Effect of Genetics, Vaccine Dosage, and Postvaccination Sampling Interval on Early Antibody Response to Salmonella enteritidis Vaccine in Broiler Breeder Chicks1 M. G. KAISER,* T. WING,† and S. J. LAMONT*,2 *Department of Animal Science, Iowa State University, Ames, Iowa 50011-3150, and †Cobb-Vantress, Inc., Siloam Springs, Arkansas 72761 postvaccination. At all vaccine dosages, there was a significant antibody-response difference between the genetic lines at 6 and 10 d postvaccination. The vaccine dosage significantly affected antibody levels in one of the two genetic lines. These results demonstrate a genetic component of early antibody response to SE vaccine in broiler breeder chicks.
ABSTRACT Broiler breeder chicks of two different genetic lines were evaluated for early antibody response to Salmonella enteritidis (SE) vaccine. Antibody responses to three dosages of SE vaccine administered at 22 d of age were measured at 3, 6, and 10 d postvaccination. Within each line, antibody levels at 10 d postvaccination were significantly higher than at either 3 or 6 d
(Key words: early antibody response, Salmonella enteritidis, broiler breeder, chick) 1998 Poultry Science 77:271–275
enhance antibody responsiveness in chickens. Previously, several chicken lines, including both broilers and White Leghorns, have been selected for enhanced immune function without pathogenic challenge (Siegel and Gross, 1980; Pevzner et al., 1981; Heller et al., 1992; Pinard et al., 1992; Kean et al., 1994). White Leghorns selected for high antibody response to GAT have correlated high antibody levels to Pasteurella multocida and Mycoplasma gallisepticum vaccinations (Cheng and Lamont, 1988). Long-term selection for immune competence in Leghorns altered both the time of peak antibody production and the duration of antibody production to both P. multocida and M. gallisepticum vaccinations (Weigend et al., 1997). Broiler chicks have been divergently selected for early antibody response to an Escherichia coli vaccine (Heller et al., 1992). The selection for high, early E. coli antibody response also increased antibody response to Newcastle disease virus and SRBC, increased phagocytic activity, and increased proliferation response to E. coli antigen and concanavalin A, phytohemagglutinin, and pokeweed mitogen (Heller et al., 1992). Broilers with higher antibody levels have greater survival against certain pathogenic challenges. Broilers with high vaccine antibody levels had a higher survival rate to challenges with P. multocida (Hofacre et al., 1986) and E. coli (Leitner et al., 1992) than did their low antibody level counterparts.
INTRODUCTION Salmonella enteritidis (SE) contamination of poultry products continues as a major food safety concern (O’Brien, 1988; Altekruse and Swerdlow, 1996). In 1991, a year-long, nationwide survey in the U.S. reported SE contamination rates of 6 to 20%, by region, in unpasteurized liquid egg (Ebel et al., 1993). An estimated 30 to 50% of all poultry carcasses are contaminated with Salmonella spp. (Waldroup, 1996). The prevalence of SE in reported Salmonella infections in humans has risen dramatically in the recent decades, from 6% in 1980 to 26% in 1994 (Anonymous, 1995). In addition to being a food safety concern, disease from SE can also contribute to a loss in broiler production efficiency and an increase in early mortality (Rampling et al., 1989). Lister (1988) proposed that vertical transmission may occur in SEinfected broiler breeding stock. Because of continuing concerns about SE in the poultry industry, additional control measures need to be initiated. Methods to control SE contamination include management practices, the use of pharmaceutical agents, and genetic selection for immune function. Genetic selection for immune function is an effective method to
Received for publication February 28, 1997. Accepted for publication October 7, 1997. 1Journal paper Number J-17255 of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa. Project Number 3290. Partly supported by BARD grant US-2317-93. 2 To whom correspondence should be addressed: email:
[email protected]
Abbreviation Key: PV = postvaccination; SE = Salmonella enteritidis.
