INFECTION
AND
Vol. 21, No.2
IMMUNITY, Aug. 1978, p. 474-479
0019-9567/78/0021-0474$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Suppression and Enhancement of Mitogen Response in Chickens Infected with Marek's Disease Virus and the Herpesvirus of Turkeys LUCY F. LEE,* J. M. SHARMA, K. NAZERIAN, AND R. L. WITTER U.S. Department ofAgriculture, Science and Education Administration-Federal Research, Regional Poultry Research Laboratory, East Lansing, Michigan 48823 Received for publication 4 May 1978
The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 72 chickens and in HVT-inoculated line 72 chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response. In susceptible chickens, Marek's disease virus (MDV) causes a progressive lymphoproliferative disease, Marek's disease (MD), which results in lymphoma formation and death (3, 13). In naturally resistant chickens, MDV induces a lymphoproliferation which is eventually overcome by the host (22). An antigenically related herpesvirus of turkeys (HVT), when inoculated into chickens, induces transient mild lymphoproliferative lesions in nerves and gonads (27), and the inoculated chickens are protected from lymphoma formation upon subsequent exposure to virulent MDV (14). The HVT-vaccinated MDVchallenged chickens harbor both viruses (27), and the MDV that is shed by the vaccinated chickens is as virulent as the inoculum virus and may cause disease in susceptible chickens (18). Infection with MDV affects host cell-mediated immune response. In chickens infected with MDV, homograft and allograft rejection was delayed and graft-versus-host reaction was either enhanced or unaffected (15, 17). T-cell responsiveness to phytohemagglutinin (PHA) or concanavalin A has been correlated with immune competence in many animal systems, including chickens; this responsiveness is impaired upon virus infection (4). Lymphoid cells from the spleen, peripheral blood, or tumor of chickens with MD all showed depression in PHA
response (1, 2, 5, 6, 8, 11, 15, 24, 25). On the other hand, no depression in mitogen response and Tcell-mediated immune response was observed in HVT-vaccinated, MDV-challenged chickens (20, 25). The altered immune response of chickens after infection with MDV may be associated with the pathological changes occurring in the thymus, spleen, and bursa of Fabricius (16). The exact nature of this relationship is not well understood. It is not known whether the impairment of immune response is a consequence of lymphoma formation or a direct consequence of virus infection and thus a probable factor in lymphoma formation. A microassay using whole blood rather than purified lymphocytes from peripheral blood and which allows a sequential monitoring of the PHA response of individual chickens following virus infection has been recently developed (9, 10). It becomes possible by use of this method to perform functional analyses of lymphocytes in infected chickens to better define alterations in the immune system associated with tumor formation. In this paper, we report the kinetics of PHA response and its correlation with disease pathogenesis in susceptible and resistant chickens infected with oncogenic MDV or nononcogenic HVT and in HVT-vaccinated, MDV-challenged susceptible chickens.
474
VOL. 21, 1978
PHA RESPONSE IN MDV- AND HVT-INFECTED CHICKENS
MATERIAIS AND METHODS Reagents. The mitogen PHA (PHA-P; Difco Laboratories, Detroit, Mich.) was reconstituted to 10 mg/ml in phosphate-buffered saline (PBS) as a stock solution and stored at -200C. Before each experiment, the stock solution was diluted in culture medium without serum. Medium RPMI 1640 with L-glutamine (Flow Laboratories, Rockville, Md.) containing 5% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) was used as the cell culture growth medium for purified lymphocytes obtained from the peripheral blood and spleen. A 1.lx concentration of Dulbecco PBS (D-PBS; pH 7.4) (21) containing 5%
fetal calf serum was used as a general buffer. Culture medium RPMI 1640 was maintained at 40C before and during use. Both D-PBS and medium RPMI 1640 contained 100 U of penicillin per ml and 100 jug of streptomycin per ml. A lymphocyte-separating agent containing carbonyl iron particles was obtained from Technicon Instruments Corp., Tarrytown, N.Y. Ficoll 400 (Pharmacia Fine Chemicals, Piscataway, N.J.)