The Influence of Major Histocompatibility Complex Genotypes on Resistance to Pasteurella multocida and Newcastle Disease Virus in Turkeys1

The Influence of Major Histocompatibility Complex Genotypes on Resistance to Pasteurella multocida and Newcastle Disease Virus in Turkeys1 K. E. NESTOR,* Y. M. SAIF,+ J. ZHU,* D. O. NOBLE,* and R. A. PATTERSON* *Department of Animal Sciences and fFood Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691 Mortality following challenge with P. multocida or NDV was higher in the F line than in its randombred control. The MHC genotypes differed in mortality following exposure to both organisms but the rankings of the genotypes were not the same for P. multocida and NDV. The increased susceptibility of the F line to both organisms could not be explained by known changes in the frequency of the MHC haplotypes.

(Key words: major histocompatibility complex, Pasteurella multocida, Newcastle disease virus, disease resistance, turkey) 1996 Poultry Science 75:29-33

INTRODUCTION

In chickens, MHC haplotypes are associated with resistance or susceptibility to disease organisms and diseases, including Rous sarcoma virus (Heinzelmann et al, 1981; Brown et al, 1982; Guyre et al, 1982; Nordskog and Gebriel, 1983; Collins et al, 1985, 1986), Marek's disease (Pevzner et al, 1981; Briles et al, 1982; Steadham et al, 1987; Martin et al, 1989; Pinard et al, 1993), Newcastle disease virus (NDV, Dunnington et al, 1992), Staphylococcus aureus (Cotter et al, 1992), avian leukosis (Yoo and Sheldon, 1992), necrotic enteritis (Siegel et al, 1993), Pasteurella multocida (Lamont et al, 1987), and Eimeria tenella (Lillehoj et al, 1989). The MHC genotypes also influence the antibody response to antigens in SRBC (Dunnington et al, 1989), Escherichia coli (Uni et al, 1993), NDV (Dunnington et al, 1992), Brucella abortus (Dunnington et al, 1992), S. aureus (Cotter et al, 1987), and Mycoplasma galliseptum (Cheng and Lamont, 1988; Kean et al, 1994). Dunnington et al (1984) and Martin et al. (1990) observed that the allelic frequencies of MHC haplotypes were different in chicken lines selected for high or low antibody response to SRBC. Uni et al. (1993) found that MHC haplotype frequency differed in lines of chickens selected for high or low antibody response to E. coli vaccination at 10 d of age. The above selection experiments suggest that the MHC haplotypes are involved in antibody response. Four haplotypes of turkey MHC Class II genes were discovered in two randombred control (RBC) lines by

The B complex was first described as a blood group by Briles et al. (1950) and was later identified as the MHC in chickens (Schierman and Nordskog, 1961). The MHC is a gene family encoding a group of glycoproteins involved in some important aspects of the immune response, such as antigen presentation and self recognition (Guillemot et al, 1988). Three classes, I, II, and IV, of the chicken MHC have been discovered (reviewed by Dietert et al, 1991). The Class I antigens are distributed on all nucleated cell membranes, and they present endogenously synthesized antigen to cytotoxic T cells. Unlike the Class I antigens, the Class II antigens are expressed mainly on antigen-presenting cells, such as B cells and macrophages, and they present internally processed antigens, which originate exogenously, to T helper cells (Guillemot et al, 1988; Dietert et al, 1991). The Class IV antigens are unique proteins in avian species with unknown functions. They were first found on red blood cells (Pink et al, 1977) and later detected in chicken intestinal epithelium (Miller et al, 1990).

Received for publication June 26, 1995. Accepted for publication September 7, 1995. Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. Manuscript Number 103-95.

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ABSTRACT Sublines homozygous for each of four MHC haplotypes were developed from randombred control populations of turkeys and challenged with Pasteurella multocida (capsular serogroup a, somatic serotype 3, 4) at 6 wk of age or Newcastle disease virus (NDV; Texas GB strain) at 4 wk of age. In addition, individuals from a randombred control line (RBC2) and a subline (F) of RBC2 long-term selected for increased 16-wk BW were included in most of the challenge trials. The duration of the challenge trials was 2 wk for both organisms.

