Inactivated North American and European H5N2 avian influenza virus vaccines protect chickens from Asian H5N1 high pathogenicity avian influenza virus

Abstract

High-pathogenicity (HP) avian influenza (AI) virus of the H5N1 subtype has caused an unprecedented epizootic in birds within nine Asian countries/regions since it was first reported in 1996. Vaccination has emerged as a tool for use in managing the infection in view of future eradication. This study was undertaken to determine whether two divergent H5N2 commercial vaccine strains, one based on a European and the other a North American low-pathogenicity AI virus, could protect chickens against a recent Asian H5N1 HPAI virus. The North American and European vaccine viruses had 84 and 91% deduced amino acid sequence similarity to the HA1 segment of haemagglutinin protein of Indonesia H5N1 HPAI challenge virus, respectively. Both vaccine strains provided complete protection from clinical signs and death. The vaccines reduced the number of chickens infected and shedding virus from the respiratory and intestinal tracts at the peak of virus replication. In addition, the quantity of virus shed was reduced by 10⁴ to 10⁵ median embryo infectious doses. The use of specific neuraminidase inhibition tests allowed identification of infected chickens within the vaccinated groups. These data indicate that the currently available H5 vaccines of European and North American lineages will protect chickens against the Asian H5N1 HPAI virus and reduce environmental contamination by the H5N1 HPAI virus. They will be an adjunct to biosecurity measures to reduce virus transmission.

Les vaccins inactivés à virus influenza aviaire H5N2 européen et nord américain protègent les poulets contre le virus influenza aviaire H5N1 asiatique hautement pathogène

Le virus influenza aviaire (AI) hautement pathogène (HP) de sous type H5N1 a causé une épizootie sans précédent chez les oiseaux de neuf régions/pays asiatiques depuis sa première déclaration en 1996. La vaccination est apparue comme un outil à utiliser pour contrôler l'infection en vue de son éradication future. Cette étude a été entreprise pour déterminer si deux souches divergentes H5N2, de vaccins du commerce, dérivées de virus AI de faible pathogénicité l'un européen et l'autre nord américain, pouvaient protéger les poulets contre un virus asiatique récent HPAI H5N1. Les virus vaccinaux nord américain et européen avaient une séquence déduite en acides aminés qui présentait respectivement 84% et 91% de similarité au niveau du segment HA1 de la protéine de l'hémagglutinine avec le virus d'épreuve HPAI H5N1 indonésien. Les deux souches vaccinales ont conféré une protection complète au regard des symptômes et de la mortalité. Les vaccins ont réduit le nombre de poulets infectés et la diffusion du virus à partir des tractus respiratoire et intestinal, au pic de réplication du virus. De plus, la quantité de virus diffusé a été réduite de 10^4–5 doses infectieuses 50 pour l'embryon (EID₅₀). L'utilisation de tests d'inhibition spécifiques de la neuraminidase a permis l'identification de poulets infectés au sein des groupes vaccinés. Ces données indiquent que les vaccins H5 actuellement disponibles des lignages européen et nord américain protègent les poulets contre le virus HPAI H5N1 asiatique et réduisent la contamination environnementale par le virus HPAI H5N1. Ils représentent un complément aux mesures de biosécurité pour réduire la transmission du virus.

Inaktivierte Vakzine mit nordamerikanischem oder europäischem H5N2 aviärem Influenzavirus schützen Hühner vor dem hochpathogenen asiatischen H5N1 aviären Influenzavirus

