INTRODUCTION. J.-H. Roh,,1 M. Kang,,1 B. Wei, R.-H. Yoon, H.-S. Seo, J.-Y. Bahng, J.-T. Kwon, S.-Y. Cha,,1 and H.-K. Jang,2

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1 Efficacy of HVT-IBD vector vaccine compared to attenuated live vaccine using in-ovo vaccination against a Korean very virulent IBDV in commercial broiler chickens J.-H. Roh,,1 M. Kang,,1 B. Wei, R.-H. Yoon, H.-S. Seo, J.-Y. Bahng, J.-T. Kwon, S.-Y. Cha,,1 and H.-K. Jang,2 Departments of Infectious Diseases and Avian Diseases, College of Veterinary Medicine and Korea Zoonosis Research Institute, Chonbuk National University; and Department of Broiler Breeder, Halim Co. LTD, South Korea ABSTRACT The production performance, efficacy, were intraocularly challenged at 24 d. Eight of 10 chickens and safety of two types of vaccines for infectious bursal disease virus (IBDV) were compared with in-ovo vaccination of Cobb 500 broiler chickens for gross and microscopic examination of the bursa of Fabricius, bursa/body weight (b/b) ratio, flow cytometry, and serologic response to Newcastle disease virus (NDV) vaccination. One vaccine was a recombinant HVT-IBD vector vaccine (HVT as for herpesvirus of turkeys) and the other was an intermediate plus live IBDV vaccine. A significant difference was detected at 21 d. Eight of 10 chickens that received the IBDV live vaccine had severe bursal lesions and a relatively low b/b ratio of 0.95, and an inhibited NDV vaccine response. On the other hand, the HVT-IBD vector vaccine resulted in mild bursal lesions and a b/b ratio of Therefore, the live vaccine had lower safety than that of the HVT-IBD vector vaccine. To determine the protective efficacy, chickens in the IBDV live vaccination group showed gross and histological lesions characterized by hemorrhage, cyst formation, lymphocytic depletion, and a decreased b/b ratio. In contrast, the HVT-IBD vector vaccinated chickens showed mild gross and histological lesions in three of 10 chickens with a b/b ratio of 1.36, which was similar to that of the unchallenged controls. Vaccinated chickens showed a significant increase in IBDV antibody titers, regardless of the type of vaccine used. In addition, significantly better broiler flock performance was observed with the HVT-IBD vector vaccine compared to that of the live vaccine. Our results revealed that the HVT-IBD vector vaccine could be used as an alternative vaccine to increase efficacy, and to have an improved safety profile compared with the IBDV live vaccine using in-ovo vaccination against the Korean very virulent IBDV in commercial broiler chickens. Key words: HVT-IBD vector vaccine, infectious bursal disease, IBDV live vaccine 2016 Poultry Science 95: INTRODUCTION Infectious bursal disease caused by infectious bursal disease virus (IBDV) is an acute and highly infectious disease in chickens at 3 wk of age and older. IBDV attacks the bursa of Fabricius and infects B lymphocytes in the bursal follicles. It causes immunosuppression due to the depletion of B lymphocytes. Immunosuppression causes a poor immune responses to vaccination, resulting in significant economic losses. Immunosuppressed chickens are highly susceptible to a variety of secondary infections leading to a reduced growth rate (Alloui et al., 2005;BechtandMüller, 1991; Zhou et al., 2010). C 2016 Poultry Science Association Inc. Received October 6, Accepted December 15, These authors contributed equally to this study. 2 Corresponding author: hkjang@chonbuk.ac.kr Vaccination is widely used to control infectious bursal disease. Commercially available attenuated live IBDV vaccines (intermediate plus) are usually used (Giambrone and Closser, 1990; Rautenschlein et al., 2005; Zhou et al., 2010). These IBDV live vaccines, however, can themselves cause a degree of vaccine-induced bursal atrophy and result in immunosuppression. In addition, they may promote negative reactions such as poor weight gain, secondary infection, and diverse diseases (Alloui et al., 2005; Snyder, 1990; Zhou et al., 2010). Moreover, commercial live vaccines can also be inhibited by maternal derived antibodies (MDA) present at the time of vaccination. The herpesvirus of turkeys (HVT) recombinant vector vaccine was generated by inserting an IBDV VP2 gene expression cassette into the HVT genome (Le Gros et al., 2009; Sultan et al., 2012). This vaccine has a safety and efficacy advantage in the presence of IBDV MDA at the time of vaccination. 1020

