Differences in the immunopathogenesis of infectious bursal disease virus (IBDV) following in ovo and post-hatch vaccination of chickens

Veterinary Immunology and Immunopathology 106 (2005) 139–150 www.elsevier.com/locate/vetimm

Differences in the immunopathogenesis of infectious bursal disease virus (IBDV) following in ovo and post-hatch vaccination of chickens Silke Rautenschlein *, Christine Haase Clinic for Poultry, School of Veterinary Medicine Hannover, Bu¨nteweg 17, 30559 Hannover, Germany Received 21 September 2004; received in revised form 19 January 2005; accepted 1 February 2005

Abstract Not much is known about IBDV-pathogenesis and immune mechanisms following in ovo vaccination. In this study, we compared the immunopathogenesis of an intermediate IBDV-vaccine in post-hatch- and in ovo-inoculated chickens. In ovovaccinated birds recovered significantly faster from lesions of the bursa of Fabricius than post-hatch vaccinated (P < 0.05). A significant accumulation of intrabursal CD8+ T cells was observed in post-hatch but not in in ovo-vaccinated chickens (P < 0.05). The innate immunity was comparable between in ovo- and post-hatch-vaccinated groups as indicated by comparable intrabursal macrophage accumulation and intrabursal IBDV-clearance. Overall, our observations indicate that IBDV in ovo vaccination may be advantageous over post-hatch. In ovo-vaccinated birds recover faster from bursa lesions and exhibit similar protection against challenge in comparison to post-hatch vaccinated. # 2005 Elsevier B.V. All rights reserved. Keywords: In ovo; Post-hatch; Infectious bursal disease; Chicken; Immunopathogenesis; Vaccination

1. Introduction

Abbreviations: B/BW, bursa to body weight ratio; CT, caecal tonsil; ELD, egg lethal dose; IBD, infectious bursal disease; IBDV, infectious bursal disease virus; PV, post vaccination; rpm, rounds per minute; S/BW, spleen to body weight ratio; SPF, specific pathogen-free; TCID, tissue culture infectious dose * Corresponding author. Tel.: +49 511 953 8763; fax: +49 511 953 8580. E-mail address: [email protected] (S. Rautenschlein).

Infectious bursal disease (IBD) is an immunosuppressive disease in chickens (Lukert and Saif, 2003). Despite vigorous vaccination strategies, IBD is still an economically important disease in commercial poultry. The most common way of infectious bursal disease virus (IBDV) vaccine delivery in the field is by drinking water post-hatch, but also in ovo vaccination has been shown to induce protective immunity (Gagic et al., 1999; Coletti et al., 2001; Corley et al., 2001; Giambrone et al., 2001; Sharma

0165-2427/$ – see front matter # 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.vetimm.2005.02.011

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et al., 2002). Not much is known about the immunopathogenesis and the mechanisms of immunity following in ovo vaccination in chicken embryos lacking a fully mature immune system (Ahmad and Sharma, 1993; Reddy et al., 1996; Karaca et al., 1998; Gagic et al., 1999; Rautenschlein et al., 1999). So far, studies comparing the efficacy of in ovo versus posthatch vaccination against IBD have focused on the induction of systemic humoral immunity and vaccine-induced lesions of the bursa of Fabricius (Gagic et al., 1999; St. Hill and Sharma, 1999; Coletti et al., 2001; Corley et al., 2001; Giambrone et al., 2001; Sharma et al., 2002), but recent studies indicated that humoral systemic immunity might not be the only mode of protection against IBDV challenge (Rautenschlein et al., 2002a). The induction of local immunity may also play an important role for protection because IBDV enters the circulation through gut-associated tissue before it is distributed to other organs (Mu¨ ller et al., 1979). The induction of local gut-associated immunity and the impact of in ovo versus post-hatch vaccination on the non-specific and specific cell-mediated immunity is not known. To optimise vaccination strategies in the field, and for the evaluation of IBDV vaccines it is essential to gain more knowledge about the modes of immune stimulation by IBDV-vaccines. Furthermore, possible differences in vaccine virus pathogenesis should be considered depending on the route and age of vaccine delivery. In this study, the immunopathogenesis of an intermediate IBDV-vaccine in post-hatch- and in ovovaccinated chickens was compared. The induction of bursa lesions and recovery, distribution of intrabursal T-cell populations and macrophage-like cells, IBDVantigen distribution and clearance, the induction of humoral gut-associated and systemic immunity, and protection against challenge with classic virulent IBDV were determined.

