Immune Response to a Killed Infectious Bursal Disease Virus Vaccine ...

IMMUNOLOGY, HEALTH, AND DISEASE Immune Response to a Killed Infectious Bursal Disease Virus Vaccine in Inbred Chicken Lines with Different Major Histocompatibility Complex Haplotypes H. R. Juul-Madsen,*1 T. S. Dalgaard,* C. M. Røntved,* K. H. Jensen,* and N. Bumstead† *Danish Institute of Agricultural Sciences, PO Box 50, DK-8830 Tjele, Denmark; and †Institute for Animal Health, Compton RG20 7NN, Berkshire, UK BG, but different on BL, indicated that part of the primary vaccine response was an MHC II restricted T-cell dependent response. The humoral response in another pair of recombinant haplotypes originating in 2 different White Leghorn chickens being BF21, BL21, BG15 (BR4) and BF15, BL15, BG21 (BR5) on the MHC locus indicated that the BG locus may perform an adjuvant effect on the antibody response as well. Vaccination of chickens at different ages and in lines with different origin indicated that age and background genes also influence the specific antibody response against inactivated IBDV vaccine.

ABSTRACT The influence of MHC on antibody responses to killed infectious bursal disease virus (IBDV) vaccine was investigated in several MHC inbred chicken lines. We found a notable MHC haplotype effect on the specific antibody response against IBDV as measured by ELISA. Some MHC haplotypes were high responders (B201, B4, and BR5), whereas other MHC haplotypes were low responders (B19, B12 and BW3). The humoral response of 1 pair of recombinants isolated from a Red Jungle Fowl (BW3 and BW4) being identical on BF and

Key words: infectious bursal disease virus, antibody response, vaccination, major histocompatibility complex 2006 Poultry Science 85:986–998

Shaw and Davison (2000) that protection was still induced in the absence of detectable serum antibodies using a recombinant vaccine containing a VP2 capsid protein of the virus (fpIBD1). This suggests the possibility of a significant role for cell-mediated immunity in protection against IBDV. Results from Sharma and coworkers support this by showing that chickens lacking B-cells are able to clear IBDV and retain immunological memory that is effective against subsequent exposure to the virus (Yeh et al., 2002) and that chickens lacking T-cells do not achieve full protection against virulent IBDV after vaccination with an inactivated IBDV vaccine (Rautenschlein et al., 2002). Furthermore, the inactivated IBDV vaccine induced higher levels of virus neutralizing and ELISA antibodies in birds with a normal complement of T-cells compared with T-cells of depleted birds. Hence, T-helper cells are needed for the production of protective antibodies in vaccinated birds. The MHC genes play an important role in the immune system of all vertebrates as restriction elements presenting antigens to T-lymphocytes and thereby initiating the specific immune response. In chickens, the MHC is called the B-system. The B-system is considered to be the strong transplantation locus. It contains 2 classical class I genes and 2 classical class II genes. In addition, the Bsystem contains genes encoding a third group of highly polymorphic molecules, the BG genes, which have only been found in birds (Guillemot et al., 1989; Kaufman et al., 1989). In the chicken, the MHC is closely associated

INTRODUCTION Infectious bursal disease virus (IBDV) is a member of the Birnaviridae family whose genome consists of 2 segments of double-stranded RNA. The IBDV causes infectious bursal disease (IBD), which is an acute, highly contagious immunosuppressive disease among young chickens (van den Berg et al., 2000; van den Berg, 2000; Lukert and Saif, 2003). The main target cells for viral replication and lysis are actively dividing IgM-bearing Blymphocytes in the bursa of Fabricius (Rodenberg et al., 1994; Sharma et al., 2000). In spite of vaccination programs in the field, IBD continues to cause economic problems largely due to the emergence of more virulent strains (Chen et al., 1998; Ture et al., 1998) causing a higher incidence of acute disease and higher mortality (Nunoya et al., 1992; van den Berg, 2000). Further, the virus-induced immune suppression results in secondary infections, growth retardation, and condemnation of carcasses at slaughter. It is generally accepted that the immunological protection against IBDV is primarily due to a long-lasting hightiter antibody level. However, it was recently shown by

2006 Poultry Science Association Inc. Received October 28, 2005. Accepted January 17, 2006. 1 Corresponding author: [email protected]