Hochpathogenes (HP) aviäres Influenza (AI)-Virus des H5N1 Subtyps hat seit der Erstbeschreibung im Jahr 1996 eine noch nie da gewesene Epizootie bei Vögeln in neun asiatischen Ländern/Regionen hervorgerufen. Die Vakzination hat sich als ein Mittel zur Bekämpfung der Infektion im Hinblick auf die spätere Eradikation erwiesen. Diese Studie wurde durchgeführt, um festzustellen, ob zwei verschiedene kommerzielle H5N2-Vakzinestämme, einer basierend auf einem europäischen und der andere auf einem nordamerikanischen schwach pathogenen AI-Virus, Hühner gegen das neue asiatische H5N1-HPAI-Virus schützen kann. Das nordamerikanische und das europäische Vakzinevirus hatten eine 84- bzw. 91 %ige Übereinstimmung der Aminosäurensequenz mit dem HA1-Segment des Hämagglutininproteins des indonesischen H5N1-HPAI-Challengevirus. Beide Vakzinestämme schützten vollständig vor Erkrankung und Tod. Zum Zeitpunkt der größten Virusvermehrung reduzierten die Vakzinen die Anzahl der infizierten und damit die Anzahl der über den Respirations- und Intestinaltrakt virusausscheidenden Hühner. Außerdem wurde die Quantität der Virusausscheidung um 10^4–5 Embryo infektiöse Dosen₅₀ (EID₅₀) vermindert. Die Anwendung von Neuraminidaseinhibitionstests erlaubte die Identifizierung infizierter Hühner innerhalb der vakzinierten Gruppen. Diese Ergebnisse weisen darauf hin, dass die zur Zeit verfügbaren H5-Vakzinen europäischer und nordamerikanischer Herkunft Hühner gegen das asiatische H5N1-HPAI-Virus schützen und die Kontamination der Umgebung mit diesem Virus reduzieren können. Sie können eine Unterstützung der Sicherheitsmaßnahmen zur Reduktion der Virusübertragung sein.

Vacunas de virus inactivados de Influenza Aviar H5N2 de América del norte y Europa protegen a los pollos frente al Virus de Influenza Aviar de Alta Patogenicidad H5N2 de Asia

El virus de influenza aviar (AI) de alta patogenicidad (HP) del subtipo H5N1 ha causado una epizootía sin precedentes en aves de nueve países/regiones Asiáticos desde su primera descripción en 1996. La vacunación se ha convertido en una herramienta en uso para el control de la infección en vistas de una futura erradicación. Este estudio se llevó a cabo para determinar si dos cepas vacunales H5N2 distintas, una basada en una cepa Europea y la otra en una cepa Norte Americana del virus AI de baja patogenicidad,, podían proteger a pollos frente al virus asiático H5N1 HPAI. Los virus vacunales de Norte América y Europa mostraban un 84% y 91% de similitud en las secuencias aminoacídicas deducidas respecto el segmento HA1 de la proteína de la hemaglutinina del virus campo de Indonesia H5N1 HPAI, respectivamente. Ambas cepas vacunales proporcionaron protección completa frente a signos clínicos y mortalidad. Las vacunas redujeron el número de pollos infectados y la excreción de virus vía tracto respiratorio e intestinal en el momento de máxima replicación viral. Además, la cantidad de virus excretado se redujo hasta 10^4–5 dosis infectivas embrionarias medias (EID₅₀). El uso de pruebas específicas de inhibición de la neuraminidasa permitió la identificación de las aves infectadas en los grupos vacunados. Estos resultados indican que las vacunas H5 disponibles actualmente, procedentes de líneas Europeas y Norte Americanas protegerán a los pollos frente al virus H5N1 HPAI Asiático y reducirán la contaminación del medio por este virus. Estas vacunas podrán sumarse a las medidas de bioseguridad para reducir la transmisión vírica.

Introduction

The first outbreak of Asian H5N1 high-pathogenicity (HP) avian influenza (AI) was reported in domestic geese in China during 1996 (Xu et al., 1999), which has been followed by outbreaks in Hong Kong and China (Sims et al., 2003a b; Chen et al., 2004). Beginning with December 2003, nine countries in Eastern and Southeastern Asia reported outbreaks of H5N1 HPAI in domestic poultry, and feral or captive non-poultry species of birds (Li et al., 2004; Lee et al., 2005). Quarantine, depopulation, cleaning and disinfection were used as the principle components in the control strategies (FAO, 2004). In addition, China, Hong Kong, and Indonesia have instituted vaccination programmes using inactivated H5 avian influenza vaccines as adjuncts for preventing or managing the disease with the intent of eventual eradication (FAO, 2004).

