Indian Journal of Experimental Biology Vol. 42, August 2004, pp. 823-829

Infiltration by CD4+ and CD8+ lymphocytes in bursa of chickens infected with Infectious Bursal Disease Virus (IBDV): strain-specific differences

Bhawna Paonia & Shiv Charan*

Department of Veterinary Microbiology, CCS Haryana Agricultural University, Hisar I25 004, India

Received 25 November 2003; revised 26 April 2004

In order to investigate if there is any definite correlation between the degree ofT-cell response in the bursa of Fabricius (BF) and the virulenco.: of Infectious Bursal Disease (lBO) virus strains, chickens were infected with strains of different virulence i.e. mild (Luken strain), intermediate (Georgia strain) or invasive intermediate (IV-95 strain). At various times post-inoculation, bursal samples were collected to study virus specific histopathological lesions, the di stribution of viral antigen and the extent ofT-cell infiltration in the bursa. Most severe bursal lesions were induced by IV-95 strain (the invasive intermediate strain), whereas Luken, the mild strain caused the least severe lesions. The number of virus positive cells in the bursa was highest in chickens infected with IV -95 strain. Substantial infiltration of CD4+ and CD8+ T-cells in the bursal follicles of virus-infected groups was observed from 4 d.p.i. onwards. The magnitude ofT-cell response was more in the birds infected with intermediate (Georgia) or invasive intermediate strains of virus than chickens inoculated with mild (Luker!) strain, even when 10-fold higher doses of the inoculums were used. PHA responses to peripheral lymphocytes were found suppressed in all the groups of chickens only transiently. The results indicate that the magnitude ofT-cell responses in BF during IBDV infection is influenced more by the virulence of virus strain rather than the quantum of viral load in BF. Over all these studies may have implications in understanding the role ofT-cells in pathogenesis and immunity in lBO.

Keywords: Bursa of Fabricius, Bursal disease, Infectious, Immunity, Pathogenesis T-cell response.

Infectious Bursal Disease Virus (IBDV) is an important pathogen of chickens, which is responsible for a highly contagious disease characterized by immunosuppression. Chickens infected with IBDV develop reduced humoral and cellular immune responses and respond poorly to vaccines 1• The virus has a cytocidal effect on B cells leading to the reduced antibody production2

·3

. The effect of virus infection on cell-mediated immunity is not fully characterized4

• Recently, CD4+ and CD8+ T-cells infiltration has been demonstrated in the bursa of Fabricius (BF) of IBDV infected chickens5

•7

• These studies showed that T-cells begin to accumu late at the site of virus replication in the bursa as early as 24 hr post-infection (pi) and persisted for at least 12 weeks after infection 7• Kim et al. 8 showed that both bursal CD4+ and CD8+ T-cells mediated suppression of mitogenic responses of normal splenocytes. Cyclosporin-A treatment in the chickens infected with IBDV resulted in increased virus replication in the

Correspondent au thor: Phone: +98120 44461; Fax : +981662234952 Emai l: sh ivcharan@hau.nic.in;

BF, suggesting a role for T-cells in anti-IBDV immunity9

.

The aim of this study is to investigate the T-cell responses in the bursa of chickens infected with three strains of IBDV having different virulence.

Materials and Methods Chickens-Day-old broiler chicks were obtained

from the poultry farm of CCS Haryana Agricultural University, Hisar. The farm had no history of lBO outbreak. The chicks were examined for anti-IBDV neutralizing antibodies before inclusion in the studies.

The chicks were raised together in departmental animal house till three weeks of age after which they were divided into different experimental groups to be kept in separate isolation units . All animal manage­ment protocols were undertaken in accordance with the requirements of 'B lue Cross Society, COVS, Hisar' (recognised by the Animal Welfare Board of India; Prevention of Cruelty to Animals Act 1960).

