Improvements to the hemagglutination inhibition test for serological assessment of recombinant fowlpox-H5-avian-influenza vaccination in chickens and its use along with an agar gel immunodiffusion test for differentiating infected from noninfected vaccinated animals

In general, avian influenza (AI) vaccines protect chickens from morbidity and mortality and reduce, but do not completely prevent, replication of wild AI viruses in the respiratory and intestinal tracts of vaccinated chickens. Therefore, surveillance programs based on serological testing must be developed to differentiate vaccinated flocks infected with wild strains of AI virus from noninfected vaccinated flocks in order to evaluate the success of vaccination in a control program and allow continuation of national and international commerce of poultry and poultry products. In this study, chickens were immunized with a commercial recombinant fowlpox virus vaccine containing an H5 hemagglutinin gene from A/turkey/Ireland/83 (H5N8) avian influenza (AI) virus (rFP-H5) and evaluated for correlation of immunological response by hemagglutination inhibition (HI) or agar gel immunodiffusion (AGID) tests and determination of protection following challenge with a high pathogenicity AI (HPAI) virus. In two different trials, chickens immunized with the rFP-H5 vaccine did not develop AGID antibodies because the vaccine lacks AI nucleoprotein and matrix genes, but 0%-100% had HI antibodies, depending on the AI virus strain used in the HI test, the HI antigen inactivation procedure, and whether the birds had been preimmunized against fowlpox virus. The most consistent and highest HI titers were observed when using A/turkey/Ireland/83 (H5N8) HPAI virus strain as the beta-propiolactone (BPL)-inactivated HI test antigen, which matched the hemagglutinin gene insert in the rFP-H5 vaccine. In addition, higher HI titers were observed if ether or a combination of ether and BPL-inactivated virus was used in place of the BPL-inactivated virus. The rFP-H5 vaccinated chickens survived HPAI challenge and antibodies were detected by both AGID and HI tests. In conclusion, we demonstrated that the rFP-H5 vaccine allowed easy serological differentiation of infected from noninfected birds in vaccinated populations of chickens when using standard AGID and HI tests.     

Use of a tetanus toxoid marker to allow differentiation of infected from vaccinated poultry without affecting the efficacy of a H5N1 avian influenza virus vaccine

Tetanus toxoid (TT) was assessed as a positive marker for avian influenza (AI) virus vaccination in chickens, in a vaccination and challenge study. Chickens were vaccinated twice with inactivated AI H5N2 virus vaccine, and then challenged three weeks later with highly pathogenic AI H5N1 virus. Vaccinated chickens were compared with other groups that were either sham-vaccinated or vaccinated with virus with the TT marker. All sham-vaccinated chickens died by 36 hours postinfection, whereas all vaccinated chickens, with or without the TT marker, were protected from morbidity and mortality following exposure to the challenge virus. Serological testing for H5-specific antibodies identified anamnestic responses to H5 in some of the vaccinated birds, indicating active virus infection.     

Influence of maternal immunity on vaccine efficacy and susceptibility of one day old chicks against Egyptian highly pathogenic avian influenza H5N1

In Egypt, continuous circulation of highly pathogenic avian influenza (HPAI) H5N1 viruses of clade 2.2.1 in vaccinated commercial poultry challenges strenuous control efforts. Here, vaccine-derived maternal AIV H5 specific immunity in one-day old chicks was investigated as a factor of vaccine failure in long-term blanket vaccination campaigns in broiler chickens. H5 seropositive one-day old chicks were derived from breeders repeatedly immunized with a commercial inactivated vaccine based on the Potsdam/H5N2 strain. When challenged using the antigenically related HPAIV strain Italy/98 (H5N2) clinical protection was achieved until at least 10 days post-hatch although virus replication was not fully suppressed. No protection at all was observed against the Egyptian HPAIV strain EGYvar/H5N1 representing a vaccine escape lineage. Other groups of chicks with maternal immunity were vaccinated once at 3 or 14 days of age using either the Potsdam/H5N2 vaccine or a vaccine based on EGYvar/H5N1. At day 35 of age these chicks were challenged with the Egyptian HPAIV strain EGYcls/H5N1 which co-circulates with EGYvar/H5N1 but does not represent an antigenic drift variant. The Potsdam/H5N2 vaccinated groups were not protected against EGYcls/H5N1 infection while, in contrast, the EGYvar/H5N1 vaccinated chicks withstand challenge with EGYvar/H5N1 infection. In addition, the results showed that maternal antibodies could interfere with the immune response when a homologous vaccine strain was used.     

