Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination

Veterinary Immunology and Immunopathology, 34 ( 1992 ) 159-172 159 Elsevier Science Publishers B.V., Amsterdam

Immunological parameters in meat-type chicken lines divergently selected by antibody response to

Escherichia coli vaccination

E. D a n H e l l e r a, G a b r i e l L e i t n e r a, A h a r o n F r i e d m a n a, Z e h a v a U n i a, M i c h a l G u t m a n b a n d A v i g d o r C a h a n e r b

aDepartment of Animal Science, Faculty of Agriculture, The Hebrew University o f Jerusalem, Rehovot 76100,Israel

bDepartment of Genetics, Faculty of Agriculture, The Hebrew University of Jerusalem, Rehovot 76100, Israel

(Accepted 31 December 1991 )

ABSTRACT

Heller, E.D., Leitner, G., Friedman, A., Uni, Z., Gutman, M. and Cahaner, A., 1992. Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination. Vet. Immunol. Immunopathol., 34: 159-172.

Our group has established two lines of meat-type chickens divergently selected for early (HC line) and late (LC line) antibody responsiveness at 10 days of age to immunization with inactivated pathogenic Escherichia coli bacteria. The question addressed in the study presented here is whether this selection has changed other immunological responses, increasing the overall 'early' immunocompetence. Broilers of the third and fourth generations ($3 and $4) of the selected lines (HC and LC) and a control, unselected line (CT) were vaccinated at 10 days of age with E. coli vaccine, Newcastle virus vaccine (NDV), sheep erythrocytes (SRBC) or bovine serum albumin (BSA), Line-HC chicks exhibited higher antibody titers to E. coli, NDV and SRBC than CT or LC chicks. At 20 days of age HC chicks demonstrated a higher total protein and a higher fl- and ),-globulin levels in their serum. At 21 days of age, HC chicks cleared carbon particles faster than LC chicks. Peripheral blood lymphocytes (PBL) from HC chicks vaccinated with E. coli vaccine, proliferated in vitro more actively in the presence of the stimulating antigen than the PBL of LC chicks. Peripheral blood lymphocytes (PBL) obtained from HC-line chicks exhibited a higher proliferative response to concanavalin A (Con A)-, phytohemagglutinin (PHA)- or pokeweed mitogen (PWM)-stimulation than LC PBL. These results demonstrate that the selection for high or low antibody response to E. coli at a young age resulted also in a significant change in the response of other parameters of the immune system. The high response to E. coli was found to be associated with a high antibody response to other antigens (NDV and SRBC), increased phagocytic activity and increased proliferative response to antigen or mitogens. The selection most probably affected early immunocompetence.

ABBREVIATIONS

BSA, bovine serum albumin; CC, carbon clearance; Con-A, concanavalin A; cpm, counts per minute; GAT, Glutamine-Alanine-Tyrosine; MHC, Major Histocompatability Complex; NDV,

Correspondence to." E. Dan Heller, Department of Animal Science, Faculty of Agriculture, The Hebrew University of Jerusalem, P.O. Box 12, Rehovot 76100, Israel.

© 1992 Elsevier Science Publishers B.V. All rights reserved 0165-2427/92/$05.00

160 E.D. HELLER ET AL.

Newcastle disease virus; PBS, phosphate-buffered saline; PC, post challenge; PHA, phytohemagglutinin; PI, post-injection; PWM, pokeweed mitogen; SRBC, sheep red blood cells.

INTRODUCTION

Diseases in livestock result from interactions between the individual's gen- otype, the pathogenic agent and the environment. Although control of the environment by sanitation and isolation, and massive use of pharmaceutical treatments such as vaccination and drugs, reduces the incidence of many diseases, the problem has not been eliminated. In addition to the high cost of these treatments, drug residues in meat have increasingly become a cause for concern among consumers. Modern intensive poultry production has created a demand for a state of continuous health in flocks, achieved by vaccination, hygienic measures, eradication of disease and medication to reduce losses ow- ing to disease. Genetic selection for higher resistance will make all these measures more efficient.

