Host genetic resistance to Marek's disease sustains protective efficacy of herpesvirus of turkey in both experimental and commercial lines of chickens.

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Host genetic resistance to Marek’s disease sustains protective efficacy of herpesvirus of turkey in both experimental and commercial lines of chickens

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Shuang Chang a,b,1,2 , Qingmei Xie a,b,c,1 , John R. Dunn a , Catherine W. Ernst b , Jiuzhou Song d , Huanmin Zhang a,∗ a

USDA, Agriculture Research Service, Avian Disease and Oncology Laboratory, East Lansing, MI 48823, USA Department of Animal Science, Michigan State University, East Lansing, MI 48824, USA c South China Agricultural University, Guangzhou 510642, China d University of Maryland, College Park, MD 20742, USA b

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Article history: Received 18 November 2013 Received in revised form 22 January 2014 Accepted 30 January 2014 Available online xxx

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Keywords: Marek’s disease Host genetics Vaccine efficacy

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1. Introduction

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Marek’s disease (MD) remains a continual threat to the poultry industry worldwide as the MD virus continues evolving in virulence. MD has been controlled primarily by intensive use of vaccines since 1969. Based on the antigenic and pathogenic differences of the viruses that the vaccines were derived from, commercially available MD vaccines are classified into three categories, MDV-1–3 vaccines. This study was designed to compare the protective efficacy of MDV-1 (CVI988/Rispens) and MDV-3 (HVT) vaccines against challenge of a very virulent plus strain of Marek’s disease virus (vv + MDV) in experimental and commercial egg-layer lines of chickens under controlled experimental conditions. The two experimental lines (63 and 72 ) of chickens carry a uniform MHC B*2 haplotype and are known to differ in resistance to MD. One of the two commercial egg-layer lines (WL and BL) segregates for three MHC haplotypes (B*2, B*15, and B*21); the other is unclear. MD incidences of the unvaccinated groups of both experimental lines and both commercial lines were 100% or close to 100% induced by the vv + MDV, 648A. Survived day patterns of the unvaccinated groups significantly differed between the two experimental lines, but did not between the two commercial lines, which suggested the two experimental lines do differ in resistance to MD but not between the two commercial lines. At manufacturers’ recommended vaccine dosage, two HVTs conveyed comparable protection for the MD resistant line 63 chickens as did both CVI988/Rispens used in this study. The two HVTs also conveyed comparable protection for both commercial lines of chickens as did one of two CVI988/Rispens (CVI988/Rispens-A). At a 2000 PFU uniform dose, HVT and CVI988/Rispens again conveyed comparable protection for the MD resistant experimental line of chickens. The findings suggest vaccine protective efficacy is modulated by factors including the types and the sources of vaccines and the genetic backgrounds of chickens. The findings also suggest HVT delivers equal protection in MD resistant lines of chickens as does the industry-recognized golden standard of MD vaccine, CVI988/Rispens. © 2014 Published by Elsevier Ltd.

Marek’s disease (MD) is an alpha-herpesvirus-induced T-cell lymphoma of chicken [1,2]. MD historically caused devastating

∗ Corresponding author. Tel.: +1 517 337 6835; fax: +1 517 337 6776. E-mail addresses: [email protected] (S. Chang), [email protected] (Q. Xie), [email protected] (J.R. Dunn), [email protected] (C.W. Ernst), [email protected] (J. Song), [email protected], [email protected] (H. Zhang). 1 The authors contributed equally >to this work. 2 Current Address: Guangdong Wen’s Foodstuff Group Co., Ltd, Guangdong 527439, China.

losses to the poultry industry worldwide and has been under control since 1969 through wide use of vaccine [3–7]. Yet MD continues to cost the world poultry industry over $1 billion annually resulting from prevention measures (management efforts and vaccinations), reduced egg production in layers (egg-type chickens), and reduced growth and condemnation in broilers (meat-type chickens). MD remains a serious threat to the poultry industry since commercial lines of chickens at large are susceptible to MD, with the exception of a limited number of lines on some farms where selection for genetic resistance to MD has been practiced, and new isolates of MDV have been emerging with escalated virulence [4,6,8–12]. Under the same strategies, which have been followed for almost half a century, current control measures using commercially