种鸡接种网状内皮组织增生病病毒弱毒疫苗的安全性和免疫效果观察

在正常饲养条件下，在肉种鸡鸡群中试用网状内皮增生病病毒（reticuloendotheliosis virus，REV）的弱毒疫苗，观察其对体重增长、产蛋生产性能、对其他疫苗应答有无影响。同时连续定期测定种鸡血清REV抗体，并测试抗体阳性鸡的后代有无病毒垂直传播。结果表明，该疫苗接种18周龄种鸡后，对生长、产蛋率、受精率和孵化率等生产性能均无不良影响，对正常疫苗免疫的抗体应答也无影响。经免疫接种REV弱毒疫苗的种鸡，在开产后及产蛋高峰期，均不表现病毒的垂直传播。免疫种鸡后，其激发的抗体可持续280d以上，且雏鸡血清中母源抗体可持续至少7d。结果表明，该REV弱毒在开产前种鸡应用时有很高的安全性，并能为雏鸡提供足够的特异性母源抗体。     

Consequence of Cryptosporidiosis on the immune response of vaccinated broiler chickens against Newcastle disease and/or avian influenza

The consequence of cryptosporidiosis on the immune response of vaccinated chickens against Newcastle disease and/or avian influenza was studied by using 240, 1 day old, male, white Hy-Line chicks and divided into 8 groups and subgroups. Each group or subgroup was consisting of 30 chicks (15?×?2 replicates). The first and second groups were kept as unvaccinated control, G1uninfected and G2 infected. G3, G4 and G5 contained 2 subgroups A&B (G3A, G3B, G4A, G4B, G5A and G5B). Chicks of subgroup A were vaccinated only while chicks of subgroup B were infected and vaccinated. These chicks were orally inoculated with 5?×?10⁵ oocysts of Cryptosporidium baileyi (C. baileyi) at 2 days of age. Chickens were vaccinated intraocular with live Newcastle disease (ND) vaccine (Hitchner on day 7th and LaSota on day 17th of chicken life) (G3) or vaccinated by subcutaneous route with Volvac®- H5N2- AI vaccine on day 10 of chicken life (G4). Last group (G5) was infected similarly and vaccinated with ND and AI vaccines with the same day, dose and route of vaccination for each one. Random blood samples were collected for 3 weeks post-vaccination for investigation of humoral immune response against Newcastle and/or avian influenza vaccines by the haemagglutination inhibition (HI) test. The results showed that H5N2 vaccine at day 10 of chicken life is effective in chickens indicated by the geometric mean of HI titer against AI virus. The findings of this study showed that the infection with Cryptosporidia in the broiler chicken has a depressive effect on the immune status of the birds vaccinated against ND and/or AI vaccination. Moreover, the obtained protection rates against challenge with virulent ND virus observed to be parallel to the results of HI- test. Also, by using 2 different antigens (one commercial and field prepared antigen) to avian influenza virus, lower Geometric mean (GM) HI titer were appeared in infected and vaccinated group than vaccinated group only. A study of the relative lymphoid organs weight such as bursa of Fabricius from the experimental chicks indicated that those organs were comparable between the groups infected-vaccinated and vaccinated only. Non significant variations in final live weight between uninfected control and infected groups were indicated. Also, H5N2-AI vaccination at 10 days old did not affect the final live weight. ND and/or AI Vaccination could not be a substitute to application of good hygienic measures and fecal examination of the birds especially for protozoal diseases such as cryptosporidiosis. It could be concluded that cryptosporidiosis could be one cause of ND and/or AI vaccination failure in poultry farms.     