ground white Leghorn, line Lohmann LSL-LITE. Chickens were hatched and reared in pressurised isolation units (Montaim Van Stratum, Kronsberg, Netherlands) following the guidelines of the Animal Care Committee for the duration of the study. The birds were given food and water ad libitum. Birds from different experimental groups were housed in separate isolation units. 2.2. Virus An intermediate vaccine strain of IBDV (Bursine 2; Sharma et al., 2000) was propagated and titrated in chicken embryo fibroblast cultures (Kim et al., 2000). Birds were inoculated with 100 ml of 103 (Experiment 1), or 104 (Experiment 2 and 3) tissue culture infectious dose50 (TCID50) of IBDV/bird per eye drop (Experiment 1) or orally (Experiment 2 and 3) at 14 days post-hatch, or in ovo at embryonation day 18. The IBDV strain IM was used as a challenge virus at a dose of 103 egg-lethal dose (ELD)50/bird inoculated by eye drop route (Kim et al., 1999). IBDV-IM was propagated in 3-week-old SPF chickens. At 5 days post-IBDV inoculation, bursae from infected birds were harvested, homogenised, and titrated in embryonated chicken eggs as previously published (Kim et al., 1999; Tanimura and Sharma, 1997). 2.3. H&E staining For the detection of histopathological lesions, the bursa of Fabricius, spleen, and caecal tonsils were collected, fixed in 10% phosphate-buffered formalin and stained with hematoxylin and eosin (H&E). Lesions were observed microscopically. Bursa lesion scores were determined and compared between groups (Kim et al., 1999; Sharma et al., 1989). The scoring was as follows: 1 = 1–25%, 2 = 26–50%, 3 = 51–75%, and 4 = 76–100% of follicles showing cellular depletion.

2. Material and methods 2.1. Chickens

2.4. Immunohistochemical detection of different immune cell populations and IBDV-antigen

Specific pathogen-free (SPF) chickens, hatched from VALO1 eggs, or embryonated eggs (VALO1) were obtained from Lohmann Tierzucht (Cuxhaven, Germany). The birds belonged to the genetic back-

For the detection of different immune cell populations, the following monoclonal antibodies were used: CVI-ChNL-74.2, which detects macrophages (Jeurissen et al., 1992); CT8 (Chan et al., 1988), specific for

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CD8-positive T cells; and CT4 (Chan et al., 1988), specific for CD4-positive T cells. A polyclonal antiIBDV Bursine 2 rabbit antiserum, which was prepared following previously published procedures (Tanimura et al., 1995), was used to detect IBDV-antigen. For immunohistochemical staining of cell surface determinants, bursae of Fabricius, spleen, and caecal tonsils were collected, snap-frozen, sectioned and processed for immunohistochemical staining as described (Rautenschlein et al., 1998; Kim et al., 1999). For the detection of IBDV-positive cells, tissues were fixed in 10% buffered formalin and processed as described by Tanimura and Sharma (1997). The group means of the numbers of IBDV-infected cells, T cells, or macrophages per microscopic field at a magnification of 400 were determined after counting 3–10 fields/tissue/bird.

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All samples were tested for IBDV-specific antibodies by using (a) a commercial IBDV antibody ELISA kit (ProFLOK1 IBD, Synbiotics Europe, Munich, Germany) detecting IBDV-specific IgG-type antibodies, and (b) virus-neutralisation test (Winterfield et al., 1972) using IBDV-Bursine 2 as a virus to be neutralised. Mean log10 ELISA and mean log2 VNIBDV antibody titres were calculated and compared between groups. 2.6. Statistical analysis Group responses within experiments were analyzed by Student’s t-test, one-way analysis of variance (ANOVA), pairwise multiple comparison procedure by the Tukey test, or x2-test. 2.7. Experimental plan

2.5. Collection of gut-lavage, serum, bile and tissue for IBDV-antibody detection At 2, 3, 4, 5, 7–8, 14, 17, and 21 days post-vaccination (PV), anti-IBDVantibody levels were determined in bile, serum, gut-lavages, bursa and spleen. To assess local antibodies in spleen and bursa of Fabricius, the tissue was removed directly post mortem and washed thoroughly in Eagles minimum essential medium (MEM) containing 250 mg/ml of Streptomycin and 250 IU/ml of Penicillin. Three 3square millimetre pieces were cut and placed together in a single tube with 1 ml medium. Tissues were incubated in medium at 4 8C overnight. The medium was collected and assayed for IBDV-specific antibodies. One millilitre of buffered saline (PBS, pH 7.2) was used as collecting medium for bile. For the collection of gut lavage from the caeca and jejunum, PBS was supplemented with 50 mM EDTA, 0.1 mg/ml soybean trypsin inhibitor (Sigma) and phenyl methyl suphonyl fluoride (PMSF) (Sigma) in absolute ethanol (final concentration 2.0 mM) (Zigtermann et al., 1993; Raj and Jones, 1996). One caecum and 5 cm of jejunum were rinsed with 1 ml of supplemented PBS, and the washes pooled for antibody detection. Solid gut material was separated from the lavage fluid by centrifugation at 2000 rpm for 10 min at room temperature and discarded. The lavage fluid was stored at 20 8C until further use.