-Hypaque-M, 75% (Winthrop Laboratories, New York, N.Y.) gradient was prepared to a specific gravity of 1.09 g/ml. Isotope [5-'2I]iodo-2-deoxyuridine (0.9 to 1.1 Ci/mmol; Amersham/Searle, Chicago, Ill.) was used at a concentration of 0.05 QCi per culture. Filter papers (Reeve Angel, Clifton, N.J.) were used for harvesting lymphocyte samples. Microtiter plates (Microtest II; Falcon-P Plastics, Los Angeles, Calif.) were used as cell culture plates. Chickens. Two White Leghorn lines of chickens from the Regional Poultry Research Laboratory were used. These chickens lacked maternal antibody to MDV and HVT. Line 72 was highly susceptible and line N was resistant to MD (23). They were housed in Horsfall Bauer isolators for the entire experimental period. Viruses. The JM strain of MDV (clone illS) (21) was used at the 11th tissue culture passage. Typically, more than 80% of the susceptible chickens developed grossly enlarged peripheral nerves characteristic of MD lymphoproliferative lesions at 3 to 6 weeks after inoculation with the virus. Strain FC 126 of HVT was similarly cloned (21) and stored. The HVT is antigenically related to MDV (26) and is widely used as a vaccine virus against MD (14). Cell culture techniques. The spleen was removed aseptically from chickens and suspended in D-PBS. Cells were released into D-PBS by repeated aspiration into a 10-ml syringe, and the cell suspension was filtered through a sterile gauze and washed once with D-PBS. The resuspended cells (9 ml) were layered over 5 ml of Ficoll-Hypaque gradient and centrifuged at 400 x g for 15 min. Viable lymphocytes recovered from the interface were washed three times with DPBS and suspended in RPMI 1640 for use in the lymphocyte stimulation test. Blood was taken from chickens by cardiac or venipuncture with a syringe containing 20 U of heparin per ml of blood. Whole blood samples were cultured in the growth medium without additional serum but with 200 U of penicillin and 100 gtg of streptomycin per ml of medium. Procedures for lymphocyte stimulaion test. Whole blood samples (10 fd) or spleen lymphocytes
475
(106) in 0.2 ml of medium RPMI 1640 plus 5% fetal calf serum were cultured with 100,ug of stock PHA per ml in each of the 96 wells on a microtiter plate. All cell cultures in the microtiter plates were incubated at 41WC in a 5% CO2 atmosphere for 48 h, followed by addition of 0.05 ,uCi of [1I]iododeoxyuridine per culture. Incubation was continued for 16 h more. Lymphocyte cultures were harvested on filter papers with an automatic 24-multiple-culture cell harvester (model M24V; Brandel, Rockville, Md.). The filters containing labeled cells were punched out and placed in disposable tubes for gamma counting in a Beckman gamma counter. The PHA stimulation results are expressed as a stimulation index that was calculated as the mean counts per minute of isotope incorporated in cells in triplicate wells treated with PHA divided by the mean counts per minute of isotope incorporated in cells in triplicate wells not treated with PHA. For all results reported in this paper, the background activity of cultures not treated with PHA did not differ significantly between control chickens and those inoculated with MDV. The mean counts per minute of ['S2I]iododeoxyuridine incorporated into lymphocyte cultures not treated with PHA was 84 ± 3. Removal of adherent and phagocytic cells from spleen. A 5-ml sample of lymphocyte suspensions containing 108 cells was incubated for 45 min at 371C in 100-mm petri dishes. The nonadherent cells were gently removed from adherent cells. For removal of adherent or phagocytic cells with carbonyl iron/magnet, lymphocytes (108) were suspended in 5 ml of the lymphocyte-separating agent containing carbonyl iron particles. The mixture was incubated for 60 min at 370C in 50-ml conical tubes. The tubes were then put on top of a magnet, and the supernatant was removed by Pasteur pipettes. This procedure was repeated six times, and the cells were washed once before cultivation. Pathology. The lesion status of control and MDVinfected chickens was monitored by gross necropsy and by histological sections of peripheral nerves and gonads. Chickens that died before the termination date were examined for MD lesions by similar procedures. Statistical analysis. All results are expressed as a mean stimulation index. Standard errors of the mean were calculated. Student's t test was used for calculating statistical significance.
RESULTS PHA response of peripheral blood lymphocytes from chickens inoculated with MDV. Two replicate tests were performed. Chickens in replicate no. 1 were inoculated at 1 week of age, and chickens in replicate no. 2 were inoculated at 3 weeks of age. In each replicate test, eight MDV-inoculated and eight uninoculated line 72 chickens were bled before inoculation to establish the reference response to PHA. The PHA response of whole blood peripheral lymphocytes was monitored at weekly intervals after virus inoculation (Table 1). At 1 week after
476
INFECT. IMMUN.