Disease resistance through genetic improvement is becoming a useful tool in livestock production. In poultry, several selection experiments have been based on the immune response to a single antigen, such as sheep red blood cells (SRBC), a non-pathogenic multi-determinant antigen (Siegel and Gross, 1980; Van der Zijpp, 1983) or an inactivated virus such as the Newcastle disease virus antigen (NDV) (Takahashi et al., 1984). However, to date, no selection experiment has resulted in general resistance or overall immunity. Negative or zero correlation between antibody production and bacteriocidal activity of macrophages in mice was reported by Biozzi et al. (1979). In chickens, no correlation was found between antibody titer to SRBC and cell- mediated immunity measured by the response to phytohemagglutinin (PHA) (Van der Zijpp, 1983). More recently, Cheng and Lamont ( 1988 ) analyzed the immune response to a variety of antigens and mitogens in already-established lines that had been selected for Major Histocompatability Complex (MHC) type and antibody response to the synthetic polypeptide Glutamine- Alanine-Tyrosine (GAT). They found a low correlation between antibody response, phagocytosis and T-cell-mediated responses. On the other hand, haplotypes exhibiting a high antibody response to GAT showed significantly higher antibody titers to Pasteurella multocida and Mycoplasma gallisepti- cure vaccines.

Early immunocompetence is especially important in controlling disease during the short lifespan of commercial broilers. One experiment involving selection at a young age was based on an index of early antibody production to E. coli and NDV (Pitcovski et al., 1987). Recently, our group has established two lines of meat-type chickens divergently selected for early and late

ANTIBODY RESPONSE TO E. COL1 VACCINATION IN CHICKEN 161

maturat ion of the immune system, based on family and individual antibody responsiveness at 10 days of age to immunizat ion with inactivated pathogenic E. coli bacteria (Leitner et al., 1992 ).

The question addressed in this study is whether our selection for early immunocompetence, based on antibody response to the E. coli vaccine at an early age, has changed 'early' overall immunocompetence, thereby making the chickens better suited to the conditions of modern management practices.

MATERIALS AND METHODS

Chickens

Experiments were conducted with chicks from the third ($3) and fourth ($4) generations of lines divergently selected for high (HC) or low (LC) response to E. coli vaccination at an early age, and from a control (CT) line (Leitner et al., 1992). For the in vitro experiments, chicks from the three lines were raised on the Rehovot campus in electrically-heated batteries. An- tibody production was determined in larger groups of $3 and $4 chicks which were raised in litters under standard commercial broiler management, in a large-scale experimental farm located elsewhere.

Immunization with various antigens

Antigens Escherichia coli serotypes 078:K80 and 02:K1 were used for immunization,

challenge and ELISA, as described by Leitner et al. ( 1989 ). Newcastle disease virus (NDV) mesogenic Komarov strain grown on chick embryos (alantoic fluid was collected), was used as an antigen following inactivation by beta- propiolacton and resuspension in a luminum hydroxide at a concentration of 10 9.3 embryonic LDs0 U ml - i . Each chick was immunized subcutaneously with 0.5 ml of this suspension. $4 chicks were vaccinated with a commercially attenuated NDV vaccine (BLT, Jerusalem, Israel) introduced via the drink- ing water.

Sheep erythrocytes were washed three times in 0.9% saline and suspended to a concentration of 5% (v /v ) . Each chick was immunized with 0.3 ml of this suspension, injected intermuscularly. Bovine serum albumin (BSA, Re- his, Phoenix, AZ) was dissolved in phosphate-buffered saline (PBS, 20 mg ml -~ ) and mixed 1 : 1 ( v /v ) with a luminum hydroxide prior to immunization. Each chick received 0.5 ml of this mixture injected into various subcu- taneous sites. For ELISA (see below), BSA was dissolved in 0.02 mol carbon- ate-bicarbonate, pH 9.6.


Antibody titer determination Chicks' humoral immune response to E. coli vaccination was evaluated by

ELISA (Leitner et al., 1990). The natural log (Ln) antibody titer of each serum was calculated from the linear regression equation

LN t i t e r=4 .90+ 1.35 ( P / N ) - 6 . 2 5

(6.25 is the optical density of a serum which is equal to that of the negative control serum, i.e. P / N = 1 ).