2 EFFICACY OF HVT-IBD VECTOR VACCINE 1021 In the past few years, the use of in-ovo IBDV vaccines has increased. The in-ovo route is known to be fast, effective in 100% of the eggs that receive it, and labor saving. However, no study has compared the safety and efficacy of HVT-IBD vector vaccine and IBDV live vaccine through the in-ovo route. Therefore, the objectives of this study were: 1) to compare the safety of the HVT-IBD vector vaccine to that of a live vaccine; 2) to determine their efficacy against a very virulent IBDV (vvibdv) isolate from Korea; and 3) to evaluate the production performance of the vaccinated flocks. MATERIALS AND METHODS Observations in the Farm IBDV Vaccinations The VAXXITEK R HVT-IBD commercial vector vaccine (Merial Inc., Duluth, GA) and an IBDV live vaccine (intermediate plus vaccine) were used in this study. The HVT-IBD vector vaccine was generated by inserting an IBDV VP2 gene (cloned from the Faragher 52/70 IBDV strain) expression cassette into the HVT genome. The parents were Cobb 500 broiler chickens with maternal antibodies to IBDV. Eggs obtained from the same parental flocks were divided into the following three groups: 1) HVT-IBD vector vaccine group; 2) IBDV live vaccine group; and 3) PBS vaccinated group. These groups were vaccinated in-ovo at 18 d of embryonic development according to the manufacturer s instructions. Farm The field trial took place in commercial facilities. The IBDV live vaccine and HVT-IBD vector vaccine flocks were at separate sites to avoid cross contamination in the same farm. The chickens had grown up under the same environment and management, and feed and water were provided ad libitum. Egg transmitted diseases were tested at 1 d in the field trial. Field exposure at 21 and 35 d were also determined. Bursa/Body Weight Ratio and Bursal Lesion Scoring The bursa of Fabricius was collected from 10 chickens in each flock at 1, 7, 14, 21, 28, and 35 d, and the bursa/body weight (b/b) ratio was recorded. Gross lesions of the bursa of Fabricius were evaluated for inflammation and hemorrhage. The bursa of Fabricius was fixed in 10% formalin solution and processed for histological paraffin sectioning, and the sections were stained with hematoxylin-eosin. Bursal lymphoid tissue lesions were scored 0 to 4 based on the degree of lymphoid necrosis and/or lymphocytic depletion (0 = 5%, 1 = 5 25%, 2 = 25 50%, 3 = 50 75%, and 4 > 75% of the lymphoid follicles) (Sharma et al., 1989). Flow Cytometry The bursa of Fabricius was taken randomly from 10 chickens at 1, 7, 14, 21, 28, and 35 d. Pools of 10 chickens per group were examined. Bursal cells were prepared by crushing the organs. Approximately cells were seeded in each tube, and washed with phosphate buffered solution (PBS). The cell pellet was resuspended with chicken serum to block nonspecific binding. Staining was performed by resuspending the cell pellet with staining buffer. After adding antibodies mouse anti-chicken IgM-FITC (SouthernBiotech, Birmingham, AL) and mouse antichicken CD3-PE (SouthernBiotech), the cells were incubated at 4 C for 30 min and washed with staining buffer. The stained cells were then fixed in 4% paraformaldehyde before profiling. Positively stained cells were analyzed by FACS Calibur flow cytometer and CellQuest software (Becton Dickinson, Mountain