LEE ET AL.
5 died of MD within 2 to 3 weeks of the initial depression after a slight recovery in PHA response, and four other birds suffered a second depression either before death or in association with the development of MD lesions. Inoculated chickens 7 and 8 survived MD, and, following an initial depression, their PHA response recovered to a level higher than that of the control. For these two chickens, virus inoculation seemed to have had a potentiating effect on their PHA responsiveness. When necropsied at the end of the 7-week experimental period, neither of the two chickens showed any gross or microscopic lesions (Table 2). Table 3 represents the results of two replicate TABLE 1. Sequential monitoring of PHA response experiments on the PHA response of lymphoof whole blood lymphocytes from genetically cytes from MDV-inoculated, genetically resistsusceptible line 72 chickens infected with the JM strain of MDVG ant line N chickens. Lymphocyte response to PHA was severely depressed at about 1 week Stimulation index' RepliAge Week after inafter virus inoculation in both experiments. Week cate test Agee ouaterion (wes) oculation no. Control Inoculated However, this initial depression was transient, and, within 2 to 3 weeks, the lymphocytes from I 1 Before 15 ± 3 20 ± 2 inoculation all the infected chickens recovered their PHA 1 5± 1** 2 32±5 response to a level comparable with that of the 2 3 82 ± 11 16 ± 2** lymphocytes from the control chickens. At ter4 3 129± 14 21±8** mination, all inoculated chickens were exam4 5 117 ± 13 12 ± 9* (5) 5 6 115± 12 24±0(1) ined, and no gross or microscopic lesions were found. 2 3 Before 42 ± 5 44 6 PHA response of peripheral blood lyminoculation phocytes from HVT-inoculated, MDV-chal4 1 106± 12 21±6** 5 2 45±7 23±7* lenged chickens. Results from two sequential 6 3 76± 16 19 7** replicate studies on the PHA response of whole 7 4 107 ± 12 27 13** blood peripheral lymphocytes from normal, 8 5 89 ± 5 22 13* (3) HVT-inoculated, MDV-inoculated, and HVTGenetically susceptible line 72 chickens were inoculated intra-abdominally with JM strain of MDV at 6 x 103 plaque- vaccinated but MDV-challenged chicken are summarized in Table 4. Eight line 72 chickens forming units per chicken. b Stimulation index ± standard error of the mean. Statistiwere used in each of the four groups. Chickens cal significance is calculated by Student's t test: *, P < 0.01; in replicate studies 1 and 2 showed a statistically l, P < 0.001. Numbers in parentheses show the number of chickens that survived MD at the time tested. The starting significant depression in PHA response 1 week number of chickens was eight for each of the inoculated and after inoculation with the nononcogenic HVT. the control groups. The response remained depressed for 2 to 3
inoculation, severe depression was observed in infected chickens, and the level of response of the inoculated chickens was significantly lower than that of the control throughout the entire experimental period. Since data from chickens individually monitored indicated that depression in PHA response was related to the severity of MD (data not shown), a third replicate experiment was conducted. As shown in Table 2, all inoculated chickens showed an initial depression in PHA response as compared to the average response of age-matched control chickens. Chickens 4 and
TABLE 2. Sequential monitoring of PHA response of whole blood lymphocytes from individual genetically susceptible line 72 chickens infected with the JM strain of MDV0 Week after MDV Control stimulation ininoculation dex (8 chickens)b
1 2 3 4 5 6
34 ± 4 (14-56) 20 ± 5 (8-44) 42 + 4 (27-0) 69 ± 8 (44-111) 77+ 14 (35-145) 69 + 9 (41-73)
1
2
8 10 31 14 5 6
2 14 19 31 20 4
Stimulation index for chicken no.: 3 4 5 6
13 10 49 64 20 Died
2 11 Died
2 8 19 Died
11 8 66 11 20 7
7
8
4 22 86 67 102 169
4 9 54 102 126 110
MD lesion' + + + + + + a Genetically susceptible line 72 chickens were inoculated intra-abdominally with JM strain of MDV at 6 x 103 plaque-forming units per chicken. b Stimulation index ± standard error of the mean and, in parentheses, range of values. 'MD status was assessed by gross and microscopic lesions.