Virus- Three strains of infectious bursal disease virus (lBDV) were used. M/s Indovax Pvt. Ltd., Hi sar, gifted the mild (Lukert), intermediate (Georgia) and invasive intermediate (IV -95), all vaccine strains

S24 INDIAN J EXP BIOL, AUGUST 2004

of IBDV. The isolates Lukert and Georgia were grown in chicken embryo fibroblast (CEF) cell culture and titrated before usi ng for experimental inoculation. The IV -95 strain grown in chick embryo was used as indi cated by the manufacturers.

Monoclonal antibodies-The FITC-conjugated anti-CD4+ and anti-CDS+ ( both of JgG 1 K isotype) monoclonal antibod ies (mAbs) were kindly gifted by Dr. A. Kaushik, University of Guelph, Canada 10

. The anti-IBDV monoclonal antibody against VP2 (lgG2a isotype) was kindly gifted by Dr. J. fgnjatovic of CSIRO, Australia 11

• Optimal diluti ons of the above monoclonals were determined using checker-board titration.

Experimental design- At the age of three weeks ch ickens (96) were divided into fo ur groups of 24 each. The chickens in different groups were inoculated with different strai ns of IBDV by oral and ocul ar routes as follows: Group I chickens were inoculated with I 06

·3 TCfD5ofml of Lukert strain ,

group II chickens with I 05·3 TCID5ofml of Georgia

strain and group III chickens with I 02·3 EID5ofml of

IV-95 strain (as this strain was adapted in chick embryo). Chickens in group IV were inoculated with phosphate buffered sali ne (PBS) and served as control. The birds were examined daily for clinical mani fes tations of IBD.

Sample collection-Blood was coll ected in heparin (used @20 IU/ml) and BF were collected in 10% neutral buffered formalin on days 0, 2, 4, 7, 10, 14, 21 and 2S pi.

Bursa was used to prepare 4 sets of paraffin­embedded tissue sections. One set was stained with Hematoxylin and Eosin to study hi stopathological changes caused by strains of IBDV diffe ring in their virulence. Second set was used for demonstration of viral antigen in affected tissue and the third and fourth sets were used to demonstrate the distribution of CD4+ and CDS+ T-lymphocyte influx in bursal ti ssue sections, respectively.

Studies conducted

Detection of IBDV antigen in the bursal sections­IBDV antigen was detected, using indirect immune­peroxidase test, by the method of Cruz-coy et al. 12

,

which was used with some modifications. Briefly , the paraffin-embedded sections were

deparaffinized in three changes of xylene followed by rehydration in graded (95, 90, 70 and 45%) ethanol. Sections were treated with 0.1 % trypsin in PBS

containing 0.1 % calcium chloride (pH 7 .S) for 15 min at 37°C followed by thorough rinsing under running water. Endogenous perox idase acti vi ty was inhibited by pl acing slides in 0.75% hydrogen peroxide in methanol for 30 min followed by rinsing in PBS. Samples were blocked with 6% bovine serum albumin solution containing equal amounts of I :20 diluti on of normal goat serum. Sections were then treated with dilution of anti-IBDV mAb ( I :40) for 60 min at 37°C followed by rinsing in PBS. Goat anti-mouse peroxidase conjugate was used in l :40 dilution for 60 min at 37°C. After thorough washing, slides were incubated with peroxidase substrate diaminobenzidine tetrahydrochloride (Sigma, USA) f r 3-5 min. Later slides were washed in tap water, and counter stained with Haematoxylin . Finally the sections were microscopically evaluated for nu 1ber of positive cells, using a score from 1 to 3, where score I indicated that only a few lightly stai ned positive cells present; score 2 indicated widely scattered positive cells and score 3 indicated numerous dark staining cell s. A total of 20 fields were counted per section and results were averaged. The slides were examined at random to exclude any personal bias.

