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.     

Comparison of electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus and virulent avian influenza virus

Electrocardiograms of chickens infected with viscerotropic velogenic Newcastle disease virus (NDV) or virulent avian influenza virus (AIV) were characterized and compared. The ECG were monitored by radiotelemetry and were recorded twice daily before virus infection and during the course of the infection. Thirteen lead II intervals, segments, and amplitudes were measured and analyzed. The ECG of NDV-infected chickens were characterized by lengthened (P less than or equal to 0.05) ST segments and increased (P less than or equal to 0.05) P amplitudes. The ECG of AIV-infected chickens were characterized by lengthened (P less than or equal to 0.05) RS intervals, ST segments, TP intervals, and PR segments and by increased (P less than or equal to 0.05) P amplitudes. The TP intervals and PR segments of ECG of AIV-infected chickens were significantly (P less than or equal to 0.05) longer than those of NDV-infected chickens. The pronounced conduction delays indicated in the ECG of AIV-infected chickens may have diagnostic importance.     

DNA vaccine encoding avian influenza virus H5 and Esat-6 of Mycobacterium tuberculosis improved antibody responses against AIV in chickens

The H5 gene of avian influenza virus (AIV) strain A/chicken/Malaysia/5744/2004(H5N1) was cloned into pcDNA3.1 vector, and Esat-6 gene of Mycobacterium tuberculosis was fused into downstream of the H5 gene as a genetic adjuvant for DNA vaccine candidates. The antibody level against AIV was measured using enzyme-linked immunosorbent assay (ELISA) and haemagglutination inhibition (HI) test. Sera obtained from specific-pathogen-free chickens immunized with pcDNA3.1/H5 and pcDNA3.1/H5/Esat-6 demonstrated antibody responses as early as 2 weeks after the first immunization. Furthermore, the overall HI antibody titer in chickens immunized with pcDNA3.1/H5/Esat-6 was higher compared to the chickens immunized with pcDNA3.1/H5 (p < 0.05). The results suggested that Esat-6 gene of M. tuberculosis is a potential genetic adjuvant for the development of effective H5 DNA vaccine in chickens.     

Evaluation of a competitive ELISA for antibody detection against avian influenza virus

Active serologic surveillance is necessary to control the spread of the avian influenza virus (AIV). In this study, we evaluated a commercially-available cELISA in terms of its ability to detect AIV antibodies in the sera of 3,358 animals from twelve species. cELISA detected antibodies against reference H1- through H15-subtype AIV strains without cross reactivity. Furthermore, the cELISA was able to detect antibodies produced following a challenge of the AIV H9N2 subtype in chickens, or following vaccination of the AIV H9 or H5 subtypes in chickens, ducks and geese. Next, we tested the sensitivity and specificity of the cELISA with sera from twelve different animal species, and compared these results with those obtained by the hemagglutination-inhibition (HI) test, the "gold standard" in AIV sera surveillance, a second commercially-available cELISA (IZS ELISA), or the agar gel precipitation (AGP) test. Compared with the HI test, the sensitivities and specificities of cELISA were 95% and 96% in chicken, 86% and 88% in duck, 97% and 100% in turkey, 100% and 87% in goose, and 91% and 97% in swine, respectively. The sensitivities and specificities of the cELISA in this study were higher than those of IZS ELISA for the duck, turkey, goose, and grey partridge sera samples. The results of AGP test against duck and turkey sera also showed significant correlation with the results of cELISA (R-value >0.9). In terms of flock sensitivity, the cELISA correlated better with the HI test than with commercially-available indirect ELISAs, with 100% flock sensitivity.     

不同剂型新城疫-禽流感二联灭活疫苗免疫SPF鸡后抗体水平对比试验

不同剂型的佐剂对疫苗的免疫效果有较大的影响,本试验旨在对比不同剂型佐剂的新城疫-禽流感(简称"新-流")二联灭活疫苗的免疫效果,从而为生产上选择最佳剂型佐剂应用于新城疫-禽流流感二联灭活疫苗提供依据。分别将油包水型、水包油型、水包油包水型佐剂与新城疫及禽流感灭活抗原乳化成不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗,将这三种不同剂型的新城疫-禽流感(H9亚型,HP株)二联灭活疫苗分别免疫一组7日龄SPF鸡。每羽颈部皮下注射0.2 m L,同时设一组未免7日龄SPF鸡作为对照组,各免疫组与对照组SPF鸡于免疫组免后6、10、15、21、28、35 d进行采血,检测新城疫与禽流感抗体水平。结果表明:免疫油包水型疫苗组SPF鸡免疫后新城疫与禽流感抗体上升最快,在免后10、15、21、28、35 d的新城疫与禽流感抗体也最高,其次是免疫水包油包水型的,而免疫水包油型疫苗组的SPF鸡新城疫与禽流感抗体上升最慢,而且在免后10、15、21、28、35 d的新城疫与禽流感抗体也最低。此外,从各免疫组可以看出,在免后6 d时新城疫抗体已开始产生,在1log2以下,而禽流感抗体仍未产生。可见免疫新-流二联苗后,新城疫抗体比禽流感抗体更早产生,油包水型新-流二联苗的免后抗体高于水包油包水及水包油型,而且能持续刺激机体产生高水平抗体,更具优势。     

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.     