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with resistance and susceptibility to a number of diseases. A difference in resistance to viral infections (Longenecker et al., 1977; Gavora and Spencer, 1979; Yoo and Sheldon, 1992; Pinard et al., 1993; White et al., 1994; Bumstead, 1998; Kaufman and Venugopal, 1998), bacterial (Cotter et al., 1992; Siegel et al., 1993; Guillot et al., 1995), protozoal (Lillehoj et al., 1989; Uni et al., 1995; Caron et al., 1997) and autoimmune diseases (Rose, 1994) has been reported. Although no relation between MHC and resistance to IBDV has been observed so far (Nielsen et al., 1998; Hudson et al., 2002), major differences were found between different chicken lines. Bumstead et al. (1993) reported that mortality rates in 11 inbred and partly inbred lines inoculated with a very virulent strain of IBDV varied considerably, being highest in a Brown Leghorn line and lowest in some White Leghorn lines. Nielsen et al. (1998) reported that a meat-type chicken line was more resistant to IBDV infection than a layer-type line, and Hassan et al. (2002) reported major differences in mortality rates in 6 genetically distant chicken lines. It has been suggested that genetic differences in antibody response to inactivated vaccines as well as to recombinant virus proteins exist in chickens (Bacon and Witter, 1994; Pitcovski et al., 2001). Shaw and Davison (2000) suggested that differences in the outcome of an IBDV challenge in VP2 recombinant fowl pox virus (fpIBD 1) vaccinated birds were a result of genetic difference in MHC and in its ability to present VP2-derived peptides to the immune response. In support of that, we have previously shown that protection against IBDV in young chickens vaccinated with attenuated live IBDV vaccine was MHC-linked (Juul-Madsen et al., 2002). Thus, a notable MHC haplotype effect on the specific antibody response against IBDV and a notable recovery from the disease, measured by histological scorings of the bursa, was found. It has been postulated that the depletion of B-cells in the bursa during IBDV infection is due to induction of apoptosis (Tanimura and Sharma, 1998). A recently discovered surface protein on B-cells is the chB6 molecules (former called BU-1) that are expressed on chicken B-cells throughout most of their development and on a subset of macrophages (Houssaint et al., 1987). The avian chB6 alloantigen was shown to trigger apoptosis (Pifer et al., 2002) and may thereby be involved in negative selection of B-cells in the bursa. Normally, less than 5% B-cells leave the bursa and enter the secondary organs (Lassila, 1989). There are currently 3 recognized forms of the chB6 alloantigen (Tregaskes et al., 1996), and genetic association of chB6 alleles and expression of MHC II have been reported (Fredericksen and Gilmour, 1985). In the present study, the immune response to a killed IBDV vaccine in inbred chicken lines with different MHCand chB6 haplotypes was investigated by measuring the specific antibody titer against IBDV up to 37 wk postvaccination in birds vaccinated at 2 different ages. Some of the lines were further analyzed for the total amount of IgG and IgM in serum, the virus neutralization (VN) titer, and the expression and percentage of positive cells for

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the leukocyte markers MHC I, MHC II, CD8, and CD4 at d 0, 5, 7, 11, 14, and 21 postvaccination.

MATERIALS AND METHODS Experimental Design Three experiments were included in the study. Experiment (Exp.) 1 was conducted to determine the specific antibody response to IBDV vaccination in birds with different MHC haplotypes. Ten animals from each of 12 MHC inbred lines were vaccinated intramuscularly with 1 dose (0.5 mL) of killed IBDV vaccine (Nobilis Gumboro INAC:VET 452037, Intervet Danmark A/S, Skovlunde, Denmark) at 8 wk of age. Serum samples were collected at 0, 1, 3, 5, 7, and 16 wk postvaccination (PV), and the serum was assessed for the amount of specific IBDV antibodies. Experiment 2 was conducted to determine the contribution from the MHC and chB6 genes as well as the background genes in 3 lines. In total, 12 to 15 animals of each of the 12 genotypes (MHC × chB6 × line) were vaccinated intramuscularly with the same vaccine as in Exp. 1 at 8 wk of age. Unstabilized blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, and 12 wk PV, and the sera were assessed for the amount of specific IBDV antibodies and VN antibodies. The serum samples from 2 of the 3 lines were further accessed for the concentration of Ig. In addition, citrate-stabilized blood samples were collected from the same 2 lines at 4, 7, 11, 14, and 21 d PV to assess white blood cell (WBC) concentration (cells/ L), the percentage of various surface marker-defined lymphocyte population, and the level of expression of lymphocyte cell-surface markers. Experiment 3 was conducted to determine the contribution from age. In total, 4 to 12 animals of each of 6 genotypes (MHC × line) representing the same 3 lines as in Exp. 2 were vaccinated intramuscularly with the same vaccine as in Exp. 1 at 15 wk of age. Unstabilized blood samples were collected at 0, 1, 2, 3, 4, 5, 6, 7, 8, 14, 24, and 36 wk PV, and the serum was assessed for the amount of specific IBDV antibodies. Control animals in Exp. 2 and 3 were mock-injected with 0.5 mL of PBS.

Birds Birds with the following B haplotypes were included in Exp. 1: B2 originates from Scandinavian White Leghorn (SWL; Simonsen et al., 1982); B4 derives from the Prague CC line (Ha´la, 1987) but was intercrossed with a SWL line containing other MHC haplotypes to maintain the haplotype; B6 derives from the GB2 line and is still kept in the GB2 line (Briles et al., 1982; Ha´la, 1987); B12 originates from the Prague CB line (Ha´la, 1987) but was intercrossed with a SWL line containing other MHC haplotypes to maintain the haplotype; B14 derives from the American H.B.14A line and is still kept in the H.B.14A line (Ha´la, 1987); B15 derives from the Cornell K line and is still kept in the Cornell K line (Cole and Hutt, 1973); B19 derives from the American H-B19 line and is still kept