2.7.1. Experiment 1 At embryonation day 18 and 14 days post-hatch, 19 embryos (group 1) and 25 chickens (group 2) received 103 TCID50 of IBDV in ovo (Sharma and Burmester, 1982) or intraconjunctivally, respectively, while 19 control embryos (group 3) and 21 chickens (group 4) were inoculated with PBS. At 2 (day 20 of embryonation for groups 1 and 3, day 16 post-hatch for groups 2 and 4), 3 (day of hatch for groups 1 and 3, day 17 posthatch for groups 2 and 4), 5, 8, and 14 (only the posthatch groups 2 and 4) days PV, four–five embryos and birds per group were exsanguinated, and the following observations made: pathological and histopathological lesions at bursa of Fabricius, spleen, and caecal tonsils (CT); immunohistochemical detection of IBDV-antigen in sections of these organs; detection of CD4, CD8, and CVI-ChNL-74.2-positive cells in immunohistochemically stained bursa sections; antibody detection by VN and ELISA tests in sera, bile, and gut lavages. 2.7.2. Experiment 2 At embryonation day 18 and 14 days post-hatch, 62 embryos (group 1) and 43 chickens (group 2) received 104 TCID50 of IBDV in ovo or orally, respectively, while 55 control embryos (group 3) and 39 chickens (group 4) were inoculated with PBS. At 2 (day 20 of embryonation for groups 1 and 3, day 16 post-hatch for groups 2 and 4), 3 (day of hatch for groups 1 and 3, day 17 post-hatch for groups 2 and 4), 4, 7, 14, 18, and

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21 days PV, three–five embryos and birds per group were exsanguinated, and the observations as indicated in Experiment 1 repeated. In addition, bursa of Fabricius and spleen were used for antibody detection as well. At 14 days PV, three–five birds per group were challenged with IBDV-IM, and morbidity and mortality rate observed. At 7 days post-challenge, surviving birds were sacrificed, macroscopical and microscopical bursa lesions determined, and IBDVantigen in bursa sections detected. 2.7.3. Experiment 3 At embryonation day 18 and 14 days post-hatch, 37 embryos (group 1) and 20 chickens (group 2) received 104 TCID50 of IBDV-B2 in ovo or orally, respectively, while 37 control embryos (group 3) and 16 chickens (group 4) were inoculated with PBS. At 14 (11 days post-hatch for groups 1 and 3, 28 days post-hatch for groups 2 and 4), 17, and 21 days PV, three–five embryos and birds per group were exsanguinated, and the observations as indicated in Experiment 2 repeated. At 14 days PV, four–five birds per group were challenged with IBDV-IM, and morbidity and mortality rate observed. At 5 days post challenge, surviving birds were sacrificed, macroscopical and microscopical bursa lesions determined, and IBDVantigen in bursa sections detected.

3. Results 3.1. Induction of lesions Vaccination with IBDV-B2 did not induce any morbidity or mortality in post-hatch-inoculated

birds. In ovo vaccination induced a significant reduction of the hatch rate (P < 0.05), which has been observed also in other experiments (Gagic et al., 1999). While 78 and 81% of the PBS-in ovoinoculated chickens hatched in Experiment 2 and 3, only 61 and 58% of the IBDV-inoculated birds hatched, respectively. No IBDV-specific macroscopical bursa lesions, such as gelatination or hemorrhages, were seen following post-hatch and in ovo vaccination. In ovo vaccination with IBDV induced a significant increase in the bursa to body weight (B/BW) ratio at 3 days PV in comparison to virus-free birds of the same age group (P < 0.05). A significant reduction in the B/ BW ratio in the in ovo-vaccinated in comparison to non-vaccinated control hatch-mates was observed beginning at 7–8 days PV (Table 1, P < 0.05). Posthatch vaccination induced only a significant reduction of the B/BW ratio in comparison to virus-free hatchmates on experimental days 4–5 PV (P < 0.05; Table 1). Differences between vaccinated and nonvaccinated birds in the spleen to body weight (S/BW) ratio was only seen following in ovo vaccination at 4–5 and 7–8 days PV (data not shown). In ovo-vaccinated birds had significantly enlarged spleens with an average S/BW ratio of 0.4  0.2 and 1.6  0.7 at 4–5 and 7–8 days PV, respectively, versus 0.2  0.1 and 0.4  0.2 in non-vaccinated hatch-mates (P < 0.05). While in ovo-vaccinated chickens showed the most significant lesion development at 4–5 days PV, the post-hatch-inoculated birds showed the severest lesions at 17–18 days PV (Fig. 1). Lesions are characterised by lymphoid depletion of bursa follicles, cyst formation, infiltration of heterophiles, and cell

Table 1 Induction of pathological lesions following in ovo and post-hatch vaccination with IBDV Groups

Vaccinated*

Bursa to body weight ratio at days PV (n = 9–11) 2

Post-hatch Post-hatch In ovo In ovo

+ +

3.7  0.9 3.5  1.2 1.3  0.3 1.3  0.3

3

4–5 a

3.2  0.7 3.7  1.0a 1.0  0.4A 1.4  0.4B

7–8 a

5.8  1.5 3.1  0.9b 1.5  0.8A 1.1  0.5A

14 a

4.4  1.1 3.9  1.5a 1.6  0.5A 1.0  0.4B

21 a

4.4  0.9 4.1  1.1a 4.0  1.7A 1.9  0.6B

4.5  1.2a 3.2  1.6ab 3.7  1.0A 2.7  0.8B

At different days PV, birds were sacrificed and the bursa to body weight ratio determined. Different superscript letters indicate significant differences between groups of hatch mates. Compared were IBDV-positive with virus-negative in ovo-inoculated birds, or IBDV-positive with virus-negative post-hatch-inoculated birds (P < 0.05; Student’s t-test). Summary of three experiments. * Fourteen-day-old chickens and 18-day-old embryos were vaccinated with IBDV.