0264-410X/$ – see front matter © 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.vaccine.2014.01.092

Please cite this article in press as: Chang S, et al. Host genetic resistance to Marek’s disease sustains protective efficacy of herpesvirus of turkey in both experimental and commercial lines of chickens. Vaccine (2014), http://dx.doi.org/10.1016/j.vaccine.2014.01.092

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available vaccines might be overwhelmed at an unpredicted time in the future, as has happened historically during the 1980s and 1990s [5,6,9]. Host genetic resistance is considered as an attractive approach to augment the current control measures against MD [5,13–16]. Host genetic resistance to MD has been recognized since the 1940s [17–19]. It was during and after the 1970s that researchers using specialized experimental lines of chickens identified and confirmed that the major histocompatibility complex (MHC) or B-haplotypes in chicken significantly contribute to genetic resistance to MD [10,16,17,20–25]. Soon after that, experimental evidence suggested genes outside of the MHC also contribute to MD resistance. Two lines of chickens (lines 63 and 72 ) developed and maintained at the Avian Disease and Oncology Laboratory (ADOL) share a common MHC (B*2) haplotype, but one is highly resistant and the other is highly susceptible to MD as a result of selection [2,22,23,26]. Genetic resistance to MD, either due to MHC or genes outside of the MHC, is associated with vaccine protective efficacy. Bacon and Witter in the 1990s reported that MHC B haplotypes affect host immunoresponse to MD vaccines. MDV-2 vaccine (SB-1) conveys better protection, and resulted in significantly lower MD incidence in chickens with B*5 haplotype than in chickens with other B haplotypes. In contrast, MDV-1 vaccine (CVI988/Rispens) conveys better protection than MDV-2 (SB-1) or MDV-3 vaccine (HVT) in chickens with B*2, B*13, B*15, or B*21 haplotype(s) [5,21,27–30]. In recent years, we have assessed non-MHC genetic effect on MD vaccine efficacy using the lines 63 and 72 chickens as well as chickens from a series of recombinant congenic strains (RCS), which all share the same MHC B*2 haplotype. Our data clearly suggested genes outside of the MHC region significantly affect MD vaccine protective efficacy [31–33]. Our data also showed for the first time there may be a chicken line by vaccine interaction modulating MD vaccine protective efficacy [32]. We further hypothesized that vaccine protective efficacy, which reportedly depends on many factors including vaccine dosage, number of vaccinations, age at vaccination, the time interval between vaccination and infection, maternal antibody, type of vaccine, and host genetics, is also partially determined by interactions among vaccine virus genome, host genome, as well as the challenge virus genome [32]. This study was designed to further assess non-MHC genetic effect on vaccine protective efficacy in experimental lines of chickens sharing an identical MHC B haplotype and to explore the genetic background effect of commercial egg-layer lines of chickens that may differ in MHC haplotypes on vaccine protective efficacy. Manufacturer recommended commercial doses for both MDV-1 and MDV-3 vaccines were given in two of the three challenge experiments using experimental or commercial egg-layer lines of chickens. Therefore, the data from this study should provide an additional piece of experimental evidence for host genetics influence on vaccine protective efficacy, and the findings should be highly relevant to the poultry industry for vaccination practice.

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2. Materials and methods

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2.1. Genetic lines of chickens