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in the H-B19 line (Ha´la, 1987). Two additional haplotypes, BW3 and BW4, originate from the Red Jungle Fowl (Bankiva; Crone and Simonsen, 1987) and were backcrossed with a SWL line (6.25% genes remained from the Red Jungle fowl); B201 originates from a single Scandinavian Rhode Island Red bird (Hedemand et al., 1993) and is a B21-like haplotype (Juul-Madsen et al., 2000). The haplotype was backcrossed with a SWL line (12.5% genes remained from Rhode Island Red). Finally, the 2 recombinants BR4 (B21R3) and BR5 (B15R1) originated from White Leghorns from Hy-Line (Briles et al., 1982; Simonsen et al., 1982; Koch et al., 1983; Miller et al., 2004). A table showing the inbred lines at the Danish Institute of Agricultural Sciences is available (http://www. agrsci.dk/afdelinger/forskningsafdelinger/sve/ medarbejdere/hjm). The chickens are hatched and kept as homozygote inbred stocks at the Danish Institute of Agricultural Sciences under normal farm conditions. The chickens are fed with diets that meet NRC requirements. Feed and water are provided ad libitum. Birds from the following 3 lines were included in Exp. 2 and 3: Line 1 consists of 6.25% Red Jungle Fowl, G. Gallus gallus, and 93.75% SWL and segregates for 2 haplotypes: BW1 isolated from the Red Jungle Fowl and characterized as a B21-like haplotype and B19 isolated from SWL. Line 131 consists of 50% White Cornish and 50% SWL and segregates for 2 haplotypes: B131 isolated from White Cornish and characterized as a B21-like haplotype and B19 isolated from SWL. Line 21 is 100% White Leghorn and segregates for 2 haplotypes: B21 derives from H-B21 and B19 derives from the American H-B19 (Ha´la, 1987). The 3 lines (1, 21, 131) are kept under the same conditions in a filtered air positive pressure house (Hedemand et al., 1993; Juul-Madsen et al., 2002). The parent flock of the experimental animals was tested serologically for antibodies against IBDV and found negative.

MHC Typing and chB6 (BU-1) Typing The animals in all experiments were serologically typed. Serological MHC typing was carried out by haemagglutination on open glass plates with specific MHC alloantisera (BF and BG) as described by Juul-Madsen et al. (1993). The animals in Exp. 1 were further MHC typed by the microsatellite LEI 0258 as described by Dalgaard et al. (2005). Typing of animals in Exp. 2 for the alloantigens chB6-a and chB6-b was performed by flow cytometry (see later) 3 wk before the vaccination with the 2 monoclonal antibodies: L22 that recognizes chB6-a and 5-11G2 that recognizes chB6-b (Veromaa et al., 1988). Only a/a and b/b homozygous animals were used.

Enzymes and Reagents Restriction enzymes, Hybond N+ filter membranes, and 32P isotopes (γATP, dCTP) were from Amersham Pharmacia Biotech (Hørsholm, Denmark).

Preparation of DNA and RFLP Analyses Genomic DNA isolation, restriction enzyme digestion, and hybridization were carried out as described previously by Juul-Madsen et al. (1993): DNA (10 ␮g/lane) was digested with Rsa I. After Southern blotting the filter was sequentially hybridized with the BF3, BLX5, and BG3 probes (Kaufman et al., 1989; Juul-Madsen et al., 1993).

Serum Antibody Titers Against IBDV The ProFLOK IBD ELISA Test Kit (Kirkegaard and Perry Laboratories, Gaithersburg, MD) was used to measure serum specific antibody titers against IBDV. The ELISA was performed according to the kit manual. Briefly, 96-well microtiter plates coated with IBDV antigen were incubated with 300-␮L diluted serum samples (1:50) and the positive and negative serum controls included in the kit, followed by incubation with a horseradish peroxidase conjugated affinity purified antibody from a pool of serum from goats immunized with chicken IgG (H+L). 2.2′-Azinodi (3-ethyl benzthiazoline sulfonic acid; ABTS) was used as chromogen and 5% sodium dodecyl sulfate as stop solution. The result was monitored as optical density at 405 nm, and the antibody titer was calculated from the following equation format: SP = (sample absorbance) − (average normal control absorbance)/corrected positive control absorbance. Inter- and intraCV were below 5.6%.

Virus Neutralizing Antibody Titer Determination A serum VN antibody test was performed in chicken embryo (CE) cell cultures as described by Juul-Madsen et al. (2000) on samples from Exp. 2 on 96-well microtiter plates. Briefly, 102.2 tissue culture infectious dose (TCID50) of the IBDV vaccine strain Poulvac Bursine 2* Vet. (Fort Dodge Animal Health, CP Weesp, Holland) was used in each CE-cell culture. The inverse of the highest dilution of each serum that was able to inhibit the growth of IBDV was designated as the VN titer of the chicken serum. In each test, dilutions of a positive and a negative control serum against IBDV were included in the range of 1:20 to 1:1,280. Furthermore, controls of CE-cells were incubated with plain medium and serum from each chicken (lowest dilution), and the IBDV inoculum was titrated on the CEcells to confirm the VN titer. All samples and controls were run as duplicates.