Detection of CD4+ and CD8+ T- cells in the bursa-The presence of CD4 + and CDS+ T­lymphocytes in formalin-fixed, paraffin-embedded BF tissue sections was demonstrated by direct fluorescent antibody test. The slides were cleparaffinized in xylene and rehydrated in graded alcohol followed by distilled water and PBS. Sections were treated with 0.1% trypsin solution containing 0.1% calcium chloride for I S min. Tissue sec tions were then incubated with predetermined dilution (1 :20) of mouse anti-chicken CD4+ or CDS+ mAbs for 60 min at 37°C. Slides were thoroughly washed in three changes of PBS, sections covered with 1: I 0 solution of glycerol in PBS, cover-slipped and observed under fluorescent microscope. The T -ce" I response in the sections of bursa was estimated on the basis of CD4+ or CDS+ T-cell accumulation by counting the percentage of affected follicles in 20 randomly selected follicles from each bursa as per the method followed by Tanimura and Sharma 5

. The response was expressed as 1 + to 3+. Results were expressed as 1+ when less than 113 rct of lymphoid follicles were affected. It was expressed as 2+ when 113rd to 2/3 rct follicles were affected. The response was expressed as 3+ when more than 2/3"1 of fo llicles had cell infiltration.

POONIA & CHARAN: INFILTRATION BY CD4+ & CD8+ IN BURSA 825

Lymphoproliferative responses ( lp )-The proli­ferative responses of peripheral blood mononuclear cells (PBMC) from infected chickens were measured by MTT (3-(4,5-dimethylthiazol-2-yl) 2,5-diphenyl tetrazolium bromide) assay. The method of Mosmann 13 was used with modifications. Blood was collected in heparinized tubes with 20 units of heparin per ml of blood and diluted in equal part of serum free RPMI medium. PBMC were separated from diluted blood by density gradient centrifugation over Histopaque-1 077 and were suspended at a concentration of 5XI06 cells/ml in RPMl complete medium (containing 20mM HEPES buffer, 2mM L­glutamine, 0.5mM 2-mercaptoethanol , 3.5% sodium bicarbonate and I% stock solution of antibiotics to give a final concentration of I 00 IU/ml of penicillin and 100 ~tg/ml of streptomycin) . PBMC suspension ( 100 ~I) was dispensed into triplicate wells of flat­bottomed 96-well microtitration plates containing 4 ~g PHA. The appropriate controls were included and the final volume of media was made to 200 ~I in each well. The plates were incubated at 37 °C in 5% C02

atmosphere for 72 hr at the end of which 15 ~I of MIT (5mg/ml) was added in each well and plates were further incubated at 37 °C for 4 hr. After incubation, 100 ~I supernatant was carefully aspirated from each well without disturbing the formazan precipitate. Finally, stop solution (3 % triton X-100 in DMSO) was added in l 00 ~I volume in each well and mixed thoroughly to dissolve dark blue formazan crystals. After a few minutes at room temperature to ensure complete dissolution of formazan crystals, plates were read on multi-well spectrophotometer (Organon Technica reader 530) at 540 nm wave length. Plates were normally read within 15 min of adding stop solution. Blastogenic responses for the assay were expressed as mean stimulation index calculated by dividing the mean 0.0. of stimulated cultures with the mean O.D. of unstimulated cultures 14

•

Statistical analysis-Duncan's multiple range tests of variables was used to assess the significance of difference between treatment groups.

Results and Discussion Clinical signs-Following experimental

inoculation with IBDV, birds showed dullness and there was decreased feed intake during the initial phase of infection, which lasted upto first-week post infection. Birds in the group inoculated with IV -95

strain were relatively more depressed. Birds from control group were healthy.

Pathology

Gross pathology-Haemorrhages on thigh and pectoral muscles appeared by 2 d.p.i. in chickens infected with Lukert, Georgia and IV -95 strains of IBDV. Bursal haemorrhages were not conspicuous in any group. The most marked change observed was bursal hypoplasia which was observed in all virus­inoculated groups from 7 d.p.i. onwards. Infection with Lukert strain caused decrease in size of bursae between 4 d.p.i. and 14 d.p.i. Lesions were milder as compared to other strains. Bursal hypoplasia was most severe in IV -95 inoculated group between l 0 and 28 d.p.i. Bursal size was maximal on day 2 p.i. in Lukert infected group and on day 4 p.i. in Georgia or IV -95 infected groups, however, enlargement of bursa was not observed.

