The Dynamics of Antibody Response to Escherichia coli Vaccination ...

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(Key words: antibody response, dynamics, chickens, early age, Escherichia coli). 2000 Poultry Science 79:1418–1423. INTRODUCTION. The immune system in ...
IMMUNOLOGY AND MOLECULAR BIOLOGY The Dynamics of Antibody Response to Escherichia coli Vaccination in Meat-Type Chicks N. Yonash,*,1,2 G. Leitner,† A. Cahaner,* and D. E. Heller* *Faculty of Agricultural, Food, and Environmental Quality Sciences, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel; and †Kimron Veterinary Institute, P.O. Box 12, Bet Dagan 50250, Israel ABSTRACT The dynamics of serum antibody (Ab) response in young broilers were studied in lines divergently selected for high (HC) or low (LC) Ab response to Escherichia coli vaccination at an early age, and their cross (HL). Chicks were divided into three vaccination-age (VA) groups: 8, 10, and 12 d of age (VA8, VA10, and VA12, respectively). Antibody response was determined five times for each chick, at 6, 8, 10, 12, and 14 d postvaccination (dPV). The effects of line, VA, and dPV on Ab titers were highly significant. The HC and LC chicks exhibited the highest and lowest mean titers, respectively, in all VA groups. The HL chicks exhibited midparent Ab values

for all VA and dPV combinations, indicating additive inheritance of early Ab production. In LC, the highest mean Ab titer was obtained on Day 26 (14 dPV of the VA12 group), whereas in HC, the same titer had already been obtained on Day 18 (VA8-10 dPV and VA10-8 dPV combinations). The VA8 and VA12 chicks differed markedly in their Ab titer dynamics curves, and the VA10 chicks exhibited an intermediate curve. The three VA groups exhibited a similar change in Ab level from 6 to 10 dPV, but they differed in Ab change from 10 to 14 dPV. This significant dPV × VA interaction suggests that the VA12 and VA10, but not VA8, chicks maintained the capability to produce persisting Ab.

(Key words: antibody response, dynamics, chickens, early age, Escherichia coli) 2000 Poultry Science 79:1418–1423

The immune system in newly hatched chicks is not completely mature (Sharma, 1976; Yonash et al., 1996), rendering them highly susceptible to diseases. Selection for early maturation of the immune system is, therefore, expected to increase the immunocompetence of young chicks. In broilers, which reach market weight at an early age (currently at about 6 to 7 wk), early maturation of the immune system and disease resistance can be crucial for survival and productivity. Selection for increased immune response has been found to improve disease resistance in egg-type (Gross et al., 1980; Dunnington et al., 1986; Pinard et al., 1993) and meat-type (Leitner et al., 1992) chickens. In most of these selection experiments, antibody (Ab) response at a single time point was used as the selection criterion; hence, individual differences in Ab response kinetics were not considered in the selection. However, in several other experiments, the dynamics of the chicken’s immune response were studied. Lindsay et al. (1988) showed differences in age susceptibility of

broiler chickens to Cryptosporidium baileyi infection by the intratracheal route. Hatkin et al. (1993) reconfirmed the importance of age of infection in susceptibility to C. baileyi and demonstrated that chicks produce at least two antiC. baileyi Ig isotypes: IgM and IgG. The kinetics of the anti-C. baileyi IgM and IgG were shown to be dependent upon the age at which chickens were exposed to C. baileyi: 14-d-old chicks produced a more rapid immune response than 1- or 7-d-old birds. Genetic differences exist between White Leghorn chickens selected for high or low Ab response in the magnitude (van der Zijpp, 1978, 1983) and kinetics (van der Zijpp, 1983) of primary Ab response to SRBC. Miller et al. (1992) also studied the dynamics of Ab response in White Leghorn lines divergently selected for high or low Ab response to SRBC antigen, and in lines divergently selected for high or low 8-wk BW. They reported a peak of Ab to SRBC at 6 to 7 d after the primary injection, and consistently higher Ab titers in the highresponse than in the low-response line, and in the highBW than in the low-BW line. Similar Ab production dynamics were found by Bacon et al. (1972) in adult, eggtype chickens, where the peak titer was also observed 6 to 7 d after primary immunization.

Received for publication July 12, 1999. Accepted for publication May 24, 2000. 1 Present address: Department of Animal Science, The University of Connecticut, Storrs, CO 06269-4040. 2 To whom correspondence should be addressed: [email protected]

Abbreviation Key: Ab = antibody; dPV = days postvaccination; HC = line selected for high Ab response of young chicks to Escherichia coli vaccination; HL = progeny of HC × LC and LC × HC crosses; LC = line selected for low Ab response of young chicks to Escherichia coli vaccination; OD = optical density; VA = vaccination age.