Detection of Chicken IgM and IgG in Serum Quantitative ELISA kits for measurement of chicken IgM and IgG from Bethyl Laboratories (Montgomery, TX) were used to measure the concentration of immunoglobulins in serum samples from Exp 2. The ELISA was performed according to the kit manual with a few exceptions. Briefly, 96-well microtiter plates were coated with the capture antibody (anti-chicken IgM or IgG) followed by


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Figure 1. The time course of the specific antibody response against killed infectious bursal disease virus vaccine measured by ELISA on the indicated weeks postvaccination (PV). Ten animals from each of 12 MHC inbred chicken lines were vaccinated at 8 wk of age, and serum samples were measured for specific antibodies from wk 0 to 16 PV.

incubation with serum samples and standards (IgM 15.6 to 250 ng/mL; IgG 12.5 to 200 ng/mL). The concentrations of IgM and IgG were detected by incubation with HRPconjugated goat anti-chicken IgM or IgG antibody. Then TMP (3,3′,5,5′-tetramethyl benzidine) was used as chromogen and 2 M H2SO4 as stop solution. The result was monitored as optical density at 405 nm. The IgM and IgG concentrations were calculated from a standard curve according to the equation suggested by the company.

Number of White Blood Cells (WBC) The total number of leukocytes in the blood were assessed by haemacytometry. Approximately 100 ␮L of citrate-stabilized blood was analyzed in a Cell-Dyn 3500 haemacytometer (Abbott Laboratories, Abbott Park, IL) using a specialized configuration for chicken blood. The apparatus was standardized daily using Cell-Dyn22 controls. The leukocytes were measured as cells × 109/L.

were obtained from the Southern Biotechnology Association Inc. (Birmingham, AL). For the unlabeled antibodies, F21-21 and 2G11, a secondary FITC-labeled antibody was used (Goat F(ab’)2 Fragment antimouse IgG (H + L) from Beckman Coulter (Fullerton, CA). The samples were analyzed on a Coulter Epics XL flow cytometer with a 488nm argon laser. A total of 10,000 mononuclear cells were counted in each sample. Analytic gates were chosen based on forward and side scatter to include small mononuclear cell population (lymphocytes and thrombocytes) and to exclude debris, dead cells, erythrocytes, and large mononuclear cells (monocytes). Contaminating thrombocytes in the leukocyte preparation were not measured. The expression of surface markers was expressed as mean fluorescence intensity. Flow cytometer alignment verification was performed using Flow-Check Fluorospheres (Beckman Coulter), and day-to-day standardization of the flow cytometer was performed using Uniform Dyed Microspheres (0.96 ␮m) from Bangs Laboratories Inc. (Fishers, IN).

Flow Cytometric Analysis The isolation of leukocytes and the flow cytometric analysis in Exp. 2 were performed as described by JuulMadsen et al. (2002). In short mononuclear cells were isolated using Lymphoprep 1.077. The primary antibodies were: anti-chicken β2-microglobulin (F21-21); antichicken BLβ (2G-11); anti-chicken CD4 (RPE-conjugated CT4); anti-chicken CD8α (FITC-conjugated CT8). The hybridoma F21-21 and 2G-11 were kindly donated by K. Skjoedt, Odense, Denmark. The antibodies CT4 and CT8

Statistical Analyses All variables were subjected to ANOVA using the maximum likelihood method in the mixed model with multiple error terms in the statistical package, SAS PROC MIXED (SAS Institute, 1996). If necessary to obtain homogenous variance, the variables were transformed by the logarithmic or the square root transformation depending on the data distribution. Individual measurements, averages or sums, were used as experimental

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units. Only data from vaccinated animals were included in the analyses except in the analysis aimed at comparing measurements from nonvaccinated birds with those from vaccinated birds. Data from Exp. 1 were analyzed by a model including line, week PV, and the interaction between these effects if significant as general fixed effects. In addition, antibody titer before vaccination was included as covariate. Week PV was treated as repeated measurements using an unstructured covariance structure. Data from Exp. 2 (IBDV, IgG, and IgM) were analyzed by a model that included line, MHC haplotype (B19 vs. B21-like), chB6 genotype (a/a vs. b/b), week PV, and all significant 2-, 3- and 4-way interactions between these effects as general fixed effects. The antibody titer and the Ig concentration before vaccination were included as covariates. In case of IBDV antibodies, the interaction between initial antibody titer and week PV also was included as covariate. Week PV were treated as repeated measurements using an unstructured covariance structure. In addition, IgM, IgG, and cellular responses in vaccinated birds were compared with nonvaccinated control birds in a comparable model including vaccination (vaccination vs. placebo). The sum of total IgG and the sum of IgM concentrations from wk 0 to 12, and the average cellular responses from d 0 to 21, were analyzed by a model including line, MHC haplotype (B19 vs. B21-like), chB6 genotype (a/a vs. b/b), and all significant 2-, 3-, and 4-way interactions between these effects as general fixed effects. Data from Exp. 3 were analyzed by a model that included line, MHC haplotype (B19 vs. B21-like), week PV, and all significant 2- and 3-way interactions between these effects as general fixed effects. Because of lack of individual identification of birds, treatment of week PV as repeated measurement and IBDV antibody titers before vaccination as covariate were omitted. Satterthwaite’s approximation was used in all models due to unbalanced data material as a result of lack of observations. Results of the analysis with the mixed procedure are given in F-values and the degree of freedom for the investigated effect (df 1) and of the error term in the denominator (df 2), and the P-value. Pairwise comparisons belonging to effects are presented by the P-value. All the means are raw means supplied with standard error. All analyses were performed as 2-tailed tests.