不同规模养鸡场禽流感灭活疫苗免疫效果的调查

对不同规模养鸡场接种禽流感疫苗的免疫效果进行了调查,结果表明:大中型种鸡场的禽流感抗体水平较高,群体几何平均效价为27.4,免疫保护率平均达93.3%。农村小型肉鸡场的禽流感抗体水平较低,群体几何平均效价为23.5,免疫保护率平均达37.2%。农村散养鸡群的禽流感抗体水平最低,群体几何平均效价为21.8,免疫保护率平均达26.1%。同时,对不同免疫状况下,鸡群接种禽流感疫苗后免疫抗体的消长动态进行了测定。     

Avian influenza adenovirus-vectored in ovo vaccination: target embryo tissues and combination with Marek's disease vaccine

We investigated embryo tissues targeted by replication competent adenovirus (Ad)-free recombinant Ad expressing a codon-optimized avian influenza (AI) H5 gene from A/turkey/WI/68 (AdH5) when injected into 18-day embryonated eggs. We also evaluated the effects of concurrent in ovo vaccination with the experimental AdH5 vaccine and commercially available Marek's disease virus (MDV) vaccine combinations Rispens/turkey herpesvirus (HVT) or HVT/SB-1. Computed tomography indicates that in ovo injection on day 18 of incubation places the solution in the amnion cavity, allantoic cavity, or both. Ad DNA was consistently detected in the chorioallantoic membranes as well as in the embryonic bursa of Fabricius, esophagus, and thymus 3 days postinoculation. H5 expression in these tissues also was detected by immunofluorescence assay. These results indicate possible swallowing of vaccine virus contained in the amnion. In contrast, vaccine localization in the allantoic fluid would have allowed bursal exposure through the cloaca. When the AdH5 vaccine was used in combination with MDV, chickens responding to the AdH5 vaccine had similar AI antibody levels compared with AdH5-only-vaccinated birds. However, combined vaccinated groups showed reduced vaccine coverage to AI, suggesting some level of interference. The combination of AdH5 with MDV Rispens/HVT affected the vaccine coverage to AI more severely. This result suggests that the replication rate of the more aggressive Rispens strain of serotype 1 may have interfered with the Ad-vectored vaccine. Increasing the Ad concentration produced similar AI antibody titers and AI vaccine coverage when applied alone or in combination with the HVT/SB-1 vaccine. Ad DNA was detected in hatched chickens 2 days after hatch but was undetectable on day 9 after hatch. MDV DNA was detected in feather follicles of all vaccinated birds at 12 days of age. Thus, Ad-vector vaccination does not interfere with the efficacy of MDV vaccination by using any of the commonly used vaccine strains.     

母源抗体对同源及异源禽网状内皮增生病病毒感染诱发免疫抑制的预防作用

蛋用型海兰褐母鸡在用网状内皮增生病病毒（REV）细胞适应毒REV-C99（p30）免疫接种后,均在2周内产生REV特异性抗体。来自免疫种鸡的雏鸡在7日龄内100%（10/10）REV母源抗体阳性。分别在有母源抗体和没有母源抗体的1日龄鸡接种与疫苗株同源的低传代毒REV-C99（p3）或异源的REV中国野毒株HA9901（p5）,以此比较母源抗体对同源和异源REV病毒感染造成的免疫抑制的预防作用。结果表明,REV母源抗体能同等有效地预防同源和异源REV感染造成的对H5和H9禽流感灭活疫苗HI抗体反应的抑制作用。     

Needle-free delivery of an inactivated avian influenza H5N3 virus vaccine elicits potent antibody responses in chickens

A needle-free delivery system was assessed as a route for providing quick, safe, and effective vaccination against avian influenza (AI). Two groups of chickens were vaccinated with a commercially available inactivated H5N3 virus vaccine delivered either with a needle-free device or with the conventional syringe-and-needle method recommended by the vaccine manufacturer. The kinetic aspects of seroconversion, peak antibody levels, and antibody titers were measured by a combination of an indirect enzyme-linked immunosorbent assay and the hemagglutination-inhibition test and were all found to be similar in the 2 groups of chickens. We conclude that the needle-free delivery system could result in effective immunization against H5N1 AI epidemics and pandemics in chickens.     

鸡新城疫—传染性支气管炎—禽流感(H9N2)三联灭活疫苗的安全性研究

为证实鸡新城疫—传染性支气管炎—禽流感(新支流)三联灭活油乳疫苗的安全性,通过颈部皮下注射接种14日龄来航鸡,每日测定鸡的体重,连续监测28 d,用t检验分析对照组和免疫组体重之间的差异显著性。试验结果表明,免疫接种组和空白对照组体重差异不显著,所制备的新支流三联疫苗不影响鸡的生长性能,对鸡安全。     

Evaluation of the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 influenza viruses in chickens