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3.3. Induction of intrabursal T-cell and macrophage accumulation

Fig. 1. Induction of histopathological bursa lesions following in ovo and post-hatch vaccination with IBDV (summary of three experiments; n = 9–11).Histological lesions are characterised by lymphoid cell necrosis, depletion of lymphocytes, infiltration of heterophiles, and edema. The development of bursa lesions was determined by comparing bursa sections of hatch mates of vaccinated and nonvaccinated birds or embryos, differences in bursa architecture between age groups were considered. *Significantly different than the other group (P < 0.03, x2-test). None of the IBDV-free birds showed any bursa lesions at any tested time point.

death. In all three experiments, it was demonstrated that in ovo-vaccinated birds recovered significantly faster from microscopical IBDV bursa lesions than post-hatch-inoculated birds (P < 0.03). No lesions were detected in spleen and CT after vaccination. None of the non-vaccinated birds showed IBDV bursa lesions. 3.2. Antigen distribution and quantitation There were no significant differences in the number of IBDV-antigen-positive cells in the bursa of Fabricius of IBDV post-hatch and in ovo-vaccinated birds (Fig. 2a). But the incidence of IBDV-positive cells in spleen and CT differed significantly between vaccinated groups. Only one of 10 post-hatchvaccinated birds showed IBDV-positive cells in the spleen at 2 days PV, and none in the CT, while at 4–5 days PV 75 and 67% of the in ovo-vaccinated birds had IBDV-antigen in spleen and CT, respectively (Fig. 2b and c; P < 0.05). Still at 7–8 days, 60 and 67% of the in ovo-vaccinated birds had IBDV-antigen in the spleen and CT, respectively. None of the nonvaccinated birds were positive for IBDV-antigen at any tested time points.

Post-hatch vaccination with IBDV induced a significant accumulation of CD4+ and CD8+ T cells in the bursa of Fabricius beginning at 7 days PV (P < 0.05, Fig. 3a and b). No significant increase in CD8+ cells was observed after in ovo vaccination in comparison to non-vaccinated controls (P > 0.05). The number of CD4+ cells was significantly increased at 21 days PV in in ovo-vaccinated birds in comparison to the virus-negative controls (Fig. 3a). The number of macrophage-like cells staining CVI-ChNL-74.2-positive was significantly increased at 7 days PV following post-hatch as well as in ovo inoculation (Fig. 3c). In Experiment 2, already at 3 days PV, two of five and five of five post-hatch and in ovo-vaccinated birds, respectively, showed a significant increase in the number of CVI-ChNL-74.2-positive cells (data not shown). In Experiment 3 at 14 days PI, in ovovaccinated birds had still a significant higher number of intrabursal macrophages than control birds (P < 0.05, data not shown). 3.4. Induction of systemic and local anti-IBDV antibodies The induction of circulating VN-anti-IBDV antibodies following vaccination post-hatch or in ovo was comparable (Fig. 4a). At 2 and 3 days post vaccination, already 25 and 40% of the post-hatch vaccinated birds had VN-titre of log2 of 3 and 3.75, while all the other tested birds were negative for VNantibodies at these time points (data not shown). At 14 days PV, VN-antibodies were detectable in 100% of the vaccinated birds (data not shown). The ELISAantibody levels of the IgG-type differed significantly between post-hatch and in ovo-vaccinated birds beginning at 14 days PV (P < 0.05). Post-hatch vaccinated birds had significantly higher ELISA antibody levels beginning at 14 days PV than the in ovo-inoculated birds (P < 0.05; Fig. 4b). While 100% of the post-hatch-vaccinated birds had developed detectable ELISA antibodies beginning 14 days PI, only 88, 67, and 75% of the in ovo-inoculated birds had IBDV-antibodies at 14, 17–18, and 21 days PI, respectively (data not shown). No ELISA-antibodies were detected in bile, gut-lavage, spleen and bursa of

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Fig. 2. Immunohistochemical detection of IBDV-antigen in the bursa of Fabricius (a), spleen (b), and CT (c) following post-hatch and in ovo vaccination. None of the non-vaccinated birds showed any detectable IBDV-antigen in any of the examined tissues. aGroup average of the number of IBDV-positive cells/microscopic field at 400. Summary of three experiments, n = 9–11. *Significantly different to the other group (P < 0.05, x2-test).