RESULTS IBDV Antibody Response to Vaccination in MHC Inbred Chicken Lines To determine whether there are differences in antibody response to vaccination against IBDV in our experimental MHC lines originating from different sources (Exp. 1), the specific IBDV IgG antibody titers were measured in 12 chicken lines as shown in Figure 1. The specific antibody response rose from 1 wk of age and peaked at 5 to 7 wk

Figure 2. The time course of the specific antibody response against inactivated infectious bursal disease virus (IBDV) vaccine measured by ELISA on the indicated weeks postvaccination (PV). The animals were 8 wk of age when vaccinated. A) The pair of recombinants BW3 and BW4. B) The pair of recombinants BR4 (F21-L21-G15) and BR5 (F15L15-G21). These 4 haplotypes are also shown in Figure 1; however, standard deviation is shown here as bars.

PV. Major differences were observed between the inbred lines. The lines containing the B21-like haplotype B201, the recombinant haplotype BR5, and the B4 haplotype showed the highest titers of approximately 7,000 compared with the lines containing the haplotypes B19 and B12 that showed the lowest titer of approximately 4,000. The titers did not decrease from 7 to 16 wk PV in any of the lines tested. There was a significant interaction between line and week (F44,107 = 2.99; P < 0.0001) reflecting the just mentioned time course as well as consistent significant differences over time between lines. Among others, significant differences between 2 haplotypes pairs were found (Figure 2). The 2 B haplotypes, BW3 and BW4, originated from a heterozygous Red Jungle Fowl (Bankiva) cock provided by the Copenhagen Zoo. Mixed lymphocyte reaction clearly distinguished these haplotypes, whereas they were indistinguishable by serology using BF and BG specific allo-antisera (Crone and Simonsen, 1987), assuming that they have identical BF and BG genes but different BL genes. The RFLP— using several restriction enzymes—confirmed that they were identical on BF and BG but different on BL. Figure 3 shows one of the digests—Rsa I. The * indicate the bands that differ between the 2 haplotypes. This MHC haplotype pair was found to differ in antibody response from 3 to 16 wk PV (P ≤ 0.0068; Figure 2a). The BR4 and BR5 pair of recombinants was found in the early 1980s in offspring from matings between heterozygous B15/B21 animals (Koch et al., 1983). In total 4,456 individual chickens were


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Figure 3. Southern blot hybridization of DNA from 7 animals with the haplotype as indicated above the x-ray of the blot. Ten micrograms of DNA/lane was digested with the restriction enzyme Rsa I and hybridized consecutively with the BF3, BLX5, and the BG3 probe as indicated below the x-ray of the blot.

MHC-typed, and only 4 recombinants were found indicating very low crossover frequency in the MHC region. Two of the recombinants originating from 2 different animals were assigned BR4 and BR5 (Briles et al., 1982). BR4 was typed to be BF21, BL21, and BG15 by serology, flow cytometry, and mixed lymphocyte reaction, whereas BR5 was typed to be BF15, BL15, and BG21 (Koch et al., 1983). Analysis of the LEI0258 microsatellite located in the BF/ BL region next to the single BG gene in that region (Zoorob et al., 1998) reveals a fragment that was identical in size with B21 (357bp) in both recombinants (data not shown), indicating that the recombination events in the original animals must have occurred on each side of the LEI258 microsatellite or be a reciprocal translocation of a segment in the nearby region of BF/BL. The antibody response differs from 3 to 7 wk PV (P ≤ 0.0217) in this haplotype pair (Figure 2b). In Exp. 2, the influence of background genes was investigated in a system where contribution from both MHC and nonMHC genes could be investigated. This system contains 3 lines of chickens with 4 different B haplotypes (Juul-Madsen et al., 2002). Two haplotypes were present in each line. One haplotype (B19) was common between the lines (see Materials and Methods). Therefore, it was possible to fix the MHC and look at the contributing effect

of the background genes. The B19 from line 1 and 131 originates from the same SWL population (Simonsen et al., 1982), whereas B19 from line 21 originates from the American H-B19 line (Ha´la, 1987). The IBDV specific IgG antibody response of the 6 genotypes (Figure 4a) had the same developmental profiles as in Exp. 1. The antibody titer started to rise between wk 1 and 2 PV and peaked 5 to 7 wk PV. Birds containing B21 or B21-like haplotypes (BW1 and B131) have the highest titers with approximately 7,000, and birds containing the B19 haplotype have the lowest titers with 4,000 to 5,000. A significant difference between B21/B21-like haplotypes and their respective B19 haplotypes within each line was found (interaction between week, line, and MHC haplotype: F16,171 = 3.59; P ≤ 0.0001) 2 to 8 wk PV. The difference persisted in lines 1 and 21 at week 12 PV (P < 0.0188), whereas the MHC haplotypes in line 131 at 12 wk PV did not differ (P = 0.2448). Analysis of the effect of genotype showed a significant interaction between genotype and week PV (F40,171 = 12.24, P < 0.0001). The L131 genotypes containing 50% meat-type genes were significantly lower that the other 4 genotypes of layer-type origin in several weeks. L131-B131 differs from L1-BW1 at 7 and 8 wk PV (P ≤ 0.0324) and from L21-B21 at 8 wk PV (P = 0.0431). L131B19 differs from the L1-B19 and L21-B19 from 6 to 8 wk