View, CA) (Carballeda et al., 2011; Khatri et al., 2005). Serology Blood samples were collected randomly from 20 birds at 1, 7, 14, 21, 28, and 35 d for serological examination. Serum samples were screened by enzymelinked immunosorbent assay (ELISA) for antibodies to IBDV using a commercial ELISA kit (PROFLOK R Plus IBD Ab test kit; Zoetis, Florham Park, NJ). IBDV antibody titers were calculated based on the manufacturer s instructions. The average antibody titer per flock was compared weekly. Production Performance Weight gain, growth rate, and feed conversion ratio (FCR) were calculated in two flocks to determine the production performance. FCR is a measure of a chicken s efficiency in converting feed mass into increased muscle (Al-Mufarrej, 2014). Experiments in the Laboratory Experimental Challenge Design To evaluate protective efficacy against vvibdv, there were seven groups (10 chickens per group): 1) PBS vaccine; 2) HVT-IBD vector vaccine; 3) IBDV live vaccine; 4) PBS vaccine+vvibdv challenge; 5) HVT- IBD vector vaccine+vvibdv challenge; 6) IBDV live vaccine+vvibdv challenge; 7) PBS vaccine+vvibdv challenge (SPF chickens). For the first six groups, the broiler chickens were used and the seventh group consisted of SPF chickens as a vvibdv pathogenicity control. Broiler chickens were moved from the farm to strictly isolated experimental rooms at 1 d and a group of SPF chickens at the same age were used. The challenge virus was administered via the intraocular route with 10 6 ELD 50 /0.2 ml per bird at 24 d using SH/92 strain (vvibdv) isolated in Korea. Mortality and clinical signs were observed for 10 d post-challenge. Then, the chickens were euthanized and necropsied. Protective efficacy was evaluated by mortality, gross and histological lesions, b/b ratios, and flow cytometry conducted as described above. All experimental procedures and animal management procedures were undertaken in accordance with the requirements of the Animal Care and Ethics Committees of Chonbuk National University (CBNU ). The animal facility of the Chonbuk National University is fully accredited by the National Association of Laboratory Animal Care. NDV Antibody Titers Measure To determine the extent of interference from ND antibody production by IBDV vaccine types, the following four groups (10 chickens per group) were used: 1) PBS vaccine;

3 1022 ROH ET AL. Table 1. Gross lesions, histological lesions, b/b ratio, and B cell population of the bursa of Fabricius in broiler chickens vaccinated with IBDV live vaccine or HVT-IBD vector vaccine in the farm. Age (days) HVT-IBD vector vaccine flock IBDV live vaccine flock Gross Tissue b/b B cell Gross Tissue b/b B cell lesion 1 lesion 2 ratio 3 population(%) lesion lesion ratio population(%) 1 0/ / / / / / / / / / / / Number of chickens with inflammatory or hemorrhagic lesions/number tested. 2 Average bursal lymphoid tissue histological lesion score (Sharma et al., 1989; 0 = 5%, 1 = 5 25%, 2 = 25 50%, 3 = 50 75%, and 4 > 75% of the lymphoid follicles). 3 Bursa/body weight ratio 1,000. Difference between IBDV live vaccine and HVT-IBD vector vaccine was statistically significant at the same age (P < ). Difference between IBDV live vaccine and HVT-IBD vector vaccine was statistically significant at the same age (P < 0.005). not tested. 