不同免疫程序的鸡群新城疫和禽流感抗体水平与免疫保护之间的相关性试验

新城疫（Newcastle disease，ND）是由新城疫病毒（NDV）引起的禽类的一种急性高度接触性传染病，禽流感（avian influenza，AI）是由A型流感病毒中一些血清亚型毒株引起各种家禽及野生禽类的全身性和／或呼吸系统综合征的传染病，两种疫病均给养禽业造成巨大的经济损失，实施严格的生物安全措施和在流行地区使用疫苗是控制两种疫病的主要方法。     

抗禽流感病毒NS1蛋白单克隆抗体的制备及其抗原表位的初步分析

为制备抗禽流感病毒(AIV)NS1蛋白的单克隆抗体(MAb)及鉴定其抗原表位鉴定,本研究以原核表达并纯化的NS1重组蛋白免疫BALB/c小鼠,采用淋巴细胞杂交瘤技术制备杂交瘤细胞,通过间接ELISA进行筛选,制备的D7和D9 2株能够稳定分泌抗NS1蛋白MAb的杂交瘤细胞,亚型鉴定均为IgG1型,轻链均为k链.Western blot鉴定表明,这2株MAbs均能够识别NS1重组蛋白.间接免疫荧光鉴定表明,这2株MAbs均能够识别真核表达的NS1蛋白.利用噬菌体展示技术得到D9对应的短肽WNLNTV,与NS1蛋白aa 182～aa 187基本匹配,提示182WNDNTV187为NS1蛋白的一个线性表位.该结果为进一步研究NS1蛋白的结构和功能以及建立诊断方法奠定了基础.     

An enzyme-linked immunosorbent assay for the detection of antibody against avian influenza virus

An enzyme-linked immunosorbent assay (ELISA) was developed for detecting antibody to type A avian influenza (AI) virus. The sensitivity and group specificity of the AI-ELISA were compared with those of the agar-gel-precipitin test (AGPT) and the hemagglutination-inhibition (HI) test under conditions of both controlled and field exposure. During the course of temporal experimental infection (0-76 days) of specific-pathogen-free (SPF) chickens with AI subtype Hav9N2, the AI-ELISA was able to detect specific AI antibody as early as 8 days postinoculation (PI), and it measured rising levels of antibody through 35 days PI, at which time the chickens were re-exposed to AI virus. Conversely, AGP tests were negative through 35 days PI, and HI tests began to detect low levels of AI antibody only at 21 days PI. Following a secondary infection at 35 days PI with the same AI subtype, all tests measured rising levels of AI-specific antibody (35-76 days PI). However, the AGP test was positive at only the 7- and 14-day samplings postsecondary immunization. Under field conditions, the AI-ELISA was able to detect serum AI antibody in flocks from which highly pathogenic AI was isolated, but the AGP tests of these sera were negative.     

Antibody and T cell responses induced in chickens immunized with avian influenza virus N1 and NP DNA vaccine with chicken IL-15 and IL-18

We had examined the immunogenicity of a series of plasmid DNAs which include neuraminidase (NA) and nucleoprotein (NP) genes from avian influenza virus (AIV). The interleukin-15 (IL-15) and interleukin-18 (IL-18) as genetic adjuvants were used for immunization in combination with the N1 and NP AIV genes. In the first trial, 8 groups of chickens were established with 10 specific-pathogen-free (SPF) chickens per group while, in the second trial 7 SPF chickens per group were used. The overall N1 enzyme-linked immunosorbent assay (ELISA) titer in chickens immunized with the pDis/N1 + pDis/IL-15 was higher compared to the chickens immunized with the pDis/N1 and this suggesting that chicken IL-15 could play a role in enhancing the humoral immune response. Besides that, the chickens that were immunized at 14-day-old (Trial 2) showed a higher N1 antibody titer compared to the chickens that were immunized at 1-day-old (Trial 1). Despite the delayed in NP antibody responses, the chickens co-administrated with IL-15 were able to induce earlier and higher antibody response compared to the pDis/NP and pDis/NP + pDis/IL-18 inoculated groups. The pDis/N1 + pDis/IL-15 inoculated chickens also induced higher CD8+ T cells increase than the pDis/N1 group in both trials (P < 0.05). The flow cytometry results from both trials demonstrated that the pDis/N1 + pDis/IL-18 groups were able to induce CD4+ T cells higher than the pDis/N1 group (P < 0.05). Meanwhile, pDis/N1 + pDis/IL-18 group was able to induce CD8+ T cells higher than the pDis/N1 group (P < 0.05) in Trial 2 only. In the present study, pDis/NP was not significant (P > 0.05) in inducing CD4+ and CD8+ T cells when co-administered with the pDis/IL-18 in both trials in comparison to the pDis/NP. Our data suggest that the pDis/N1 + pDis/IL-15 combination has the potential to be used as a DNA vaccine against AIV in chickens.     