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A total of 865 chickens were used in this study. Of these, 483 chickens were sampled from two highly inbred specific-pathogen free experimental lines, which were developed and have been maintained at the USDA-Agricultural Research Service, Avian Disease and Oncology Laboratory (ADOL), East Lansing, Michigan [23,34]. One of the two experimental lines is known as line 63 and is relatively resistant to MD; the other is line 72 and is highly susceptible to MD. Both of the experimental lines of chickens share a common MHC B*2 haplotype [23]. The remaining 382 chickens were

sampled from two commercial egg-layer lines. One is designated as white layer (WL), and the other, brown-layer (BL). The WL line has been selected for MD resistance and are known to segregate for MHC B*2, B*15, and B*21 haplotypes. The BL line lacked information on MD susceptibility and MHC background. 2.2. Cells, vaccines and viruses Line 0 primary chicken embryonic fibroblasts were used in preparation of one of the two cell-associated HVT FC126 [23,35] vaccines and the very virulent plus (vv+) strain of MDV 648A [36]. One other HVT and two CVI988/Rispens vaccines were kindly provided by different vaccine manufacturers for this study. 3. Challenge experiments and layouts Three challenge experiments were conducted. Experiment 1 was conducted in chickens sampled from the two experimental inbred lines (63 and 72 ) known to differ in MD resistance. A total of 310 SPF chickens from the two lines were sampled for experiment 1. Fifteen were housed as negative control to monitor the experimental condition for non-specific mortality, and the rest from each line were randomly divided into five treatment groups. Two groups were vaccinated with CVI988/Rispens from two separate commercial companies (designated as CVI988/Rispens-A and CVI988/Rispens-B) at the manufacturers’ recommended doses, and two groups were vaccinated with HVT of different sources (designated as HVT-A and HVT-B). All vaccines were given to the chicks on the day of hatch. The other group was not vaccinated (unvaccinated). Experiment 2 was conducted following the same design in commercial egg-layer type of chickens under the same controlled conditions at ADOL. The commercial egg-layer types of chickens were randomly sampled from two commercial genetic lines, WL and BL. The chickens from each line were randomly assigned into five treatment groups. Four groups were vaccinated either with CVI988/Rispens or HVT, and the other group was not vaccinated (unvaccinated). The third experiment was conducted again using the two experimental lines (63 and 72 ) of chickens vaccinated either with CVI988/Rispens-B or HVT-B at a 2000 PFU uniform dose for fair comparison of protective efficacy between the MDV-1 and MDV-3 vaccines. An unvaccinated group was also included for each line of chickens in experiment 3. All treatment groups of chickens in each experiment (except the negative control groups) were inoculated on day 5 post hatch with the vv + MDV, 648A, intra-abdominally at a dose of 500 PFU per bird (for the experiment layouts and the number of birds used for each treatment group in each experiment, please see the Supplementary Table 1in Appendix A Supplementary Data). All chickens used in this study were housed in negatively pressured BSL-2 isolators during each experiment. Feed and water were supplied ad libitum. The chickens were observed daily throughout the entire duration of each experiment. The challenge experiments were approved by USDA, Avian Disease and Oncology Laboratory Animal Care and Use Committee (ACUC). The ACUC guidelines established and approved by the ADOL ACUC (April 2005) and the Guide for the care and use of Laboratory Animals by Institute for Laboratory Animal Research (2011) were closely followed throughout the experiments. 4. Pathological examination Chickens that died during or were euthanized at the end of each experiment were examined for gross MD lesions, which include enlarged peripheral nerves, and visceral lymphomas. Histological analysis was performed on tissues with non-definitive gross


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lesions. All experimental birds were pathologically categorized either as MD or normal according to necropsy records. MD refers to those with a gross tumor or tumors, or histologically-confirmed micro-tumors, and/or nerve enlargement(s), or died during the period between 8 day post MDV infection and before experimental termination. Normal refers to those that survived throughout the challenge experiment period and were free of any pathological MD symptoms. 5. Statistical analysis Since the lines of chickens used or the dosage of vaccine given differed between the experiments, the data of each experiment were analyzed separately. MD incidence was calculated for each treatment group as a ratio between the number of MD birds and the total number of challenged birds within that treatment group in each line. The protective index (PI) for each vaccine was calculated following a similar equation as described by Sharma and Burmester [37].