2) NDV vaccine; 3) NDV+HVT-IBD vector vaccine; 4) NDV+IBDV live vaccine. The PBS, HVT-IBD vector vaccine and IBDV live vaccine groups were vaccinated in ovo at 18 d of incubation and each group was vaccinated with the NDV vaccine (VG/GA strain; Avinew, Rhone Merieux, France) at 1 d by spray and by the intraocular route at 18 d. Chickens were moved from the farm to strictly isolated experimental rooms at 1 d. Serum samples were collected at 0, 7, 14, 21, and 28 d after the final NDV vaccination. Sera were examined for hemagglutination (HA)-specific antibodies by HA inhibition (HI) test using NDV antigen. Serial 2- fold dilutions of serum in PBS were mixed with equal volumes (25 μl) of four hemagglutinating units (HAU) of the ND virus. A 25 μl aliquot of washed chicken red blood cells was added and incubated at room temperature for 40 min. The titer was defined as the reciprocal of the last dilution of serum that completely inhibited HA. Statistical Analysis All statistical analyses were performed by one-way analysis of variance and Student t-test using SPSS 11.0 software (SPSS Inc., Chicago, IL). P values < 0.05 were considered statistically significant. Detection of IBDV Antibody Titers The IBDV ELISA antibody titers remained comparable between the two flocks. Antibody titers of the IBDV live vaccine and the HVT-IBD vector vaccine flock decreased rapidly for 1 to 4 d showing clear serological conversion in both flocks. The antibody titers increased gradually, with titers of 9,096 and 7,526 for the IBDV live vaccine and the HVT-IBD vector vaccine flock, respectively, at 35 d (data not shown). Production Performance Body weight and feed intake were recorded for the entire experimental period to evaluate flock production performance. The HVT-IBD vector vaccine flock had a higher body weight and a satisfactory growth rate compared to those of the IBDV live vaccine flock. The FCR was calculated as the amount (in grams) of feed consumed to produce 1 g live weight. The HVT-IBD vector vaccine flock had a better FCR than that of the IBDV live vaccine flock (Table 2). Overall, significantly better performance was observed for the HVT-IBD vector vaccine flock in all data, including average weight gain, growth rate, and FCR. RESULTS Bursal Lesions, b/b Ratio, and B Cell Populations on the Farm No damage to the bursa of Fabricius was detected until 14 d. At 21 d, the IBDV live vaccine flock showed bursal lesions and atrophy (Table 1). The flow cytometry evaluation revealed a notable reduction in the percentage of B cells. In contrast, the HVT-IBD vector vaccine flock was not different from the PBS group and two of 10 chickens had mild inflammatory lesions at 28 d. Table 2. Averages of body weight, growth rate and feed conversion ratio of flocks in the farm. Flock Averages of body weight (kg) Growth rate 1 (%) FCR 2 HVT-IBD vector vaccine flock IBDV live vaccine flock Growth rate = (Last weight first weight)/first weight FCR (feed conversion ratio) = Feed intake/weight gain. HVT-IBD vector vaccine flock was significantly different from IBDV live vaccine flock (P < ).

4 EFFICACY OF HVT-IBD VECTOR VACCINE 1023 Table 3. Protective efficacy against IBDV challenge in the laboratory. Group 10 d post challenge Gross Tissue b/b B cell Mortality lesion 1 lesion 2 ratio 3 population (%) Broiler chickens PBS vaccine 0/10 0/10 0/ a 64.8 HVT-IBD vector vaccine 0/10 0/10 0/ a 44.8 IBDV live vaccine 0/10 5/10 6/ b 26.4 PBS vaccine +vvibdv challenge 1/10 6/10 10/ b 11.3 HVT-IBD vector vaccine +vvibdv challenge 0/10 3/10 3/ a 43.7 IBDV live vaccine +vvibdv challenge 0/10 6/10 8/ b 27.8 SPF chickens PBS vaccine +vvibdv challenge 5/10 9/10 10/ a Number of chickens with lesions of inflammation or hemorrhage/tested. 2 Number of chickens with lymphocytic depletion more than 25% of the lymphoid follicles/tested. 3 Bursa/body weight ratio 1,000. a,b Different letters in same column indicate significant difference by the test of Tukey (P < 0.001). Protective Efficacy against vvibdv Challenge in the Lab Sixty broiler chickens and 10 SPF chickens were used to examine the protective efficacy of the IBDV live vaccine and HVT-IBD vector vaccine in broiler chickens (Table 3). By 10 d post vaccination (dpv), mortality was observed only in the challenged control group. The IBDV live vaccine control group and IBDV live vaccine+vvibdv challenge group showed gross and histological lesions of the bursa in >50% of chickens, including