IMMUNE SYSTEM FUNCTION AND DEVELOPMENT IN BROILERS

Gisela F. Err

Center of Excellence for Poultry Science

University of Arkansas, Fayetteville, AR 72701

Phone: 501-575-8664, Fax: 501-575-7139

gfeff@comp.uark.edu

Introduction

Significant progress has been made during the past 10-15 years in understanding the

development and function of the immune system in avian species. However, most of the

research in avian immunology has focused on egg-type chickens. This is due, in part, to

the availability of genetically defined lines, biomedical animal models, and specific-

pathogen-free (SPF) individuals within the egg-type chicken strains. Considering both the

differences in the genetic selection pressures imposed on meat-type versus egg-type

chickens, as well as, the observation that body weight is negatively correlated with immune

function (Han and Smyth, Jr., 1972, Parmentier et al., 1996), basic immunological norms

established in egg-type chickens may not be readily applied to meat-type chickens

(broilers).

With the continued increase in the economic importance of broilers, a better

understanding of the development and function of the immune system in these birds is

necessary. This knowledge will find direct application in breeder selection programs,

which have to include selection for early immunocompetence and high disease resistance to

meet the demands of modern intensive broiler production systems. Although current

information on basic broiler immunology is limited, it will be the focus of this discussion.

In the first section of this paper, the basic concepts of chicken immunology will be

presented. In the second section, studies on specific (humoral and cell-mediated) immunity

in broilers will be discussed.

Abbreviations: BCR, B cell receptor; BW, body weight; MHC, major histocompatibility

complex; SCWL, Single Comb White Leghorn; SPF, specific-pathogen-free; SRBC, sheep

red blood cell; TCR, T cell receptor (TCR1 = y6 TCR, TCR2 = otfll TCR, TCR3 = cxB2

TCR); FITC, fluorescein isothiocyanate; PE, phycoerythrin.

109

The Avian Immune System I

Vertebrates, including chickens, have basically two types of defense systems against

infectious agents. These are innate (non-specific) immunity and adaptive (specific)

immunity. Non-specific immunity is comprised of a broad spectrum of defense

mechanisms. These include: 1) physical and biochemical barriers designed to prevent

invasion by both infectious and non-infectious agents (antigens), and 2) soluble and cellular

components capable of eliminating foreign substances (antigens) which have successfully

invaded the body tissues. The cellular components of the non-specific immune system

include monocytes/macrophages, heterophils (neutrophils in mammals), basophils,

eosinophils, and natural killer cells. For the most part, non-specific immunity is very

effective in eliminating antigens. However, when non-specific immunity is not effective, it

becomes necessary to specifically focus immune reactivity on the antigen. At this point,

specific immunity is activated.

Specific immunity encompasses two aspects of immunity. These are humoral

immunity, which is carried out by antibodies, and cell-mediated immunity, which refers

primarily to T cell activity. Lymphocytes are the cellular components of specific

immunity. There are various subpopulations of lymphocytes which are morphologically

indistinguishable but Which differ in site of development, tissue location, phenotypic

expression of cell surface molecules, and functional abilities. Chickens lymphocytes

consist of B cells, which develop in the bursa of Fabricius, and T cells, which develop in

the thymus. Both B cells and T cells express antigen receptors on their surface. Each B

cell and T cell has a homogeneous set of antigen-receptors specific for a given antigen.

Collectively, the B cell and the T cell compartment each have a repertoire of nearly 109

different antigen-specificities.

Upon the first encounter with an antigen, for example a pathogen "A", there are

relatively few T and B cells with receptors specific for the antigen. Before pathogen "A"

can be eliminated by specific immune components, T and B cells with pathogen "A"-

specific receptors have to proliferate and differentiate into effector cells (e.g. antibody-

producing plasma cells). However, proliferation and differentiation take time,, often giving

pathogen "A" the opportunity to cause disease. Instead of becoming effector cells, cells

lFor references concerning some basic aspects of avian immunology, see Davison et al.,

1996 (general concepts); Higgins, 1996 and Ratcliffe et al., 1996 (B cells); and G6bel,

1996 (T cells).

ii0

recognizing pathogen "A"can also differentiate into long-lived, fast-reactingmemorycells.Hence, during a T or B cell response to pathogen "A", the numberof pathogen "A'-

specific cells increases, producinglarge numbersof pathogen "A"-specific effector cells

and memory cells. The effector cells will participatein the eliminationof pathogen "A",whereas the memory cells will be set aside, ready to effectively respond to pathogen "A"