PI =

MD% of unvaccinated challenged group − MD% of vaccinated challenged group MD% of unvaccinated challenged group

× 100

The MD variable (MD and normal) is a nominal binary variable and was analyzed under a reduced nominal logistic model to examine chicken line, vaccine, and line by vaccine interaction effects on the MD incidence for each experiment. The effect likelihood ratio tests were used to assess the statistical significance of differences between lines, vaccines, and line by vaccine interaction providing the interaction effect was statistically significant in a reduced model for each experiment. The differences among the trends of survived day patterns were examined with Log-Rank and Wilcoxon tests. Bonferroni corrections were applied in multiple comparisons. All statistical analyses were performed using Statistical Analysis Software JMP version 10 (SAS Institute Inc., NC, USA) [38,39].

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6. Results

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6.1. Experiment 1

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The experimental lines 63 and 72 chickens sharing a single uniform MHC B*2 haplotype were vaccinated with commercial doses recommended by the vaccine manufacturers followed with vv + MDV challenge. Statistical analyses showed MD incidence was affected by the genetic line and by vaccination. A line by vaccine interaction was also detected as a significant factor affecting MD incidence in this experiment (P < 0.001). Survival day analyses suggested the overall survived day patterns for all vaccinated groups significantly differed (P < 0.0001) between the genetic lines based on both Log-rank and Wilcoxon tests. The survived day patterns of the vaccinated groups within each line, however, did not differ (P > 0.05). 6.2. MD incidences between the vaccine treatment groups in experiment 1 MD incidences of all vaccinated groups were significantly lower than the unvaccinated control group (P < 0.01) except the HVT vaccinated line 72 groups. MD incidences of both CVI988/Rispens and HVT vaccinated line 63 groups were relatively low and were not significantly different from one another (P > 0.05). The MD incidence of vaccinated line 72 groups varied. The CVI988/Rispens-B group was significantly lower (P < 0.001) than the other line 72 groups. The two HVT groups were higher, and the CVI988/Rispens-A group was in between in MD incidence (Fig. 1). 6.3. Survival day patterns of the two lines of chickens within each treatment group in experiment 1 The survived day patterns of unvaccinated line 63 and 72 groups were significantly different from each other (P < 0.0001; Fig. 2A). All vaccinated groups were significantly different between the two lines (P < 0.001; Fig. 2B–D) except CVI988/Rispens-B groups, which

Fig. 1. MD incidence of experimental line 63 and 72 chickens induced by vv + MDV with or without prior vaccination in experiment 1. HVT from two separate sources (HVT-A and HVT-B) significantly reduced MD incidence as did the two CVI988/Rispens (CVI988/Rispens-A and CVI988/Rispens-B) from two separate companies at manufacturers’ recommended doses in the MD resistant line 63 chickens. CVI988/Rispens also significantly reduced MD incidence in the MD susceptible line 72 chickens. Both HVTs, however, conveyed minimal protection against MD incidence for the line 72 chickens. The histogram bars not sharing a common letter (top of each bar) are significantly different in MD incidence from one other (P < 0.01).


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Fig. 2. Survival plots between the experimental line 63 and line 72 chickens in experiment 1. Based on both Log-rank and Wilcoxon tests, survived day patterns between the experimental line 63 and line 72 were significantly different from each other (plot A: unvaccinated; plot B: CVI988/Rispens-A: P < 0.01; plot C: HTV-A vaccinated, plot D: HVT-B vaccinated, P < 0.0001). The survived day patterns of the CVI988/Rispens-B groups between the two lines were not significantly different (plot not shown, P > 0.05).