477
PHA RESPONSE IN MDV- AND HVT-INFECTED CHICKENS
VOL. 21, 1978
slightly increased at about 2 weeks after MDV challenge. As shown in replicate 2, Table 4, a statistically significant increase in PHA responsiveness was observable by week 5 after HVT inoculation and stayed at that level until termination. As expected, none of the eight TABLE 3. Sequential study of PHA response of HVT-inoculated chickens had developed gross whole blood peripheral lymphocytes from genetically or microscopic MD lesions at termination. No resistant line N chickens infected with the JM depression of PHA response was observed in strain of MDVHVT-vaccinated chickens when challenged by Stimulation index' the virulent JM strain of MDV (Table 4). In Replicate Age Week after inocaddition, the lymphocytes from the HVT-vacInocuulation test no. (weeks) Control cinated MDV-challenged chickens, like the lymphocytes from HVT-inoculated chickens, ap48 +±7 52 + 8 Before 1 3 peared to be more PHA responsive than the inoculation 1 58± 12 6±2* 4 normal lymphocytes. All eight chickens survived 2 66±12 40±10 5 the entire experimental period, and none had 3 91±20 46±13 6 developed gross or microscopic lesions when ex4 75± 10 50± 10 7 amined at termination. In contrast, six of eight 5 51± 8 48±7 8 6 100±17 94±14 9 MDV-inoculated chickens in replicate studies 1 and 2 either died of MD or had developed gross ± 27 ± 22 3 3 1 Before 2 or microscopic MD lesions at termination. inoculation Depression in PHA response in MDV 7±1* 1 38±5 2 44±7 30±6 2 3 spleen cells. The PHA response of spleen cells 62 10 46±6 3 4 from the normal and inoculated line 72 chickens 86 10 71 ± 12 4 5 was studied, and the results are summarized in a Genetically resistant line N chickens were inoculated inTable 5. As shown, MDV spleen cells were untra-abdominally with JM strain of MDV at 6 x 103 plaque- responsive to PHA at 1 week after inoculation. units per chicken. forming I Stimulation index ± standard error of the mean. Statisti- However, removal of adherent cells by adhercal significance is calculated by Student's t test: i, P < 0.001. ence treatment or by carbonyl iron/magnet
weeks before recovery. As shown in replicate 1, Table 4, the PHA response after recovery not only was comparable with the response of the normal lymphocytes but also appeared to be
even
TABLE 4. Sequential study of PHA response of whole blood lymphocytes from the normal, HVT-inoculated, MDV-inoculated, and HVT-vaccinated and MDV-challenged chickens Replicate test no.
1
Week after HVT vaccination
2 3 4
5 6 9 MD lesions 2
1 2 3 4 5 6 7 MD lesions
Week after MDV challenge
Response (stimulation index)b HVT
MDV
HVT/MDV
57 ± 18 112 ± 21 69±8 46±5
9±2* 25±5 46± 4 97 ± 12 80±5 44±9
40 ± 11** 66±17 31±8
105 ± 15 92±15 44± 11
0/8
0/8
Normal 17±1 31±10
Before inoculation 1 2 3 Before inoculation 1
2 3 4 5
6
6/8
0/8
31 ± 5 34±7 20±5 42±4
13 ± 3* 18±3* 24±3 65±7*
6±2** 11±2* 46±10
69±8
91± 12*
44± 12
53±9 97±6*
77±14 64+9
112±15* 95±16*
54±25 36±21
103±8* 85±18
35±17 21±3
0/8 6/8 0/8 0/8 x 6 with 1 of at with 103 vaccinated HVT plaqueday age aGenetically susceptible line 72 chickens were forming units per chicken. The vaccinated chickens were challenged with the JM strain of MDV at 6 x 103 plaque-forming units per chicken at 4 weeks after HVT vaccination for replicate 1 and at 2 weeks after vaccination for replicate 2. The starting number of chickens was eight for each of the inoculated and the control groups. b Stimulation index ± standard error of the mean. Statistical significance is calculated by Student's t test: *, P
AND
Vol. 21, No.2
IMMUNITY, Aug. 1978, p. 474-479
0019-9567/78/0021-0474$02.00/0 Copyright i 1978 American Society for Microbiology
Printed in U.S.A.