Fabricius either from vaccinated, or from nonvaccinated birds (data not shown). Locally, there were significant differences in the induction of antibodies in bile between the in ovoand post-hatch-vaccinated birds. When birds were vaccinated at 14 days post-hatch, they showed

already at 2 days PV VN-anti-IBDV antibodies in bile (Table 2). In ovo-vaccinated birds showed for the first time, antibodies in bile at 4–5 days PV. Posthatch-vaccinated birds developed higher detectable VN-antibody levels in spleen and bursa of Fabricius at 14 and 21 days post-vaccination. At earlier time

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Fig. 3. Detection of (a) CD4+ T cells, (b) CD8+ T cells, and (c) macrophage-like cells (CVI-ChNL-74.2-positive) in the bursa of Fabricius following in ovo and post-hatch vaccination with IBDV. The number of positive cells was evaluated counting immunohistochemically stained cells at a magnification of 400 in 3–10 fields per bird. The group average was calculated based on the average number of positive cells/bird. Data from one representative experiment (n = 3–5). Different superscript letters indicate significant differences between vaccinated and nonvaccinated hatch-mates (P < 0.05, Student’s t-test).

points, no antibodies were detectable in the bursa, and only 20% of the in ovo-vaccinated birds had detectable VN-antibodies in the spleen (Fig. 5). The induction of VN-antibodies in the gut-lavage did not differ significantly between vaccinated groups (P > 0.05; Fig. 5).

3.5. Induction of protection following in ovo and post-hatch vaccination When non-vaccinated birds were challenged, all of them developed clinical signs, such as depression and ruffled feathers. The mortality rate of non-vaccinated

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Fig. 4. Induction of IBDV-VN (a) and ELISA (b) antibodies following post-hatch and in ovo vaccination with IBDV; n = 6-14 (summary of three experiments). *Significantly different between groups (P < 0.05). No IBDV-antibodies were detected in non-vaccinated chickens.

and challenged birds was 0% in Experiment 2, 67% in Experiment 3, and macroscopical lesions, such as bursa gelatination and mottled spleens were seen in these birds. None of the vaccinated birds died after challenge, showed clinical signs, or macroscopical lesions. But in Experiment 2, only one of five in ovo-vaccinated birds was protected against challenge virus-induced histological bursa lesions. Four of five in ovo-vaccinated and challenged birds developed bursa lesions of score 4

(Table 3), although no significant increase of IBDVpositive cells was detected in bursa sections in comparison to in ovo-vaccinated birds without challenge. No significant differences were seen between post-hatch-vaccinated and post-hatch-vaccinated and challenged birds in Experiment 2. In Experiment 3, in ovo- and post-hatch-vaccinated birds were not protected against challenge virus-induced bursa lesions and intrabursal IBDV-replication (Table 3).

Table 2 Induction of VN-IBDV antibodies in bile following in ovo and post-hatch vaccination Groups

Post-hatch Post-hatch In ovo In ovo

IBDV

+ +

Birds with IBDV-specific virus-neutralizing activity in bile at days PV (n = 9–11) (%) 2

3

4–5

7–8

14

17–18

21

0 88* 0 0

0 100* 0 0

0 75 0 29

0 67 0 20

0 30 0 25

0 78* 0 0

0 63 0 38

Fourteen-day-old chickens and 18-day-old embryos were vaccinated with IBDV. At different days PV, bile samples were tested for IBDVantibodies in the VN-test. Summary of three experiments. * Significantly different to the other groups (P < 0.03; x2-test).

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Fig. 5. Induction of IBDV-VN antibodies in bursa of Fabricius, spleen, and gut-lavage (summary of three experiments, n = 6–14). *Significantly different between post-hatch- and in ovo-vaccinated birds (P < 0.05). No IBDV-antibodies were detected in non-vaccinated chickens. aGroup average of VN-antibody titre (log2); dpv = days post-vaccination.

Table 3 Induction of protection against IBDV-IM challenge following in ovo and post-hatch vaccination Groups

Vaccinated

Post-hatch Post-hatch

+ +

In ovo In ovo

Challenged

Group average bursa lesion score  S.D.*

Group average of IBDV-positive cells/ field in the bursa of Fabricius Experiment 2

Experiment 3

+

4.0  0.0 0.0  0.0 1.3  1.3 1.0  1.4 4.0  0.0 0.0  0.0 3.0  1.7 0.0  0.0

126.0  53a 0.0  0.0b 1.5  3.0b 0.4  0.5b 96.0  76.0a 0.0  0.0b 0.1  0.3b 0.1  0.1b

12.5  9.4a 0.0  0.0b 2.9  2.6ab 0.0  0.0b 32.9  17.9a 0.0  0.0b 47.9  3.4a 0.1  0.4b

+ +

+ +

+

14-day-old chickens and 18-day-old embryos were vaccinated with IBDV. At 14 days PV, birds were challenged with IBDV-IM. At 7 (Experiment 2) or 5 (Experiment 3) days post-challenge, birds were sacrificed and histological bursa lesions and IBDV-antigen in the bursa were detected. Different superscript letters indicate significant differences between hatch-mate groups (ANOVA; P < 0.05). * Experiment 2 as a representative experiment.