Early maturation of the immune system, better immunity of the young chick, and an early Ab response to antigen stimulation are not the only relevant factors; the ability to continuously produce Ab over a longer period of time is also important. Divergent selection of meattype chickens for high (HC) or low (LC) Ab response to Escherichia coli vaccination at an early age (Leitner et al., 1992) resulted in early or late immunocompetence, respectively. The two selected lines differed in the earliest age at which young chicks could produce detectable Ab titers against antigen, as well as in the titer’s subsequent rate of increase (Yonash et al., 1996). A similar level of heritability was found for early immune response in the two divergent lines, but this variation was fully expressed 4 d earlier in the HC line than in the LC line. In addition to the significant difference in the selection criterion (i.e., Ab response to E. coli vaccination), the two chicken lines differed in their Ab response at an early age to immunization with Newcastle disease virus and SRBC and in the total IgG level in the serum at 20 d of age (Heller et al., 1992). The objective of this work was to study Ab response dynamics in young meat-type chicks from the divergently selected HC and LC lines and their cross (HL).

MATERIALS AND METHODS Chicks and Experimental Procedures Chicks were produced by mating males and females from the fifth (S5) generation of meat-type chicken lines, HC and LC (Leitner et al., 1992; Yonash et al., 1996). All the chicks (430 males and females) were produced in one hatch by matings within lines (9 males with 18 females in HC, and 7 males with 19 females in LC) and between lines (9 HC males with 21 LC females, and 7 LC males with 17 HC females). All chicks were wing-banded and raised together on the floor in one litter pen, under standard broiler management. The chicks from each dam family were divided into three groups that differed in vaccination age (VA). The first group was vaccinated at 8 d of age (VA8), the second at 10 d of age (VA10), and the third at 12 d of age (VA12). Antibody response was determined five times for each chick, at 6, 8, 10, 12, and 14 d postvaccination (dPV).

Vaccination and Antibody Determination Escherichia coli serotype O2:K1 was used for vaccination and for ELISA, as described by Leitner et al. (1989). For the vaccination and the Ab determination, the bacterial suspension was sonicated using an ultrasonic disintegrator at 1.2 A for 15 min. The suspension was stored at −20 C. Before vaccination, a frozen, sonicated sample was thawed and mixed 1:1 with aluminum hydroxide (109 bacteria/mL). Each chick received 0.5 mL of suspension injected subcutaneously. Chick Ab response was evaluated by measuring Ab production in vivo using ELISA. When an assay was performed, 100 µL of tested serum diluted 1:400 was


added in duplicate and incubated for 1 h at 37 C. The plates were washed three times, and a 1:2,000 dilution of a rabbit anti-chicken IgG heavy-and-light-chain peroxidase conjugate was added to each well and incubated for 1 h at 37 C. The bound Ab were detected by adding 100 µL of 2,2′-azino-di[3-ethylbenzthiazolinesulfonate (6)] peroxidase substrate after washing. The respective plates were read in a microplate autoreader at 405 nm (Leitner et al., 1990). The natural log of the Ab titer (Yi) in each serum was calculated as described by Leitner et al. (1992) using the following linear regression equation: Yi = 0.32(Pi/N) − 0.32 where Yi is the Ab titer of the ith sample, Pi is the sample’s optical density (OD) reading, and N is the OD of the low (negative) standard in each plate.

Statistical Analysis Homogeneity of variances among the nine groups of chicks (line × VA combinations) was tested by O’Brien test using JMP (SAS Institute, 1995). Ab-response dynamics were expressed by the titer at 6, 8, 10, 12, and 14 dPV. Changes in Ab titer were calculated for four consecutive 2-d periods (6 to 8, 8 to 10, 10 to 12, and 12 to 14 dPV) and two consecutive 4-d periods (6 to 10 and 10 to 14 dPV). The significance of titer differences between lines (L), sex (S), VA (V), dPV (P) and their interactions was determined by a 4-way model according to a split-plot design with line, sex, and VA assigned to main plots (chicks), and dPV assigned to secondary plots (Ab measurement at a given dPV). The ANOVA was calculated using JMP (SAS Institute, 1995). The effects of line, VA, sex, and their interactions were tested against the between-chicks error term (ec), and the effects of dPV and its interactions were tested against the between-measurements error term (em): Y=µ+L+V+S+L∗V+L∗S+V∗S + L ∗ V ∗ S + ec + P + P ∗ L + P ∗ V + P ∗ S +P∗L∗V+P∗L∗S+P∗V∗S + P ∗ L ∗ V ∗ S + em.


Model 1 was also used to analyze Ab titer changes during the four 2-d periods and during the two 4-d periods, with dPV (P in Model 1) indicating the periods.

RESULTS The degrees of freedom and significance levels of all the main effects and their interactions are presented in Table 1. Due to the high degrees of freedom of the error term (approximately 300), only effects with P(F) < 0.01 were considered significant, whereas those with marginal significance (0.01 < P(F) < 0.05) were regarded as nonsignificant. The interactions with sex, as well as the sex main effect, were not significant, and therefore averages of male


YONASH ET AL. TABLE 1. An ANOVA of the main effects of line,1 vaccination age (VA),2 sex, and days postvaccination (dPV),3 and their interactions on antibody (Ab) response at each dPV, on Ab change in each of four 2-d periods, and on Ab change in each of two 4-d periods dPV3

2-d period4

4-d period5

Source of variation







Line VA Sex Line × VA Line × sex VA × sex Line × VA × sex dPV dPV × line dPV × VA dPV × sex dPV × line × VA dPV × line × sex dPV × VA × sex dPV × line × VA × sex

2 2 1 4 2 2 4 4 8 8 4 16 8 8 16

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