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Reducing Risk of Avian Influenza Outbreaks in Commercial Chickens by Genetic Resistance

Livant, Emily; Ewald, Sandra
Auburn University
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The long-term goal of this project is to reduce the risk of avian influenza (AI) outbreaks by increasing genetic resistance in the commercial chicken population. We hypothesize that, as in other species, an allele of the chicken Mx gene would limit AI viral replication and/or disease outbreaks in chickens. Thus, genetic stocks can be selected for increased resistance against AI. The objectives of the proposed research are

  1. To determine if chicken embryo fibroblast (CEF) cultures differing in Mx genotype differ in susceptibility to infection with low pathogenic AI strains (LPAI).
  2. To determine the effect of interferon-alpha inducers (IFN-a) on Mx expression and resistance of cells to AI infection and to determine if biological response stimulators may work in concert with antiviral Mx to increase resistance to AI
  3. To determine whether commercial chickens that have the antiviral Mx allele are resistant to challenge with LPAI viruses of two different subtypes.
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Non-Technical Summary- Avian influenza poses a serious threat to the poultry industry. The incidence of both low pathogenicity and high pathogenicity avian influenza outbreaks has increased since 1997. The purpose of this study is to evaluate the potential of selecting commercial chicken lines for genetic resistance to avian influenza. A chicken gene that is a strong candidate for contributing to avian influenza resistance has naturally occurring variants candidate gene that are found in some commercial chicken lines. These genetic variants will be evaluated for ability to protect cells and chickens against low pathogenicity strains of avian influenza virus.

APPROACH: A dimorphism in the gene encoding amino acid residue 631 of the chicken Mx gene was shown by a Japanese laboratory to determine antiviral activity against avian influenza (AI) virus and vesicular stomatitis virus. Experiments by this group demonstrated that transfected mouse 3T3 cells constitutively expressing chicken Mx-631 Asn are resistant to infection with AI virus, whereas transfected cells expressing chicken Mx 631-Ser are not protected. The investigators of the current project developed a method to type chickens for the residue 631 dimorphism by PCR amplifying and sequencing Mx exon 13 from genomic DNA. Preliminary results demonstrated that some commercial broiler breeder chicken lines are segregating for Mx alleles with both dimorphisms (henceforth referred to as Mx+ for antiviral alleles, and Mx- for alleles lacking antiviral activity). For the proposed activities, an SPF flock of broiler chickens will be established, in which Mx+ and Mx- alleles are segregating. Embryonic cell cultures will be established from several individual embryos, followed by typing for Mx exon 13 genotype. Individual cultures of different Mx genotypes will be infected with one of two different low pathogenicity strains of avian influenza virus. Viral replication will be compared to determine if Mx genotype influences resistance or susceptibility. Because the Mx gene is not ordinarily expressed in most cells in the absence of infection, we will also evaluate expression of Mx and ability of Type I interferon (IFN) to enhance Mx protection of cells against viral infection. This objective will be approached by determining the kinetics of Mx mRNA up-regulation in chicken macrophages or embryonic fibroblast cells after AI infection or treatment with synthetic IFN stimulators. Mx expression will be determined by quantitative real-time RT-PCR. Additionally, we plan to determine if CEF cultures or macrophages pre-induced to express Mx will show enhanced resistance to AI replication. For the third objective, we plan to determine whether chickens of the three Mx genotypes differ in resistance to challenge with LPAI. There has not been a formal demonstration in chickens that Mx alleles that have antiviral activity in vitro confer relative resistance to AI (or other related viruses) in vivo. The approach will be to evaluate chickens from a commercial line for Mx allele associations with resistance or susceptibility to AI challenge. Chicks in the study will be typed for Mx 631 dimorphism and challenged with LPAI. Because we expect no mortality or disease symptoms from these AI strains, we will evaluate duration and severity of infection by 3 criteria: 1) persistence of virus in the respiratory and intestinal tract, 2) histopathological changes and histomorphometry of the nasal and tracheal mucosa, and 3) specific antibody response. Persistence of virus in the respiratory and intestinal tract will be determined by virus re-isolation attempts.

