Antibody Responses to Sheep Red Blood Cell and Brucella abortus Antigens in a Turkey Line Selected for Increased Body Weight and Its Randombred Control1 Z. Li,* K. E. Nestor,*,2 Y. M. Saif,† and J. W. Anderson* *Department of Animal Sciences and †Food Animal Health Research Program, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, Ohio 44691 ABSTRACT Turkeys from a randombred control line (RBC2) and its subline (F) selected for increased 16-wk BW were tested for primary and secondary antibody responses to SRBC antigen and Brucella abortus antigen (BA). Previous studies have shown that the F line was more susceptible to Pasteurella multocida and Newcastle disease virus than was the RBC2 line. Individuals from the RBC2 and F lines were intravenously injected with 1 mL 5% SRBC antigen or 0.1 mL undiluted BA at 4 and 6 wk of age; blood samples were collected at 0, 4, 7, and 10 d post-immunization. Total, IgG, and IgM titers were measured by agglutination assays. Compared with the RBC2 line, the F line had generally higher total anti-SRBC titers; the differences were significant at 14 d postprimary immunization (PPI) (females);
at 10 d postsecondary immunization (PSI) (males); and at 4, 7, and 10 d PSI (females) (P ≤ 0.05). The F line also had higher IgM titers at 14 d PPI (females) and at 10 d PSI (males) (P ≤ 0.05). For IgG titers, a line difference was evident in females at 4 and 10 d PSI (P ≤ 0.05); the F line had higher titers than did the RBC2 line. For the antibody response to BA in males, the F line had lower total and IgM titers at 10 d PPI (P ≤ 0.05) than did the RBC2 line. No significant line differences in response to the BA were found in total and IgM titers in female turkeys or in IgG titers in both sexes at any time. These results suggest that selection for fast growth rate of turkeys might have resulted in changes in humoral immunity to the SRBC antigen and BA.
(Key words: turkey, sheep red blood cell antigen, Brucella abortus antigen, growth selection, antibody response) 2000 Poultry Science 79:804–809
been selected long-term for increased 16-wk BW; the line exhibits BW approximately 75% greater than that of a corresponding randombred control base population (RBC2) (Nestor, 1977b, 1984; Nestor et al., 1996a). The F line had higher mortality either during natural outbreaks of erysipelas and fowl cholera (Saif et al., 1984) or during experimental challenges with virulent Newcastle disease virus (Tsai et al., 1992) and Pasteurellla multocida (Sacco et al., 1991). The changes in resistance to Newcastle disease virus and P. multocida in the F line could not be explained by changes in frequency of MHC Class II haplotypes (Nestor et al., 1996c). Many kinds of antigens have been used to monitor immune responsiveness in poultry. Nonpathogenic antigens include synthetic glutamic acid-alanine-tyrosine (Cheng and Lamont, 1988), BSA (Heller et al., 1992; Parmentier et al., 1994, 1997), SRBC (Siegel and Gross, 1980; Van der Zijpp and Nieuwland, 1986), and Brucella abortus (BA) (Munns and Lamont, 1991; Scott et al., 1994; Nelson et al., 1995; Dix and Taylor, 1996). The SRBC antigen
INTRODUCTION Commercial turkey breeders should have a goal of improving genetic resistance to disease along with improvements in growth rate, conformation, and reproduction. Improving genetic resistance to disease would reduce the cost of vaccination and other disease prevention procedures and reduce the mortality and loss in performance during disease outbreaks. Selection for increased BW has been shown to be genetically associated with a reduction in immunocompetence and disease resistance in chickens (Han and Smyth, 1972, 1973; Mauldin et al., 1978; Qureshi and Havenstein, 1994) and turkeys (Saif et al., 1984; Sacco et al., 1991; Nestor et al., 1996a,b; Bayyari et al., 1997). A line (F) of turkeys has
Received for publication April 26, 1999. Accepted for publication March 6, 2000. 1 Salaries and research support provided by state and federal funds appropriated to the Ohio Agricultural Research and Development Center, The Ohio State University. 2 To whom correspondence should be addressed: Karl E. Nestor, Department of Animal Sciences, Ohio Agricultural Research and Development Center, The Ohio State University, Wooster, OH 44691; email: [email protected]
Abbreviation Key: BA = Brucella abortus antigen; MER = mercaptoethanol-resistant; MES = mercaptoethanol-sensitive; PPI = post-primary immunization; PSI = post-secondary immunization; RBC = randombred control; TD = thymus-dependent; TI = thymus-independent.