Previously, commercial and experimental inactivated H5 AI, recombinant fowlpox vaccine with a H5 AI haemagglutinin gene insert and baculovirus-expressed haemagglutinin protein vaccines have shown protection in chickens against the 1997 H5N1 HPAI viruses isolated from humans and chickens in Hong Kong (Swayne et al., 2000a b 2001). In April 2002, Hong Kong instituted a vaccination programme for chickens raised on some Hong Kong farms for sale in the live poultry markets following three waves of H5N1 HPAI outbreaks beginning in 1997 (Sims et al., 2003a). An existing commercial inactivated vaccine containing an H5N2 low-pathogenicity (LP) AI virus strain isolated in 1994 from chickens in Mexico was used. The vaccination programme was expanded in 2003 to include all chickens imported from mainland China (Ellis et al., 2004c). Following the institution of the vaccination programme, outbreaks of morbidity and mortality in chickens have not been seen (Ellis et al., 2004b c).

Unlike the need to frequently change human influenza vaccine strains to provide adequate protection, H5 AI vaccines for chickens have shown the ability to provide protection against H5 AI viruses isolated over many years (Swayne et al., 1999 2000a b). However, recent re-evaluation of an inactivated H5N2 vaccine used in Mexico for 9 years has shown sufficient change in the field viruses for it to no longer be protective against H5N2 LPAI viruses circulating in central America (Lee et al., 2004). The current study was undertaken to determine whether two current commercial vaccines with diversely different backgrounds would protect against a 2003 Asian H5N1 virus isolated from chickens in Indonesia.

Materials and Methods

Viruses

The vaccine viruses were provided as commercial vaccines and were based on LPAI virus strains, A/duck/Potsdam/1402/86 (H5N2) (Potsdam/96) (Matrosovich et al., 1999) and A/chicken/Mexico/232/94 (H5N2) (Mexico/94) (Lee et al., 2004). An Asian HPAI virus, A/chicken/Indonesia/7/03 (H5N2) (Indonesia/03), was used as the challenge virus. This virus was isolated from a diagnostic specimen submitted from a farm experiencing high mortality in broiler chickens during early December 2003. All AI viruses were propagated by allantoic sac inoculation of 9-day-old embryonating chicken eggs by standard methods (Swayne et al., 1998).

Quantification of AI virus in vaccines

RNA extraction for vaccine virus quantification

A method was developed specifically for extracting RNA from oil emulsion vaccines. The vaccine was mixed at medium speed for 30 min at room temperature on an orbital shaker, 100 µl was removed and added to 150 µl nuclease free water and 750 µl Trizol LS reagent (Invitrogen, Inc., Carlsbad, California, USA). The sample was mixed by vortexing and incubated for 7 min at room temperature, after which 200 µl chloroform was added. Samples were incubated for 7 min at room temperature, and then were centrifuged for 15 min at 14 000×g. The top two phases were removed together, then added to an equal volume of RLT buffer (Qiagen, Inc., Valencia, California, USA) and an equal volume of 70% ethanol and mixed by inversion. The sample was then applied to a Qiagen RNeasy Column (Qiagen, Inc.), washed and eluted in nuclease-free water in accordance with the kit instructions. Each RNA sample was extracted from the oil emulsion vaccine in replicates of six to ensure reproducibility.

Quantitative real-time reverse transcriptase-polymerase chain reaction

Homologous virus isolates were used as the quantitative standards. Allantoic fluid virus stocks were diluted in brain heart infusion broth (Becton-Dickinson, Sparks, Maryland, USA) and titrated in embryonated chicken eggs at the time of dilution according to standard methods (Swayne et al., 1998). Whole virus RNA was extracted along with the vaccine samples, by the same method. Real-time reverse transcriptase-polymerase chain reaction (RRT-PCR) for the influenza matrix gene was performed (Spackman & Suarez, 2005). All six RNA extractions were run separately for each sample. RNA extracted from an oil–emulsion vaccine as described previously, which did not contain AI virus, was used as a negative control. The quantity of haemaggluttinin (HA) protein in the vaccine was calculated using previous data reporting infectious titre and HA protein quantity in the vaccine (Swayne et al., 1999).