Suppression and Enhancement of Mitogen Response in Chickens Infected with Marek's Disease Virus and the Herpesvirus of Turkeys LUCY F. LEE,* J. M. SHARMA, K. NAZERIAN, AND R. L. WITTER U.S. Department ofAgriculture, Science and Education Administration-Federal Research, Regional Poultry Research Laboratory, East Lansing, Michigan 48823 Received for publication 4 May 1978
The kinetics of phytohemagglutinin (PHA) response of peripheral blood lymphocytes from chickens infected with oncogenic Marek's disease (MD) virus (MDV) or nononcogenic herpesvirus of turkeys (HVT) was studied with a whole blood microassay. At about 7 days after inoculation, a depression in PHA response was observed in MDV-inoculated resistant line N or susceptible line 72 chickens and in HVT-inoculated line 72 chickens. All chickens initially regained their PHA responsiveness. Susceptible chickens that died of MD or developed MD lymphoma in later stages of virus infection showed a second severe depression in PHA response. No depression was observed in HVT-vaccinated chickens when challenged with MDV. The PHA response of MDV-inoculated chickens that survived MD, HVT-inoculated chickens, and HVT-vaccinated MDV-challenged chickens showed evidence of enhancement. The depression of PHA response was studied and was attributed to the suppressive effect of macrophages on T-cell response, a finding consistent with our previous studies on MDV suppression of PHA response. In susceptible chickens, Marek's disease virus (MDV) causes a progressive lymphoproliferative disease, Marek's disease (MD), which results in lymphoma formation and death (3, 13). In naturally resistant chickens, MDV induces a lymphoproliferation which is eventually overcome by the host (22). An antigenically related herpesvirus of turkeys (HVT), when inoculated into chickens, induces transient mild lymphoproliferative lesions in nerves and gonads (27), and the inoculated chickens are protected from lymphoma formation upon subsequent exposure to virulent MDV (14). The HVT-vaccinated MDVchallenged chickens harbor both viruses (27), and the MDV that is shed by the vaccinated chickens is as virulent as the inoculum virus and may cause disease in susceptible chickens (18). Infection with MDV affects host cell-mediated immune response. In chickens infected with MDV, homograft and allograft rejection was delayed and graft-versus-host reaction was either enhanced or unaffected (15, 17). T-cell responsiveness to phytohemagglutinin (PHA) or concanavalin A has been correlated with immune competence in many animal systems, including chickens; this responsiveness is impaired upon virus infection (4). Lymphoid cells from the spleen, peripheral blood, or tumor of chickens with MD all showed depression in PHA
response (1, 2, 5, 6, 8, 11, 15, 24, 25). On the other hand, no depression in mitogen response and Tcell-mediated immune response was observed in HVT-vaccinated, MDV-challenged chickens (20, 25). The altered immune response of chickens after infection with MDV may be associated with the pathological changes occurring in the thymus, spleen, and bursa of Fabricius (16). The exact nature of this relationship is not well understood. It is not known whether the impairment of immune response is a consequence of lymphoma formation or a direct consequence of virus infection and thus a probable factor in lymphoma formation. A microassay using whole blood rather than purified lymphocytes from peripheral blood and which allows a sequential monitoring of the PHA response of individual chickens following virus infection has been recently developed (9, 10). It becomes possible by use of this method to perform functional analyses of lymphocytes in infected chickens to better define alterations in the immune system associated with tumor formation. In this paper, we report the kinetics of PHA response and its correlation with disease pathogenesis in susceptible and resistant chickens infected with oncogenic MDV or nononcogenic HVT and in HVT-vaccinated, MDV-challenged susceptible chickens.