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did not differ in survived day patterns (survival plot not shown; P > 0.05). 6.4. Experiment 2 Chickens from two commercial egg-layer (WL and BL) lines with varied MHC haplotype backgrounds were vaccinated at the manufacturers’ recommended doses followed with vv + MDV challenge. Statistical analyses showed MD incidences were not different between the WL and BL lines (P > 0.10) but differed among the vaccine treatment groups (P < 0.0001). Survival day analyses suggested that the survived day patterns significantly differed among vaccinated groups of both lines (P < 0.0001) and among vaccinated groups within each of the commercial lines based on Log-rank and Wilcoxon tests (P < 0.01). 6.5. MD incidences between vaccine treatment groups in experiment 2 All vaccinated groups of the WL and BL lines were significantly lower in MD incidence than the unvaccinated groups (P < 0.01). MD incidences of both WL and BL line chickens vaccinated with CVI988/Rispens-B were the lowest among and significantly differed from all the treatment groups (P < 0.01). The MD incidences of CVI988/Rispens-A, HVT-A, and HVT-B vaccinated groups of both WL and BL lines were not different from one another (P > 0.05; Fig. 3). 6.6. Survival day patterns between vaccinated groups of each commercial line in experiment 2 The survival day patterns of the CVI988/Rispens-B and HVT-B vaccinated WL line were comparable to each other and survived significantly longer than the CVI988/Rispens-A vaccinated WL group (P < 0.01). The survived day pattern of the HVT-A vaccinated WL group lay between the CVI988/Rispens-B and HVT-B groups and the CVI988/Riepens-A group, and was not different statistically from either of the groups (P > 0.05; Fig. 4 top panel). Similar differences

in survived day patterns were observed between vaccinated groups in the BL line except the HVT-B group. The survival day pattern of the HVT-B vaccinated BL group showed relatively poor survival and was comparable with the CVI988/Rispens-A vaccinated group (Fig. 4 bottom panel). 6.7. Experiment 3 To verify the protective efficacy of HVT and CVI988/Rispens at an equal dosage, the experimental line 63 and 72 chickens were vaccinated with CVI988/Rispens-B or HVT-B at a uniform dosage of 2000 PFU per bird followed by the same vv + MDV challenge in experiment 3. Again, statistical analyses showed the overall MD incidence significantly differed between the genetic lines and vaccine groups (P < 0.01). A line by vaccine interaction was also detected, which significantly affected the MD incidence outcomes (P < 0.05) based on effect likelihood ratio tests. Survival day patterns were significantly different among the vaccinated and unvaccinated groups of both lines (P < 0.001). 6.8. MD incidence of the vaccinated and unvaccinated groups in experiment 3 High MD incidences were observed in the unvaccinated groups of both lines 63 and 72 (close to or equal to 100%) and were not significantly different from each other (P > 0.05). The lowest MD incidences were observed in the CVI988/Rispens-B vaccinated line 63 and 72 groups and the HVT-B vaccinated line 63 group, which were similar to one another and significantly different from the unvaccinated groups as well as from the HVT-B vaccinated line 72 group (P < 0.0001; Fig. 5). 6.9. Survived day patterns between chicken lines and vaccine treatment groups in experiment 3 The survived day patterns of the unvaccinated line 63 and 72 groups significantly differed from each other (P < 0.0001; see


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Fig. 3. MD incidence of commercial white egg-layer (WL) line and brown egg-layer (BL) line chickens induced by vv + MDV with or without prior vaccination in experiment 2. CVI988/Rispens-A and both HVTs conveyed comparable protection and significantly reduced MD incidences compared with the unvaccinated groups. CVI988/Rispens-B conveyed significantly better protection in both WL line and BL line chickens and resulted in the lowest MD incidence among the vaccinated groups (P < 0.0001).

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Supplementary Fig. 1 top panel). Line 63 chickens survived more days than line 72 chickens post MDV challenge. The survived day patterns of both CVI988/Rispens-B and HVT-B vaccinated line 63 groups were comparable from each other and survived significantly more days post MDV challenge than the HVT-B vaccinated line 72 group (P < 0.0001). The survived day pattern of CVI988/RispensB vaccinated line 72 chickens lay between the line 63 vaccinated groups and HVT-B vaccinated line 72 group, and was not statistically different from the other groups (Supplementary Fig. 1 bottom panel).

