Research Note - PubAg - USDA

5 downloads 0 Views 87KB Size Report
Received 24 April 2009; Accepted and published ahead of print 27 September 2009 ... In addition, the mean death times varied greatly between groups, ranging ...
AVIAN DISEASES 54:572–575, 2010

Research Note— Major Histocompatibility Complex and Background Genes in Chickens Influence Susceptibility to High Pathogenicity Avian Influenza Virus Henry D. Hunt,A Samadhan Jadhao,B and David E. SwayneBC A

Avian Disease and Oncology Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 3606 East Mount Hope Road East, East Lansing, MI 48823 B Southeast Poultry Research Laboratory, Agricultural Research Service, U.S. Department of Agriculture, 934 College Station Road, Athens, GA 30605 Received 24 April 2009; Accepted and published ahead of print 27 September 2009 SUMMARY. The chicken’s major histocompatibility complex (MHC) haplotype has profound influence on the resistance or susceptibility to certain pathogens. For example, the B21 MHC haplotype confers resistance to Marek’s disease (MD). However, non-MHC genes are also important in disease resistance. For example, lines 6 and 7 both express the B2 MHC haplotype, but differ in non-MHC genes. Line 6, but not line 7, is highly resistant to tumors induced by the Marek’s disease herpesviruses and avian leukosis retroviruses. Recently, survival in the field by Thai indigenous chickens to H5N1 high-pathogenicity avian influenza (HPAI) outbreaks was attributed to the B21 MHC haplotype, whereas the B13 MHC haplotype was associated with high mortality in the field. To determine the influence of the MHC haplotype on HPAI resistance, a series of MHC congenic white leghorn chicken lines (B2, B12, B13, B19, and B21) and lines with different background genes but with the same B2 MHC haplotype (Line 63 and 71) were intranasally challenged with low dose (10 mean chicken lethal doses) of reverse-genetics–derived rg-A/chicken/ Indonesia/7/2003 (H5N1) HPAI virus. None of the lines were completely resistant to lethal effects of the challenge, as evidenced by mortality rates ranging from 40% to 100%. The B21 line had mortality of 40% and 70%, and the B13 line had mortality of 60% and 100% in two separate trials. In addition, the mean death times varied greatly between groups, ranging from 3.7 to 6.9 days, suggesting differences in pathogenesis. The data show that the MHC has some influence on resistance to AI, but less than previously proposed, and non-MHC background genes may have a bigger influence on resistance than the MHC. RESUMEN. Nota de Investigacio´n—Influencia del complejo mayor de histocompatibilidad y de otros genes en la susceptibilidad del pollo al virus de la influenza aviar de alta patogenicidad. El haplotipo del complejo mayor de histocompatibilidad (con las siglas en ingle´s MHC), tiene una profunda influencia en la resistencia o susceptibilidad a ciertos pato´genos. Por ejemplo el haplotipo B21 del MHC confiere resistencia a la enfermedad de Marek. Sin embargo, genes no relacionados con el MHC tambie´n son importantes en la resistencia a la enfermedad. Por ejemplo, las lı´neas 6 y 7, ambas expresan el haplotipo B2 MHC, pero son diferentes en los genes no relacionados con el MHC. La lı´nea 6, a diferencia de la lı´nea 7, es altamente resistente a los tumores inducidos por el herpesvirus de la enfermedad de Marek y por los retrovirus de la leucosis aviar. Recientemente, la supervivencia en el campo por los pollos auto´ctonos tailandeses durante los brotes del virus de la influenza aviar de alta patogenicidad se atribuyo´ a la presencia del haplotipo B21 del MHC, mientras que el haplotipo B13 del MHC estuvo asociado con alta mortalidad en el campo. Para determinar la influenza del haplotipo de MHC en la resistencia a la influenza aviar de alta patogenicidad, series de lı´neas de aves conge´nicas tipo leghorn blancas (B2, B12, B13, B19 y B21) y lı´neas con diferentes genes de fondo pero con el mismo haplotipo B2 del MHC (Lı´nea 63 and 71) fueron desafiadas de manera intranasal con una dosis baja (10 dosis letales medias en pollo) de un virus de alta patogenicidad producido por reversio´n gene´tica, rg-A/pollo/Indonesia/7/2003 (H5N1). Ninguna de las lı´neas fue completamente resistente a los efectos letales del desafı´o como lo evidenciaron los porcentajes de mortalidad que estuvieron dentro el rango del 40% al 100%. En dos estudios separados, la lı´nea B21 mostro´ porcentajes de mortalidad del 40% y del 70%, mientras que la lı´nea B13 mostro´ mortalidades del 60% y del 100%. Adema´s, los tiempos medios de mortalidad variaron significativamente entre los grupos, con un rango de 3.7 a 6.9 dı´as, lo que sugiere diferencias en la patoge´nesis. Los datos muestran que el MHC tiene una influencia en la resistencia a la influenza aviar, pero menor de lo que se propuso previamente y otros genes no relacionados con el MHC pueden tener una influencia mayor en la resistencia en comparacio´n con el MHC. Key words: avian influenza virus, major histocompatibility complex, B-complex, B congenics Abbreviations: B-complex 5 chicken’s major histocompatibility complex; DPI 5 days postinoculation; HI 5 hemagglutination inhibition; HPAI 5 highly pathogenic avian influenza; MHC 5 major histocompatibility complex; MD 5 Marek’s disease; MDT 5 mean death time; MST 5 mean survival time