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Because of the age at which broilers are marketed, it is most effective that genetic selection for humoral immunity enhance early antibody responsiveness. The ability to respond to an antigen is age-dependent in broiler chicks. Leitner et al. (1992) demonstrated that broiler chicks became progressively more responsive to E. coli vaccine from 1 to 10 d of age. Furthermore, their study illustrated that levels of E. coli antibody increased over time to a maximum at about 10 d postvaccination (PV). Lines selected for early high or low levels of E. coli antibody differ in their ability to produce antibody, in both the maximum level and the kinetics of antibody production (Yonash et al., 1996). Multiple components of the immune system impact the host response to Salmonella (Corrier et al., 1991; Arnold and Holt, 1995). The appearance of antibodies to Salmonella typhimurium after pathogenic challenge suggests that rapid antibody response may be an important component in protection against Salmonella (Lee et al., 1981; Brito et al., 1993). In contrast to many measures of cell-mediated immunity, quantifying serum antibody levels provides a method to measure immune response in birds that can continue in a reproductive population, thus allowing direct genetic selection for antibody response. It is possible to successfully select chicken lines for enhanced antibody response to bacterial vaccines and thereby improve resistance to disease. Genetic enhancement of SE resistance would help minimize losses in broiler production and possibly provide poultry products with less microbial contamination. The objective of this study was to evaluate the effects of genetics, vaccine dosage, and PV sampling interval on rapid, early antibody response to SE vaccination in broiler breeder chicks.
MATERIALS AND METHODS
Chickens In each of two commercial broiler breeder male lines, 95 1-d-old chicks were divided into three vaccine-dosage groups and one unvaccinated control group. Chicks were assigned to the groups so that sire families were equally represented within vaccine dosage groups. The chicks received a standard vaccination program of Marek’s disease/infectious bursal disease3 vaccine at hatch, and coccidiosis vaccine4 at 3 to 5 d of age. Chicks were housed together by line in floor pens and were given ad libitum
3HVT/SB1/Standard
IBD (MGSF-3215), Select Laboratories, Gaines-
ville, GA 30506. 4Coccivac-D, Sterwin Laboratories, Millsboro, DE 19966. 5Biommune, Lenexa, KS 66215. 6Salmonella enteritidis (antibody test), IDEXX Laboratories, Inc., Westbrook, ME 04092. 7 Model EL340 Biokinetics reader, Biotechnical Instruments, Winooski, VT 05440-0998.
access to feed and water under standard commercial broiler conditions. Diets met or exceeded National Research Council (1994) requirements.
Salmonella enteritidis Vaccination and Sera Collection Salmonella enteritidis vaccine,5 a commercially prepared bacterin, was administered s.c. in the neck at 22 d of age. Each vaccinated group received one of three dosages (0.2 or 0.5 mL injected at a single site, or 0.3 mL injected at two sites for a total of 0.6 mL). The manufacturer’s recommended dosage is 0.5 mL per adult bird. A much lower dosage was also used, to increase the opportunity to detect possible differences in genetic control of response (Gross, 1979). Blood samples (1.0 mL) were obtained from all chicks from the wing vein at 3, 6, and 10 d PV. Prevaccination samples were obtained from 7 to 10 chicks per line to monitor pre-existing SE antibody levels. The clotted blood was centrifuged (16,000 × g, 5 min) and the sera were collected, aliquoted, and stored frozen until assayed.
ELISA The ELISA assays were performed by using commercial SE antibody ELISA test kits.6 The assay was a competitive ELISA that used a monoclonal antibody to Salmonella G and M flagellin. The assay was conducted following the manufacturer’s directions except that a serum dilution of 1:100, rather than the manufacturer’s recommended dilution of 1:250 to 1:500, was used to consistently detect the relatively low antibody levels generated in the young chicks. Optical density of samples was read on an automated ELISA reader7 at 630 nM. Results were reported as the ratio S/N = sample OD/NCX where NCX = the negative control mean. The SE antibody ELISA assay is a competitive assay; therefore, lower ELISA values reflect higher serum antibody levels. An S/ N value of 1.00 would thus be identical to the negative control.
