hour incubation durations. The PHA-M in concentration of 50 micronl/ml produced greater stimulation than did that of 25 or 100 micronl/ml. The transformation response to PHA-M was significantly affected by the lot of bovine fetal serum used to supplement the RPMI 1640 culture medium. The specific antigen, purified protein derivative (PPD) of tuberculin, stimulated significant transformation in cultures of 1 X 10(6) leukocytes from Mycobacterium tuberculosis-sensitized fowl. The PPD at concentration of 50 microng/ml was superior to 100 or 150 microng/ml, and 68-hour incubation was significantly better than 92-hour incubation. The magnitude of the in vitro transformation response to PPD was greater at 6 weeks after sensitization than at 2 or 4 weeks, and it was not directly related to the magnitude of the in vivo wattle test response in sensitized chickens.