Selection for disease-resistance traits remains a priority for commercial animal breeders and the identification and selection of ‘‘natural resistance’’ traits is an ongoing objective for poultry breeders. Progeny testing, a method of selecting resistant progeny derived from sires that produced chickens surviving placement in C

Corresponding author. E-mail: [email protected]

housing environments intentionally contaminated with very virulent pathogens, has been quite successful (8). This method is relatively effective but is expensive and the process takes at least 4 yr for resistance genes to appear in commercial production. Progeny testing coupled with the selection of known resistant genetic loci has been quite successful in selecting for Marek’s disease (MD) resistance primarily due to the remarkable resistance afforded by B21 major histocompatibility complex (MHC or B-complex) haplotype (5).

572

MHC genes and resistance to HPAI virus in chickens

The success of progeny testing and selection of resistant B haplotypes for MD serves as a paradigm for investigating genetic resistance to other viral diseases (8). The B-complex and other genetic systems have been investigated for their natural resistance to avian influenza, infectious bursal disease virus, infectious bronchitis virus, and infectious laryngotracheitis virus to name a few (9,16). For some, such as avian influenza, infectious bursal disease virus, and infectious laryngotracheitis virus, the B haplotype appears important in disease resistance (3,4,7,10,16); in others, such as infectious bronchitis virus, the B haplotype appears less important (6,9). Both the B-complex haplotype and the Mx alleles have been analyzed for their ability to protect birds against highly pathogenic avian influenza (HPAI) viruses (3,4,11,12). The use of genetic markers to select for disease resistant is currently gaining favor and there are genetic markers for both the B haplotype and Mx alleles. The ability to molecularly identify resistant traits such as the Mx alleles or the B-complex haplotype and quickly move them into production is attractive however many of these genomic regions, including the B-complex, appear epistatic in nature. For instance the linkage of Ly6E (Sca2) to MD resistance is dependent on the heterozygous nature of the B-complex with the B homozygous chickens more susceptibility to MD (14). Thus, epistatic effects can be observed even in situations where there is strong linkage of resistance to a single locus such as with the B-complex and MD resistance. These epistatic interactions confuse our ability to assess the effects individual genes have on disease resistance with outbred populations of chickens. Because of these epistatic effects it is not surprising that researchers, using different lines of outbred chickens, come to different conclusions regarding a genes effect on disease resistance. For instance, several groups have evaluated the Mx alleles with regard to resistance to AI and come to very different conclusions (13,18). The retrospective study identifying the B21 haplotype as resistant to HPAI exposure in Thai indigenous chickens is also subject to epistatic effects since these outbred groups of chickens would have widely different background genes. To evaluate the effects of the B-complex in the absence of epistatic effects we used the B congenic lines of inbred chickens. These lines have been developed to identify the impact of only the B-complex on disease resistance by eliminating the epistatic effects of other genetic systems. The B congenic lines all have the same background genes obtained from the 15I5 inbred chicken line. The different B haplotypes were bred into the 15I5 background and then selected by backcross mating to the 15I5 line. This creates a series of different B haplotypes with identical (greater than 99.99%) background genes, thus eliminating the differential epistatic effects segregating in outbred populations. These B congenic lines were evaluated for their resistance or susceptibility to HPAI. MATERIALS AND METHODS Chickens. To characterize the effects of different B-complex haplotypes on HPAI resistance, four 15-B congenic lines, 15.C-12 (B12), 15.P-13 (B13), 15.P-19 (B19), 15.N-21 (B21), and the parental 15I5 (B15) lines were used. The 15-B congenic lines were developed by 10 or 11 backcross generations of mating to inbred line 15I5 (1,17). In the second experiment we repeated trial 1 (trial 2a) and included some new groups as trial 2b, B congenic line 15.7-2 (B2), inbred line 71 (B2), and inbred line 63 (B2) to compare the effects of different background genes on the resistance or susceptibility of HPAI in the B2 haplotype. Briefly, the B congenic birds were developed as follows. Male breeders heterozygous for an introduced B haplotype were selected for 10–11 backcrosses to 15I5, B heterozygous parents were mated and chickens homozygous for the introduced gene were selected. Since development, 6 males and 30–50 females have been used to reproduce each line for 19 generations. Before each backcross the males and females of each line are