- upon a repeatencounter- before pathogen"A" can cause disease. However, pathogen

"A"-specific memory cells will notprotect the individualfrom a differentpathogen (e.g.

pathogen "B"). This concept of expandingthe pool of antigen-specific cells and producingantigen-specific memory cells is directlyutilized in vaccinationprograms, whereby a non-

pathogenic form of a pathogen is introduced to an individual's immune system. The

immune system will mount a response to specific components of the pathogen as described

above. Upon a later encounter with the pathogenicform of the pathogen, the immune

system will be ready to respond and eliminate the pathogen before it can cause disease.

B cells and antibodies

The B cell antigen-receptor (B cell receptoror BCR) consists of antigen-specific,

membrane-bound immunoglobulin and BCR-associatedsignal proteins. When a B cell

encountersthe antigen for which it has a specific BCR, it can bind to the antigen via the

BCR. The binding of the antigen to the BCRprovides a first signal for B cell activation.

In most B cell-antigen interactions, the B cell requiresa second signal from T cells, in the

form of soluble factors or through direct T cell-B cell contact, before it can become fully

activated. Once activated, the B cell multipliesand differentiateseither into antibody-

producing plasmacells or memory cells. A B cell responsethat requires a second signal

from T cells is called a T-dependentresponse. Certainantigens, or so-called T-

independent antigens, are able to activate B cells without additional factors from T cells (T-

independent response).Antibodies, rather than the B cell itself, are the actual effectors of the humoral

immune response. Due to their ability to specifically interact with antigen, antibodies can

greatly enhance the effectiveness of non-specificimmunecomponents. For example,

: antibodies increase the ability of phagocytic cells to capture and eliminate antigens, activatethe complement system, and prevent antigens from binding to and infecting cells. In

chickens, three classes of antibody molecules (immunoglobulins; Ig) have been identified.

These axe IgM, IgG ClgY),and IgA.

Depending on the type and stage of a humoral immune response, different classes

of antibodies predominate. The majority of antibodies during a primary immune response

iii

are of the class IgM. A switch from IgM to IgY or IgA can be observed towards the end

of a primary immune response, however, Ig class switching to IgY or IgA is most apparent

during a second or repeated exposure to the same antigen. Additionally, different classes

of antibodies have different functional abilities. To mention a few, IgM has the ability to

easily agglutinate large antigens and to cause the precipitation of soluble antigens, thus 7greatly enhancing the immune systems ability to remove antigens through phagocytosis.

IgA is found in secretions and functions at mucosal surfaces, and IgY can be transferred

from the peripheral circulation of the hen into the egg (maternal antibody).

T cell populations and_functions

Similarly to B cells, each T cell has a homogeneous set of antigen receptors (T cell

receptor; TCR) specific for a given antigen. Independent of antigen-specificity, a T cell's

TCR can be belong to one of three classes. These are: TCR1, a heterodimer consisting of

a gamma (y) and a delta (_f)protein chain; TCR2, a heterodimer consisting of an alpha (o0

and a beta (13)protein chain which is encoded in part by a VB1 gene; and TCR3, which is

also an od3heterodimer but it's 13chain is encoded in part by a V132gene. Regardless of

which type of TCR a T cell expresses, TCR expression is always associated with a group

of signalling proteins, collectively referred to as the CD3 complex. T cells first express

their TCR during thymic T cell development. At this stage, most T cells already express

or start to express CD4 and CD8 cell surface molecules. In healthy egg-type (Davidson

and Boyd, 1992) and broiler (Erf et al., 1997) chickens, double-positive (CD4+CD8 +)

lymphocytes in the thymus (thymocytes) constitute the majority of thymocytes (> 60%).

The majority of CD4+CD8 + thymoeytes express od3TCR (> 50%), a small percentage

express y_ TCR (< 10%), and the remainder do not yet express a TCR. Double-positive

thymocytes with od]TCR go through two selection processes. First, they are selected for

their ability to recognize self major histocompatibility complex (MHC) molecules, and then

thymocytes specific for an individual's own body components are deleted or rendered

unresponsive. The majority of surviving od3TCR thymocytes will mature into

od3TCR+CD4+CD8" or od]TCR+CD4"CD8 + T cells and enter the periphery. Minor

populations of mature od3T cells expressing both or neither CD4 and CD8 have also been

observed in the periphery (Luhtala et al., 1997; Err et al., 1997). Selection processes

regarding thymoeytes with y/5TCR (TCRI +) are less understood but do appear to exist

(Buoy et al., 1991).