Previous infection with virulent strains of Newcastle disease virus reduces highly pathogenic avian influenza virus replication,disease, and mortality in chickens

Highly pathogenic avian influenza virus (HPAIV) and Newcastle disease virus (NDV) are two of the most important viruses affecting poultry worldwide and produce co-infections especially in areas of the world where both viruses are endemic; but little is known about the interactions between these two viruses. The objective of this study was to determine if co-infection with NDV affects HPAIV replication in chickens. Only infections with virulent NDV strains (mesogenic Pigeon/1984 or velogenic CA/2002), and not a lentogenic NDV strain (LaSota), interfered with the replication of HPAIV A/chicken/Queretaro/14588-19/95 (H5N2) when the H5N2 was given at a high dose (10^6.9 EID₅₀) two days after the NDV inoculation, but despite this interference, mortality was still observed. However, chickens infected with the less virulent mesogenic NDV Pigeon/1984 strain three days prior to being infected with a lower dose (10^5.3–5.5 EID₅₀) of the same or a different HPAIV, A/chicken/Jalisco/CPA-12283-12/2012 (H7N3), had reduced HPAIV replication and increased survival rates. In conclusion, previous infection of chickens with virulent NDV strains can reduce HPAIV replication, and consequently disease and mortality. This interference depends on the titer of the viruses used, the virulence of the NDV, and the timing of the infections. The information obtained from these studies helps to understand the possible interactions and outcomes of infection (disease and virus shedding) when HPAIV and NDV co-infect chickens in the field.

Electronic supplementary material

The online version of this article (doi:10.1186/s13567-015-0237-5) contains supplementary material, which is available to authorized users.     

Antiviral efficacy of oseltamivir against avian influenza virus in avian species

Avian influenza is one of the most contagious viral diseases in bird species and, increasingly, interspecies transmission to mammalian species has been reported. Prevention and eradication of avian influenza virus (AIV) infection in birds may require vaccines as part of a comprehensive program including biosecurity, culling, diagnostics, and surveillance. However, for valuable bird species in zoos, novel eradication strategies are needed, including antiviral treatments. The present study evaluated the anti-influenza efficacy of the potent neuraminidase inhibitor oseltamivir in avian species using the orders Galliformes (chickens) and Anseriformes (ducks). Viral replication of low pathogenic AIV was significantly reduced in the chicken model and completely reduced in the duck model. Anti-influenza drug administration to valuable bird species with an appropriate extrapolation approach could be useful for control of AIV in combination with active surveillance and vaccination strategies. Further, evaluation of oseltamivir against highly pathogenic avian influenza (HPAI) using avian models would be needed to optimize the oseltamivir application guideline for HPAI control.     

Evaluation of the kidney as a potential site of avian influenza virus persistence in chickens.

One-day-old chickens were inoculated intravenously with one of three low-pathogenicity avian-origin influenza isolates. On day 5 postinoculation (PI), the frequency of influenza virus isolation from cloacal swabs following challenge with each isolate ranged from 83% to 100% for clinically normal euthanatized chickens. Influenza virus was also frequently isolated from kidneys of these chickens (47%) and from chickens that died (100%). Kidneys positive for virus isolation had lesions of nephrosis and/or acute nephritis, and influenza viral nucleoprotein was demonstrated in nuclei and cytoplasm of necrotic renal tubule epithelium. On sampling days 28 and 45/60 PI, influenza virus was neither isolated from nor immunohistochemically demonstrated in kidneys (0/125); however, the kidneys (47%) did have chronic histologic lesions that suggested previous influenza virus infection of the kidneys. Influenza virus was isolated from cloacal swabs of two of 44 chickens on day 28 PI, but all cloacal swabs were negative for virus recovery on sampling day 45/60 PI (0/81). These results indicate that replication of influenza virus in renal tubule epithelial cells did not result in persistence of type A influenza virus in this immunologically privileged site.     