4. Discussion In the present study, we examined the immunopathogenesis of an intermediate IBDV-vaccine following in ovo and post-hatch inoculation. Our results demonstrate that IBDV-pathogenesis varies significantly between in ovo- and post-hatch-inoculated birds, which may be related to age differences of inoculated birds. The inoculation route may also affect the pathogenesis, but based on previous observations, substances inoculated in ovo are transported from the amnion via the mouth and trachea to the lungs and the

intestine (Sharma et al., 1984; Jochemsen and Jeurissen, 2002), which makes it comparable to the oral or conjunctival route used in the post-hatchvaccinated birds. In all the conducted experiments, bursa lesion development in in ovo-vaccinated birds took place faster, reaching its peak at 4–5 days PI than in posthatch-vaccinated birds with 17 days PV. For the first time, it was demonstrated that in ovo-inoculated birds recovered faster from bursa lesions. Although the bursa to body weight ratio of in ovo-vaccinated birds was lower than from the virus-free controls at

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21 days PV, none of the IBDV-inoculated birds showed any histological bursa lesions. Post-hatchinoculated birds still had in 50% of the birds, significant bursa lesions of scores between 1 and 3 at 3 weeks PV. The faster recovery may be due to less functional T cells in younger birds. As shown in previous studies, T cells have a negative impact on follicular recovery of the bursa (Rautenschlein et al., 2002b). When chickens were depleted of functional T cells, they recovered significantly faster from bursa lesions (Rautenschlein et al., 2002b). In these experiments, we observed a significant accumulation of CD4+ and CD8+ T cells in the bursa of posthatch-vaccinated birds (Tanimura and Sharma, 1997). The accumulated intrabursal T cells, which are activated as demonstrated by Kim et al. (2000), may interfere with recovery of depleted bursa follicles in post-hatch-vaccinated birds. There was no significant increase in CD8+ T cells in bursae of in ovo-vaccinated chicken in comparison to virusfree birds, and CD4+ T cells were only increased in numbers at 21 days PV. These observations differ from Jeurissen et al. (1998), who observed an increase in CD3+ cells following in ovo vaccination. This difference may be due to the more virulent vaccine strain or the different chicken line she used in her experiments. The first line of defence mechanisms may be comparable between in ovo- and post-hatch-vaccinated birds. In both age groups, IBDV induced a transient intrabursal accumulation of macrophage-like cells beginning at 3 days PI, and reaching its peak at 7 days PV (Jeurissen et al., 1998). The macrophage activity may play the major role in clearance of intrabursal IBDV (Rautenschlein et al., 2002b), because both age groups cleared virus infection from the bursa in a comparable manner. Although being similar in intrabursal virus replication, IBDV replicated in extrabursal tissue of in ovo-vaccinated birds more vigorously than in post-hatch-vaccinated birds (P < 0.05). The immaturity of the chickens’ immune system may play an important role in systemic IBDVcontrol not being as efficient in controlling virus distribution as in the post-hatch-vaccinated ones. The more extensive IBDV-replication in the spleen may have been the reason for splenomegaly in in ovoinoculated birds, which was not observed following post-hatch vaccination.

Further indication for immature T cells in in ovovaccinated birds is provided by the lower IgG-IBDVantibody response. Although the VN-antibody levels, which are most important for IBDV-protection (Rautenschlein et al., 2002a), were comparable between groups, the ELISA antibodies differed significantly between age groups. At 21 days PV, not all in ovo-vaccinated birds had developed IBDVELISA antibodies, and they had significantly lower titres in the serum than post-hatch-vaccinated birds. Antibody differences were also seen in the bursa of Fabricius, bile and the spleen where post-hatchvaccinated birds had significantly higher VN-antibody levels than in ovo-inoculated birds. The meaning of this observation for protection against challenge is not clear, but the challenge data indicate that the differences in IBDV-immunopathogenesis between different age groups did not significantly affect protection efficacy. None of the vaccinated groups showed morbidity or mortality in comparison to 100% morbidity and 0–67% mortality in non-vaccinated controls. Both age groups showed comparable challenge virus replication. Interestingly, in ovovaccinated birds developed significant lesions after challenge and post-hatch vaccinated did not. This may possibly be due to the fact that post-hatch-vaccinated birds still had significant bursa lesions at the time of challenge while in ovo-vaccinated birds did not. These bursal lesions at time of challenge may have interfered with lesion development induced be the challenge virus. Similar observations were also made in broilers where vaccine virus-induced lesions affected challenge virus-induced lesion development (Rautenschlein et al., 2004). Overall, this study demonstrated for the first time differences in lesion development and recovery, IBDV-replication, and B- and T-cell stimulation between post-hatch and in ovo IBDV-inoculated chickens. Based on our data, we speculate that the innate immune system may be comparable between the two age groups because both showed accumulation of macrophages in the bursa. Furthermore, both had comparable clearance of IBDV from the bursa, despite differences in T-cell numbers, indicating that nonspecific immunity may be involved in intrabursal viral clearance as speculated before (Rautenschlein et al., 2002b). The lower number of intrabursal T cells in in ovo-inoculated birds may explain the faster bursal