BODY WEIGHT SELECTION AND ANTIBODY RESPONSE
is classified as a thymus-dependent (TD) antigen that obviously needs the help of T lymphocytes to produce antibodies; BA is a thymus-independent (TI) antigen that stimulates B cells with little assistance from T helper cells (Mosier and Subbarao, 1982). The antibody responses to vaccines for Newcastle disease and fowl cholera have been previously studied in the F and RBC2 lines (Sacco et al., 1994). Those researchers found that the F line had higher 9- and 15-wk anti-Newcastle disease titers, but lower 15-wk anti-P. multocida titers, than did the RBC2 line. The objective of the present study was to compare the antibody responses to SRBC antigen and BA in the F and RBC2 lines to determine whether these two lines have similar responses to the different types (TD and TI) of antigens.
MATERIALS AND METHODS Two lines (F and RBC2) of turkeys were used in the present study. Line RBC2 was a randombred control line (Nestor, 1977a); Line F was a subline of RBC2 selected for increased 16-wk BW (Nestor, 1977b, 1984; Nestor et al., 1996a). The birds were given access to water and feed ad libitum. Birds were provided the first two rations in a five-ration feeding system with declining protein (Naber and Touchburn, 1970). The birds of both lines were grown intermingled, sexes separate, and not vaccinated before the experiment. Ten males and 10 females from each line were injected i.v. either with 1 mL 5% SRBC antigen suspended in 0.9% saline or 0.1 mL undiluted BA3 per bird at 4 and 6 wk of age. Blood samples were collected before injection and at 4, 7, and 10 d postprimary (PPI) and postsecondary immunization (PSI). Sera were harvested and stored at −20 C until all samples were analyzed simultaneously. The agglutination assays for both SRBC antigen and BA were carried out as described by Munns and Lamont (1991). Sera were analyzed for total titers, mercaptoethanol-resistant (MER) titers, and mercaptoethanol-sensitive (MES) titers. The MER and MES titers are presumably IgG and IgM, respectively, in turkeys as in other species (Delhanty and Solomon, 1966; Qureshi and Havenstein, 1994).
Statistical Analysis Antibody titers were transformed to reciprocal log2 units of greatest dilution showing 100% agglutination prior to analysis. The IgM titers were calculated individually by subtracting the IgG titers from the total antibody titers. Analysis of variance of the transformed data was performed using the General Linear Models procedure (SAS Institute, 1988) in a factorial design with line and sex as factors. Means were separated using the LSD test. A difference with a probability of P ≤ 0.05 was considered significant.
Difco Laboratories, Detroit, MI 48232.
FIGURE 1. Total anti-SRBC titers (log2) in male turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days differ significantly (P ≤ 0.05).
RESULTS Response to SRBC Antigen The anti-SRBC titers for total and IgG antibodies were measured by a hemagglutination test. Except for IgG titers at 4 and 10 d PSI, significant interactions were not found between line and sex. However, there were significant sex differences in total titers at 4, 7, and 14 d PPI, in IgM titers at 4 and 14 d PPI, and in IgG titers at 4 and 10 d PSI (data not shown). Therefore, the means of the total, IgG, and IgM titers were compared between the two sexes separately for simplicity of presentation. Compared with the RBC2 line, the F line had significantly higher total anti-SRBC titers at 10 d PSI (males; Figure 1); at 14 d PPI; and at 4, 7, and 10 d PSI (females; Figure 2). The F line also had significantly higher IgM titers at 10 d PSI (males; Figure 3) and at 14 d PPI (females; Figure 4). The F line had significantly higher IgM titers than the RBC2 line but only for females at 4 and 10 d PSI (Figures 3 and 4). In general, female birds in the F line had apparently higher total and IgM anti-SRBC titers than did female birds in the RBC2 line, peaking at 7 d PPI and 4 d PSI, respectively.