Despite the long-term vaccination programs implemented in China, H9N2 avian influenza viruses (AIVs) continue to persist in chicken populations, even in vaccinated flocks. We previously demonstrated that H9N2 AIV isolated from chickens in China also underwent antigenic drift and evolved into distinct antigenic groups (C, D and E). To understand whether antigenic drift of viruses away from the vaccine strain partially contributed to the circulation of H9N2 AIV in China, we evaluated the protective efficacy of a commercial vaccine against different antigenic groups of H9N2 AIV. Challenge experiments using vaccinated chickens indicated that the vaccine prevented shedding of antigenic group C viruses, but not those of the more recent groups D and E. Vaccinated chickens, even those with vaccine-induced HI titers of 1:1024, shed virus after being infected with A/chicken/Shandong/ZB/2007, a representative virus of antigenic group D. Genetic analysis showed that the representative viruses of antigenic groups D and E possessed greater numbers of amino acid substitutions in the hemagglutinin protein compared to the vaccine strain and the antigenic group C virus, and many of which were located in antigenic sites. Our results indicated that the persistence of H9N2 AIV in China might be due to incomplete vaccine protection, and that the avian influenza vaccine should be regularly evaluated and updated to maintain optimal protection. Furthermore, the avian influenza vaccination policy also needs to be re-assessed, and increased veterinary biosecurity on farms, rather than vaccine application alone, should be implemented to prevent and control avian influenza.     

H9N2 AIV HA蛋白S145N变异毒株的抗原性和免疫原性

在研究1998-2008年中国H9N2亚型禽流感病毒（AIV）分离株HA基因的进化时,发现在25个毒株中有2个致病性最强的毒株因HA基因第145位氨基酸的突变导致产生1个潜在的糖基化位点,从而使其不与单抗H6、F6等反应。为进一步探究这类变异毒株HA基因变异对H9亚型AIV的抗原性和免疫原性的影响,本试验对12株HA蛋白S145N变异的H9N2AIV进行了交叉中和试验和交叉攻毒试验。结果显示,不同H9N2S145N变异株与疫苗株间在抗原性上变化不大,或无显著差异（0.5≤R≤0.67）。但参照现有的H9灭活疫苗效力检验方法对HP疫苗免疫鸡进行攻毒,用HP株攻毒对照组0/5保护,免疫组保护≥9/10,达到了H9灭活疫苗质量标准要求;但用S145N变异株N3攻毒,仅保护2/10～6/10,且随免疫量剂量的增加,抗体水平的提高,攻毒保护也依次升高。对H9变异株疫苗（N1、N2、N3、N8）免疫鸡用N3攻毒,仅保护2/5～4/5,N3同源抗体也不能有效地阻止其攻毒后的排毒。用N3、N6 2个变异株交叉攻毒,采用与疫苗株攻毒相同的剂量作攻毒试验也得到类似结果。表明高于6log2的抗体能抵抗疫苗株和大多数流行毒株攻毒后的排毒,但不能抵抗S145N变异株攻毒后的排毒。这类毒株免疫原性上的变化与病毒HA基因的变异密切相关。因HA基因145～147aa位增加了1个NGT,导致三维空间构象的变化,并影响其邻近的受体结合位点,从而使这类毒株致病性提高,免原性发生改变。虽然这一类变异株或免疫逃逸毒株仅占当前流行毒株总数的5%～7%,但在强大的免疫压力和自然选择下有可能逐步成为优势毒株,造成更大的危害,这为该病的防控提出了新的挑战。     

Protection of chickens against highly pathogenic avian influenza virus (H5N2) by recombinant fowlpox viruses.

Two recombinant fowlpox viruses containing the avian influenza H5 hemaglutinin (HA) gene were evaluated for their ability to protect chickens against challenge with a highly pathogenic isolate of avian influenza virus (H5N2). Susceptible chickens were vaccinated with the parent fowlpox vaccine virus or recombinant viruses either by wing-web puncture or comb scarification. Following challenge 4 weeks later with highly pathogenic avian influenza virus, all birds vaccinated by the wing-web method were protected by both recombinants, while 50% and 70% mortality occurred in the two groups of birds vaccinated by comb scarification. Birds vaccinated with the unaltered parent fowlpox vaccine virus or unvaccinated controls experienced 90% and 100% mortality, respectively, following challenge. Hemagglutination-inhibition (HI) antibody levels were low, and agar-gel precipitin results were negative before challenge. Very high HI titers and positive precipitating antibody responses were observed in all survivors following challenge.     