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recovery in the younger birds, because previously it was shown that activated T cells delay follicular recovery in IBDV-inoculated birds. Lack of full T-cell maturity may also explain the significant lower IgGtype serum IBDV-antibody response, and lower local humoral immunity in in ovo-vaccinated birds in comparison to the post-hatch vaccinated (P < 0.05). These observations indicate that application of IBDVvaccines in ovo may be advantageous over later times. Birds may recover faster from lesions, and possibly immunosuppression induced by some vaccine viruses (Gagic et al., 1999; Coletti et al., 2001; Corley and Giambrone, 2002). This adds up to the arguments for in ovo vaccination, such as an early immune response beginning already before the bird encounters a contaminated environment after hatching, stress-free, easy-to-handle method of low labour cost with an evenly distributed vaccine delivery for the whole flock (Sharma and Burmester, 1982; Ricks et al., 1999).

Acknowledgements The authors thank Lieselotte Peiser and Christian Kraemer for their technical assistance, and Sonja Bernhard and Martina Koschorrek for their excellent animal care. Furthermore, we thank Ulrich Neumann and Martin Ryll for critically reviewing the manuscript. This work was supported by the Deutsche Forschungsgemeinschaft (DFG).

References Ahmad, J., Sharma, J.M., 1993. Protection against hemorrhagic enteritis and Newcastle disease in turkeys by embryo vaccination with monovalent and bivalent vaccines. Avian Dis. 37, 485– 491. Chan, M.M., Chen, C.L., Ager, L.L., Cooper, M.D., 1988. Identification of the avian homologues of mammalian CD4 and CD8 antigens. J. Immunol. 140, 2133–2138. Coletti, M., Del Rossi, E., Franciosini, M.P., Passamonti, F., Tacconi, G., Marini, C., 2001. Efficacy and safety of an infectious bursal disease virus intermediate vaccine in ovo. Avian Dis. 45, 1036–1043. Corley, M.M., Giambrone, J.J., Dormitorio, T.V., 2001. Detection of infectious bursal disease vaccine viruses in lymphoid tissues after in ovo vaccination of specific-pathogen-free embryos. Avian Dis. 45, 897–905.

149

Corley, M.M., Giambrone, J.J., 2002. Immunosuppression in specific-pathogen-free broilers administered infectious bursal disease virus vaccines by in ovo route. Avian Dis. 46, 810–815. Gagic, M., St. Hill, C.A., Sharma, J.M., 1999. In ovo vaccination of specific-pathogen-free chickens with vaccines containing multiple agents. Avian Dis. 43, 293–301. Giambrone, J.J., Dormitorio, T., Brown, T., 2001. Safety and efficacy of in ovo administration of infectious bursal disease viral vaccines. Avian Dis. 45, 144–148. Jeurissen, S.H., Claassen, E., Janse, E.M., 1992. Histological and functional differentiation of non-lymphoid cells in the chicken spleen. Immunology 77, 75–80. Jeurissen, S.H., Janse, E.M., Lehrbach, P.R., Haddad, E.E., Avakian, A., Whitfill, C.E., 1998. The working mechanism of an immune complex vaccine that protects chickens against infectious bursal disease. Immunology 95, 494–500. Jochemsen, P., Jeurissen, S.H., 2002. The localization and uptake of in ovo injected soluble and particulate substances in the chicken. Poult. Sci. 81, 1811–1817. Karaca, K., Sharma, J.M., Winslow, B.J., Junker, D.E., Reddy, S., Cochran, M., McMillen, J., 1998. Recombinant fowlpox viruses coexpressing chicken type I IFN and Newcastle disease virus HN and F genes: influence of IFN on protective efficacy and humoral responses of chickens following in ovo or post-hatch administration of recombinant viruses. Vaccine 16, 1496–1503. Kim, I.J., Gagic, M., Sharma, J.M., 1999. Recovery of antibodyproducing ability and lymphocyte repopulation of bursal follicles in chickens exposed to infectious bursal disease virus. Avian Dis. 43, 401–413. Kim, I.J., You, S.K., Kim, H., Yeh, H.Y., Sharma, J.M., 2000. Characteristics of bursal T lymphocytes induced by infectious bursal disease virus. J. Virol. 74, 8884–8892. Lukert, P.D., Saif, Y.M., 2003. Infectious bursal disease. In: Saif, Y.M., Barnes, H.J., Glisson, J.R., Fadly, A.M., McDougald, L.R., Swayne, D.E. (Eds.), Diseases of Poultry. 11th ed. Iowa State University Press, Ames, Iowa, pp. 161–180. Mu¨ ller, R., Weiss, I.K., Reinacher, M., Weiss, E., 1979. Immunofluorescence studies of early virus propagation after oral infection with infectious bursal disease virus (IBDV). Zbl. Veterinaermed. Med. (B) 26, 345–352. Raj, G.D., Jones, R.C., 1996. Local antibody production in the oviduct and gut of hens infected with a variant strain of infectious bronchitis virus. Vet. Immunol. Immunopathol. 53, 147–161. Rautenschlein, S., Suresh, M., Neumann, U., Sharma, J.M., 1998. Comparative pathogenesis of haemorrhagic enteritis virus (HEV) in turkeys and chickens. J. Comp. Pathol. 119, 251–261. Rautenschlein, S., Sharma, J.M., Winslow, B.J., McMillen, J., Junker, D., Cochran, M., 1999. Embryo vaccination of turkeys against Newcastle disease infection with recombinant fowlpox virus constructs containing interferons as adjuvants. Vaccine 18, 426–433. Rautenschlein, S., Yeh, H.Y., Sharma, J.M., 2002a. The role of T cells in protection by an inactivated infectious bursal disease virus vaccine. Vet. Immunol. Immunopathol. 89, 159–167. Rautenschlein, S., Yeh, H.Y., Njenga, M.K., Sharma, J.M., 2002b. Role of intrabursal T cells in infectious bursal disease virus