573

typed to insure the correct B haplotype. Each of the 15.B congenic lines are greater than 99.9% identical to the inbred parental 15I5 chicken line, but each is homozygous for the unique set of genes defining the individual B haplotypes (2). The 63 and 71 chickens are highly inbred and derived from the original commercial lines obtained by the Avian Disease and Oncology Laboratory in 1939. They were originally selected to be resistant (line 6) or susceptible (line 7) to lymphoid leukosis and then inbred since that selection process (19,21). All of these genetic lines are maintained specific pathogen free at the Avian Disease and Oncology Laboratory. For the following experiments, the chicks were hatched at the Avian Disease and Oncology Laboratory and shipped overnight to Southeast Poultry Laboratory for housing and HPAI virus challenge. Viruses and challenge experiments. The reverse-genetic–derived strain rgA/chicken/Indonesia/7/2005 (H5N1) HPAI virus was used as the challenge virus administered at a minimal chicken lethal dose that would produce 100% mortality; i.e., 10 mean chicken lethal doses (CLD50). This virus strain was derived by using the previously published eight-plasmid system (15). Based on preliminary experiments, 10 CLD50 was equivalent to 103 mean embryo infectious doses (EID50). In the two trials, based on backtitrations, each 3-wk-old chicken received 103.1 (trial 2) or 103.3 (trial 1) EID50 of virus in 0.1 ml via inoculation through the choanal slit into the middle nasal chamber. The chickens were observed for clinical signs for 14 days and deaths were recorded. At 14 days postchallenge, all survivors were bled and euthanatized with intravenous pentobarbital (100 mg/kg of body weight). Sera were tested in standard hemagglutination inhibition tests to confirm all birds were negative for H5 antibodies on day of inoculation and to determine serological status of survivors at 14 days postinoculation (20).