Histopathology-Changes in the bursa included mild depletion of lymphocytes and vesicle formation in the follicles along with the presence of heterophils in bursal follicles during early stages of infection. From 7 to 21 d.p.i., follicular lesion was characterized by decrease in size of bursal follicles and an increase in interfollicular connective tissue in Georgia virus infected group. However, few vesicles were detected in follicles. Most severe lesions were observed in birds infected with IV-95 virus. In this group, necrosis of lymphocytes along with presence of heterophils was observed as early as 2 d.p.i. From 4 to 7 d.p.i., a decrease in follicular size along with an increase in interfollicular connective tissue was observed. Later, severe decrease in interfollicular connective tissue along with decrease in size of follicles and cyst like structures were seen in the follicles.

Lesions in chickens inoculated with Lukert strain were mild when compared to other two groups. Bursae from control group of chickens did not show any lesions.

IBDV antigen positive cells in the bursa-The antigen positive cells were marked by intense brown colour in immunoperoxidase reaction. Viral antigen was detected as early as 2 d.p.i. in the bursa of infected chickens. The lesion scores based on intensity of colour and area involved are shown in Table l. The frequency of positive cells as well as intensity of colour was maximum in IV-95 group. By 4 d.p.i., IBDV antigen could be detected in the cortex and medulla of most of the bursal follicles. Maximum

826 INDIAN J EXP BIOL, AUGUST 2004

reaction was observed at day 4 p.i. in all virus inoculated groups. After day 10 p.i., the frequency of antigen positive cells diminished and at day 21 p.i. only a few positive cells could be detected in IV -95 infected group. In Georgia virus infected group no positive cell could be detected after 14 d.p.i. No IBOV antigen was observed in bursa of control chickens.

Detection ofT-cells in the bursa-In uninoculated control chickens, a few T-cells were always present in the bursal follicles. In IBOV inoculated chickens, an increase in both C04+ and C08+ T-cells was observed as early as I d.p.i. The C04+ cells were located at the boundary area between cortex and medulla, whereas, cog+ cells were scattered throughout the lymphoid follicles. By 4 d.p.i., more than two-thirds of the lymphoid follicles were affected and a large proportion of lymphoid cells were T-cells. Although both C04+ and cog+ T-cells increased in numbers, there was a greater proportion of cog+ T-cells on several occasions (Table 2). Infection with Lukert strain resulted in milder T-cell reaction compared to infection with the other two strains between days 2

Table 1- lmmuno-histochemical evaluation of antigen positive cells in bursae of IBDV infected chickens

Frequency of antigen positive cells in the bursa DPI upon inoculation of chickens with

Luker! Georgi a IV-95

2 1.0 1.0 2.0

4 3.0 3.0 3.0

7 1.6 2.0 3.0

10 0.6 1.3 1.6

14 0.6 0.6 1.0

21 0.3 0.0 0.6

2S 0.0 0.0 0.0

and 10 p.i. [Fig. 1 a-d)]. However, the degree ofT­cell response was not distinguishable between Georgia and IV -95 strains, though IV -95 produced more severe lesions and higher frequency of antigen positive cells in immunoperoxidase assay.

Lymphoproliferative respollses ( lp )-A transient depression in the proliferative responses was observed upon infection of chickens with IBOV within the first two weeks (Table 3). There was considerable vanat10n in the blastogenic responses among individual chickens within groups. Infection with Lukert strain resulted in SI values of 2.56±0.24 and 2.24±0.76 against control values of 7.6 and 9.9g±o.2g employing MTT assay on days 2 and 7 respectively . Similarly, infection with Georgia strain resulted in an early suppression of lymphoproliferative responses with SI value of 2.34±0.16 on 2 d.p.i. and 2.55±0.25 on 7 d.p.i. However, infection with IV-95 strain caused a late suppression of lp responses starting at 7 d.p.i. Maximum suppression was observed in this group at 14 d.p.i. with an SI val e of 2.26±0.21 against a control value of 6.g±l.6. Control birds had high proliferative responses on all intervals.