禽流感油乳剂灭活疫苗的研制

以禽流感病毒（AIV）H5N4株，H7N3株为抗原，分别研制了H5N4、H7N3油乳剂灭活疫苗，并对其物理性状、安全性、免疫效力、保存期及抗体消长规律进行了检测。结果表明，3种抗原含量不同的H5N4疫苗在免疫后3周－9个月对AIV－H5N4攻击均获8／8保护，H7N3疫苗在免疫后3周与3个月时对AIV－H5N4攻击则分别保护6／8与3／7，疫苗4℃保存15个月，其免疫效力没有下降。     

Intraocular vaccination with an inactivated highly pathogenic avian influenza virus induces protective antibody responses in chickens

Because it is expected to induce cross-reactive serum and mucosal antibody responses, mucosal vaccination against highly pathogenic avian influenza (HPAI) is potentially superior to conventional parenteral vaccination. Here, we tested whether intraocular vaccination with an inactivated AI virus induced protective antibody responses in chickens. Chickens were inoculated intraocularly twice with 10⁴ hemagglutination units of an inactivated H5N1 HPAI virus. Four weeks after the second vaccination, the chickens were challenged with a lethal dose of the homologous H5N1 HPAI virus. Results showed that most of the vaccinated chickens mounted positive antibody responses. The median serum hemagglutination inhibition titer was 1:80. Addition of CpG oligodeoxynucleotide 2006 or cholera toxin to the vaccine did not enhance serum antibody titers. Cross-reactive anti-hemagglutinin IgG, but not IgA, was detected in oropharyngeal secretions. In accordance with these antibody results, most vaccinated chickens survived a lethal challenge with the H5N1 HPAI virus and did not shed the challenge virus in respiratory or digestive tract secretions. Our results show that intraocular vaccination with an inactivated AI virus induces not only systemic but also mucosal antibody responses and confers protection against HPAI in chickens.     

Comparative susceptibility of chickens and turkeys to avian influenza A H7N2 virus infection and protective efficacy of a commercial avian influenza H7N2 virus vaccine

During the spring of 2002, a low pathogenic avian influenza (LPAI) A (H7N2) virus caused a major outbreak among commercial poultry in Virginia and adjacent states. The virus primarily affected turkey flocks, causing respiratory distress and decreased egg production. Experimentally, turkeys were more susceptible than chickens to H7N2 virus infection, with 50% bird infectious dose titers equal to 10(0.8) and 10(2.8-3.2), respectively. Comparison of virus shedding from the cloaca and oropharynx demonstrated that recent H7N2 virus isolates were readily isolated from the upper respiratory tract but rarely from the gastrointestinal tract. The outbreak of H7N2 virus raised concerns regarding the availability of vaccines that could be used for the prevention and control of this virus in poultry. We sought to determine if an existing commercial avian influenza (AI) vaccine prepared from a 1997 seed stock virus could provide protection against a 2002 LPAI H7N2 virus isolated from a turkey (A/turkey/Virginia/158512/02 [TV/02]) in Virginia that was from the same lineage as the vaccine virus. The inactivated AI vaccine, prepared from A/chicken/ Pennsylvania/21342/97 (CP/97) virus, significantly reduced viral shedding from vaccinated turkeys in comparison with sham controls but did not prevent infection. The protective effect of vaccination correlated with the level of virus-specific antibody because a second dose of vaccine increased antiviral serum immunoglobulin G and hemagglutination inhibition (HI) reactivity titers in two different turkey age groups. Serum from CP/97-vaccinated turkeys reacted equally well to CP/97 and TV/02 antigens by HI and enzyme-linked immunosorbent assay. These results demonstrate the potential benefit of using an antigenically related 1997 H7N2 virus as a vaccine candidate for protection in poultry against a H7N2 virus isolate from 2002.     