Phylogenetic analysis and sequence comparison

Sequences for the vaccine viruses were obtained from Genbank (AF082042 and AY497063) and the sequence of the challenge virus was submitted to Genbank (Genbank accession number pending). The HA1 portion of the haemagglutinin gene from all three viruses was aligned with the Clustal W algorithm (Lasergene, DNASTAR, Inc., Madison, Wisconsin, USA). Phylogenetic analysis performed with PAUP* 4.0b10 (Sinauer Associates, Inc. Sunderland, Massachusetts, USA) using the maximum parsimony tree building method by heuristic search with 500 bootstrap replicates.

Animals and housing

Three-week-old specific pathogen free White Leghorn chickens were obtained from flocks maintained at the Southeast Poultry Research Laboratory. For the vaccination and challenge portion of the studies, chickens were housed in negative-pressure stainless steel isolation cabinets ventilated with HEPA-filtered air and provided with continuous lighting. All challenge experiments were carried out in a USDA-certified biosafety level 3 agriculture facility. Water and feed were provided ad libitum.

Experimental design

Groups of 10 chickens were immunized subcutaneously in the nape of the neck with 0.5 ml each of the three vaccines: Nobilis Hepatitis + ND Inactivated (Intervet, Boxmeer, The Netherlands) (Sham), Nobilis I.A. Inactivated (Mexican/94) and Nobilis Influenza, H5N2 (Potsdam/86). At 3 weeks post vaccination, chickens were challenged by intranasal inoculation with 10^6.0 median embryo lethal doses (ELD₅₀) of Indonesia/03 HPAI virus. Clinical response was recorded for 14 days post-challenge (p.c.) to ascertain the protection provided by the vaccines. Birds were observed for clinical signs of lethargy, hunched posture, reluctance or failure to rise and drooping wings, and death.

Sera were collected from each bird on the day of vaccination, challenge, and day 14 p.c. Sera were tested for the presence of antibodies against the nucleoprotein/matrix protein (agar gel precipitin [AGP] test), haemagglutinin (haemagglutination inhibition [HI] test) and neuraminidase (neuraminidase inhibition [NI] test) (Van Deusen et al., 1983; Swayne et al., 1998). For HI tests, a 1:10 or higher titre was considered positive. Oropharyngeal and cloacal swabs were taken at day 2 p.c. for attempted virus isolation and titration in 10-day embryonating specific pathogen free chicken eggs using standard procedures (Swayne et al., 1998). In preliminary studies, titres of virus isolated were highest on day 2 p.c. Titres were expressed as the ELD₅₀ per millilitre of swab fluid.

Statistical analyses

Frequency of morbidity, mortality, virus isolation and detection of anti-influenza virus antibodies were analysed for significance (P<0.05) by Fisher's exact test on personal computer-based software (SigmaStat 2.0; Jandel Scientific, San Rafael, California, USA). Virus isolation and HI serological titres were tested for normal distribution. Normally distributed data sets were further tested by parametric one-way analysis of variance (ANOVA). Those ANOVA data sets with significant differences were further analysed by Student–Neuman–Keuls multiple comparison test. Data sets not normally distributed were analysed by non-parametric analysis of variance test (Kruskal–Wallis), and for significantly different groups (P < 0.05) Dunn's multiple comparison test was performed. Normality, ANOVA, Kruskal–Wallis, Student–Neuman–Keuls and Dunn's tests were performed on personal computer-based software (SigmaStat).

The minimum virus titre detected by virus isolation procedures in this study was 10^1.0 ELD₅₀/ml. Thus, for statistical purposes, all oropharyngeal and cloacal swabs from which virus was not isolated were given a numeric value of 10^0.9 ELD₅₀/ml, which represents the lowest detectible level of virus if the virus isolation procedure were modified to use four instead of three embryonating chicken eggs per sample.

Results

Quantification of AI virus in vaccines

Based on the AI virus standard titre curves and quantitative RRT-PCR, the vaccines containing Potsdam/86 and Mexico/94 AI virus isolates had virus titres equivalent to 10^5.1 and 10^5.9 EID₅₀/ml vaccine, respectively. Based on prior titrations for haemagglutinin protein (Swayne et al., 1999), this would be equivalent to 0.025 and 0.16 µg HA protein/ml or 0.0125 and 0.08 µg HA protein/dose for Potsdam/86 and Mexico/94 AI virus isolates, respectively.