Statistical Analysis Statistical analyses were conducted by using the General Linear Models procedure (GLM) of SAS (SAS Institute, 1988). The model was: y = line + PV interval + dosage + sex + body weight + plate (assay date) + assay date + line × PV interval + dosage × PV interval + e where y = S/N ratio and e = error. Probability values of P ≤ 0.05 values were considered significant. Prevaccination samples and samples from chicks receiving no vaccine
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EARLY ANTIBODY RESPONSE TO VACCINATION IN BROILER CHICKS TABLE 1. Salmonella enteritidis antibody ELISA values1 of two broiler breeder lines by line, vaccine dosage, and postvaccination sample interval
Line
Dosage
1 1
(mL) . . . 0
1
0.2
1
0.5
1
0.6
2 2
. . . 0
2
0.2
2
0.5
2
0.6
Postvaccination interval
n
Mean + SD2
(d) 0 3 6 10 3 6 10 3 6 10 3 6 10 0 3 6 10 3 6 10 3 6 10 3 6 10
7 4 4 4 26 26 28 29 26 27 11 10 12 10 5 4 5 28 27 25 26 28 25 12 11 12
0.98 1.03 0.93 1.08 1.03 1.02 0.79 0.98 0.99 0.83 1.06 1.14 0.82 0.90 1.01 0.95 1.00 1.04 0.98 0.61 1.05 0.97 0.60 1.04 1.01 0.71
± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±
0.06 0.15a 0.04a 0.17a 0.10a 0.15a 0.21b 0.09a 0.16a 0.21b 0.10a 0.14a 0.20b 0.16 0.16a 0.15a 0.10a 0.21a 0.13a 0.18b 0.14a 0.15b 0.13c 0.06a 0.08a 0.16b
Range 0.91 0.86 0.89 0.94 0.86 0.83 0.49 0.80 0.78 0.47 0.95 0.98 0.48 0.50 0.87 0.85 0.88 0.39 0.74 0.27 0.83 0.75 0.39 0.94 0.86 0.50
to to to to to to to to to to to to to to to to to to to to to to to to to to
1.10 1.20 0.98 1.28 1.27 1.39 1.30 1.13 1.43 1.24 1.21 1.41 1.17 1.07 1.25 1.16 1.11 1.48 1.30 0.92 1.41 1.31 0.85 1.14 1.13 1.07
a–cMeans within dosage within line with no common superscript differ significantly (P ≤ 0.05; Duncan’s test). 1ELISA values = sample OD/negative control OD. The SE antibody ELISA is a competitive assay; thus, lower values indicate higher serum antibody levels.
were not included in the antibody response data analyzed, except where specified.
RESULTS The SE antibody ELISA value means, standard deviations, and ranges, by line, dosage, and PV interval, are presented in Table 1. Unvaccinated chicks did not produce measurable SE antibody over the time of the experiment, nor did prevaccination antibody levels differ from the negative control. Two variables related to the conduct of the antibody assay (day of assay and plate on which the sample was run) had significant effects on antibody values (data not shown) and thus are included in the model for the ANOVA summary presented in Table 2. Overall main effects (data not shown) of genetic line, vaccine dosage, and PV interval and interactions of line by PV interval and dosage by PV interval on postvaccination antibody levels in chicks that received vaccine were significant. Main effects of sex and body weight on antibody response were nonsignificant (data not shown). The genetic line effect was evident at all doses and at 6- and 10-d PV sampling intervals. There was a significant (P ≤ 0.002) genetic line by PV sampling interval interaction on the SE antibody response for 0.2- and 0.5-mL dosages (Table 2).