禽流感疫苗免疫方法对北京油鸡生长发育、免疫效力和屠体性状的影响

本研究主要比较不同禽流感免疫方法对北京油鸡生长发育、免疫效力和屠体性状的影响。试验选用180只1日龄肉用型北京油鸡公鸡,随机分为6个处理组,每组30只,每10只为1个重复。禽流感疫苗H5N1来自2个厂家,注射部位有颈部皮下、胸肌、腿肌3个部位,注射剂量有0.3mL、0.5mL两种。测定0、8、16周龄时鸡只个体重,7周龄ND、H9抗体效价,9、14周龄H5、新城疫(ND)和H9抗体效价,以及16周末胸肌率、腿肌率、腹脂率的变化。结果表明:①禽流感疫苗颈部皮下注射可以获得和胸肌注射、腿肌注射相似的免疫效力,同时应激降低;②禽流感二次重免时可适当降低免疫剂量,即2周龄采用H5N10.3mL,3周龄采用ND-H9二联苗0.3mL,5周龄采用H5N10.3mL,均采取颈部皮下注射方法,可获得较好的免疫效力;③较高的禽流感免疫效力往往伴随着屠体性状的降低。     

三种禽流感灭活疫苗对农村散养鸭的免疫效果比较

采用重组禽流感灭活疫苗(H5N1亚型,Re-1株)、禽流感灭活疫苗(H5亚型,N28株)、禽流感H5-H9二价灭活疫苗三种疫苗免疫农村散养鸭,结果表明,在一次免疫和加强免疫中,重组禽流感灭活疫苗、禽流感灭活疫苗的抗体效价都分别明显高于H5-H9二价灭活疫苗同期的H5抗体效价,因此对水禽免疫宜使用重组禽流感灭活疫苗或禽流感灭活疫苗;加强免疫明显比一次免疫效果好,应用禽流感灭活苗对散养鸭免疫,必须加强方可达到理想的免疫效果。     

Pandemic H1N1 influenza virus in Chilean commercial turkeys with genetic and serologic comparisons to U.S. H1N1 avian influenza vaccine isolates

Beginning in April 2009, a novel H1N1 influenza virus caused acute respiratory disease in humans, first in Mexico and then around the world. The resulting pandemic influenza A H1N1 2009 (pH1N1) virus was isolated in swine in Canada in June 2009 and later in breeder turkeys in Chile, Canada, and the United States. The pH1N1 virus consists of gene segments of avian, human, and swine influenza origin and has the potential for infection in poultry following exposure to infected humans or swine. We examined the clinical events following the initial outbreak of pH1N1 in turkeys and determined the relatedness of the hemagglutinin (HA) gene segments from the pH1N1 to two H1N1 avian influenza (AI) isolates used in commercial turkey inactivated vaccines. Overall, infection of turkey breeder hens with pH1N1 resulted in -50% reduction of egg production over 3-4 weeks. Genetic analysis indicated one H1N1 AI vaccine isolate (Alturkey/North Carolina/17026/1988) contained approximately 92% nucleotide sequence similarity to the pH1N1 virus (A/Mexico/4109/2009); whereas, a more recent AI vaccine isolate (A/ swine/North Carolina/00573/2005) contained 75.9% similarity. Comparison of amino acids found at antigenic sites of the HA protein indicated conserved epitopes at the Sa site; however, major differences were found at the Ca2 site between pH1N1 and A/ turkey/North Carolina/127026/1988. Hemagglutinin-inhibition (HI) tests were conducted with sera produced in vaccinated turkeys in North Carolina to determine if protection would be conferred using U.S. AI vaccine isolates. HI results indicate positive reactivity (HI titer > or = 5 log2) against the vaccine viruses over the course of study. However, limited cross-reactivity to the 2009 pH1N1 virus was observed, with positive titers in a limited number of birds (6 out of 20) beginning only after a third vaccination. Taken together, these results demonstrate that turkeys treated with these vaccines would likely not be protected against pH1N1 and current vaccines used in breeder turkeys in the United States against circulating H1N1 viruses should be updated to ensure adequate protection against field exposure.     

Characterization of recombinant raccoonpox vaccine vectors in chickens

Raccoonpox virus (RCN) has been used as a recombinant vector against several mammalian pathogens but has not been tested in birds. The replication of RCN in chick embryo fibroblasts (CEFs) and chickens was studied with the use of highly pathogenic avian influenza virus H5N1 hemagglutinin (HA) as a model antigen and luciferase (luc) as a reporter gene. Although RCN replicated to low levels in CEFs, it efficiently expressed recombinant proteins and, in vivo, elicited anti-HA immunoglobulin yolk (IgY) antibody responses comparable to inactivated influenza virus. Biophotonic in vivo imaging of 1-wk-old chicks with RCN-luc showed strong expression of the luc reporter gene lasting up to 3 days postinfection. These studies demonstrate the potential of RCN as a vaccine vector for avian influenza and other poultry pathogens.