474
VOL. 21, 1978
PHA RESPONSE IN MDV- AND HVT-INFECTED CHICKENS
MATERIAIS AND METHODS Reagents. The mitogen PHA (PHA-P; Difco Laboratories, Detroit, Mich.) was reconstituted to 10 mg/ml in phosphate-buffered saline (PBS) as a stock solution and stored at -200C. Before each experiment, the stock solution was diluted in culture medium without serum. Medium RPMI 1640 with L-glutamine (Flow Laboratories, Rockville, Md.) containing 5% fetal calf serum (Grand Island Biological Co., Grand Island, N.Y.) was used as the cell culture growth medium for purified lymphocytes obtained from the peripheral blood and spleen. A 1.lx concentration of Dulbecco PBS (D-PBS; pH 7.4) (21) containing 5%
fetal calf serum was used as a general buffer. Culture medium RPMI 1640 was maintained at 40C before and during use. Both D-PBS and medium RPMI 1640 contained 100 U of penicillin per ml and 100 jug of streptomycin per ml. A lymphocyte-separating agent containing carbonyl iron particles was obtained from Technicon Instruments Corp., Tarrytown, N.Y. Ficoll 400 (Pharmacia Fine Chemicals, Piscataway, N.J.)-Hypaque-M, 75% (Winthrop Laboratories, New York, N.Y.) gradient was prepared to a specific gravity of 1.09 g/ml. Isotope [5-'2I]iodo-2-deoxyuridine (0.9 to 1.1 Ci/mmol; Amersham/Searle, Chicago, Ill.) was used at a concentration of 0.05 QCi per culture. Filter papers (Reeve Angel, Clifton, N.J.) were used for harvesting lymphocyte samples. Microtiter plates (Microtest II; Falcon-P Plastics, Los Angeles, Calif.) were used as cell culture plates. Chickens. Two White Leghorn lines of chickens from the Regional Poultry Research Laboratory were used. These chickens lacked maternal antibody to MDV and HVT. Line 72 was highly susceptible and line N was resistant to MD (23). They were housed in Horsfall Bauer isolators for the entire experimental period. Viruses. The JM strain of MDV (clone illS) (21) was used at the 11th tissue culture passage. Typically, more than 80% of the susceptible chickens developed grossly enlarged peripheral nerves characteristic of MD lymphoproliferative lesions at 3 to 6 weeks after inoculation with the virus. Strain FC 126 of HVT was similarly cloned (21) and stored. The HVT is antigenically related to MDV (26) and is widely used as a vaccine virus against MD (14). Cell culture techniques. The spleen was removed aseptically from chickens and suspended in D-PBS. Cells were released into D-PBS by repeated aspiration into a 10-ml syringe, and the cell suspension was filtered through a sterile gauze and washed once with D-PBS. The resuspended cells (9 ml) were layered over 5 ml of Ficoll-Hypaque gradient and centrifuged at 400 x g for 15 min. Viable lymphocytes recovered from the interface were washed three times with DPBS and suspended in RPMI 1640 for use in the lymphocyte stimulation test. Blood was taken from chickens by cardiac or venipuncture with a syringe containing 20 U of heparin per ml of blood. Whole blood samples were cultured in the growth medium without additional serum but with 200 U of penicillin and 100 gtg of streptomycin per ml of medium. Procedures for lymphocyte stimulaion test. Whole blood samples (10 fd) or spleen lymphocytes
475
(106) in 0.2 ml of medium RPMI 1640 plus 5% fetal calf serum were cultured with 100,ug of stock PHA per ml in each of the 96 wells on a microtiter plate. All cell cultures in the microtiter plates were incubated at 41WC in a 5% CO2 atmosphere for 48 h, followed by addition of 0.05 ,uCi of [1I]iododeoxyuridine per culture. Incubation was continued for 16 h more. Lymphocyte cultures were harvested on filter papers with an automatic 24-multiple-culture cell harvester (model M24V; Brandel, Rockville, Md.). The filters containing labeled cells were punched out and placed in disposable tubes for gamma counting in a Beckman gamma counter. The PHA stimulation results are expressed as a stimulation index that was calculated as the mean counts per minute of isotope incorporated in cells in triplicate wells treated with PHA divided by the mean counts per minute of isotope incorporated in cells in triplicate wells not treated with PHA. For all results reported in this paper, the background activity of cultures not treated with PHA did not differ significantly between control chickens and those inoculated with MDV. The mean counts per minute of ['S2I]iododeoxyuridine incorporated into lymphocyte cultures not treated with PHA was 84 ± 3. Removal of adherent and phagocytic cells from spleen. A 5-ml sample of lymphocyte suspensions containing 108 cells was incubated for 45 min at 371C in 100-mm petri dishes. The nonadherent cells were gently removed from adherent cells. For removal of adherent or phagocytic cells with carbonyl iron/magnet, lymphocytes (108) were suspended in 5 ml of the lymphocyte-separating agent containing carbonyl iron particles. The mixture was incubated for 60 min at 370C in 50-ml conical tubes. The tubes were then put on top of a magnet, and the supernatant was removed by Pasteur pipettes. This procedure was repeated six times, and the cells were washed once before cultivation. Pathology. The lesion status of control and MDVinfected chickens was monitored by gross necropsy and by histological sections of peripheral nerves and gonads. Chickens that died before the termination date were examined for MD lesions by similar procedures. Statistical analysis. All results are expressed as a mean stimulation index. Standard errors of the mean were calculated. Student's t test was used for calculating statistical significance.
