Klinik für Geflügel, Bünteweg 17 D Hannover Germany. *Corresponding Author: Silke Rautenschlein. Tel.: Fax:

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1 A COMPARISON OF THE EFFECTS ON THE HUMORAL AND CELL-MEDIATED IMMUNITY BETWEEN AN HVT-IBD VECTOR VACCINE AND AN IBDV- IMMUNE COMPLEX VACCINE AFTER IN OVO VACCINATION OF COMMERCIAL BROILERS Silke Rautenschlein 1 *, Stéphane Lemiere 2 Birgid Simon 3 and Francesco Prandini 2 1 Klinik für Geflügel, Bünteweg 17 D Hannover Germany 2 Merial S.A.S., 29 avenue Tony Garnier F Lyon cedex 07 France 3 Merial GmbH., Am Soeldnermoos 6, Hallbergmoos Germany *Corresponding Author: Silke Rautenschlein Tel.: Fax: Silke.Rautenschlein@tiho-hannover.de Resumen Las vacunas vectorizadas contra el virus de la enfermedad infecciosa de la bolsa (IBDV, por sus siglas en ingles) basadas en el herpes virus recombinante de pavos (HVT- IBD, por sus siglas en inglés) y las vacunas de complejo inmune-ibdv (Icx-IBD) han sido autorizadas para usarse aún en la presencia de anticuerpos maternos (mab, por sus siglas en inglés). Comparamos los efectos de una vacuna recombinante HVT- IBD y una Icx-IBD sobre la inmunidad humoral y la inmunidad mediada por células en pollos de engorda comerciales. Después de una vacunación in ovo los pollos de engorda se inocularon con una vacuna de virus de enfermedad de Newcastle inactivado a los días postnacimiento, cuando se alcance el título de rompimiento de los niveles de mab de IBDV, o a los días post-nacimiento. Se determinaron los efectos diferentes de las dos vacunas sobre los niveles de anticuerpos de IBDV y NDV, sobre los números de células B y T intrabursales y circulantes y sobre la respuesta mitogénica de los leucocitos en la sangre periférica. Ninguna de las vacunas afectó la respuesta mitogénica de las células T periféricas. Icx- IBD redujo el número de células B circulantes en comparación con el grupo vacunado con HVT-IBD y el grupo control no inoculado. Introduction Infectious bursal disease virus (IBDV), a member of the Birnaviridae family, cause an acute, highly contagious and immunosuppressive disease in young chickens (Eterradossi & Saif, 2008). The main target cells for IBDV replication are dividing B lymphocytes; thus infection leads to the destruction of lymphoid cells in the bursa of Fabricius and, to a lesser degree, in other lymphoid organs. The acute phase lasts one to two weeks and is often accompanied by variable mortality. In birds that survive and control IBDV infection depleted bursal follicles become repopulated with B cells and immune competence may return to normal levels after some time (Kim et al., 1999; Withers et al., 2006; Withers et al., 2005). Replication of IBDV in the bursa is accompanied by an influx of activated T cells that control viral replication but also delay recovery of infected animals (Kim et al., 2000; Rautenschlein et al., 2002; Tanimura & Sharma, 1997). Systemically T cell immunity 810