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The estimated protective indices of CVI988/Rispens and HVTs at manufacturer-recommended commercial doses or at a 2000 PFU uniform dose against vv + MD challenge in experiments 1–3 are given in Table 1. At manufacturer-recommended commercial doses, both HVT-A and HVT-B conveyed comparable protection as the CVI988/Rispens-A and CVI988/Rispens-B did in the experimental line 63 chickens. CVI988/Rispens-B conveyed significantly better protection in line 72 chickens than both HTVs and CVI988/RispensA (Table 1, experiment 1). CVI988/Rispens-B clearly conveyed better protection for both WL and BL lines of chickens (75.5 and 92.1, respectively) than the rest of the vaccines (CVI988/RispensA, HVT-A and HVT-B), which all conveyed lower and comparable protection for WL and BL chickens (Table 1, experiment 2). At the 2000 PFU dose, HVT-B again conveyed comparable protection for line 63 chickens as did the CVI988/Rispens-B, but significantly poorer protection for line 72 chickens than the CVI988/Rispens-B (Table 1, experiment 3).

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7. Discussion

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It is common knowledge to the poultry industry and research communities that vaccination is one of the most vital control measures that have been implemented to protect birds against various infectious diseases including MD, which once devastated and still remains a serious threat to the poultry industry worldwide [7]. It is also hard to imagine that the poultry industry could have enjoyed the success and prosperity as it has been during the last

40 years without the invention and proper implementation of vaccines to control a variety of infectious diseases of poultry [40,41]. Yet the challenge for continuation of such successes and, even more, improving the control of infectious diseases remains. For instance, emerging MDV of higher virulence historically overwhelmed MD vaccine protective efficacy and a next generation MD vaccine had to be implemented from time to time to control MD outbreaks [9,42,43]. It is a serious concern that the CVI988/Rispens has been used as one of the last in the commercially available line of effective vaccines against MD induced by MDV of varied virulence. Furthermore, there are no signs indicating that the long lasting evolution of MDV toward higher virulence has stopped or may cease evolving in the near future. Emerging MDV with increasing virulence has the potential to pose catastrophic effects on the poultry industry, food security, and chicken welfare [6,9,42]. The emerging strains of MDV with escalated virulence have appeared to parallel the implementation of different generations of MD vaccines derived from higher virulent MDV [9]. Therefore, it is suggested that the historical implementations of new MD vaccines could be, in turn and in part, the driving force behind the MDV evolution toward higher virulence [6,8]. So far, there has been no obvious and practical solution to this paradox between the virus evolution and use of MD vaccines derived from more pathogenic MDV. This continuous race between MDV, MD vaccine, and host remains a serious challenge for the poultry industry for the foreseeable future [9]. Therefore, efforts on re-evaluation of the current strategies and re-innovation of the infectious disease control system to break the vicious cycle may be worthy of pursuit. We recently showed non-MHC genetic variation, in addition to MHC B haplotypes that were primarily reported by Bacon and Witter in the 1990s [21,27–30], also plays an important role in modulating MD vaccine efficacy in experimental lines of White leghorns under controlled conditions [31–33]. The findings of host genetics background, including both MHC and non-MHC genomics effects, on MD vaccine protective efficacy are in good agreement with reports of studies both in humans and other animals [44,45]. Under controlled conditions, this study using chickens from two experimental lines and two commercial egg-layer lines evaluated MD vaccine protective efficacy at the manufacturers’


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Fig. 4. Survival plots showing survived day patterns between vaccine treatment groups within each commercial egg-layer line. The survival patterns showed the white egg-layer (WL) chickens vaccinated with CVI988/Rispens-B or HVT-B survived more days than the same line of chickens vaccinated with CVI988/Rispens-A (top panel). In the brown egg-layer (BL) groups, however, only the CVI988/Rispens-B group survived significantly more days than the CVI988/Rispens-A and the HVT-B groups (bottom panel) (P < 0.01).