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Figure 4. The time course of the specific antibody response against killed infectious bursal disease virus vaccine measured by ELISA on the indicated weeks postvaccination (PV). Three lines with 4 haplotypes are shown. Line 1 contains the B21-like haplotype BW1 and B19, line 21 contains B21 and B19, and line 131 contains the B21-like haplotype B131 and B19. A) Twenty-four to 30 animals from each of the 6 genetic groups were vaccinated at 8 wk of age, and serum samples were measured for specific antibodies from wk 0 to 12 PV. B) Four to 12 animals from each of the 6 genetic groups were vaccinated at 15 wk of age, and serum samples were measured for specific antibodies from wk 0 to 36 PV. Standard deviation is shown as bars.

PV (P ≤ 0.0384). Finally, L21-B19 had a significantly higher titer than L1-B19 at 3 wk PV (P = 0.0009) and L131-B19 at 3 and 4 wk PV (P ≤ 0.0148). To study the influence of the B-cell marker chB6 on the vaccination response, the animals from Exp. 2 were further divided into 2 different chB6 genotypes a/a and b/b within each MHC genotype. This had a small but significant influence on the development of the antibodies (interaction between chB6 genotype and week F8,171 = 2.04; P = 0.0446). At 3 and 4 wk, PV b/b tended to be lower than a/a (P ≤ 0.0924). In addition, the chB6 genotype and line interacted (F2,173 = 3.51, P = 0.0320). The L131 b/b

genotype was significant lower than L1 and L21 genotypes (P < 0.0356). To investigate the effect of age on the IBDV vaccination (Exp. 3), new animals from the same 3 lines as in Exp. 2 were vaccinated at 15 wk of age and tested for IBDVspecific IgG antibodies (Figure 4b). The results show the same profiles as in Exp. 2 but with a higher overall antibody titer in all genotypes. The results were, however, not as clear as in Exp. 2 (Figure 4a). The statistical analyses showed an overall difference between genotypes (interaction between line and MHC haplotype: F2,573 = 32.87, P < 0.0001). The L21-B21 had a significantly higher IBDV


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Figure 5. The time course of the concentration of IgG and IgM in infectious bursal disease virus vaccinated birds in the indicated weeks postvaccination (PV). The birds were 8 wk of age when vaccinated. A) The concentration of IgM in 24 to 30 animals from each of the indicated 4 genetic groups from wk 0 to 12 PV. B) The concentration of IgG in 24 to 30 animals from each of the indicated 4 genetic groups from wk 0 to 12 PV. Standard deviations are shown as bars.

antibody titer than other genotypes (P < 0.0001), followed by L1-BW1 (P < 0.0001). In addition L1-B19 significantly differed from other genotypes (P < 0.0001), whereas L131B131, L131-B19, and L21-B19 did not differ from each other (P > 0.05). The response profiles could be explained by MHC haplotype (interaction between MHC haplotype and week PV: F11,573 = 7.61, P < 0.0001) as well as line (interaction between line and week PV: F22,573 = 1.87, P < 0.0094). In general, B21 and B21-like haplotypes responded more than B19 haplotypes, and within haplotypes the response decreased from L21 to L1 to L131. Serum samples from L1 and L131 in Exp. 2 were analyzed for the concentration of IgG and IgM in serum

(Figure 5). An increase in the concentration of both Ig from 2 wk until 12 wk PV was observed—most pronounced in L131 (interaction between line and week; IgG F4,89 = 4.47, P = 0.0024; IgM: F4,89 = 5.83, P = 0.0003). In L1 the IgG and IgM antibody concentrations increased significantly from wk 2 to 4, and from wk 8 to 12 IgM increased and IgG decreased (P ≤ 0.01). In L131 the IgG antibody concentration increased significantly between consecutive sampling points except from wk 4 to 6 (P ≤ 0.0006), whereas the IgM antibody concentration increased at all times (P < 0.0001). The increase was probably not a result of the vaccination but more an age effect because the unvaccinated birds of different genotypes had the same

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Figure 6. The virus-neutralizing (VN) antibody titer in 24 to 30 animals from each of the 6 indicated genetic groups performed on serum samples 7 wk postvaccination. The animals were vaccinated at 8 wk of age. The VN antibody test was performed in chicken embryo cells, and the values were log transformed for statistical evaluation. Standard deviations are shown as bars. No significant difference was observed (P > 0.05).