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NESTOR ET AL

MATERIALS AND METHODS Four MHC haplotypes were identified and characterized by Emara et al. (1992) and found to be functionally different by Emara et al. (1993). Homozygotes of the four MHC haplotypes were obtained by selective matings in the RBC1 and RBC2 lines (Emara et al, 1992). The RBC1 and RBC2 lines do not differ in susceptibility to either P. multocida (Saif et al, 1984; Sacco et al, 1991) or NDV (Tsai et al, 1992). Homozygotes of the four MHC haplotypes were produced each generation by inter se mating of homozygotes. The accuracy of pedigreeing was checked periodically using RFLP analysis (Zhu et al, 1995).

Challenge with P. multocida Individuals with four different MHC genotypes (homozygotes of the MHC haplotypes) were challenged with P. multocida in four trials over a 2-yr period. In three of the trials, individuals from the F line were included because this line has been shown to be highly susceptible to the organism (Saif et al, 1984; Sacco et al., 1991). The RBC2 line was included in two of the trials. Details of the development, maintenance, and performance of the F and RBC2 lines were given by Nestor (1984). The breeder flocks used were not vaccinated for fowl cholera. One-day-old poults were wing-banded by MHC genotype or line and brooded intermingled with feed and water given on an ad libitum basis. Birds were bled at about 40 d of age via the brachial vein before being moved to isolation facilities. The prechallenge serological status of each poult was determined using an ELISA procedure (Sacco et al, 1991). Control birds for each challenge trial were kept in conventional housing to determine whether an infection was evident at the time of challenge.

2

Difco Laboratories, Detroit, MI 48223.

Inoculated poults were housed in wire cages in isolation rooms. Poults were challenged at 42 d of age by subcutaneous injection of 1 mL of inoculum in the back of the neck. The inoculum was prepared from a field isolate of P. multocida, capsular serogroup A, somatic serotype 3, 4. The field isolate produced mucoid colonies on tryptose blood agar, no growth on MacConkey agar, positive acid reactions in triple sugar iron slants, a negative urease reaction, fermented glucose without gas formation, and fermented sucrose, lactose, and mannitol. The bacteria were grown in veal infusion broth 2 for 24 h at 37 C. The inoculum was washed three times with sterile PBS (pH 7.3), divided into aliquots, and stored at -70 C. Bacteria were enumerated after thawing by plating serial dilutions on tryptose blood agar. The inoculum contained 1.2 x 10 7 bacteria/mL. The duration of each trial was 14 d. Birds were observed twice daily. Poults exhibiting severe clinical signs of fowl cholera were euthanatized and counted as dead. Euthanatized and dead birds were examined for gross lesions. Swabs taken from the liver, spleen, and lungs during necropsy were subjected to the same biochemical tests described for the inoculum. The sex of each bird was determined by visual inspection of the gonads.

Challenge with Newcastle Disease Virus Individuals of the four MHC genotypes and the F and RBC2 lines were challenged with NDV in three trials in the same year. The management of the birds was similar to that used with the P. multocida trials. A virulent NDV (Texas GB strain) from the National Veterinary Services Laboratory, Ames, IA 50010, was used in the infection trials. Four-week-old poults were inoculated with 0.1 mL of Texas GB strain NDV containing 107-7 mean embryo infectious doseso by the cloacal route. Birds were observed twice daily for 2 wk. Poults exhibiting severe clinical signs of Newcastle disease and judged to be terminally ill were euthanatized and counted as dead. Euthanatized and dead birds were examined for gross lesions. Birds that were clinically normal and survived to Day 14 postinfection were euthanatized, bled, and examined for gross lesions. The sex of the bird was recorded. All serum samples were tested for antibodies to NDV after challenge by hemagglutination-inhibition (HI) and ELISA by the method of Tsai et al. (1992). La Sota strain, a lentogenic NDV live vaccine virus, was used in these tests.