RESULTS AND DISCUSSION

A retrospective survey of the MHC haplotypes obtained from live and dead indigenous chickens in regions of Thailand known to have outbreaks of H5N1 HPAI reported that 100% of the B21 haplotype was present in survivors and none in any of the fatal cases (3). The authors conclude that the MHC B21 haplotype is highly resistant to HPAI strain H5N1. Although the infection status of individual chickens was not determined, the results suggested that the MHC haplotype may be linked to resistance or susceptibility to H5N1. To test the influence of the B haplotype on resistance to HPAI, we challenged two different generations of B congenics with low dose of an H5N1 HPAI virus strain. The hypothesis was that a low HPAI virus exposure dose might enhance survival if genetic resistance were present; a high-exposure dose might overwhelm any genetic resistance to lethal effects of HPAI virus. The MHC haplotypes B2, 5, 12, 13, 19, and 21 were challenged and monitored for disease and mortality induced by the H5N1 challenge. Table 1 gives the percent mortality, infection status of the survivors, the number of birds alive per days postinfection, and the mean time to death for the different B congenic haplotypes in the two trials (trial 1 and trial 2a). In the first trial, mortality ranged from 50% (B15) to 100% (B13), with 70% mortality observed in the B21 haplotype. In the second trial, the morality ranged from 40% (B19 and B21) to 80% (B15) haplotype. Thus in both trials the B21 haplotype had more survivors following HPAI virus infection than the B13 haplotype; however, this difference was not statistically significant in either trial (Table 2). In trial 1 the B13 haplotype was statistically more susceptible than the B15 or B12 haplotypes but this significance did not repeat in the second trial (Table 2). The variability between the two trials does not allow the ranking of the different B haplotypes with regard to resistance or susceptibility; note that the most resistant B haplotype (B15) in the first trial was the most susceptible in the second trial. The survival curves between the B15 and B19 haplotypes were statistically different in both trials (Table 2);

574

H. D. Hunt et al.

Table 1. Mortality and survival data for different chicken lines following intranasal challenge with 103.1–3.3 EID50 of reverse-genetics–derived A/ chicken/Indonesia/7/2003 (H5N1) HPAI virus. Serum samples taken 14 DPI and tested by HIA test.B Number alive (DPI)

Number challenged

Mortality (10 DPI)

Mortality (%)

HI serology (titer of positive)

3

4 5

6

7

8

9

10

MDTC

Rank (MDT.)

15.P-13 (B13) 15.C-12 (B12) 15.15I5 (B15) 15.P-19 (B19) 15.N-21 (B21)

10 10 10 10 10

10a 6ab 5b 9ab 7ab

100 60 50 90 70

– 1/4 (16) 0/5 0/1 0/3

5 8 9 7 8

2 4 6 2 4

2 4 5 2 3

1 4 5 2 3

1 4 5 2 3

0 4 5 1 3

0 4 5 1 3

0 4 5 1 3

4.1 3.7 4.0 4.1 3.9

4 1 3 4 2

Trial 2a Line 15.P-13 (B13) Line 15.C-12 (B12) Line 15.15I5 (B15) Line 15.P-19 (B19) Line 15.N-21 (B21)

10 10 10 10 10

6a 6a 8a 6a 4a

60 60 80 40 40

0/4 0/4 0/2 0/4 1/6 (64)

10 9 9 10 9

8 7 5 8 6

7 7 4 7 6

6 6 3 7 6

4 5 3 7 6

4 5 2 6 6

4 5 2 6 6

4 5 2 6 6

5.5 4.8 4.8 5.3 3.8

4 2 2 3 1

Trial 2b Line 15.7-2 (B2) Line 63 (B2) Line 71 (B2)

10 10 10

4a 7a 9a

40 70 90

0/6 0/3 1/1 (256)

9 7 6 6 9 8 7 7 10 9 7 7

6 5 4

6 6 5 5 3 2

6 5 1

4.0 5.2 6.9

1 2 3

Genetic line (B haplotype)

Trial 1 Line Line Line Line Line

A

HI 5 hemagglutination inhibition. Different lower-case letters denote significant differences in mortality within each trial, Fisher’s exact test, P , 0.05. C MDT 5 mean death time—average day when death identified. B

however, as noted above, in trial 1 the B15 was the most resistant, whereas in trial two B15 was the most susceptible. The results do show that all B haplotypes tested in this study, including the B21 haplotype, are susceptible to death induced by H5N1 challenge. In the second trial (Table 1, trial 2b), we investigated the effects of background genes on resistance or susceptibility to H5N1. Chicken lines 15.7-2, 63, and 71 have the B2 MHC haplotype but vary significantly in their background genes. Within the different B2 haplotypes, mortality ranged from 40% in the 15.7-2 line to 90% in line 71, with 70% mortality in line 63, but the survival curves were not statistically different. This suggests, but does not prove, due to the variability between the trials, that background genes can influence the resistance or susceptibility to H5N1 HPAI virus challenge. We conclude that the B21 haplotype is not highly resistant to mortality of H5N1 HPAIV virus following controlled challenge by a minimal chicken lethal dose of virus, as was suggested in the field data from Thailand (3,4). However, the MHC could have some minor impact on susceptibility to HPAI virus, which would require additional