profiles (data not shown). For both IgM and IgG the effect could not be ascribed to the MHC haplotype, the chB6 haplotypes, or the line alone. However, the integrated levels of IgG antibodies and IgM antibodies over time were affected by an interaction between MHC haplotype and line (IgG: F1,69 = 7.26, P = 0.0088; IgM: F1,78 = 7.78, P < 0.0066). For IgM, L131-B19 was significantly different from L131-B131, L1-BW1, and L1-B19 (P ≤ 0.0038). Furthermore, L131-B131 was significantly different from L1B19 (P = 0.0267). The ranking order of the 4 genotypes was L131-B19 > L131-B131 > L1-BW1 > L1-B19. For IgG, L131-B19 was significantly higher than L131-B131 and L1-B19 (P ≤ 0.0345). Finally, serum samples from the 3 lines—investigated in Exp. 2—were assessed for their ability to neutralize IBDV at 7 wk PV. The results of each genetic group are shown in Figure 6. Lower titer was found in L131 when compared with the other 2 lines, but due to a large variation within each group the difference was not found to be significant (P = 0.3908), nor were differences between the 2 haplotypes within each line. Nonvaccinated birds had no VN antibodies (data not shown).

Blood Lymphocyte Profiles Following Vaccination in MHC Inbred Chicken Lines To investigate the change in lymphocyte population profiles in response to vaccination 4 of the 6 genotypes— used in Exp. 2—were further subjected to flow cytometric analysis (L1-BW1, L1-B19, L131-B131, L131-B19). The number of WBC and the surface expression of MHC I, MHC II, CD4, and CD8 including the percentage of positive cells were measured at 0, 5, 7, 11, 14, and 21 d PV. No response due to the vaccination was observed during the sampling time because the flow cytometric profiles of the vaccinated birds did not differ significantly from the profiles obtained from the control birds (P > 0.05).

The results from the 6 sampling days from each individual were therefore pooled and analyzed in relation to the immunophenotypes only (Figure 7). The expression of the surface markers analyzed was not influenced by the chB6 genotype except for the percentage of MHC II positive cells (interaction between chB6 genotype, MHC haplotype and line: F1,48 = 9.22, P = 0.0039). The ChB6 a/a had a lower percentage of MHC II positive cells in L131B131 (P < 0.0001). For the WBC, the percentage of MHC II positive cells, the percentage of CD8 single positive cells, the expression of CD4 on CD4 single positive cells, and the percentage of CD4 single positive cells, the observed difference could not be ascribed to the MHC haplotype or the line. In all cases—except for the percentage of MHC II positive cells—L1-B19 was lower than the other genotypes. For the expression of MHC I (F1,52 = 422,61, P < 0.0001) and MHC II (F1,52 = 7.6, P = 0.0080), the difference could be ascribed to the MHC haplotypes being a B19 haplotype or a B21/B21-like haplotype. For the expression of MHC I (F1,52 = 7.26, P = 0.0095), expression of CD8 on CD8 single positive cells (F1,52 = 8.37, P = 0.0056), the expression of CD8 on CD4CD8 double positive cells (F1,52 = 8.86, P = 0.0044), and the expression of CD4 on CD4CD8 double positive cells (F1,52 = 6.20, P = 0.016), the difference could also be ascribed to the line.

DISCUSSION This study aimed at defining genetic differences in inbred chicken lines with different MHC haplotypes in response to vaccination against IBDV. The study focused on the role of MHC in this process using a killed virus vaccine for IBDV as the model system. Twelve MHC inbred chicken lines with different background genes were assessed for the amount of specific IBDV antibodies. Enormous differences between the lines were found indicating that the MHC was a major determinant of the primary IBDV humoral vaccination response (Figure 1). In short, the B21-like haplotype B201, B4, and BR5 were high responders, and the haplotypes B19, B12, and BW3 were low responders. The Red Jungle Fowl haplotypes BW3 and BW4 that differ on BL only showed different levels of specific antibodies against IBDV vaccination (Figure 2a) 3 to 16 wk PV, indicating that part of the primary vaccine response was an MHC II-restricted T-cell-dependent response since the lines containing these 2 haplotypes have identical background genes. The expected antibody response in the 2 recombinants BR4 and BR5 was that, based on their BF/BL genotypes, BR4 would follow B21 and B21-like haplotypes whereas BR5 would follow the B15 haplotype. That was not the case (Figure 2b). BR5 had an antibody response very similar to B21, and BR4 had an antibody response very similar to B15. The 2 recombinants differ from 2 to 7 wk PV. Interestingly, this supports the idea that BG genes may have an adjuvant effect in the antibody response as previous suggested by Kaufman and Salomonsen (1992) as BR5 is BG21 and BR4 is BG15. After 20 yr of investigation the exact function of BG is still a mystery, and no mamma-


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Figure 7. The mean percentage of positive cells within the small mononuclear cell population as well as the expression of the surface marker for MHC I, MHC II, CD4 single positive cells, CD8 single positive cells, and CD4CD8 double positive cells as indicated above each diagram in IBDV-vaccinated birds. The birds were 8 wk of age when vaccinated. Twenty-four to 30 animals from each of the 4 indicated MHC genetic groups are shown in each diagram except for the percentage of MHC II positive cells in which the chB6 genotype (a/a or b/b) is also shown. Six measurements (d 0, 5, 7, 11, 14, and 21) from each animal were pooled. The means without a common letter are statistically different (P < 0.05). WBC = white blood cells; MFI = mean fluorescence intensity.