Statistical Analysis Mortality data were analyzed by chi-square after adjustment for difference in numbers (Snedecor, 1959). Multiple chi-square tests were used to separate means. In response to challenge with P. multocida, analyses were performed on data for the MHC genotypes only and for

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Emara et al. (1992) using restriction fragment length polymorphism (RFLP) analysis. The haplotypes were found in linkage disequilibrium with allelic specificities by mixed lymphocyte reaction, graft-versus-host reaction, and skin graft reaction (Emara et al, 1993). The frequency of these and other haplotypes differed in lines of turkeys selected long term for increased egg production or 16-wk body weight (F) and their corresponding RBC (RBC1 and RBC2) (Zhu et al, 1995). The turkey lines also differed in susceptibility to P. multocida (Saif et al, 1984; Sharaf et al, 1988; Sacco et al, 1991) and NDV virus (Tsai et al, 1992). The purpose of the present study was to measure the influence of the newly discovered turkey MHC haplotypes on resistance to P. multocida and NDV to evaluate whether the line differences in susceptibility to these disease organisms could be explained by the change in frequency of the MHC haplotypes.

RESISTANCE TO FOWL CHOLERA AND NEWCASTLE DISEASE

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TABLE 1. Mortality of turkeys of different MHC genotypes following challenge with Pasteurella

multocida

MHC genotype Trials

Variable

AA

BB

CC

DD

1992

No. birds % mortality No. birds % mortality 3 % mortality

77 3.9y 38 31.6cy 13.6y

76 11.8" 39 48.7b" 24.7"

70 8.6*y 41 31.7cy I6.6y

64 12.5" 38 52.6"" 26.4"

1993 Weighted average

RBC21

39 38.5^

F2 8 62.8 40 67.5" 64.2

a_c

Means within a row with no common superscript differ significantly (P < 0.05). "•yMHC genotype means within a row with no common superscript differ significantly (P < 0.05). 2 RBC2 = randombred control line. 2 F = subline of RBC2 selected for increased 16-wk BW. differences among genetic groups were significant (P < 0.004).

RESULTS AND DISCUSSION Mortality to challenge with P. multocida was higher in the 1993 trials than in the 1992 trials even though the same dosage of P. multocida was used both years (Table 1). In the 1993 trials when a sample of the RBC2 and F lines were included in each trial with birds from the four MHC genotypes, the differences among the genetic groups were highly significant (P < 0.004). Mortality was higher in the F line than in the RBC2 line. Similar results have been observed previously (Sacco et al, 1991). Mortality in the F line was higher than mortality in any of the MHC genotypes. Mortalities of the BB and DD genotypes were similar and higher than those of the AA and CC genotypes, which did not differ in mortality. The relationships among MHC genotypes for mortality occurring after challenge with P. multocida was consistent in rank in both years and overall. N o mortality due to P. multocida was observed in any control birds.

The MHC sublines were inbred but not congenic. Therefore, genes other than those from the MHC could differ among the MHC subline and influence the results. With these limitations, the results presented indicate that MHC genotype influences resistance to P. multocida. A more desirable experimental design would be to compare segregates within families. However, with this design, the RFLP method used for identification of the MHC haplotypes would become prohibitively expensive with the number of tests involved. In chickens, Lamont et al. (1987) reported that the MHC influences resistance to P. multocida. Zhu et al. (1995) found that the frequencies of Haplotypes A, B, C, and D changed in the F line relative to the RBC2 line. Haplotypes A and B decreased whereas Haplotypes C and D greatly increased in the F line. Haplotype D had the highest frequency (46.8%) in the F line. Even though the DD genotype had the highest mortality among the MHC genotypes after exposure to P. multocida and this haplotype increased greatly in the F line, changes in susceptibility of the F line cannot be explained solely by changes in MHC haplotypes. The frequency of MHC Haplotype B, which is also susceptible to P. multocida, decreased whereas the frequency of resistant Haplotype C increased in the F line. Mortality following exposure to NDV was higher in the F line than in the RBC2 line (Table 2). Tsai et al. (1992) reported similar results. Differences among MHC genotypes were also noted in mortality following

TABLE 2. Mortality of turkeys of different MHC genotypes following challenge with Newcastle disease virus MHC genotype Variable