Phylogenetic analysis and sequence comparison

The HA1 gene sequences were compared between the Indonesia/03 challenge virus and the two vaccine AI viruses. The Indonesia/03 challenge virus was more closely related to the Potsdam/86 than to the Mexico/94 isolate (Figure 1). The Potsdam/86 isolate had 89.4% nucleotide and 91.9% amino acid sequence identity with the Indonesia/03 challenge virus. The Mexico/94 isolate had 78.5% nucleotide and 84.8% amino acid identity with Indonesia/03 challenge virus. All three isolates have the same potential N-linked glycosylation sites at positions 11 and 286.

Figure 1.  Phylogenetic tree with the selected H5 subtype HA1 segment of the haemagglutinin gene. The tree was constructed with PAUP 4.0b10 with the maximum parsimony tree building method by heuristic search with 500 bootstrap replicates. Sequence distances are noted on the tree. The vaccine viruses used in this study are underlined and the challenge strain appears in bold-face type. CK, chicken; DK, duck; TK, turkey. States are denoted by their standard two-letter postal code.

Serology

All birds were negative for AGP, H5 HI, and N1 and N2 NI antibodies on the day of vaccination. Chickens in the sham vaccine group were negative for anti-AI antibodies on the day of challenge (Table 1), while the Mexican/94 and Potsdam/86 H5N2 vaccines induced AGP and HI antibodies in 100 and 90% of chickens 3 weeks post vaccination, respectively, and 100% of survivors had AGP and HI antibodies on day 14 p.c. With the two AI vaccine groups, the average HI titres were not significantly different on the day of challenge. At 14 days p.c., an anamnestic response was seen with titres rising over three-fold in the two AI vaccine groups. The average titre was numerically higher for the Mexico/94 group but was not significantly different from the average titre of the Potsdam/86 group (Table 1). For NI antibodies, 100% of the H5N2 vaccinated birds had antibodies against N2 both pre-challenge and post-challenge, while N1 antibodies were seen only in a small percentage of birds and only after challenge with the H5N1 HPAI virus.

Table 1.  Serological data from chickens vaccinated at 3 weeks of age and challenged intranasally at 6 weeks of age with 10^6.0 EID₅₀ Indonesia/03 HPAI virus

	AGP serology^a (number positive/total)		HI serology^a (number positive/total) (GMT)		NI serology^a (number positive/total)
					Pre-challenge		Post-challenge
Vaccine group	Pre-challenge	Post-challenge	Pre-challenge	Post-challenge	N1	N2	N1	N2
Sham	0/10^A	NA	0/10^A (<8)^3a	NA	0/10^A	0/10^A	NA	NA
Mexican/94	10/10^B	10/10^A	10/10^B (128)^b	10/10^A (776)^a	0/10^A	10/10^B	2/10^A	10/10^A
European/86	9/10^B	9/9^A	9/10^B (120)^b	9/9^A (445)^a	0/10^A	9/10^B	1/9^A	9/9^A
Pre-challenge was 3 weeks after vaccination and post-challenge was 2 weeks after challenge.
^aAll birds were negative for anti-AI viral antibodies on the day of vaccination. HI titres reported as the geometric mean titre (GMT). Different uppercase superscripts denote significance between treatment groups; Fisher's exact test, P<0.05. Different lowercase superscripts denote significance (P<0.05) between treatment groups using one-way ANOVA, or Kruskal–Wallis one-way ANOVA on ranks when the normality test failed, or Mann–Whitney rank sum test when comparing groups 2 and 3 of HI serology p.c.

Morbidity, mortality and infectivity

All chickens in the sham vaccinated group developed clinical signs and died following Indonesia/03 challenge (Table 2). On day 2 p.c., in all sham-vaccinated chickens, high titres of virus were recovered from the oropharynx and cloaca. The two vaccines provided 90 to 100% protection from clinical signs and death. One chicken vaccinated with Potsdam/86 exhibited clinical signs and died. This chicken lacked anti-AI antibodies on the day of challenge.