hemorrhage, cyst formation, lymphocytic depletion, and decreased b/b ratio. In contrast, the HVT-IBD vector vaccine group remained normal without any gross bursal lesions. The HVT-IBD vector vaccinated group followed by challenge showed mild gross and histological lesions in three of 10 chickens. Their b/b ratios were comparable to those of the PBS vaccine group. The B cell populations in the IBDV live vaccine+vvibdv challenge group and the HVT-IBD vector vaccine+vvibdv challenge group were 27.8% and 43.7%, respectively. The B cell population (27.8%) in the IBDV live vaccine+vvibdv challenge group was significantly lower than that of the PBS vaccine group (64.8%, Table 3). ND HI Titer The effect of the IBDV vaccines on antibody titers of ND vaccines was examined in a laboratory experiment. Serum antibody titers to the ND vaccine were measured at five time points by HI until 28 d after the booster vaccination. At 14 dpv, the antibody titers were 4.2 log2 and 6.1 log2 in the ND+IBDV live vaccine group and the ND+HVT-IBD vector vaccine group, respectively. These antibody levels were maintained until 28 dpv (Figure 1). DISCUSSION This study aimed to compare the production performance, efficacy and safety of two types of IBDV vaccines (HVT-IBD vector vaccine and IBDV live vaccine) with in-ovo vaccination of Cobb 500 broiler chickens. Our results revealed that the IBDV live vaccine flock had bursal gross lesions in eight of 10 chickens with histological lesion score of more than 2.5 and reduced b/b ratios as well as a reduced B cell population. In contrast, the HVT-IBD vector vaccine flock had histological lesion scores of less than 0.3 at 21, 28, and 35 d. Previous research with IBDV live vaccine using post-hatch vaccination had shown bursal lesions with significantly low b/b ratios (Le Gros et al., 2009). Therefore, the IBDV live vaccine produced pathological bursal lesions when administered either in ovo or on the day of hatch. The IBDV live vaccine may also interfere with other vaccines (Mazariegos et al., 1989). Consistent with this finding, our results also showed that the NDV+IBDV live vaccine had a lower NDV antibody titer at 14 dpv than the PBS group and the NDV+HVT-IBD vector vaccine group. Protective efficacy was evaluated by IBDV Figure 1. Serum antibody titers to NDV vaccine by hemagglutination inhibition test after the booster vaccination.

5 1024 ROH ET AL. antibody titer and lab experimental challenge in this study. Clear serological conversion to the IBDV antibody was evidenced in both the HVT-IBD vector vaccine flock and the IBDV live vaccine flock with an expected decrease in maternal immunity. The HVT-IBD vector vaccine+vvibdv challenge group developed mild gross and histological lesions in the bursa of Fabricius. This group had a similar b/b ratio and B cell population to the HVT-IBD vector vaccine control group, confirming its protective efficacy. In contrast, the IBDV live vaccine group and the IBDV live vaccine+vvibdv challenge group had bursal lesions of significantly low b/b ratios, and these results were similar to that of the PBS vaccine+vvibdv challenge group. The bursal damage in the IBDV live vaccine group might be due to the residual pathogenicity of the vaccine strain or the live virus challenge. Significantly better production performance was observed for the HVT-IBD vector vaccine flock. In other field trials, IBDV live vaccination had lower performance than those of a non-vaccinated control (Sarachai et al., 2013). Our results were also worse in production performance following the IBDV live vaccine flock than the HVT-IBD vector vaccine flock. Effective vaccination can be achieved in the presence of MDA (Bublot et al., 2007). Our results also revealed that the HVT-IBD vector vaccine through the in-ovo route could achieve high efficacy. In conclusion, IBDV live vaccine and HVT-IBD vector