AA

BB

CC

DD

RBC21

F2

No. birds % mortality

107 0.9
106 4.7c

71 9 . 9 ab

no

58 6.9bc

60 11.7^

a d

" Means within a row with no common superscript differ significantly (P < 0.05). RBC2 = randombred control line. 2 F = subline of RBC2 selected for increased 16-wk BW. X

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the F and RBC2 lines with the MHC genotypes. For the NDV challenge trials, individual antibody titers were converted to log e prior to analysis. After conversion, the antibody data were subjected to an analysis of variance with line, sex, challenge number, and all possible interactions as sources of variations. The error term was based on individuals within line, sex, and challenge number. Means were separated by Tukey's test (Snedecor, 1959).

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NESTOR ET AL. 1

TABLE 3. Antibody titers of birds surviving challenge with Newcastle disease virus MHC genotype Titer AA BB CC DD 4 HI 47.4b 40.0b 44.7b 40.0b ELISA 6,048a 3,741b 3,997** 4,333ab a b < Means within a row with no common superscript differ significantly (P < 0.05). ^ a t a were analyzed as log,, titer. For presentations, means were converted to actual titers. 2 RBC2 = randombred control line. 3 F = subline of RBC2 selected for increased 16-wk BW. 4 HI = hemagglutination-inhibition titer.

REFERENCES Briles, W. E., R. W. Briles, D. L. Polock, and M. Patterson, 1982. Marek's disease resistance of B (MHC) heterozygotes in a cross of purebred leghorn lines. Poultry Sri. 61:205-211.

P 103.9> 6,593*

Briles, W. E., W. H. McGibbon, and M. R. Irwan, 1950. On multiple alleles affecting cellular antigens in the chicken. Genetics 35:633-652. Brown, D. W., W. M. Collins, P. H. Ward, and W. E. Briles, 1982. Complementation of major histocompatibility haplotypes in regression of Rous sarcoma virus-induced tumors in noninbred chickens. Poultry Sri. 61:409^113. Cheng, S., and S. J. Lamont, 1988. Genetic analysis of immunocompetence measures in a White Leghorn chicken line. Poultry Sci. 67:989-995. Collins, W. M., W. R. Dunlop, R. M. Zsigray, R. W. Briles, and R. W. Fite, 1986. Metastasis of Rous sarcoma tumors in chickens is influenced by the major histocompatibility (B) complex and sex. Poultry Sci. 65:1642-1648. Collins, W. M., N. P. Zervas, W. E. Urban, Jr., W. E. Briles, and P. A. Aeed, 1985. Response of B complex haplotypes B22, B24, and B26 to Rous sarcoma. Poultry Sci. 64:2017-2019. Cotter, P. F., R. L. Taylor, Jr., and H. Abplanalp, 1992. Differential resistance to Staphylococcus aureus challenge in major histocompatibility (B) complex congenic lines. Poultry Sci. 71:1873-1878. Cotter, P. F., R. L. Taylor, Jr., T. L. Wing, and W. E. Briles, 1987. Major histocompatibility (B) complex-associated differences in the delayed wattle reaction to staphylococcal antigen. Poultry Sci. 66:203-208. Dietert, R. R., R. L. Taylor, Jr., and M. F. Dietert, 1991. Biological function of the chicken major histocompatibility complex. Crit. Rev. Poult. Biol. 3:111-129. Dunnington, E. A., R. W. Briles, W. E. Briles, W. B. Gross, and P. B. Siegel, 1984. Allelic frequencies in eight alloantigen systems of chickens selected for high or low antibody response to sheep red blood cells. Poultry Sri. 63: 1470-1472. Dunnington, E. A., A. Martin, R. W. Briles, W. E. Briles, W. B. Gross, and P. B. Siegel, 1989. Antibody responses to sheep erythrocytes for White Leghorn chickens differing in haplotypes of the major histocompatibility complex. Anim. Genet. 20:213-216. Dunnington, E. A., C. T. Larsen, W. B. Gross, and P. B. Siegel, 1992. Antibody responses to combinations of antigens in White Leghorn chickens of different background genomes and major histocompatibility complex genotypes. Poultry Sci. 71:1801-1806. Emara, M. G., K. E. Nestor, and L. D. Bacon, 1993. The turkey major histocompatibility complex: Characterization by mixed lymphocyte, graft-versus-host splenomegaly, and skin graft reactions. Poultry Sci. 72:6(H)6. Emara, M. G., K. E. Nestor, D. N. Foster, and S. J. Lamont, 1992. The turkey major histocompatibility complex: Identification of class II genotypes by restriction fragment

Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 12, 2014

challenge with NDV. Genotypes BB and CC were more susceptible than Genotypes AA and DD. No mortality due to NDV was observed in control birds. Antibody titers (HI and ELISA) in surviving individuals were not significantly influenced by sex, challenge number, or the interactions among the main effects. Surviving individuals of the F line had higher HI and ELISA titers to NDV than the other genetic groups (Table 3). For the HI titer, mean titer for the F line was significantly greater than the other genetic groups, which did not differ from each other. For ELISA titers, the values for the F line and MHC Genotype AA were significantly higher than those for MHC Genotype BB. The ELISA titers for MHC Genotypes CC and DD and Line RBC2 did not differ from the other genetic groups. Tsai et al. (1992) also observed that following challenge with the same NDV virus, HI titers of surviving birds were significantly higher in the F line than in the RBC2 line and there was no significant line difference for ELISA titers. Overall, no relationship existed between mean HI and ELISA titers of surviving birds and mortality occurring during challenge with NDV. Genotype BB was susceptible to both P. multocida and NDV (Tables 1 and 2), which may indicate that this genotype has a general susceptibility to disease. On the other hand, Genotype DD was susceptible to P. multocida but resistant to NDV. The changes in haplotype frequencies of the F line observed by Zhu et al. (1995) cannot be used to explain the difference in resistance of the RBC2 and F lines to NDV. In summary, it appears that MHC genotype influences resistance to P. multocida and NDV. However, changes in susceptibility of the F line to both diseases cannot be explained by known changes in frequencies of MHC haplotypes. Other genetic changes associated with the long-term selection for increased 16-wk BW in the F line are probably responsible for the reduction in disease resistance of the F line.

RBC22 39.0b 4,860ab

RESISTANCE TO FOWL CHOLERA AND NEWCASTLE DISEASE

selection for immune responsiveness and of major histocompatibility complex on resistance to Marek's disease. Poultry Sci. 72:391^102. Pink, J.R.L., W. Droege, K. Hala, V. C. Miggiano, and A. Ziegler, 1977. A three-locus model for the chicken major histocompatibility complex. Immunogenetics 5:203-216. Sacco, R. E., Y. M. Saif, K. E. Nestor, N. B. Anthony, D. A. Emmerson, and R. N. Dearth, 1991. Genetic variation in resistance of turkeys to experimental challenge with Pasteurella multocida. Avian Dis. 35:950-954. Saif, Y. M., K. E. Nestor, R. N. Dearth, and P. A. Renner, 1984. Case report—Possible genetic variation in resistance to erysipelas and fowl cholera in turkeys. Avian Dis. 28: 770-773. Schierman, L. W., and A. W. Nordskog, 1961. Relationship of blood type to histocompatibility in chickens. Science 134: 1008-1009. Sharaf, M. M., K. E. Nestor, Y. M. Saif, R. E. Sacco, and G. B. Havenstein, 1988. Research note: Response of two lines of turkeys to challenge with Pasteurella multocida. Poultry Sci. 67:1807-1809. Siegel, P. B., A. S. Larsen, C. T. Larsen, and E. A. Dunnington, 1993. Research Note: Resistance of chickens to an outbreak of necrotic enteritis as influenced by major hi stocompatibility genotype and background genome. Poultry Sci. 72: 1189-1191. Snedecor, G. W., 1959. Statistical Methods. The Iowa State College Press, Ames, IA. Steadham, E. M., S. J. Lamont, I. Kujdych, and A. W. Nordskog, 1987. Association of Marek's disease with Ea-B and immune response genes in subline and F2 populations of the Iowa State SI leghorn line. Poultry Sci. 66:571-575. Tsai, H. J., Y. M. Saif, K. E. Nestor, D. A. Emmerson, and R. A. Patterson, 1992. Genetic variation in resistance of turkeys to experimental infection with Newcastle disease virus. Avian Dis. 36:561-565. Uni, Z., M. Gutman, G. Leitner, E. Landesman, D. Heller, and A. Cahaner, 1993. Major histocompatibility complex class IV restriction fragment length polymorphism markers in replicated meat-type chicken lines divergently selected for high or low early immune response. Poultry Sci. 72: 1823-1831. Yoo, B. H., and B. L. Sheldon, 1992. Association of the major histocompatibility complex with avian leukosis virus infection in chickens. Br. Poult. Sci. 33:613-620. Zhu, J., K. E. Nestor, and S. J. Lamont, 1995. Survey of major histocompatibility complex class II haplotypes in four turkey lines using restriction fragment length polymorphism analysis with a nonradioactive probe. Poultry Sci. 74:1067-1073.