Table 2.  Morbidity, mortality and virus isolation data from 3-week-old chickens vaccinated with inactivated AI vaccine at 3 weeks of age and intranasally challenged at 6 weeks of age with 10^6.0 EID₅₀ Indonesia/03 HPAI virus

Vaccine group	Morbidity (number ill/total)	Mortality (number dead/total) (mean time to death [days])	Virus isolation, day 2 p.c. (number positive/total, log10 EID50 titre/ml)
			Oropharyngeal swab	Cloacal swab
Sham	10/10^A	10/10^A (2.2)	10/10^A (6.16)^3a	10/10^A (5.82)^a
Mexican/94	0/10^B	0/10^B	5/10^B (1.23)^b	3/10^B (1.00)^b
Potsdam/86	1/10^B	1/10^B (2.0)	6/10^AB (1.78)^b	3/10^B (1.53)^b
Different uppercase superscripts denote significance between treatment groups; Fisher's exact test, P < 0.05. Different lowercase superscripts denote significance (P < 0.05) between treatment groups using one-way ANOVA or Kruskal–Wallis one-way ANOVA on ranks when the normality test failed.

When examined at the peak of virus shedding (i.e. day 2 p.c.), both vaccines reduced the number of chickens infected and shedding the challenge virus. For cloacal swabs, the reduction in number of vaccinated chickens shedding was significantly less when compared with the sham group, but the number of chickens shedding virus between vaccine groups was not significantly different. For oropharyngeal swabs, the reduction was significant for sham verses Mexico/94 groups, but no difference was found between the two vaccine groups (Table 2). The two vaccines significantly reduced the quantity of challenge virus shed by 10^4-5 EID₅₀/ml compared with the sham group, but the quantity of challenge virus shed was no different between vaccine groups (Table 2).

Discussion

In the current study, both commercial H5N2 AI vaccines provided equal protection in chickens following a high challenge dose with a 2003 Indonesian H5N1 HPAI virus strain. This protection included prevention of death and clinical signs, a reduction in the number of chickens infected and in the quantity of challenge virus shed from the respiratory and intestinal tracts on day 2 p.c., and production of anti-influenza virus antibodies. Previous studies have shown not only a reduction in the number of chickens infected and in the quantity of LPAI or HPAI challenge virus shed from respiratory and intestinal tracts on the day of peak virus replication, but also on days before and after the peak (Stone, 1987; Kodihalli et al., 1994). However, such protection does not indicate that vaccinated birds cannot become infected at a low frequency and excrete a low quantity of virus. The presence of birds without clinical disease that are infected (i.e. “silent infections”) necessitates utilization of biosecurity measures in management strategies to prevent transmission, spreading and maintenance of the AI virus in the field (Swayne et al., 1997; Swayne & Akey, 2005). In a previous study, effective immunization translated into reduced contact transmission of HPAI virus among poultry (Swayne et al., 1997).

Adequate quantity of HA is critical for potent vaccines, but quantification of HA content is problematic using the established testing methods, which measure HA protein, haemagglutinating units or infectious titre prior to formulation with oil and emulsification of the final product. Measuring the HA protein content is the most direct method, but is a cumbersome and relatively insensitive process, requiring growth of large quantities of virus, concentration by ultracentrifugation and assaying with the radial single immunodiffusion test (Wood et al., 1985; Swayne et al., 1999). Haemagglutinating units have been used as an indirect measurement of HA protein content, but correlate poorly with HA protein content, while infectious titre has good correlation with HA content (Swayne et al., 1999). By comparison, the indirect quantitative RRT-PCR is a technically precise measure of RNA quantity that correlates with HA content and can be used pre-formulation or post-formulation of vaccine. The quantitative RRT-PCR test is expected to be robust as results among the six replicates for each sample were within the normal range of standard deviation of 1.5 cycles for this test (Spackman & Suarez, 2005). Furthermore, the impact of RNA degradation in the vaccine is expected to be minimal due to the small size of the RRT-PCR product, which is 99 base pairs in length. For the current study, the HA content was measured in the vaccines using quantitative RRT-PCR assay and compared with the infectious titre of virus to derive HA protein content.