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recommended-doses and at a 2000 PFU uniform dose against MD induced by vv + MDV. Since the MDV used for disease challenge was a highly pathogenic and very virulent plus strain, high MD incidences (close to 100% or 100%, respectively) were induced in the unvaccinated groups of the MD resistant line 63 and susceptible line 72 chickens (Figs. 1 and 5). However, the survived day patterns of the unvaccinated line 63 and line 72 groups significantly differed (P < 0.0001; Fig. 2A and Supplementary Fig. 1 top panel), indicating that the two experimental lines do differ in resistance to MD. The

unvaccinated groups of the two commercial egg-layer lines, WL and BL, lacked significant differences either in MD incidence (Fig. 3) or in survived day patterns (plot not shown). Both HVTs (HVT-A and HVT-B) significantly reduced MD incidence and conveyed comparable protection against the vv + MDV challenge as did both CVI988/Rispens (CVI988/Rispens-A and CVI988/Rispens-B) at the manufacturers’ recommended-doses in the MD resistant line 63 chickens (Fig. 1, Table 1), which was partially replicated in experiment 3 at a uniform 2000 PFU dose for

Table 1 Estimated protective index (%) for each of the vaccine treatment groups challenged with vv + MDV in experiments 1–3. Vaccine treatment group CVI988/Rispens-A CVI988/Rispens-B HVT-A HVT-B a b

Experiment 1a

Experiment 2a

Experiment 3b

Line 63

Line 72

White layer

Brown layer

Line 63

Line 72

68.2 72.5 82.2 84.7

27.6 69.0 0 7.1

45.9 75.5 39.5 53.8

50 92.1 56.8 46

ND 86.7 ND 91.1

ND 80.0 ND 25.0

Dose of vaccination was given following manufacturer recommended commercial dosages. A uniform dose of 2000 PFU was given to each bird for each vaccine.


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Fig. 5. MD incidence of experimental line 63 and 72 chickens vaccinated at a uniform 2000 PFU dose followed by vv + MDV challenge in experiment 3. At a 2000 PFU dose, HVT conveyed comparable protection for the line 63 chickens as did the CVI988/Rispens-B for both line 63 and line 72 chickens and resulted in low MD incidence significantly different from the unvaccinated groups and the HVT-B vaccinated line 72 group. The histogram bars not sharing a common letter (top of each bar) are significantly different in MD incidence from one other (P < 0.001).

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both CVI988/Rispens-B and HVT-B and resulted in comparable protection (Fig. 5, Table 1). However, the CVI988/Rispens-B conveyed significantly better protection than the CVI988/Ripens-A and both HVTs in both of the commercial egg-layer lines, WL and BL (Fig. 3, Table 1). We were aware that the titer of the CVI988/Rispens-B per recommended-dose was the highest, close to twice as much higher than that of the CVI988/Rispens-A, and about three times higher than the HVTs’. However, no superior protection was observed in the experimental line 63 chickens vaccinated by the CVI988Rnspens-B in experiments 1 and 3 (Figs. 1 and 5; Table 1). This suggests a complicated system underlies vaccinal immunity, and host genetic background, including both MHC and genes located outside of MHC regions, is indeed heavily involved in modulation of vaccine protective efficacy. Genetic and epigenetic bases underlying vaccinal immunity are vaguely understood. The highly inbred experimental lines 63 and 72 share a common MHC B*2 haplotype but differ in genomics contents outside of the MHC region. The striking difference in CVI988/Rispens and HVT protective efficacy between the line 63 and line 72 chickens should be attributable to the host genetic and, perhaps, also epigenetic variation outside of the MHC region. Identifying genetic and epigenetic factors underlying the differences in vaccinal protective efficacy between the two genetic lines of chickens should advance the basic understanding in genetic and epigenetic mechanisms modulating vaccinal immunity and, consequently protective efficacy. This effort is ongoing. In summary, this study provides one more piece of experimental evidence that host genetics plays an important role in vaccinal protective efficacy. The host genetic variation modulating vaccinal protective efficacy should include MHC and non-MHC genomic contents. Thus, it is evident that vaccine protective efficacy in general is modulated by factors including the types and the sources of vaccines and the genetic backgrounds of chickens. While CVI988/Rispens, the industry-recognized golden-standard MD vaccine, protects relatively well across different lines of chickens of varied genetic background (at least CVI988/Rispens-B did in this study), HVT also conveyed comparable protection even against the vv + MDV challenge in lines of chickens relatively resistant to MD. In vaccination practice, should HVT only be selectively used to vaccinate lines of chickens that are known relatively resistant to