Downloaded from http://ps.oxfordjournals.org/ at National Chung Hsing University Library on April 12, 2014

length polymorphism analysis of deoxyribonucleic acid. Poultry Sci. 71:2083-2089. Guillemot, F., C. Auffray, H. T. Orr, and J. L. Strominger, 1988. MHC antigen genes. Pages 18-143 in: Molecular Immunology. B. D. Hames and D. M. Glover, ed. IRL Press, Oxford, UK. Guyre, P. M., R. M. Zsigray, W. M. Collins, and W. R. Dunlop, 1982. Major histocompatibility complex (B): Effect on the response of chickens to a second challenge with Rous sarcoma virus. Poultry Sci. 61:829-834. Heinzelmann, E. W., K. K. Clark, W. M. Collins, and W. E. Briles, 1981. Host age and major histocompatibility genotype influence on Rous sarcoma regression in chickens. Poultry Sci. 60:2171-2175. Kean, R. P., W. E. Briles, A. Cahaner, A. E. Freeman, and S. J. Lamont, 1994. Differences in major histocompatibility complex frequencies after multitrait, divergent selection for immunocompetence. Poultry Sci. 73:7-17. Lamont, S. J., C. Bolin, and N. Cheville, 1987. Genetic resistance to fowl cholera is linked to the major histocompatibility complex. Immunogenetics 25:284-289. Lillehoj, H. S., M. D. Ruff, L. D. Bacon, S. J. Lamont, and T. K. Jeffers, 1989. Genetic control of immunity to Eimeria tenella. Interaction of MHC genes and non-MHC genes influences levels of disease susceptibility in chickens. Vet. Immunol. Immunopathol. 20:135-148. Martin, A., E. A. Dunnington, W. E. Briles, R. W. Briles, and P. B. Siegel, 1989. Marek's disease and major histocompatibility complex haplotypes in chickens selected for high or low antibody response. Anim. Genet. 20:407-414. Martin, A., E. A. Dunnington, W. B. Gross, W. E. Briles, R. W. Briles, and P. B. Siegel, 1990. Production traits and alloantigen systems in lines of chickens selected for high or low antibody response to sheep erythrocytes. Poultry Sci. 69:871-878. Miller, M. M., R. Goto, S. Young, J. Liu, and J. Hardy, 1990. Antigens similar to major histocompatibility complex B-G are expressed in the intestinal epithelium in the chicken. Immunogenetics 32:45-50. Nestor, K. E., 1984. Genetics of growth and reproduction in the turkey. 9. Long-term selection for increased 16-week body weight. Poultry Sci. 63:2114-2122. Nordskog, A. W., and G. M. Gebriel, 1983. Genetic aspects of Rous sarcoma-induced tumor expression in chickens. Poultry Sci. 62:725-732. Pevzner, I. Y., I. Kujdych, and A. W. Nordskog, 1981. Immune response and disease resistance in chickens. II. Marek's disease and immune response to GAT. Poultry Sci. 60: 927-932. Pinard, M.-H., L.L.G. Janss, R. Moatman, J.P.T.M. Noordhuizen, and A. J. van der Zijpp, 1993. Effect of divergent

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