The PV sampling interval had a significant (P = 0.0001) effect on antibodies at all dosages and in both lines (Table 2). The antibody response at 10 d PV was consistently, significantly higher (lower competitive ELISA values) than that at 3 and 6 d PV (Table 1). Antibody levels at 3 and 6 d PV did not differ from each other nor from prevaccination levels for all line by dose
TABLE 2. ANOVA summary of early antibody response to Salmonella enteritidis vaccine in breeder broiler chicks for variables of line, postvaccination (PV) interval, and dosage and for two-way interactions P values
Level Dosage, mL 0.2 0.5 0.6 Line 1 2 PV interval, d 0 3 6 10
Line 0.004 0.001 0.040 Dosage 0.0003 0.076 Line 0.690 0.703 0.009 0.0001
PV interval 0.0001 0.0001 0.0001 PV interval 0.0001 0.0001 Dosage 0.749 0.404 0.006 0.015
Line × PV interval 0.002 0.0001 0.2108 Dosage × PV interval 0.093 0.700 Line × Dosage . . . 0.954 0.183 0.240
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comparisons (Table 1). The effect of vaccine dosage on the level of antibody response to SE vaccination was significant (P = 0.0003) in Line 1 and approached significance (P = 0.076) in Line 2 (Table 2).
DISCUSSION The control of SE in broiler breeding and production is a continuing concern. The between-line differences demonstrated in this study indicate that there is a genetic component to the control of SE antibody response and the within-line phenotypic variation demonstrates that there is sufficient variation between or within families to allow within-line selection for enhanced rapid antibody response to SE vaccination. Both lines exhibited a wide phenotypic range in the response to SE vaccine at an early age. This study was not designed to identify the time at which maximum antibody levels are reached in the two genetic lines but, rather, to identify an early time point at which significant antibody level differences within lines were detectable. In a separate experiment, the broad-sense heritability (calculated from the dams’ variance component) of SE antibody at 10 d PV was 0.26 and 0.25 in the two broiler breeder lines (Kaiser et al., 1997). Serum antibody levels are associated with the chicken’s ability to resist pathogenic bacterial challenge (Hofacre et al., 1986; Leitner et al., 1992). Through genetic selection for specific antibody production, broilers can become more resistant to bacterial challenge (Leitner et al., 1992). The current study has thus laid the foundation for improving genetic resistance to SE by demonstrating that SE vaccine antibody levels are under genetic control. The PV time required to elicit a protective antibody response is an important consideration for animals such as broilers that are marketed at a relatively young age. Leitner et al. (1992) demonstrated that the age of a chick at vaccination and the PV sampling time are important parameters in antibody production to E. coli in young broilers. The present study demonstrated that the antibody response to SE is time-dependent and that genetic differences became detectable over time. Within each line, SE antibody response at 10 d PV was significantly different from the response at the other sampling intervals. It is possible that antibodies continued to increase after 10 d PV, the last measurement interval. When compared by PV sampling interval, there was a significant difference between the lines at 6 and 10 d PV with the level of significance increasing with time, illustrating the increasing ability to detect the genetic effect over time. The antibody response differed over the threefold range of dosages used in this study, significantly in Line 1 and approaching significance in Line 2. There was also a line by PV sampling interval interaction at the two lower dosages. The highest dose may have obscured the ability to detect this interaction. Appropriate choice of antigen dosage is important in studies to detect genetic
differences in the immune response. Gross (1979) demonstrated that dosage of SRBC influenced the ability to detect genetic differences in the immune response. Schat et al. (1981) noted that use of strains of pathogen (Marek’s disease virus) of different oncogenicity revealed different line rankings of genetic resistance. This study demonstrated a genetic effect on the early SE antibody response in commercial broiler breeder chicks. Genetic selection for rapid and high antibody response to SE vaccination could provide greater protection against SE infections. The extent to which the antibody response to SE vaccination can be modified and the level to which this will provide better resistance to SE infection, and perhaps reduced product contamination, has yet to be determined. However, improvement of animal welfare, production efficiency, and food safety via genetic selection for rapid, early antibody response to SE appears to be a feasible goal.
ACKNOWLEDGMENTS The authors thank Ervin Johnson and Kevin Esch for expert technical assistance, and IDEXX Laboratories, Inc. for a donation of some of the ELISA kits used in this study.
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