Response to BA The total and IgM anti-BA titers were obtained by an agglutination assay. Except at 10 d PPI (P ≤ 0.05), a significant interaction was not observed between line and sex. Significant sex effects were found at 7 d PPI in total titers, at 7 d PPI and 4 d PSI in IgG titers, and at 7 d PSI in IgM titers (data not shown). Males in the F line had significantly lower total titers (Figures 5 and 6) and IgM titers at 10 d PPI than did males in the RBC2 line (Figure 7). No significant differences were found in the total titers
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FIGURE 2. Total anti-SRBC titers (log2) in female turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days differ significantly (P ≤ 0.05).
and IgM titers in female turkeys or in the IgG titers in both sexes at any time of measurement (Figures 6, 7, and 8). Total titers peaked at 10 d (males) or 7 d (females) for the primary response and then peaked at 7 d for the secondary response for both sexes.
DISCUSSION The present results show that the total anti-SRBC titers for both sexes in the F line were generally higher than
FIGURE 3. IgG and IgM anti-SRBC titers (log2) in male turkeys of Lines F and RBC. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days differ significantly (P ≤ 0.05).
FIGURE 4. IgG and IgM anti-SRBC titers (log2) in female turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days and antibody type differ significantly (P ≤ 0.05).
those in the RBC2 line and that total titers peaked at 7 d for the primary response and at 4 d for the secondary response. However, total anti-BA titers for male turkeys in the F line were lower than those for male turkeys in the RBC2 line, and the total titers peaked at 7 d for both sexes for the secondary response. Because the requirement of T cells is the main difference between TD and TI antigens, the evaluation of T lymphocytes subpopulations
FIGURE 5. Total anti-Brucella abortus titers (log2) in male turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days differ significantly (P ≤ 0.05).
BODY WEIGHT SELECTION AND ANTIBODY RESPONSE
FIGURE 6. Total anti-Brucella abortus titers (log2) in female turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization.
may provide some clues in understanding the variation of the antibody responses between the two lines. In a separate study, turkeys (16 or 24 wk of age) in the F line had a higher proportion of CD4+CD8− T lymphocyte subsets (helper T cells) in the peripheral blood than did turkeys in the RBC2 line (Li et al., 2000). It seems that growth selection might have resulted in correlated changes of T cell subpopulations, therefore affecting antibody production. Examination of other steps in the immu-
FIGURE 7. IgG and IgM anti-Brucella abortus titers (log2) in male turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization. Antibody means with different letters within days and antibody types differ (P ≤ 0.05).
FIGURE 8. IgG and IgM anti-Brucella abortus titers (log2) in female turkeys of Lines F and RBC2. F = Line of turkeys selected for increased 16-wk BW; RBC2 = randombred control line (base population of the F line); PI = primary immunization; SI = secondary immunization.