vaccine by in-ovo vaccination led to effective antibody titers. In-ovo vaccination of IBDV live vaccine or HVT-IBD vector vaccine could achieve high efficacy even in the presence of MDA. In contrast, persistent bursal lesion and atrophy were induced by the IBDV live vaccine through the in-ovo route. In addition, this IBDV live vaccine interfered with the NDV vaccine with reduced vaccine safety. Besides its safety issues, the production performance of the IBDV live vaccine flock was lower than that of the HVT-IBD vector vaccine flock. From these observations, the HVT-IBD vector vaccine is recommended as the vaccine of choice to avoid the safety problems associated with the IBDV live vaccine. ACKNOWLEDGMENTS This research was supported by a grant ( , , ) from the Bio-industry Technology Development Program, Ministry of Agriculture, food, and Rural Affairs, a grant (PJ907105) from the Cooperative Research Program, Agriculture Science, and Technology, Rural Development Administration, and Research of Animal and Plant Quarantine Agency, South Korea. REFERENCES Al-Mufarrej, S. I Immune-responsiveness and performance of broiler chickens fed black cumin (Nigella Sativa L.) powder. J. Saudi Soc. Agric. Sci. 13: Alloui, M. N., S. Sellaoui, and S. Djaaba Morphometrical and anatomo-pathological survey of the bursa of fabricius in broiler chickens. Int. Soc. Anim. Hygiene. 2: Becht, H., and H. Müller Infectious bursal disease-b cell dependent immunodeficiency syndrome in chickens. Behring Inst. Mitt. 89: Bublot, M., N. Pritchard, F.-X. Le Gros, and S. Goutebroze Use of a vectored vaccine against infectious bursal disease of chickens in the face of high-titred maternally derived antibody. J. Comp. Path. 137: Carballeda, J. M., S. C. Zoth, E. Gómez, M. J. Gravisaco, and A. Berinstein Activation of the immune response against Infectious Bursal Disease Virus after intramuscular inoculation of an intermediate strain. Immunobiology. 216: Giambrone, J., and J. Closser Efficacy of live vaccines against serologic subtypes of infectious bursal disease virus. Avian Dis. 34:7 11. Khatri, M., J. M. Palmquist, R. M. Cha, and J. M. Sharma Infection and activation of bursal macrophages by virulent infectious bursal disease virus. Virus Res. 113: Le Gros, F., A. Dancer, C. Giacomini, L. Pizzoni, M. Bublot, M. Graziani, and F. Prandini Field efficacy trial of a novel HVT-IBD vector vaccine for 1-day-old broilers. Vaccine. 27: Mazariegos, L. A., P. Lukert, and J. Brown Pathogenicity and immunosuppressive properties of infectious bursal disease intermediate strains. Avian Dis. 34: Rautenschlein, S., C. Kraemer, J. Vanmarcke, and E. Montiel Protective efficacy of intermediate and intermediate plus infectious bursal disease virus (IBDV) vaccines against very virulent IBDV in commercial broilers. Avian Dis. 49: Sarachai, C., N. Chansiripornchai, and J. Sasipreeyajan Efficacy of infectious bursal disease vaccine in broiler chickens receiving different vaccination programs. Thai J. Vet. Med. 40:9 14. Sharma, J. M., J. E. Dohms, and A. L. Metz 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: Snyder, D Changes in the field status of infectious bursal disease virus. Avian Pathol. 19: Sultan, H., H. A. Hussein, A. G. Abd El-Razik, S. El-Balall, S. M. Talaat, and A. A. Shehata Efficacy of HVT-IBDV Vector Vaccine Against Recent Egyptian vvlbdv in Commercial Broiler Chickens. Int. J. Poult. Sci. 11: Zhou, X., D. Wang, J. Xiong, P. Zhang, Y. Li, and R. She Protection of chickens, with or without maternal antibodies, against IBDV infection by a recombinant IBDV-VP2 protein. Vaccine. 28:

Infectious Bursal Disease, Immunosuppression and the role of VAXXITEK HVT+ IBD

Research note Infectious Bursal Disease, Immunosuppression and the role of VAXXITEK HVT+ IBD Grogan K. 1 1 Poultry Chicken Scratch, LLC, 30019 Dacula GA United States of America [from Hoerr F.J., 2010,