Antibodies to NDV were determined in a standard hemagglutination-inhibition assay (Brugh et al., 1978), antibodies to SRBC were examined by a standard hemagglutination test, and antibodies to BSA were examined by ELISA (Leitner et al., 1989).

Experimental procedures Antibody response to immunizat ion was studied in generations $3 and $4,

raised in litters under standard broiler management. There were two hatches, 10 days apart, in generation $3 with a total of 642 chicks, and one hatch in $4 with 304 chicks. Chicks in all three hatches were vaccinated with E. coli antigen at 10 days of age. Antibody titer was determined in blood samples taken 10 days post-vaccination in $3, and 11 days post-vaccination in $4. Third generation S3-chicks were immunized against NDV antigen at 20 days of age, and antibody titers were determined in blood sampled 10 days later. Fourth generation $4 chicks received the standard commercially attenuated NDV vaccine at 10 days of age, and antibody titers were determined in the blood samples taken at 20 days of age.

From the first $3 hatch, 191 chicks were inoculated with SRBC, while 113 chicks from the second hatch were inoculated with BSA. Both antigens were introduced at 23 days of age. Antibody titers for both antigens were determined 7 days post-inoculation.

Blood proteins and immunoglobulins

Total serum proteins, albumin and a-, fl- and y-globulins were determined in blood serum samples. Total protein was determined by Progress selective chemistry analyzer (Kone, Finland ). Albumin and globulin levels were determined by electrophoresis using a Titan Serum Proteins Kit (Helena Labora- tories, Beaumont, TX). Eighty-three 20-day-old chicks were sampled from the $4 generation: 28 LC chicks showed the lowest E. coli titers, 30 HC chicks showed the highest E. coli titers, and 25 randomly selected CT chicks repre- sented the entire range of titers. Approximately equal numbers of males and females were taken from each line.

ANTIBODY RESPONSE TO E. COLI VACCINATION IN CHICKEN 163

Carbon clearance test

In vivo phagocytic activity was evaluated by a carbon clearance (CC) assay according to Cheng and Lamont (1988), with some modifications. A supernatant fraction (3000×g for 10 min) of India ink (Peliken 518.21A 862, Hannover, Germany) was injected into the brachial vein at a vol. of 3 ml kg -I body weight. At 2, 4, 6 and 15 min post-injection (PI), 0.2 ml of blood was drawn from the right jugular vein and transferred into 1.8 ml of 3.8% sodium citrate. After centrifugation (50 X g for 5 min ), the relative amounts of carbon remaining in the supernatant of each sample were spectrophoto- metrically estimated at 675 nm (Spectronic 70, Bausch and Lomb, Roches- ter, NY). This experiment was conducted with 40 3-week-old chicks, includ- ing ten males and ten females from HC and LC lines, respectively.

In vitro antigen-specific T-lymphocyte proliferation

An in vitro antigen-specific T-lymphocyte proliferation test was performed according to Leitner et al. (1989). Three-week-old chicks from HC and LC lines (four males and four females from each) were challenged with 1 X 106 CFU of 02:KI E. coli bacteria. Peripheral blood lymphocytes (PBL) were collected 48, 96 and 120 h post-challenge (PC) and their proliferative response to the antigen was measured.

Response to mitogens

Peripheral blood T-lymphocyte response to mitogenic stimulation by concanavalin A (Con-A) (Bio-Yeda 3X Cryst, Rehovot, Israel) ( 10 ~g m l - ' ), and PHA (HA 16, Wellcome, Dartford, UK) (20/~g ml - 1 ), was tested as described by Lassila et al. (1979). B-lymphocyte stimulation by pokeweed mitogen (PWM) (GIBCO, Grand Island, NY) (1:20 v /v ) was performed according to Lee (1984). The experiment consisted of 24 and 18 5-week-old chicks from LC and HC lines, respectively, with an equal number of males and females in each group.