In a previous study, 0.016 µg HA protein of the Mexico/94 vaccine strain was a 50% protective dose (PD₅₀) against the Mexican H5N2 HPAI challenge virus (A/chicken/Queretaro/14588-19/94 [H5N2]) (Swayne et al., 1999). In the current study, 0.0125 and 0.08 µg HA protein of the Mexico/94 and Potsdam/86 vaccines, respectively, were superior, providing 90 to 100% protection from clinical signs and death, and greater reduction in Indonesia/03 challenge virus replication (10⁴ to 10⁵ versus 10¹ to 10^2.1 to EID₅₀/ml). This superior protection may have resulted from the proprietary adjuvant system because the age of chickens, route and site of immunization and challenge virus dose were the same between studies. This superior protection was evident despite only 89.4 and 91.9% HA1 amino acid sequence similarity between vaccines and challenge virus of the current study verses 98.2% HA1 amino acid sequence similarity between Mexico/94 vaccine and H5N2 HPAI challenge virus strains in the previous study (Swayne et al., 1999). A previous recombinant fowlpox-H5-AI virus vaccine study in chickens demonstrated a significant correlation between increasing reduction in oropharyngeal titres and increasing HA sequence similarity (87.3 to 100%) between the challenge viruses and vaccine strain, but another study, using inactivated virus vaccines, demonstrated good reduction in oropharyngeal replication irrespective of HA amino acid sequence similarity (91.7 to 100%) of vaccine and challenge viruses (Swayne et al., 1999 2000a).

The Mexican/94 vaccine strain has been used in Hong Kong since 2002 and has been shown to be protective in chickens and beneficial in controlling AI in the field (Ellis et al., 2004a b). Other vaccines, including traditional inactivated North American AI vaccine strains, H5N1 reverse genetic AI vaccine strain and fowlpox recombinant vaccines with H5 AI gene inserts, have been shown to protect against diverse Asian H5N1 HPAI strains in the laboratory (Swayne et al., 2001; Liu et al., 2003; Qiao et al., 2003; Swayne, 2004; Swayne & Beck, 2005). This indicates that the need for frequent change in H5 AI vaccine strains for poultry, as occurs with human influenza A subtype H1 and H3 and influenza B vaccine strains, because of rapid drift, is not necessary. However, evaluation of AI vaccine viruses in a poultry challenge model to assess protection against circulating field viruses should be done at least biennially. If protection is inadequate, vaccine strains should be changed. In addition, only high-quality vaccines with adequate antigen content should be licensed and used (Garcia et al., 1998).

Surveillance is critical in assessing the prevalence of AI virus infections in poultry populations and adequate specific serological surveillance is crucial for assessing the success of individual components in a control strategy (Swayne & Suarez, 2000). Use of vaccination without properly designed serological surveillance will prevent detection of infections in vaccinated flocks and hamper implementation of science-based changes to control programmes, thus greatly limiting eradication efforts (Capua et al., 2003; Swayne, 2003). Traditional AGP, ELISA and HI serological tests detect antibodies against nucleoprotein/matrix protein and haemagglutinin, respectively, induced by vaccination with inactivated whole AI vaccines. To detect infections in such vaccinated populations, special provisions must be pre-planned and implemented, such as the use of AGP and HI tests in unvaccinated sentinels or the use of special tests to detect antibodies to non-structural protein or heterologous neuraminidase in vaccinated birds (Beard, 1987; Swayne & Suarez, 2000; Capua et al., 2003; Swayne, 2003; Tumpey et al., 2005). In the current study, AGP, H5 HI, and N2 NI antibodies were demonstrated in H5N2 AI vaccinated birds. N1 NI antibodies were only demonstrated in a few vaccinated birds following challenge with H5N1 HPAI virus, although HI anamnestic response ≥3log₂ was seen in six of 10 birds given Mexico/94 vaccine. This suggests the NI test is less sensitive than HI anamnestic response, but the test is useful at the farm level and not as an individual bird test for detecting infection in birds immunized with inactivated vaccines. Both unvaccinated sentinels and heterologous neuraminidase testing of vaccinated poultry have been used successfully in the field (Halvorson, 2002; Capua et al., 2003; Swayne & Akey, 2005). Finally, although further evaluation is needed, quantitative RRT-PCR, which is unaffected by antigenic drift, unlike the antibody-based radial immunodiffusion assay, may be used to evaluate HA content in oil emulsion vaccines, with the added advantage that quantity can be assessed in the final product.

Acknowledgments

The authors thank Joan Beck, Scott Lee and James Doster for technical assistance with the studies.