2 may be also compromised as indicated by a suppressed mitogenic response, which is mediated by soluble factors released by IBDV-activated macrophages (Kim et al., 1998). A variety of vaccines are commercially or experimentally available to protect chickens against IBDV (Haygreen et al., 2006; Hsieh et al., 2010; Ivan et al., 2005; Lemiere et al., 2011; Omar et al., 2006; Perozo et al., 2009; Rautenschlein et al., 2005; Wang et al., 2007). Attenuated IBDlive vaccines and IBD-immune complex (Icx) vaccines may have the disadvantage of transient destruction of bursa follicles and residual immunosuppressive abilities (Corley & Giambrone, 2002; Rautenschlein et al., 2005). Furthermore, maternal antibodies may interfere with the replication of the IBDV vaccine strain delaying the onset of protection (Block et al., 2007). IBD-immune complex vaccines are designed to by-pass the neutralizing activity of maternal antibodies (Ivan et al., 2005; Jeurissen et al., 1998). In maternal antibody negative birds IBD-Icx vaccines were also shown to have residual immunosuppressive abilities (Corley & Giambrone, 2002). New generation vaccines such as recombinant herpes virus of turkeys (HVT) expressing immunodominant IBDV peptides such as VP2, which induces IBDneutralizing antibodies, circumvent the problem of interference with IBD-maternal antibodies at the time of vaccination (Bublot et al., 2007; Le Gros et al., 2009; Lemiere et al., 2011; Perozo et al., 2009; von Bulow & Klasen, 1983). It is also speculated that this HVT does not induce significant immunosuppression (Islam et al., 2002; Reddy et al., 1996), but this aspect has not been investigated for HVT expression vectors in combination with inserts of different viruses. Our objective was to compare under experimental conditions the effects of an HVT-IBD vector vaccine with a live IBD immune complex vaccine (Icx-IBD) on the immune system and the induction of immune responses in commercial broilers. Materials and Methods Eggs/animals 400 Ross 308-type broiler eggs were obtained from a commercial ery (BWE-Brüterei Weser-Ems, Visbek, Germany). Eggs were incubated and ed at the clinic for poultry. Broiler chicks were provided with feed and water ad libitum. They were raised based on the German welfare regulations on litter in different isolation rooms at the Clinic for Poultry. IBDV vaccines A commercially available recombinant HVT- IBD vaccine was used (Le Gros et al., 2009) and compared with a commercially available immune complex vaccine (Icx-IBD). Both vaccines were handled based on the instructions of the manufacturers. Experimental plan Groups of broiler eggs were incubated and in ovo vaccinated with one dose of the HVT- IBD or the Icx-IBD vaccine at 18 days of embryonation. One group of broiler eggs received diluent by the in ovo route. After birds were placed in different isolated units. At or days post, part of each group (n = 21-24) received a commercially available ND-inactivated vaccine by the intramuscular route. Serum samples were collected at 1, 5, 7, 14, 21, 28, 35 days ph for detection of IBDV and NDV antibodies. At,, 21, 28, and 35 days post, 5-7 birds per group were sacrificed and the following parameters investigated: pathological lesions, bursa to body weight ratio, bursa histopathology, flow cytometric analysis and mitogenic response of peripheral blood leukocytes, detection of CD3+ cell in the bursa of Fabricius by immunohistochemistry, and intrabursal detection of IBDV by quantitative RT-PCR. 811

3 Quantitative real-time RT-PCR The qrt-pcr was conducted following previously published procedures using the qrt-pcr Brilliant II Master Mix Kit (Stratagene, Agilent, Waldbronn, Germany; (Eldaghayes et al., 2006). Flow cytometric analysis Three ml of blood were collected per bird in Alsever solution, and leukocytes were isolated using Biocoll (Biochrom AG, Berlin). 106 cells were stained with antibodies against CD4, CD8, CD3, and Bu-1 labelled with FITC, Biotin or unlabelled, PE, and FITC, respectively (Southern Biotech. Birmingham, Alabama, USA,). Antibodies were used at dilutions of 1:100, 1:100, 1:50, 1:200, respectively. The CD8-Bio-stained cells were further incubated with Streptavidin (Sigma; 1:50) or a polyclonal anti-mouse-fitc antibody (Sigma; 1:200) cells/sample were analysed with an EPICS-XL flowcytometer (Beckmann Coulter, Galway / Ireland), and CXP analysis software (Beckmann-Coulter) was used for data processing. An electronic gate was set by forward scatter (FS) and sideward scatter (SS) characteristics to exclude cell debris and erythrocytes. Stained lymphocyte subpopulations were discriminated by increased fluorescence signal. The percentage of lymphocyte subpopulations within gated cells was calculated. Detection of serum antibodies against NDV and IBDV For the detection of ELISA antibodies the following kits from Synbiotics Corporation, San Diego, CA were used: IBD ProFLOKplus, ND ProFLOKplus. Histology and immunohistochemistry For the detection of histopathological lesions, the bursa of Fabricius was collected, fixed in 10 % phosphate-buffered formalin and stained with hematoxilin 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 = %, 3 = %, 4 = % of follicles showing cellular depletion. Monoclonal antibodies against chicken CD3+T cells were purchased from Southern Biotechnology Associates (Southern Biotech, Birmingham, AL, USA) and used at a dilution of 1:200. For immunohistochemical staining of cell surface determinants bursae of Fabricius were collected, snap frozen, sectioned and processed for immunohistochemical staining as described (Tanimura & Sharma, 1997). The group means of the numbers CD3+ T cells per field at 400 x magnification were determined after counting 5 fields/tissue/bird. Mitogenic assay The mitogenic assays were conducted with peripheral blood leukocytes following standard procedures. Briefly, after isolation of leukocytes as indicated for the flow cytometric analysis, 7.5 x 10 6 cells/ml were cultured in medium or in addition stimulated with 10 µg/ml of the T cell mitogen Concanavalin A type V (Sigma) for 48h at 40.5 o C. After addition of 20 µl MTT (10 mg/ml) for 4 h, cells were lysed and the OD values determined at 550 nm. The stimulation indices were determined. Statistical methods Data are expressed as mean per group ± standard deviation (SD). Statistical analysis was performed by Statistix 7.0 software (Analytical Software, Tallahassee / USA), using One-way analysis of variance (ANOVA) and comparison of means by Tukey Test, or student s t-test. P values of <0.05 were considered as significant. 8