Improved immune responses against avian influenza virus following oral vaccination of chickens with HA DNA vaccine using attenuated Salmonella typhimurium as carrier

This study evaluates the immune responses of single avian influenza virus (AIV) HA DNA vaccine immunization using attenuated Salmonella enterica sv. Typhimurium as an oral vaccine carrier and intramuscular (IM) DNA injection. One-day-old specific-pathogen-free (SPF) chicks immunized once by oral gavage with 10(9) Salmonella colony-forming units containing plasmid expression vector encoding the HA gene of A/Ck/Malaysia/5858/04 (H5N1) (pcDNA3.1.H5) did not show any clinical manifestations. Serum hemagglutination inhibition (HI) titer samples collected from the IM immunized chickens were low compared to those immunized with S. typhimurium.pcDNA3.1.H5. The highest average antibody titers were detected on day 35 post immunization for both IM and S. typhimurium.pcDNA3.1.H5 immunized groups, at 4.0±2.8 and 51.2±7.5, respectively. S. typhimurium.pcDNA3.1.H5 also elicited both CD4(+) and CD8(+) T cells from peripheral blood mononuclear cells (PBMCs) of immunized chickens as early as day 14 after immunization, at 20.5±2.0 and 22.9±1.9%, respectively. Meanwhile, the CD4(+) and CD8(+) T cells in chickens vaccinated intramuscularly were low at 5.9±0.9 and 8.5±1.3%, respectively. Immunization of chickens with S. typhimurium.pcDNA3.1.H5 enhanced IL-1β, IL-12β, IL-15 and IL-18 expressions in spleen although no significant differences were recorded in chickens vaccinated via IM and orally with S. typhimurium and S. typhimurium.pcDNA3.1. Hence, single oral administrations of the attenuated S. typhimurium containing pcDNA3.1.H5 showed antibody, T cell and Th1-like cytokine responses against AIV in chickens. Whether the T cell response induced by vaccination is virus-specific and whether vaccination protects against AIV infection requires further study.     

重组禽流感病毒H5亚型二价灭活疫苗(H5N1,Re-5+Re-4)免疫鸡和鸭后抗体消长规律

禽流感(AI)是由A型流感病毒引起的一种禽类传染病.根据致病性的不同将禽流感分为高致病性禽流感(HPAI)和低致病性禽流感(IPAI),HPAI被世界动物卫生组织(OIE)列为A类传染病,我国将其列为一类动物疫病 [1].近几年来,禽流感在世界各地反复出现,2004年亚洲发生了高致病性禽流感,2006年底和2007年初东南亚又出现疫情,并发现了人感染禽流感.我国养禽业也一直受到禽流感的威胁.因此,它不仅给畜牧业造成了巨大的损失,而且给人们的公共卫生安全带来了严重的威胁.     

不同禽流感病毒含量的新-流二联灭活疫苗免疫SPF鸡后抗体水平的监测

将未浓缩的新城疫抗原分别与未浓缩的、浓缩3倍、浓缩6倍的禽流感抗原混合,并制备成三组鸡新城疫、禽流感（H9N2 HP株）二联灭活疫苗（简称新-流二联灭活疫苗）,分别免疫21日龄SPF鸡,每羽0.3 mL,同时设置未免疫的空白对照组,免疫组与对照组均在免疫前及免疫后7、14、21、28、35 d进行采血,检测新城疫和禽流感抗体。结果发现,各免疫组在免后不同日龄的新城疫抗体基本一致,禽流感病毒抗原浓缩倍数越高（即禽流感病毒含量越高）的新-流二联灭活疫苗,免后14、21 d的抗体也越高;从免后21 d开始,各免疫组的禽流感抗体水平差异逐渐减小,免疫后禽流感抗体水平的高低可以反映该疫苗的免疫效果。试验结果表明,该疫苗可以通过浓缩提高抗原病毒含量的方法来提高免后早期抗体水平,取得良好的早期免疫效果。     

抗禽流感H9亚型血凝素单克隆抗体的抗原结合特性分析

为了解和确定抗H9亚型禽流感病毒血凝素单克隆抗体所针对的抗原表位及今后的确切用途，用竞争性结合ELISA试验结合Western-blotting分析，对10株单克隆抗体的抗原结合特性进行了分析。抗体反应增值结果表明，10株单抗针对的是同一大的抗原决定簇，根据增值结果可将10株单抗分成4个亚群。除2A1外，其他9株单抗的ELISA反应增值结果与Western-blot-ting分析结果一致，认为可能是由于2A1与其他9株单抗的免疫原不同所致。