MD, like the line 63 or even the WL and BL lines, to minimize the cost and to maximize the protection of vaccination? Results from this study may provide veterinarians with evidence for considering alternative strategies for selection and use of vaccines. Author’s contributions SC and QX conducted the experiments, analyzed the data and drafted the manuscript. JRD performed necropsy, pathological analyses and revised the manuscript. CWE coordinated and supported the challenge experiments and revised the manuscript. JS supported data analyses and revised the manuscript. HZ conceived and designed the vaccination and challenge experiments, supervised the project, edited and finalized the manuscript. All authors provided input in interpretation and presentation of the results, critically revised and approved the final manuscript. Conflicts of interest statement The authors declare there are no conflicts of interest. Acknowledgments The authors thank Bernice Li for her technical assistance in conducting the challenge trials. This work was supported in part by a USDA AFRI grant (2010-65205-20588) and a Specific Cooperative Agreement (3635-31320-009-04S) between USDA-Agriculture Research Service and Michigan State University. Appendix A. Supplementary data Supplementary material related to this article can be found, in the online version, at http://dx.doi.org/10.1016/j.vaccine. 2014.01.092. References [1] Osterrieder N, Kamil JP, Schumacher D, Tischer BK, Trapp S. Marek’s disease virus: from miasma to model. Nat Rev Microbiol 2006;4(4):283–94.


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Horizontal transmission of turkey herpesvirus to chickens. 3. Transmission in three different lines of chickens.

Safety and efficacy of a turkey herpesvirus vector laryngotracheitis vaccine for chickens.

Horizontal transmission of turkey herpesvirus to chickens. 5. Airborne transmission between chickens.

Vaccination of chickens against Marek's disease with the turkey herpesvirus vaccine using a pneumatic vaccinator.

[Vaccination of field and experimental flocks with lyophilized turkey herpesvirus: antibody response of vaccinated chickens (author's transl)].

Effects of strain of chickens and vaccination with turkey herpesvirus on Marek's disease and lymphoid leukosis in breeding stocks.

Effect of aflatoxin B1 on the efficacy of turkey herpesvirus vaccine against Marek's disease.

Application of the cell-free turkey herpesvirus vaccine by the aerosol route for the prevention of Marek's disease in chickens.

Marek's disease in commercial turkey flocks.

Host resistance mechanisms to Newcastle disease virus in immunodeficient chickens (38540).

Interaction of adenine arabinoside and host defense factors in experimental infections due to Herpesvirus hominis.

Integrated genomic approaches to enhance genetic resistance in chickens.

Differential resistance to Staphylococcus aureus challenge in two related lines of chickens.

Efficacy of five commercial disinfectants and one anionic surfactant against equine herpesvirus type 1.

Protective effects of genetic inhibition of Discoidin Domain Receptor 1 in experimental renal disease.

A molecular analysis of the patterns of genetic diversity in local chickens from western Algeria in comparison with commercial lines and wild jungle fowls.

Studies of Marek's disease herpesvirus and turkey herpesvirus specific common antigen which stimulates the production of neutralizing antibodies.

Genetic factors in susceptibility and resistance to experimental autoimmune uveoretinitis.

New Insight for the Genetic Evaluation of Resistance to Ostreid Herpesvirus Infection, a Worldwide Disease, in Crassostrea gigas.

Efficacy of turkey herpesvirus vaccine when administered simultaneously with fowl pox vaccine.

Vaccination of chickens against coccidiosis ameliorates drug resistance in commercial poultry production.

Genetic basis of leg health and its relationship with body weight in purebred turkey lines.

Protective Efficacy of Recombinant Turkey Herpes Virus (rHVT-H5) and Inactivated H5N1 Vaccines in Commercial Mulard Ducks against the Highly Pathogenic Avian Influenza (HPAI) H5N1 Clade 2.2.1 Virus.

Outbreaks of Virulent Infectious Bursal Disease in Flocks of Battery Cage Brooding System of Commercial Chickens.