4 Results and Discussion At one day and five-six days post 20 serum samples were tested for IBDV antibodies to determine based on the Deventer formula the estimated break through titer for the Icx-IBD vaccine. This tentative break through time point was used to extrapolate on the possible time, when the replicating intermediate IBD vaccine, which is released by the immune complex vaccine, may induce some transient immunosuppression. Based on the serum antibody levels (Table 1), day post was chosen as the first and day as the second day of inactivated NDVvaccination. Necropsy was conducted at and days post, and 21, 28, and 35 days post to determine vaccine induced lesions and other parameters to evaluate the effects of the vaccines on the immune system and the IBD-immune response. Gelatination of the bursa of Fabricius was detected in 6-30 % of the Icx-inoculated chickens between and 28 days post. No other macroscopical lesions at spleen, bursa of Fabricius or gut were detected at any time point in any of the investigated groups. While the bursa to body weight ratio was not affected in the diluent and HVT-IBD groups, Icx-IBD inoculated birds showed a reduction of the bursa weight beginning at 21 days post (P < 0.05, Table 2). Histopathological bursa lesions were detected only in the Icx-IBD inoculated birds starting at days post (Table 3). There was a high variation in the onset time of lesion development between Icx-IBD inoculated birds allowing the speculation that the levels of maternal antibodies varied between individuals per group and influenced the onset of infection. Regeneration of bursa follicles began at 28 days post in individual birds. The development of histological lesions in the Ixc-IBD group was accompanied by infiltrating T cells (Figure 1). No significant changes in the number of intrabursal CD3+ T cells were seen over time in the diluent and HVT-IBD inoculated groups. The effect of the vaccines on circulating B and T cell populations was investigated by flow cytometric analysis of the peripheral blood leukocytes. As expected from previous research Icx-IBD induced a significant reduction in the number of circulating B cells at 21 days post (Figure 2; P < 0.05) (Corley & Giambrone, 2002). CD4+ T cell numbers were affected at 21 days post in the Icx-IBD inoculated group. The percentage of this cell population increase significantly in comparison to the diluent inoculated group (P < 0.05). The other T cell population was not affected in either group at any investigated time point (data not shown). The mitogenic response was not affected by the two vaccines in comparison to the diluent inoculated group at any of the investigated time points (data not shown). These results demonstrate that neither vaccine had a strong systemic effect on the investigated activity of systemic T cell populations. At, and 21 days post, bursa of Fabricius and spleen samples were further investigated by qrt-pcr for VP2 of IBDV. As indicated in Figure 3, the detection rate of IBDV in Icx-IBD inoculated birds in the bursa of Fabricius and spleen increased clearly over time. The detection rates for VP2 in HVT-IBD inoculated birds were lower, but it was detected in spleen as well as in bursa of Fabricius at almost all investigated time points (Figure 3) without affecting the numbers of local T cell populations or the bursa architecture. Both vaccines induced IBDVantibodies (Table 1). The IBDplus ELISA system detected IBDV antibodies at the same time and about the same titer in both vaccinated groups (Table 1), which was expected based on previous experiments (Le Gros et al., 2009). 813

5 NDV-antibodies were detected after vaccination of both IBDV-vaccinated groups in the ELISAc (data not shown). Due to high individual variation in the actual onset of vaccine take for Icx-IBD inoculated birds the NDV-antibody levels did no vary significantly between vaccinated groups. But as indicated in Figure 4, the birds of the Icx-IBD-group, which had received the NDV-vaccine at days post, when IBDV-induced lesion development clearly started as demonstrated by the histological and immunohistochemical investigations, showed lower antibody titers in individual birds as compared to the other two NDVvaccinated groups. Overall, this study clearly shows that the Icx-IBD vaccine affected the humoral immune system by reduction of circulating and intrabursal B cells, and by slightly suppressing the antibody response after NDV-vaccination. The onset of the vaccine take varied between birds, which may explain, why the vaccine impact on the humoral immune response is not significant at the early time points post NDVvaccination. Interestingly, HVT-IBD was detected not only in the spleen but also in the bursa by qrt-pcr, but it induced no detectable effect on the T cells or B cell immune response to inactivated NDV. Both vaccines induced a strong humoral IBDVantibody response detectable in the IBDplus ELISA. Acknowledgements The authors like to thank Christine Haase, Sonja Bernhardt, Katja Stolpe, Sabrina Techel for their technical assistance and animal care. 814