Table 2--CD4+ and CDS+ T-cells in the bursa of Fabricius of chickens infected with IBDV strains of different virulence

CD4+ and CDS+ T-cells reaction in the bursa of chickens DPI infected with different strains of IBDV

Lukert Georgia IV-95

CD4+ cos+ CD4+ CD8+ CD4+ cos+

+ + + + + + 2 + + + ++ + ++ 4 ++ +++ +++ +++ +++ +++

7 + ++ +++ +++ ++ +++

10 + ++ ++ +++ ++ +++ 21 + + ++ ++ + ++

Table 3--Mitogen (PHA) specific lymphoproliferative responses of chickens inoculated with different strains of IBDV as measured by MTT assay

DPI Stimulation indices (mean±SE) of PBMC from chickens opon inoculation with

Lukert Georgia IV-95 PBS

2 2.56± 0.248 2.34 ±0.168 7.15± 0.35A 7.6a

7 2.24± 0.76c 2.55 ±0.25c 6.15± 1.058 9.9S± 0.2SA

14 J0.25± 1.65A 4.6± 0.5 8C 2.26 ±0.2lc 6.S± 1.6A8

21 5.49± 0.698 9.15± 1.25AB 7.75± 0.45A8 6.43± 1.47A8

2S 7.15± 0.S5A 6.75a S.7± 0.45A 7.5a

Values with different superscripts (A, B, C, D) on a day are significantly different. Superscript 'a' indicates a single observation

POONIA & CHARAN: INFILTRATION BY C04+ & COS+ IN BURSA 827

Infiltrati on of CD4+ and CD8+ T-cells in the BF has been reported in response to IBDV infecti on s-?_ In the present study, IBDV pathogenicity and T-cell response in the bursa of Fabricius was compared in chickens infected wi th three IBDV strain s of different virulence with a view to correlate severity of les ions in the bursa with the degree of T-cell infiltration. A comparison of pathogenicity showed that Lukert strain , which is a mild vacci ne strain , caused least severe pathological changes in the infected chickens in spite of the fact that a I 0-fold higher amount of the inoculum was used to infect thi s group of chi ckens. The dose of the inoculum was adjusted so as to induce comparable virus load in the bursa based upon pil ot ex periments (results not shown). Hi stopatholog ical

changes in the bursa of chickens inoculated with Georgia were more severe when compared to Lukert infected birds. Most severe pathological changes were observed after inoculation of chi ckens with IV -95 , the in vasive intermediate vaccine strain.

The results of MTT assay show a significant depression of proliferative responses of PBMC from infected chi ckens between 2 and 14 d.p.i . The suppression was, however, trans ient and proliferative responses returned to normal after a period in a ll infected groups. A transient suppress ion of lympho­proliferative responses of lymphocytes from IBDV infected birds has been reported in the fir-s t two weeks of infection using IBDV strains of different viru lence 16

-19

. In the present as well as other studi es it

Fig. 1-C0 4+ nne! CDR+ T-ce ll s distribution in bursn of Fabri cius of chickens infected with !BOY strains of di fferent virulence (paraffin­embcclclecl bursal ti ssue sections stninecl with FITC-conjugnted monoclonal antibodies.400X) (a) C04+ T -cell in the bursa of chicken inoculated with Lukert strain (mild) at 4 dpi ; (b) C0 4+ T -cell in the bursa of chicken inoculated w ith IY-95 strain ( invasive intermediate) at 4 clpi ; (c) COS+ T-cell in the bursa of chicken inocu lated with Georgin strain ( intermediate) at 4 dpi ; (d) COS+ T-cell in the bursn of control chicken at 4 dpi.

S2S INDIAN J EX P BIOL, AUGUST 2004

was no t possible to show any coiTelation between the vi rul ence of the virus and the degree or duration of suppress ion of cell mediated immunity employing T­cell proliferation assays.