studies with larger groups of congenic chickens and more replicates. Additional, genetic studies are necessary to determine the influence of epistatic genes on HPAI virus resistance. Finally, in examining the infection status in the 43 survivors irrespective of MHC and non-MHC group, 40 chickens had no evidence of infection, as indicated by seronegativity to H5 hemagglutinin at termination (Table 1). However, three chickens were infected without clinical disease and survived. Such survival of HPAI virus infection in chickens is unusual in experimental studies and field cases. REFERENCES 1. Bacon, L. D. Influence of the major histocompatibility complex on disease resistance and productivity. Poult. Sci. 66:802–811. 1987. 2. Bacon, L. D., H. D. Hunt, and H. H. Cheng. A review of the development of chicken lines to resolve genes determining resistance to diseases. Poult. Sci. 79:1082–1093. 2000.

Table 2. Statistical analysisA of number of live birds on each DPI. Trial 1 Comparison

Mean rank

Difference

B13 B13 B13 B13 B12 B12 B12 B15 B15 B19

217.125 224.375 23.500 211.875 27.250 13.625 5.250 20.875 12.500 28.375

* *** NS NS NS NS NS ** NS NS

vs. vs. vs. vs. vs. vs. vs. vs. vs. vs.

B12 B15 B19 B21 B15 B19 B21 B19 B21 B21

Trial 2a P value

P P P P P P P P P P

, , . . . . . , . .

0.05 0.001 0.05 0.05 0.05 0.05 0.05 0.01 0.05 0.05

Mean rank

Difference

21.688 10.625 29.000 23.688 12.313 27.313 22.000 219.625 214.313 5.313

NS NS NS NS NS NS NS ** NS NS

P value

P P P P P P P P P P

. . . . . . . , . .

0.05 0.05 0.05 0.05 0.05 0.05 0.05 0.01 0.05 0.05

A Dunn’s multiple comparisons test with P values based on criteria established with the Kruskal-Wallis test performed with Graphpad InStat 3.0 software. There was no statistical (NS) difference between lines 15.7-2, 63, and 71 (see Table 1, trial 2b). *P , 0.05. **P , 0.01. ***P , 0.001.