RESULTS PHA response of peripheral blood lymphocytes from chickens inoculated with MDV. Two replicate tests were performed. Chickens in replicate no. 1 were inoculated at 1 week of age, and chickens in replicate no. 2 were inoculated at 3 weeks of age. In each replicate test, eight MDV-inoculated and eight uninoculated line 72 chickens were bled before inoculation to establish the reference response to PHA. The PHA response of whole blood peripheral lymphocytes was monitored at weekly intervals after virus inoculation (Table 1). At 1 week after
476
INFECT. IMMUN.
LEE ET AL.
5 died of MD within 2 to 3 weeks of the initial depression after a slight recovery in PHA response, and four other birds suffered a second depression either before death or in association with the development of MD lesions. Inoculated chickens 7 and 8 survived MD, and, following an initial depression, their PHA response recovered to a level higher than that of the control. For these two chickens, virus inoculation seemed to have had a potentiating effect on their PHA responsiveness. When necropsied at the end of the 7-week experimental period, neither of the two chickens showed any gross or microscopic lesions (Table 2). Table 3 represents the results of two replicate TABLE 1. Sequential monitoring of PHA response experiments on the PHA response of lymphoof whole blood lymphocytes from genetically cytes from MDV-inoculated, genetically resistsusceptible line 72 chickens infected with the JM strain of MDVG ant line N chickens. Lymphocyte response to PHA was severely depressed at about 1 week Stimulation index' RepliAge Week after inafter virus inoculation in both experiments. Week cate test Agee ouaterion (wes) oculation no. Control Inoculated However, this initial depression was transient, and, within 2 to 3 weeks, the lymphocytes from I 1 Before 15 ± 3 20 ± 2 inoculation all the infected chickens recovered their PHA 1 5± 1** 2 32±5 response to a level comparable with that of the 2 3 82 ± 11 16 ± 2** lymphocytes from the control chickens. At ter4 3 129± 14 21±8** mination, all inoculated chickens were exam4 5 117 ± 13 12 ± 9* (5) 5 6 115± 12 24±0(1) ined, and no gross or microscopic lesions were found. 2 3 Before 42 ± 5 44 6 PHA response of peripheral blood lyminoculation phocytes from HVT-inoculated, MDV-chal4 1 106± 12 21±6** 5 2 45±7 23±7* lenged chickens. Results from two sequential 6 3 76± 16 19 7** replicate studies on the PHA response of whole 7 4 107 ± 12 27 13** blood peripheral lymphocytes from normal, 8 5 89 ± 5 22 13* (3) HVT-inoculated, MDV-inoculated, and HVTGenetically susceptible line 72 chickens were inoculated intra-abdominally with JM strain of MDV at 6 x 103 plaque- vaccinated but MDV-challenged chicken are summarized in Table 4. Eight line 72 chickens forming units per chicken. b Stimulation index ± standard error of the mean. Statistiwere used in each of the four groups. Chickens cal significance is calculated by Student's t test: *, P < 0.01; in replicate studies 1 and 2 showed a statistically l, P < 0.001. Numbers in parentheses show the number of chickens that survived MD at the time tested. The starting significant depression in PHA response 1 week number of chickens was eight for each of the inoculated and after inoculation with the nononcogenic HVT. the control groups. The response remained depressed for 2 to 3
inoculation, severe depression was observed in infected chickens, and the level of response of the inoculated chickens was significantly lower than that of the control throughout the entire experimental period. Since data from chickens individually monitored indicated that depression in PHA response was related to the severity of MD (data not shown), a third replicate experiment was conducted. As shown in Table 2, all inoculated chickens showed an initial depression in PHA response as compared to the average response of age-matched control chickens. Chickens 4 and
TABLE 2. Sequential monitoring of PHA response of whole blood lymphocytes from individual genetically susceptible line 72 chickens infected with the JM strain of MDV0 Week after MDV Control stimulation ininoculation dex (8 chickens)b
1 2 3 4 5 6
34 ± 4 (14-56) 20 ± 5 (8-44) 42 + 4 (27-0) 69 ± 8 (44-111) 77+ 14 (35-145) 69 + 9 (41-73)
1
2
8 10 31 14 5 6
2 14 19 31 20 4
Stimulation index for chicken no.: 3 4 5 6
13 10 49 64 20 Died
2 11 Died
2 8 19 Died
11 8 66 11 20 7
7
8
4 22 86 67 102 169
4 9 54 102 126 110
MD lesion' + + + + + + a Genetically susceptible line 72 chickens were inoculated intra-abdominally with JM strain of MDV at 6 x 103 plaque-forming units per chicken. b Stimulation index ± standard error of the mean and, in parentheses, range of values. 'MD status was assessed by gross and microscopic lesions.