The results of indirect immunoperoxidase test usi ng an anti-IBDV mAb directed against VP2 protein of the virus were similar with all four strains of IBDV with maximum number of antigen positive cells seen in bursal tissues on 4 d .p.i. However, stronger stain intensity suggestive of higher degree of virus replication was observed with IV-95 strain, which a lso caused most severe histopatho logical lesions in the bursa of Fabricius. At 4 and 7 cl.p.i., virus was mostly detected at the border of cortex and medulla in the bursal follicles . At around the same time, micro­scopic lesions consisted of depletion of lymphocytes in the cortical area. Thus, immunoperoxidase positive cells, which represent areas of viral replication, were associated with microscopic lesions. Similar results have been reported earlier 12

·20 where it was shown that

severity of lesions in bursal follicl es correlated with strong immunoperoxidase reaction .

An increase in T-cell accumulation in the bursa l follic les of IBDV infected ch ickens as early as I d.p .i. Both CD4+ and CDS+ T-cells increased in numbers. Maximum T-cell influx was observed between clays 4 and 7 p.i. for all the three virus inoculated groups. It has been demonstrated that JBDV replication in the bursal and caecal tonsils is accompanied by a rapid and profound appearance of CD3+ T-cells and was suggested that IBDV infection in the bursa recruited T-cells circulating in the blood or lymph vessels5

.

Similarly, rapid increase in both CD4+ and CDS+ T­cells from 2 to 5 cl .p. i. has been observed by others6

.

Kim et a /.7 noted intra-follicular T-cell s within the first few days of JBDV in fection, wh ich persis ted for at least 12 weeks p.i. In the present experiments it was observed that CD4+ T-cells were mostly present at the boundary area of cortex and medulla, however, cos+ T-cells were distributed throughout the follicl es. The number of CDS+ T-cells was hi gher than CD4+ T-cells as observed earlier6

. The number ofT-cells remained higher in infected chickens compared to age matched contro ls till 21 d.p.i. Max imum antigen detection as we ll as maximum T-cell reacti on was observed around the same time i.e., around 4 d .p.i. after infection of chickens, indicating that these T-cells accumu late in response to infection . There was no marked difference in the ex tent ofT-cell reaction in response to Georgia or IV -95 stra ins. It was

interesting to observe that in spite o f almost simi lar number of IBDV antigen positive ce lls in the bursae of chickens inoculated with strains o f inte rmediate or mild virulence, lower number ofT-cell accumulation occwTed in the bursa of chickens inoculated with the mild strain . Between 4 and 10 d.p .i .. on ly one third of lymphoid fo llicles in Lukert infected ch ickens showed an increased accumulation of T-ce lls compared to two-thirds affected follicles during the same time in chickens inoculated with other two strains of IBDV. Taken together with the resu lts of Poonia and Charan9

where it was shown by cyc losporin-A treatment of fBDV infected chickens that T-ce ll s play a role in limiting the severity of disease and replication of IBDV in the bursa, the present findings suggest a definite correlation between the virulence of JBDV strain and degree of T-cell reactio n in the bursa. However, phenotype of the infiltrating cells did not show any correlation with the viru lence of IBDV strain used for infection. The extent of T -cell accumulation seems to be influenced by their requirement at the si te of pathology, however, all T­ce ll s that infiltrate the bursa in response to IBDV infection may or may not be crucial. In experimental encephalomyeliti s (EAE) in mice, it has been shown that in addition to the few T-cell clones which are responsible for inciting the disease, a whole lot of inflammatory T-cells infiltrate bra in and spinal cord on the onset of paralysis. When treated with an altered peptide ligand, the heterogeneous T-cell infiltrate disappears from the brain, with only the T-cells that incited the disease remaining in the origi nal lesion 21

.

Similarly, whether all T-cells that accumulate in the BF during IBDV infection contribute towards limiting the severity of disease or virus replication and what proportion of these ce ll s are antigen-specific would be interesting to investigate further.

Acknowledgement The first author (BP) was a rec tptent of Senior

Research Fellowship of the CSIR, ew Delhi , Indi a.

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