nological response, such as in B cell clones, T cell arming, and antigen processing and presentation, might help explain the line differences in antibody production. The MER and MES antibodies are commonly used, respectively, to measure IgG and IgM responses in chickens. Primary and secondary responses are expected to consist basically of IgM and IgG, respectively. The results in the primary responses to SRBC antigens and BA followed this pattern, consisting mainly of IgM; the secondary responses consisted of increasing but not predominant IgG. This phenomenon may be related to the shorttime interval between the primary and secondary injections. The IgG concentration in the secondary responses was reported to be greatly influenced by the number of weeks between the primary and secondary injections (Davis and Glick, 1988). However, total titers in the secondary responses are lower than those in the primary responses if birds are re-injected with antigens after a long time interval PPI in fast growing turkeys (M. A. Qureshi, Department of Poultry Science, North Carolina State University, Raleigh, NC 27601; personal communication). In addition, isotype switching is usually not expected in the responses to most complete TI antigens (Abbas et al., 1994). However, a large proportion of IgG was also found in the secondary responses to BA. Therefore, the results in present studies suggest that BA may be a partial TI antigen. Similarly, Trout et al. (1996) observed the increase of CD4+ T cells in the spleen following BA injection and suggested that there might be some CD4+ T lymphocyte participation in the responses to BA. In the present study, the total titers against SRBC antigens by i.v. administration tended to be higher than those by i.m. administration of antigens in a previous report, but the titers against BA were not significantly affected
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by either route of antigen administration (Li et al., 1997). Similar observations were described before (Van der Zijpp et al., 1986; Nelson et al., 1995). Van der Zijpp et al. (1986) postulated that the ellipsoid-associated cells were more effective in presenting SRBC antigen to immunocompetent cells than the antigen-presenting cells of the tissue or the peritoneal cavity, but both kinds of cells were equally effective in processing the BA. A sex effect was evident in antibody response to SRBC antigen in the F line. Compared with males, females seemed to have higher total and IgM titers in primary responses and higher IgG titers in secondary responses. This result is in agreement with previous results in chickens (Leitner et al., 1992; Sarker et al., 1999). The lower relative ratio of antigen dose to BW in males cannot account for this sex difference in antibody response because similar results were obtained in a group administrated with different doses of SRBC antigen according to individual BW (unpublished data). Sex hormones may be responsible for the differences in antibody response between the sexes. Eiginger and Garrett (1972) and Krzych et al. (1981) reported a distinct influence of estrogen and androgen on the immune system in mammalian species. Females usually exhibit a higher capacity for antibody formation after immunization (Paavonen et al., 1981). A negative genetic correlation between growth rate and antibody titers to SRBC antigen had been reported in chickens (Siegel and Gross, 1980; Martin et al., 1990; Miller et al., 1992; Qureshi and Havenstein, 1994). Other studies reported no adverse association between BW and antibody production in Japanese quail (Takahashi et al., 1984) or in chickens (Pitcovski et al., 1987). A theory on the allocation of available energy resources toward either growth or the immune system had been suggested (BoaAmponsem et al., 1991; Klasing and Johnstone, 1991; Qureshi and Havenstein, 1994). However, Klasing (1998) pointed out that the resources needed for immune processes should be substantially less than the resources needed for growth. Results obtained in the present study revealed an opposite relationship between BW and antiSRBC titers in turkeys than in chickens. The F line selected for increased BW had higher antibody response to SRBC than did the smaller RBC2 line. However, the F line was more susceptible to infectious diseases (Saif et al., 1984; Sacco et al., 1991; Tsai et al., 1992; Nestor et al., 1996a). Areas of the immune system other than humoral immunity, such as cellular immunity, phagocytosis, and cytokines, are being investigated in the F and RBC2 turkey lines. In other studies, the F line was found to have lower stimulation index to concanavalin A and higher frequency of turkey CD8α polymorphism than the RBC2 line (Li et al., 1999a,b). Selection pressure to increase 16wk BW in the F line might have altered T cell subpopulations and, therefore, might have resulted in changes in cellular and humoral immunity and influenced the disease resistance of this line. It would be helpful to elucidate the relationship of disease resistance and growth rate in turkeys further and improve both factors maximally and simultaneously by genetic selection.
ACKNOWLEDGMENTS The authors thank British United Turkeys of America, PO Box 727, Lewisberg, WV 14901, and Hybrid Turkeys Inc., 650 Riverbend Drive, Suite C, Kitchner, Ontario, Canada N2K 3S2, for financial support.
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