lian counterparts have been isolated. It would be interesting to further investigate the BG genes in these 2 recombinants in relation to the proposed adjuvant effect. The influence of the background genes was analyzed in Exp. 2 (Figure 4a). Within each line, animals with the B21 or the B21-like haplotypes had a significantly higher antibody titer than animals with the B19 haplotype, indicating a major influence of the MHC haplotype on the primary IBDV antibody response. B21 has also been found to be a superior antibody responder to other antigens such as sheep red blood cells (SRBC; Dunnington

et al., 1989; Parmentier et al., 2004). The statistical analysis of the specific IBDV antibody titer, however, also reveals an interaction between the MHC and line. An interaction between MHC and background genes has also been shown for SRBC by Siegel and coworkers (Dunnington et al., 1989). The antibody titer in L131-B19 was significantly lower than in L21-B19 and L1-B19 from wk 6 to 8 PV, and this was in spite of a higher concentration of IgM (Figure 5a) in L131-B19 from wk 0 to 12 PV. L131-B19 consists of 50% meat-type genes, and it has been reported before that chickens with a higher body weight have a

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lower SRBC primary antibody response (Miller et al., 1992) and that broiler-type chickens generate a higher IgM antiTNP titer but a lower IgG antiTNP titer than layer-type chickens (Koenen et al., 2002). In Exp. 3 the data suffer from a low number of animals in some of the genetic groups, but the same ranking of genetic groups was found; animals containing the B21 or the B21-like haplotypes had a much higher antibody titer than animals from the same line containing the B19 haplotype. The specific antibody titer was just higher, peaking at 12,000, indicating that age influences the antibody response against vaccine IBDV, too. An effect of background genes was observed as well. The VN antibody test performed on sera collected 7 wk PV did not reflect the specific antibody titer measured by ELISA. No significant difference in the VN titer was found between the 2 MHC haplotypes within each line (Figure 6), but a lower level in L131 consisting of 50% meat-type genes was found even though that was not statistically different. This may indicate that birds of layer origin have a higher amount of VN antibodies against IBDV than birds of broiler-type origin. Line difference was also found in another experiment in which the same 3 lines were first vaccinated with live attenuated IBDV and then later challenged with IBDV (Juul-Madsen et al., 2002). However, there was a discrepancy between the results. In Juul-Madsen et al. (2002) the ranking of the lines was L1 > L131 > L21. This may be due to the fact that in the Juul-Madsen et al. (2002) experiment a live attenuated vaccine strain was used followed by a challenge with live IBDV and therefore showed a secondary antibody response. In this study, only the primary antibody response to a killed vaccine was measured. In conclusion, no statistical correlation was found between specific ELISA antibodies and VN antibodies, but a tendency between lines was seen. No cellular response differences as a result of the vaccination were detected from d 0 to 21 PV. However, immunophenotypic differences between the genotypes were present (Figure 7). The expression of MHC I was ascribed to the MHC haplotype as previously suggested (Kaufman et al., 1995; Juul-Madsen et al., 2000), B21 and B21-like haplotypes having low expression and B19 having very high expression. The expression of MHC II could also be ascribed to the MHC haplotype. In this case, the expression was the inverse of the MHC I expression—B21 and B21-like haplotypes having a high expression and B19 a low expression. The difference in these 2 parameters correlates with the difference in antibody response and may therefore have influenced the specific IBDV antibody response even though no difference during sampling time was observed. The 6 genotypes in Exp. 2 were further divided into the chB6-a type and the chB6-b type (Veromaa et al., 1988), but no major general effects of this marker were observed for the specific antibody response or the expression of MHC II. For the percentage of MHC II positive cells (mainly B-cells), a significant interaction between chB6 genotype and MHC haplotype was found

because of differences in the chB6 genotypes in L131B131 only. It was not possible to make any conclusions about the involvement of chB6 in the T-cell response against the IBDV vaccine in this study. In conclusion, these results suggest that the MHC haplotype plays a major role in the T-helper cell-mediated production of specific antibodies in birds vaccinated with a killed IBDV vaccine and that B21 or B21-like haplotypes seem to be superior to other haplotypes in antibody response to the vaccine.

ACKNOWLEDGMENTS This work was supported by a grant from the EU FAIR 3 program (FAIR3 PL96-1502) and a special Danish Institute of Agricultural Sciences effort: Housing and Management systems in relation to the cessation of antibiotic growth-promoting drugs. The authors wish to thank K. Skjødt (University of Southern Denmark, Odense, Denmark) for the antibodies F21-21 and the 2G-11, and O. Vainio (Turku University, Turku, Finland) for the antibodies 2-6 and 11-39. Furthermore, the authors wish to thank M. M. Miller and J. Kaufman for valuable discussions, K. V. Østergaard for critical reading of the manuscript, and L. R. Dal, L. Nielsen, H. Svenstrup, and D. Thomassen for technical assistance. The experimental procedures were conducted under the protocols approved by the Danish Animal Experiments Inspectorate and complied with the Danish Ministry of Justice Law no. 382 (10 June 1987) and Acts 739 (6 December 1988) and 333 (19 May 1990) concerning animal experimentation and care of experimental animals.

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