Data analysis

A three-way ANOVA (GLM procedure, SAS Institute, 1987 ) with line, sex and hatch as main effects, revealed interactions with hatch. Therefore, antibody titers to the various antigens were analyzed separately for each hatch, using a two-way ANOVA with line and sex as main effects. A similar two-way ANOVA was applied to irnmunoglobulin data. Differences between lines in their response to each mitogen were tested by one-way analysis, since sex of chicks was not recorded. Phenotypic correlations between different antibody


responses were calculated within each l ine/hatch combination. Nested analyses of variance and covariance (NESTED procedure, SAS Institute, 1987) of sire families within lines over the two $3 hatches, were used to estimate genetic correlation between levels of response to different antigens.

RESULTS

Antibody response to immunization was studied in generations $3 and $4. There was no significant interaction between line and sex; therefore, line means over sexes within hatches are presented in Table 1. In all three hatches, the LC lines exhibited significantly lower E. coli antibody titers than the CT line, while the HC lines had the highest titers. The lines ranked similarly in their response to N D V vaccination in all three hatches (Table 1 ). Moreover, the differences between hatches in the absolute level of response to N D V cor- responded to those to E. coli antibody. The mean response to SRBC, tested in the first $3 hatch, also increased from LC through CT to HC lines, although only the latter's response was significantly higher. The selected lines did not differ however, in their mean antibody response to BSA, which was tested in only one hatch (Table 1 ).

The direct and correlated effects of selection on the distribution of individual antibody titers within lines are demonstrated in Fig. 1. The individual

TABLE1

Means (over sexes) of antibody response to E. coli, NDV, SRBC and BSA, of 20 to 30-day-old chicks from the low, high and control lines in $3 (two hatches) and $4 generations

Generation Hatch Antigen Line means ~ SD P(F) values

Low Control High Line Sex LXS

$3 l E. coli 1.39c 2.33b 3.27a 1.56 <0.00l 0.17 0.99 NDV 4.87b 5.01b 6.55a 1.93 <0.001 0.35 0.61 SRBC 2.36b 2.68b 3.71a 1.29 <0.001 0.73 0.06

$3 2 E. coli 0.96c 1.62b 2.07a .97 <0.001 0.57 0.80 NDV 3.75c 4.36b 5.68a 1.90 <0.001 0.01 0.19 BSA 11.6 - 11.9 1.44 0.350 0.12 0.86

$4 1 E. coli 1.37c 1.91b 2.41a 0.96 <0.001 0.59 0.54 NDV 4.71c 5.27b 5.92a 1.67 <0.001 0.27 0.98

No of chicks $3

$3

$4

1 E . c o l i + N D V 53 155 61 SRBC 70 45 76

2 E. c o l i + N D V 80 216 77 BSA 51 - - 62

1 E. c o l i + N D V 128 72 104

ILine means followed by different letters within a row are significantly different (P< 0.05 ).

ANTIBODY RESPONSE TO E. COLI VACCINATION IN CHICKEN ] 65

4 o - D

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9 11 13 15

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E. COLI TITER

Fig. 1. D i s t r i b u t i o n o f i n d i v i d u a l a n t i b o d y t i t e r s to ( A ) E. coli, ( B ) N D V , ( C ) S R B C a n d ( D )

B S A o f t h e c o n t r o l a n d s e l e c t e d l i n e s i n $3 g e n e r a t i o n .


titers of E. coli antibody in the CT line ranged from 0 to 9, with skewed distribution. While selection increased the proportion of medium and high titers in HC lines, it eliminated titers above 4 from the LC lines (Fig. 1 (A) ) . The correlated changes in immune response to other antigens are demonstrated by the similar changes observed in the distribution of antibody titers to NDV and SRBC, mainly in the HC lines, but also to a lesser extent in LC lines (Figs. 1 (B) and 1 (C)) .

Phenotypic correlations between antibody titers to E. coli and NDV were very low and generally non-significant (Table 2). However, the estimate of genetic correlation was positive (0.241) and significant, in agreement with the observed correlated response in N D V antibody at the line means level. The phenotypic correlations between E. coli antibody and antibodies to SRBC and BSA were also non-significant in most cases, although the genetic correlation with BSA was significant, but quite low (0.136 ).