477
PHA RESPONSE IN MDV- AND HVT-INFECTED CHICKENS
VOL. 21, 1978
slightly increased at about 2 weeks after MDV challenge. As shown in replicate 2, Table 4, a statistically significant increase in PHA responsiveness was observable by week 5 after HVT inoculation and stayed at that level until termination. As expected, none of the eight TABLE 3. Sequential study of PHA response of HVT-inoculated chickens had developed gross whole blood peripheral lymphocytes from genetically or microscopic MD lesions at termination. No resistant line N chickens infected with the JM depression of PHA response was observed in strain of MDVHVT-vaccinated chickens when challenged by Stimulation index' the virulent JM strain of MDV (Table 4). In Replicate Age Week after inocaddition, the lymphocytes from the HVT-vacInocuulation test no. (weeks) Control cinated MDV-challenged chickens, like the lymphocytes from HVT-inoculated chickens, ap48 +±7 52 + 8 Before 1 3 peared to be more PHA responsive than the inoculation 1 58± 12 6±2* 4 normal lymphocytes. All eight chickens survived 2 66±12 40±10 5 the entire experimental period, and none had 3 91±20 46±13 6 developed gross or microscopic lesions when ex4 75± 10 50± 10 7 amined at termination. In contrast, six of eight 5 51± 8 48±7 8 6 100±17 94±14 9 MDV-inoculated chickens in replicate studies 1 and 2 either died of MD or had developed gross ± 27 ± 22 3 3 1 Before 2 or microscopic MD lesions at termination. inoculation Depression in PHA response in MDV 7±1* 1 38±5 2 44±7 30±6 2 3 spleen cells. The PHA response of spleen cells 62 10 46±6 3 4 from the normal and inoculated line 72 chickens 86 10 71 ± 12 4 5 was studied, and the results are summarized in a Genetically resistant line N chickens were inoculated inTable 5. As shown, MDV spleen cells were untra-abdominally with JM strain of MDV at 6 x 103 plaque- responsive to PHA at 1 week after inoculation. units per chicken. forming I Stimulation index ± standard error of the mean. Statisti- However, removal of adherent cells by adhercal significance is calculated by Student's t test: i, P < 0.001. ence treatment or by carbonyl iron/magnet
weeks before recovery. As shown in replicate 1, Table 4, the PHA response after recovery not only was comparable with the response of the normal lymphocytes but also appeared to be
even
TABLE 4. Sequential study of PHA response of whole blood lymphocytes from the normal, HVT-inoculated, MDV-inoculated, and HVT-vaccinated and MDV-challenged chickens Replicate test no.
1
Week after HVT vaccination
2 3 4
5 6 9 MD lesions 2
1 2 3 4 5 6 7 MD lesions
Week after MDV challenge
Response (stimulation index)b HVT
MDV
HVT/MDV
57 ± 18 112 ± 21 69±8 46±5
9±2* 25±5 46± 4 97 ± 12 80±5 44±9
40 ± 11** 66±17 31±8
105 ± 15 92±15 44± 11
0/8
0/8
Normal 17±1 31±10
Before inoculation 1 2 3 Before inoculation 1
2 3 4 5
6
6/8
0/8
31 ± 5 34±7 20±5 42±4
13 ± 3* 18±3* 24±3 65±7*
6±2** 11±2* 46±10
69±8
91± 12*
44± 12
53±9 97±6*
77±14 64+9
112±15* 95±16*
54±25 36±21
103±8* 85±18
35±17 21±3
0/8 6/8 0/8 0/8 x 6 with 1 of at with 103 vaccinated HVT plaqueday age aGenetically susceptible line 72 chickens were forming units per chicken. The vaccinated chickens were challenged with the JM strain of MDV at 6 x 103 plaque-forming units per chicken at 4 weeks after HVT vaccination for replicate 1 and at 2 weeks after vaccination for replicate 2. The starting number of chickens was eight for each of the inoculated and the control groups. b Stimulation index ± standard error of the mean. Statistical significance is calculated by Student's t test: *, P