Although exceptions exists, the expression of certain cell-surface molecules on T

cells are related to the T cell's functional characteristics. In chickens, as in mammals,

112

matureCD4+CD8 - T cells constitute the subsetof T cells which recognizeexogenous

antigens (phagocytosed antigens) in associationwith MHC class II molecules on antigen-

presenting cells (e.g. macrophages). Moreover,CD4+CD8 - T cells include the cytokine-

producingregulatoryT cell subset (helperT cell) requiredfor the antigen-specificactivation of B cells, otherT cells, and non-specificimmunecells (e.g. macrophages).Mature chicken CD4-CD8+ T cells constitute the subset of T cells that recognize

endogenousantigens (viral or tumorantigens) in association with MHC class I. Once

activated, CD4-CD8+ T cells become cytotoxic, capableof the antigen-specific killing ofvirus-infected cells and tumorcells. The proportionbetween CD4+CD8 - and CD4-CD8+

T cells (CD4:CD8 ratio) has been used as an end-point in assessing the state of an

individual's immune system. In chickens, the CD4:CD8 ratio has been shown to be

positively related to the amountof antibodiesproducedin response to sheep red blood cells(SRBC, a T-dependentantigen) (Parmentieret al., 1995).

Mature T cells can also be divided into subpopulationsbased on the class of TCR

they express. In young, healthy egg-type (Chertet al., 1991) and broiler (Eft et al., 1997)

chickens, TCR1+, TCR2+, and TCR3+ T cells make up 15-30%, 45-55% and 10-15%

of lymphocytes in the blood and 15-35%, 30-40%, and 10-20% of lymphocytes in the

spleen, respectively. TCR defined T cell populationsdiffer in tissue locatibn, with orbT

cells (TCR2+ and TCR3+) preferentiallyhomingto the white pulp area in the spleen,whereas, TCR1+ T cells are most abundantwithin the red pulpof this organ. Unlike

TCR1+ and TCR2+ T cells, TCR3+ T cells are rarelyfound within the gut

microenvironment. Typically, matureT cells that express CD4 or CD8 also express TCR2

or TCR3. However, CD8 molecules may also be found on TCR1+ T cells, depending on

the cell's peripherallocation (e.g. spleen, IEL; Bucy et al., 1991). Lastly, TCR defined T

cell subpopulationsappearto differ in their functional capacities. For example, as shown

by Cihaket al. (1991), selective in rive depletionof TCR2+ T cells resultedin severe IgA

deficiency. Hence, TCR2+ T cells appearto be requiredfor the immunoglobulinisotypeswitch from IgM to IgA and/or the expansionof IgA-producingB cells. The physiological

role of T cells expressingTCR1 is notwell defined at this time.

Althoughfunctionaldifferenceswithin the TCR defined T cell subsetsare not

: completely understood, T cells expressing TCR1, TCR2, or TCR3 nevertheless constitutedifferent subsets of T cells, and may constitute a diagnostic tool to assess immunesystem

state.

113!

Aspects of Immune Development and Function in Commercial Broilers

Compared to literature on the immune system in egg-type chickens, data

specifically addressing immune development and function in broilers are limited. Research

focus on commercial egg-type chickens may be driven, in part, by the relatively long life

of layers, requiring health maintenance from the time of hatch throughout egg-production. °

Commercial broilers, on the other hand, go to market often before they are 7 weeks of

age. The short life of commercial broilers has, to some extent, taken away interest in

studying immune development and function in this type of chicken. This of course,

completely ignores the broiler breeder side of broiler production. However, recently,

Qureshi and Havenstein (1994) examined aspects of non-specific immunity in the 1991