PROGRESS: 2006/01 TO 2009/01
OUTPUTS: Results of the experiments demonstrating enhanced resistance to highly pathogenic avian influenza in chickens with the Mx Asn631 variant, were disseminated to the two commercial broiler breeder companies that participated in the project. These two companies provide almost 90% of the commercial genetic stock for broilers world-wide. Results of the experiments have also been disseminated at international meetings on avian immunology and influenza.
PARTICIPANTS: The PI directed all the research conducted at Auburn University, and coordinated with partner organizations and collaborators at other agencies. The co-P.I. contributed to direction, developed protocols, helped supervise the Research Associate, and conducted the experiments involving in vitro infection with LPAIV, and Mx gene expression. The Research Associate prepared in vitro cultures, was most involved in hatching and rearing of chicks for in vivo challenge. Both were involved in typing chickens of breeder flocks, chicks for in vivo challenge, and embryo cultures. Partner organizations provided chicks for breeder flocks, and DNA from genetic lines or crosses for Mx 631 SNP typing. Collaborators were avian influenza scientists at the Southeast Poultry Research Laboratory (Athens, Georgia). One graduate student associated with the project received training.
TARGET AUDIENCES: Reports were made to the two broiler breeder companies regarding results obtained with their genetic lines. We have also had meetings with their geneticists. Presentations were made at two international scientific meetings.
PROJECT MODIFICATIONS: The only major change was to challenge chickens differing in Mx 631 variant, with a highly pathogenic rather than a low pathogenic avian influenza virus strain for all but one experiment. The change was made because the highly pathogenic virus is more relevant.

IMPACT: 2006/01 TO 2009/01
The Mx gene encodes a protein that is expressed in cells after viral infection. In experiments evaluating the antiviral activity of chicken Mx protein by in vitro transfection of Mx cDNA into cell lines, it was reported that Mx alleles encoding Asn at position 631 conferred protection against infection with highly pathogenic avian influenza virus (HPAIV) strain, whereas Ser at 631 had no activity (Ko et al. 2002, 2004). Our experiments were designed to compare the response to infection with a strain of HPAIV of chickens (rather than cells) differing at Mx 631. Chickens from two commercial broiler breeder lines, obtained from two different companies, were evaluated. Each line was segregating for Mx Asn631 and Mx Ser631 variants. We selected chickens that were heterozygous for the Mx 631 dimorphism as breeders. From their progeny, we then selected homozygous Mx Asn631 and Mx Ser631 chicks for challenge with A/chicken/Queretaro/95 H5N2 HPAIV. These experiments were performed in collaboration with scientists at the Southeast Poultry and Disease Laboratory (Athens, GA). In Line 1 chickens (from Company A)infected with 1,000,000 EID-50 virus, the mean survival time was 2 days in the Mx Ser631 group compared with 4 days in the Mx Asn631 group. At day 2 post-infection (D2PI), significantly more Ser631 chickens were positive for viral shedding. Also at D2PI, Asn631 group had significantly higher numbers of transcripts for the cytokines IL-1beta and IFN-alpha in spleens (by RRT-PCR). A similar difference in cytokines was seen in Line 1 chickens infected with a lower dose of the virus, 10,000 EID-50. In Line 2 chickens (from Company B), morbidity as well as mortality was measured in birds infected with 50,000 EID-50 HPAIV. Among Asn631 chickens, mortality was 0%, whereas the Ser631 group had about 30% mortality. Over the 14 day observation period, Asn631 chicks had no morbidity, whereas some of the Ser631 chicks exhibited morbidity from D1 to D7 PI. Morbidity at D4PI in the two groups was significantly different(0% in Asn631 compared with 70% in Ser631 group). Although in vivo differences to infection with HPAIV were observed in chickens of the two Mx 631 variants, in vitro infection of chicken embryo kidney cells differing in Mx 631 with a low pathogenic avian influenza virus demonstrated no difference in virus yield. Nucleotide sequence of the complete Mx cDNA was determined for both Mx Asn631 and Mx Ser631 alleles in both lines. Results demonstrated that the Mx Asn631 alleles from Lines 1 and 2 were different at several amino acid positions. Multiple splice variants of the Mx cDNA were also observed in Asn631 and Ser631 variants.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens
Risk Assessment, Management, and Communication
Meat, Poultry, Game