Line-LC chicks had significantly lower levels of total serum proteins than HC chicks. Similar albumin levels were observed in the three lines, while the o~-globulin level in HC chicks was lower than that of CT chicks, but not significantly different from that of LC chicks. The three lines differed significantly in their fl- and y-globulin levels, with HC chicks having the highest mean, and LC chicks the lowest (Table 3 ).

TABLE2

Phenotypic (rp) and genetic ~ (ro) correlations between the antibody response to E. coli and NDV, SRBC, or BSA, in the $3 generation

Antigens rp (within lines) ro

Low Control High Lines combined

E. coli-NDV r 0.023 0.107 0.075 0.241 P ( r = 0 ) 0.790 0.039 0.379 <0.001 (df ) (130) (369) (136) (51,588)

E. coli-SRBC r 0.061 P ( r = 0 ) 0.616 (df) (68)

E. coli-BSA r 0.187 P ( r = 0 ) 0.189 (df) (49)

0.340 0.154 0.022 0.185

(43) (74)

2

0.195 0.136 0.132 0.010

(60) (24,88)

~Genetic correlations were estimated from analyses of variance and covariance of half-sibling sire families within lines. :There was no 'between-sire' within-line variance component for the SRBC antibody; hence rG could not be calculated.


TABLE 3

Means ( M + F ) of total protein, albumin, o~-, fl- and y-globulin content, and means and range of antibody response to E. coli and NDV, in the serum of 3-week-old chicks from the low, high and control lines, $4 generation

Line means ~ SD P ( F ) of effect

Low Control High Line Sex L × S

Content in serum (mg ml - j ) Total proteins 2.77b 3.12a 3.17a 0.46 0.003 0.06 0.77 Albumin 1.78 1.93 1.80 0.30 0.171 0.14 0.53 c~-globulin 0.489ab 0.513a 0.401b 0.16 0.031 0.20 0.97 fl-globulin 0.353c 0.494b 0.606a 0.22 0.001 0.39 0.53 y-globulin 0.136b 0.186b 0.354a 0.10 <0.001 0.82 0.24

Ln antibody titer E. coli 0.70c 2.17b 3.94a 0.83 < 0.001 0.84 0.81 NDV 3.88c 4.83b 5.72a 1.25 <0.001 0.78 0.67

No. of chicks 28 25 30

~Means are significantly different (P< 0.05 ) within a row where followed by different letters.

21 o HC male & female

"E ~ ~ i * LC male & female

~D

Q O

O "-'--" ' ~ , ~ . . . . . . . . . . - ? 0 6 ~5

Tine postinjection (min)

Fig. 2. Rate of carbon clearance from the blood of chicks of the selected lines. Means are significantly different ( P < 0.05) where followed by different letters.

In vitro phagocytic activity o f 3-week-old chicks was evaluated by CC assay. Male and female clearance rates did not differ significantly, nor were there any interactions between sex and line or time. Therefore, levels o f carbon remaining in the blood at 2, 4, 6 and 15 min PI were averaged for males and females within each selected line and plotted accordingly (Fig. 2) . The


6C

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120 t_ 13_

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6 0

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~m LC

1 Fig. 3. In vitro proliferation of PBL obtained from chicks of selected lines, after E. coli antigen stimulation. PBL were assayed for their proliferative response (A) 48 h, (B) 96 h, and (C) 120 h after challenge. Each bar represents an individual bird.

mean CC in HC chicks was significantly higher ( P < 0.05 ) than in LC chicks, except at 15 min PI.

To assess the involvement of cell-mediated immunity in the differences observed between selected lines, a specific assay of response to the E. coli antigen, was performed in vitro. All six HC chicks responded by proliferation as expressed in counts per minute (cpm) of incorporated 3H-thymidine as early as 48 h post challenge (PC) , while in the LC lines only two out of five chicks responded (Fig. 3 (A) ). This difference was found to be significant ( P < 0.05 ).


TABLE 4

Mean response (in cpm X 10 3 ) of non-specific T-lymphocytes to the mitogens PWM, Con-A and PHA of 5 week-old chicks (males and females) from the low, high and control lines, in the $3 generation

Mitogen Line means t SD P(F) of the line effect

Low Control High

Con-A 174.5a 169.0a 267.3b 70.9 0.0001 PHA 194.0a 224.2ab 273.4b 61.5 0.0002 PWM 27.4a 32.6ab 45.8b 15.8 0.0006 N 26 6 24

~Means are significantly different where followed by different letters.