150

S. Rautenschlein, C. Haase / Veterinary Immunology and Immunopathology 106 (2005) 139–150

(IBDV) infection: T cells promote viral clearance but delay follicular recovery. Arch. Virol. 147, 285–304. Rautenschlein, S., Kraemer, C., Vanmarcke, J., Montiel, E., 2004. Evaluation of IBDV-vaccine efficacy in Broilers: A difficult approach. American Veterinary Medical Association Meeting, AAAP/AVMA Scientific Program, Denver, USA, 19–23 July. Reddy, S.K., Sharma, J.M., Ahmad, J., Reddy, D.N., McMillen, J.K., Cook, S.M., Wild, M.A., Schwartz, R.D., 1996. Protective efficacy of a recombinant herpesvirus of turkeys as an in ovo vaccine against Newcastle and Marek’s diseases in specificpathogen-free chickens. Vaccine 16, 469–477. Ricks, C.A., Avakian, A., Bryan, T., Gildersleeve, R., Haddad, E., Ilich, R., King, S., Murray, L., Phelps, P., Poston, R., Whitfill, C., Williams, C., 1999. In ovo vaccination technology. Adv. Vet. Med. 41, 495–515. Sharma, J.M., Burmester, B.R., 1982. Resistance to Marek’s disease at hatching in chickens vaccinated as embryos with the turkey herpesvirus. Avian Dis. 26, 134–149. Sharma, J.M., Lee, L.F., Wakenell, P.S., 1984. Comparative viral, immunologic, and pathologic responses of chickens inoculated with herpesvirus of turkeys as embryos or at hatch. Am. J. Vet. Res. 45, 1619–1623. Sharma, J.M., Dohms, J.E., Metz, A.L., 1989. Comparative pathogenesis of serotype 1 and variant serotype 1 isolates of infectious bursal disease virus and their effect on humoral and cellular immune competence of specific-pathogen-free chickens. Avian Dis. 33, 112–124.

Sharma, J.M., Kim, I.J., Rautenschlein, S., Yeh, H.Y., 2000. Infectious bursal disease virus of chickens: pathogenesis and immunosuppression. Dev. Comp. Immunol. 24, 223–235. Sharma, J.M., Zhang, Y., Jensen, D., Rautenschlein, S., Yeh, H.Y., 2002. Field trial in commercial broilers with a multivalent in ovo vaccine comprising a mixture of live viral vaccines against Marek’s disease, infectious bursal disease, Newcastle disease, and fowl pox. Avian Dis. 46, 613–622. St. Hill, C.A., Sharma, J.M., 1999. Response of embryonic chicken lymphocytes to in ovo exposure to lymphotropic viruses. Am. J. Vet. Res. 60, 937–941. Tanimura, N., Tsukamoto, K., Nakamura, K., Narita, M., Maeda, M., 1995. Association between pathogenicity of infectious bursal disease virus and viral antigen distribution detected by immunohistochemistry. Avian Dis. 39, 9–20. Tanimura, N., Sharma, J.M., 1997. Appearance of T cells in the bursa of Fabricius and cecal tonsils during the acute phase of infectious bursal disease virus infection in chickens. Avian Dis. 41, 638–645. Winterfield, R.W., Fadly, A.M., Bickford, A., 1972. Infectivity and distribution of infectious bursal disease virus in the chicken. Persistence of the virus and lesions. Avian Dis. 16, 622–632. Zigtermann, G.J.W.J., Vandeven, W., Van Geffen, C., Loeffen, A.H.C., Panhuijzen, J.H.M., Rijke, E.O., Vermeulen, A.N., 1993. Detection of mucosal immune response in chickens after immunization or infection. Vet. Immunol. Immunopathol. 36, 281–291.