MHC genes and resistance to HPAI virus in chickens

3. Boonyanuwat, K., S. Thummabutra, N. Sookmanee, V. Vatchavalkhu, and V. Siripholvat. Influences of major histocompatibility complex class I haplotypes on avian influenza virus disease traits in Thai indigenous chickens. Anim. Sci. J. 77:285–289. 2006. 4. Boonyanuwat, K., S. Thummabutra, N. Sookmanee, V. Vatchavalkhu, V. Siripholvat, and T. Mitsuhashi. Influences of MHC class II haplotypes on avian influenza traits in Thai indigenous chicken. J. Poult. Sci. 43:120–125. 2006. 5. Briles, W. E., H. A. Stone, and R. K. Cole. Marek’s disease: effects of B histocompatibility alloalleles in resistant and susceptible chicken lines. Science 195:193–195. 1977. 6. Bumstead, N., M. B. Huggins, and J. K. Cook. Genetic differences in susceptibility to a mixture of avian infectious bronchitis virus and Escherichia coli. Br. Poult. Sci. 30:39–48. 1989. 7. Fadly, A. M., and L. D. Bacon. Response of B congenic chickens to infection with infectious bursal disease virus. Avian Dis. 36:871–880. 1992. 8. Fulton, J. E. Selection for avian immune response: a commercial breeding company challenge. Poult. Sci. 83:658–661. 2004. 9. Joiner, K. S., F. J. Hoerr, S. J. Ewald, V. L. van Santen, J. C. Wright, F. W. van Ginkel, and H. Toro. Pathogenesis of infectious bronchitis virus in vaccinated chickens of two different major histocompatibility B complex genotypes. Avian Dis. 51:758–763. 2007. 10. Juul-Madsen, H. R., O. L. Nielsen, T. Krogh-Maibom, C. M. Rontved, T. S. Dalgaard, N. Bumstead, and P. H. Jorgensen. Major histocompatibility complex-linked immune response of young chickens vaccinated with an attenuated live infectious bursal disease virus vaccine followed by an infection. Poult. Sci. 81:649–656. 2002. 11. Ko, J. H., H. K. Jin, A. Asano, A. Takada, A. Ninomiya, H. Kida, H. Hokiyama, M. Ohara, M. Tsuzuki, M. Nishibori, M. Mizutani, and T. Watanabe. Polymorphisms and the differential antiviral activity of the chicken Mx gene. Genome Res. 12:595–601. 2002. 12. Lee, S. H., and S. M. Vidal. Functional diversity of Mx proteins: variations on a theme of host resistance to infection. Genome Res. 12:527–530. 2002. 13. Li, X. Y., L. J. Qu, J. F. Yao, and N. Yang. Skewed allele frequencies of an Mx gene mutation with potential resistance to avian influenza virus in different chicken populations. Poult. Sci. 85:1327–1329. 2006.

575

14. Liu, H. C., M. Niikura, J. E. Fulton, and H. H. Cheng. Identification of chicken lymphocyte antigen 6 complex, locus E (LY6E, alias SCA2) as a putative Marek’s disease resistance gene via a virus–host protein interaction screen. Cytogenet. Genome Res. 102:304–308. 2003. 15. Neumann, G., T. Watanabe, H. Ito, S. Watanabe, H. Goto, P. Gao, M. Hughes, D. R. Perez, R. Donis, E. Hoffmann, G. Hobom, and Y. Kawaoka. Generation of influenza A viruses entirely from cloned cDNAs. Proc. Natl. Acad. Sci. U. S. A. 96:9345–9350. 1999. 16. Poulsen, D. J., D. R. Thureen, and C. L. Keeler, Jr. Research notes: comparison of disease susceptibility and resistance in three lines of chickens experimentally infected with infectious laryngotracheitis virus. Poult. Sci. 77:17–21. 1998. 17. Shen, P. F., E. J. Smith, and L. D. Bacon. The ontogeny of blood cells, complement, and immunoglobulins in 3- to 12-week-old 15I5-B congenic white Leghorn chickens. Poult. Sci. 63:1083–1093. 1984. 18. Sironi, L., J. L. Williams, A. M. Moreno-Martin, P. Ramelli, A. Stella, H. Jianlin, S. Weigend, G. Lombardi, P. Cordioli, and P. Mariani. Susceptibility of different chicken lines to H7N1 highly pathogenic avian influenza virus and the role of Mx gene polymorphism coding amino acid position 631. Virology 380:152–156. 2008. 19. Stone, H. A. Use of highly inbred chickens in research. USDA Agricultural Research Service Technical Bulletin, p. 1–21. 1975. 20. Swayne, D. E., D. A. Senne, and D. L. Suarez. Avian influenza. In: Isolation and identification of avian pathogens, 5th ed. L. Dufour-Zavala, D. E. Swayne, J. R. Glisson, J. E. Pearson, W. M. Reed, M. W. Jackwood, and P. R. Woolcock, eds. American Association of Avian Pathologists, Jacksonville, FL. pp. 128–134. 2008. 21. Waters, N. F., and C. O. Prickett. The development of families of chickens free of lymphomatosis. Poult. Sci. 23:321–333. 1944.

ACKNOWLEDGMENTS The authors thank David L. Suarez and Chang-Won Lee for providing the plasmids used in the challenge virus construction; and Raj Kulkarni, Joan Beck, Lames Doster, and Kira Moresco for technical support. This project was funded by Current Research Information System projects 6612-32000-048-00D and 6612-32000-051-00X.