At 96 and 122 h PC, all the examined chicks responded by proliferation, but HC chicks exhibited a higher mean response, which was found to be significantly different from that of LC chicks after log transformation of the data (Figs. 3 (B) and 3 (C) ).

The non-specific response of peripheral blood lymphocytes to Con-A, PHA and PWM was studied. The individual and mean proliferative responses, given in cpm X 103 of males and females in each line, for the three mitogens appear in Table 4. Line HC chicks exhibited a significantly higher response than LC chicks to all three mitogens, while CT chicks had an intermediate response to PHA and PWM, and a low response to Con-A.

DISCUSSION

The research described here was motivated by a need for highly disease- resistant chicks, especially in modern intensive broiler production systems. Today's poultry flocks depend upon protective vaccines to combat most vi- rological, as well as some bacteriological, diseases. Vaccination will no doubt continue to provide adequate control of certain diseases at cost-effective levels, although some problems are already emerging from the practice, e.g. losses in vaccinated flocks to Marek's disease or Infectious Bursal disease (Van den Berg et al., 1991 ). We therefore set out to determine whether appropriate genotypes could improve the efficiency of vaccination programs aimed at increased disease resistance.

The significance of the present study lies in its demonstration that the immune system can be selected for earlier responsiveness, i.e. its rate of maturation is influenced by the selection process. We showed that selection for high or low antibody response to E. coli antigen vaccination at an early age results in changes in other immunological characteristics as well. These include the reticuloendothelial system, as measured by CC, T-cell response to a specific antigen, lymphocyte responses to stimulation by different mitogens, and some components of blood serum proteins. This may indicate that selection causes a general change in the major genes controlling the early immune response, thereby providing a potential way of manipulating the immune response to


achieve an overall increase in early maturation and, consequently, increased viability.

In order to achieve total immunocompetence and disease resistance, all components of the immune system must function in complete cooperation and coordination. It was therefore believed that all components need to be considered simultaneously when selecting for general disease resistance (Biozzi et al., 1979). In the present study, the humoral immune response of two selected lines (HC and LC) was compared by using three different types of antigens: bacterial (E. coli), viral (NDV) and two non-pathogenic complex antigens (SRBC and BSA). The lines differed significantly in their antibody production following E. coli vaccination at 10 days of age, indicating a direct genetic response to selection. The lines also differed correspondingly in their mean antibody production to NDV and SRBC vaccination, both of which are also T-dependent antigens, i.e. HC chicks exhibited high antibody titers, while those from the LC lines showed a low response. No differences could be detected in the lines' antibody response to BSA. This could be attributed to the time lapse between vaccination and bleeding. Very high antibody titer levels to BSA were observed in both lines, indicating that the inductive phase, in which differences had previously been observed (Leitner et al., 1989), had been passed. The humoral immune system as a whole cooperates more effi- ciently and earlier in the chick's life in the HC line regardless of the antigen introduced. These results agree with those of Cheng and Lamont ( 1988 ), who showed that chickens selected for high response to GAT also had higher antibody titers to P. multocida and M. gallisepticum vaccines. They suggested that it is possible to improve antibody response to complex antigens by selecting for the response to a less complex one.

The overall humoral response in young chicks, based on the level of globulins at 20 days of age when maternal antibodies decrease to a min imum, dem- onstrates that y-globulin levels in HC chicks were twice as high as those of LC chicks. Rees and Nordskog ( 1981 ) also showed that y-globulin levels can be affected by genetic selection.

Our study of the effect of selection on specific T-cell response in vitro to the E. coli antigen revealed that T-cells from HC chicks responded with higher proliferation rates than cells from LC chicks. In addition, when polyclonal response of lymphocytes to the three mitogens (PHA, Con-A, PWM) was studied, it was found that HC chick response was significantly higher than that of LC chicks.