6 Table 1: IBDV-Antibody detection by the Synbiotics IBD+ ELISA kit Groups indv- Vaccination at days post 1 ( n = 20) Average IBD-ELISA antibody level ± SD at days post 6 (n = 20) * (n = (n = 21 (n= 9-28 (n = 3-10) 10) 10) 10) iindv-i indv-ii 35 (n = 3-10) Virusfree HVT-IBD Icx-IBD 6363 ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± 1 34 ± ± 31 ND 9401 ± ± ± ± ± 1845 *First necropsy date; indv-i = vaccination with an inactivated NDV vaccine at days post ; indv-ii = vaccination with an inactivated NDV vaccine at 25 days post. Table 2: Bursa-body weight ratio at days after macroscopical bursa Groups Virusfree HVT- IBD Icx-IBD indvvaccination at days post Average bursa body weight ratio ± SD at days post (acute lesions in birds/total number of birds per group) (n = 3-5) 2.2 ± ± ± ± ± ± ± ± ± 0.5 (1/7) 2.2 ± ± ± ± ± ± ± 0.2* 1.1 ± 0.3* (1/5) 1.5 ± 0.9 (2/5) 2.1 ± ± ± ± ± ± ± 0.2* 0.8 ± 0.2* 1.0 ± 0.2* (1/5) 2.1 ± 0.8 (0/3) 1.8 ± ± 0.6 ND 2.0 ± 0.7 (0/4) 2.3 ± ± 0.3* 0.8 ± 0.5* (0/4) 0.7 ± 0.2* 8

7 Acute bursa lesions such as gelatination; 1. NDV-vaccination at days post ; 2. NDV vaccination at days post ; *significantly different to the other groups of the experimental day (P < 0.05). ND = not done. Table 3: Development of histological bursa lesions after IBDV vaccination birds/group Groups indv- Vaccination at days post days post Development of histological bursa lesions in birds/total number of (average lesion score ± SD) days post 21 days post 28 days post 35 days post Virus-free 0/7 (0.0 ± 0) 0/7 (0.0 ± 0) 0/7 (0.0 ± 0) 0/3 (0.0 ± 0) HVT-IBD 0/7 (0.0 ± 0) 0/7 (0.0 ± 0) 0/7 (0.0 ± 0) 0/3 (0.0 ± 0) ND 0/4 (0.0 ± 0) Icx-IBD 1/7 (0.6 ± 1.5) 5/7 (1.5 ± 1.7)* 3/7 (1.3 ± 1.9)* 5/5 (3.0 ± 1.4)* 5/5 (3.4 ± 1.3)* 5/5 (3.6 ± 0.5)* 5/5 (3.4 ± 0.9)* 5/5 (3.2 ± 1.3)* 5/5 (3.4 ± 1.3)* 5/5 (2.8 ± 1.6)* 3/4 (2.5 ± 1.9)* 5/5 (3.2 ± 1.3)* No spleen lesions were observed at the indicated time points in either group. *Significantly different to the HVT-IBD and diluent inoculated birds (P < 0.05). ND = not done 816

8 Average number of intrabursal CD3+ T cells/group at 40 x Virus-free HVT-IBD Icx-IBD 0 day ph day ph 21 day ph Figure 1: Detection of CD3+ T cells in the bursa of Fabricius after vaccination with HVT-IBD and Icx-IBD. Cryosections were investigated at different time points after. Indicated is the average number of cells ± SD and in percent the number of birds/total with >100 T cells/microscopic field (400 x). 817

9 % Bu-1 positive B cells/total lymphocytes a ab b Virus-free HVT-IBD Icx-IBD Figure 2: Flow cytometric analysis of peripheral blood leukocytes. Persented is the percent of Bu-1 positive B cells detected within total lymphocytes of PBL at 21 days post. Different superscript letters indicate significant differences between groups (P < 0.05) dph dph 21 dph 40-Ct 10 5 cut off 0 HVT-IBD Ixc-IBD (a) 818

10 dph dph 21 dph Ct 10 5 cut-off 0 HVT-IBD Icx-IBD (b) Figure 3: Detection of IBDV or VP2 by qrt-pcr. Presented is the average ± SD of the 40-CT as indicated for detected viral load in (a) spleen and (b) bursa of Fabricius. 819

11 7000 NDV-ELISA antibody level/ animal Virus- HVT-IBD Icx-IBD (a) 000 NDV-ELISA antibody titer/ animal Virus- HVT-IBD Icx-IBD (b) free Figure 4: Distribution of NDV-ELISA Antibody levels. (a) 28th day post, 13 days after NDV-Vaccination at days post ; (b) 35th day post, 20 days after NDV- Vaccination at days post. 820

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