When phagocytic activity was tested in vivo by CC assay, HC chicks also cleared the carbon particles from their circulation significantly faster than LC chicks. These results agree with those in a previous report (Pitcovski et al., 1987), but not with those found by Van der Zijpp (1983) and Cheng and Lamont ( 1988 ), who reported that most of the correlations between antibody response, phagocytosis and T-cell-mediated responses were not significant.


The higher response of the HC line to the three specific antigens and higher total >globulin levels suggest that the humoral response rate of development to many antigens at a young age is controlled by the same, or by linked genes. The similar ranking of the selected lines with regard to all three components of the immune system suggests a common genetic factor controlling the immune system's rate of maturation. This factor may be connected to, or part of, the MHC.

ACKNOWLEDGEMENTS

This work was funded by the Israel-USA Binational Agriculture Research and Development Fund (BARD), and was performed by G. Leitner as par- tial fulfillment of the Ph.D. degree requirements.

REFERENCES

Biozzi, G., Mouton, D., Sant'Anna, O.A., Passos, H.D., Gennari, M., Reis, M.H., Ferreira, V.C.A., Heumann, A.M., Bouthiller, Y., Ibanez, O.M., Stiffel, C. and Sigueira, M., 1979. Genetics of immunoresponsiveness to natural antigens in the mouse. Curr. Top. Microbiol. Immunol., 85: 31-99.

Brugh, M., Beard, C.W. and Wilkins, W.S., 1978. The influence of test conditions of Newcastle disease hemagglutination-inhibition titers. Avian Dis., 22: 320-328.

Cheng, S. and Lamont, S.J., 1988. Genetic analysis ofimmunocompetence measures in a White Leghorn chicken line. Poultry Sci., 67: 989-995.

Lassila, O., Normi, T. and Eskola, J., 1979. Genetic differences in the mitogenic response of PBL in chickens. Immunogenetics, 6: 37-42.

Lee, L.F., 1985. Proliferative response of chicken T and B lymphocytes to mitogens. In: M. Kendle et al. (Editors), Chemical Regulation of Immunity in Veterinary Medicine. Alan R. Liss, PA, pp. 41-52.

Leitner, G., Heller, E.D. and Friedman, A., 1989. Sex-related differences in immune response and survival rate of broiler chickens. Vet. Immunol. Immunopathol., 21: 249-260.

Leitner, G., Melamed, D., Drabkin, N. and Heller, E.D., 1990. An enzyme-linked immunoab- sorbent assay for detection of antibodies against Escherichia coli: association between indi- rect hemagglutination test and survival. Avian Dis., 34: 58-62.

Leitner, G., Uni, Z., Cahaner, A. and Heller, E.D., 1992. Replicated divergent selection of meat- type chickens for high or low early antibody response to E. coli vaccination. Poultry Sci., 71: 27-37.

Pitcovski, J., Heller, E.D., Cahaner, A. and Peleg, B.A., 1987. Selection for early responsiveness of chicks to Escherichia coli and Newcastle disease virus. Poultry Sci., 66: 1276-1282.

Rees, M.J. and Nordskog, A.W., 1981. Genetic control of serum immunoglobulin G levels in the chicken. J. Immunogenet., 8:425-431.


SAS Institute, 1987. SAS/STAT Guide for Personal Computers, Version 6th Edn. SAS Institute, Cary, NC.

Siegel, P.B. and Gross, E.B., 1980. Production and persistence of antibodies in chickens to sheep erythrocytes. 1. Directional selection. Poultry Sci., 59: 1-5.

Takahashi, S., Inooka, S. and Mizuma, Y., 1984. Selective breeding for high and low antibody responses to inactivated Newcastle disease virus in Japanese quails. Poultry Sci., 63: 595- 599.

Van den Berg, T.P., Gonze, M. and Meulemans, G., 1991. Acute infectious bursal disease in poultry: isolation and characterisation of a high virulent strain. Avian Pathol., 20:133-144.

Van der Zijpp, A.J., 1983. Breeding for immune responsiveness and disease resistance. World's Poult. Sci. J., 39:118-131.

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Immunological parameters in meat-type chicken lines divergently selected by antibody response to Escherichia coli vaccination