Arbor Acres feather-sexable broiler strain and the 1957 Athens-Canadian Randombred

control strain (meat-type chicken). Examination of macrophage function and natural killer

cell activity revealed little or no differences between the 1957 and 1991 strains of chickens,

suggesting that genetic selection for enhanced growth performance traits had not

significantly altered the non-specific arm of the immune system in broilers. However,

when 1957 and 1991 chicks were injected with SRBC at two-weeks of age and tested for

anti-SRBC antibody levels 7 days later, the 1957 strain had higher levels of anti-SRBC

antibodies (total Ig and IgM) than the 1991 strain (Qureshi and Havenstein, 1994). At 14

days post-SRBC injection, these strain differences in anti-SRBC antibody levels were no

longer apparent. This drop in responsiveness to a T-dependent antigen in the 1991 broiler

strain as compared to the 1957 strain appears to be the direct result of genetic selection for

enhanced growth performance traits (Qureshi and Havenstein, 1994). The impact of this

reduction in humoral immune function on the overall livability of broilers may be

significant, as 2- to 3-week-old chicks are not fully immunocompetent and have also lost

protection from maternal antibodies (McCorkle and Glick, 1980; Peleg et al., 1985;

Pitcovski et al., 1987).

On the other hand, the modern broiler was found to have a superior ability to

produce anti-SRBC antibodies when compared to a strain of meat-type chickens which have

been solely selected on the basis of increased body weight for 36 generations (Praharaj et

al., 1995). The incorporation of immune performance as a selection criterion within a

breeding program for growth performance traits therefore appears to have beneficial

effects. Moreover, the selection of broiler chicks for early immune response to E. coli was

shown to result in an overall increase in early immune system maturation, including 1)

increased humoral immune response to other T-dependent antigens (e.g. SRBC or bovine

114

serum albumin), 2) enhanced in vitro T cell proliferation in response to E. coli or T cell

mitogens, and 3) increased phagocytic activity (I-Idler et al., 1992).

Information concerning cell-mediated immunity in modern commercial broilers is

sparse. Recently, we conducted a study involving SPF Single Comb White Leghorn

(SCWL) chickens and commercial broilers raised in Anderson isolators 0aiocontainment

= level P2). Although the intent of the study was not to compare SCWL and broilers, data

from the experimental controls can be used to gain insight into differences between

chickens that were selected for egg-production traits as compared to growth performance

traits. In this study, blood was collected from 12 to 15 chicks per strain when the chicks

were 1, 2, and 4 weeks of age. The chicks then were euthanized, body weight was

determined, and thymus, bursa, and spleen were collected. The weight of the bursa and

spleen was also determined for each bird. Cell-suspensions from the thymus, spleen, and

blood were immunofluorescently stained to detect the expression of CD4 and/or CD8

molecules. The percentage of cells in each cell suspension expressing CD4 and/or CD8

was determined using a flow cytometer (FACSort, Becton Dickinson Immunocytometry

Systems, San Jose, CA).

When adjustments for body weight (BW) differences were made, the SCWL chicks

had higher bursal weights (mg/g BW) at all ages (Figure la) and higher spleen weights

(mg/g BW) at 4 weeks of age (Figure lb) as compared to age-matched broilers. The

proportions among CD4 and/or CD8 defined T cell populations in the thymus were not

different at 1 or 2 weeks of age in SCWL and broilers (Table 1). However, by 4 weeks of

age, the broilers had a lower percentage of double-positive (CD4 +CD8 +) thymocytes and

a higher percentage of CD4-CD8 + thymocytes than the SCWL chicks. Spleens from the

broilers consistently contained a lower percentage of CD4+CD8 - T cells than those from

SCWL chickens, whereas no strain differences were observed in the percentages of CD4-

CD8 + and CD4+CD8 + T ceils (Table 2). In the peripheral blood, the percentages of

CD4 +CDS" T cells were lower in 2-week-old broilers than inage-matched SCWL chicks,

whereas the percentages of CD4-CD8 + T cells were the same between broilers and SCWL

chickens (Table 3).

Determination of CD4:CD8 ratios revealed differences between SCWL and broilers

consistent with those observed when the percentage of CD4 and/or CD8 defined T cell

subsets were examined (Figure 2). These data show that selection for egg production

versus growth performance traits has altered the proportions among T cell subsets.

Considering the different functional abilities of CD4 + CDS- and CD4-CD8 + T cells

(regulatory versus cytotoxic), the strain differences observed here likely reflect differences

in the general immunocompetence of SCWL as compared to broilers. For example, low

115

CD4:CD8 ratios have been associated with lower antibody responses to SRBC and higher

body weights (Parmentier et al., 1995). Hence, based on the data presented above,

broilers would appear to be less immunocompetent than SCWL chickens during their first 4

weeks of life. The conclusion that young broilers have below optimal immune system

function is further corroborated by the observation that management practices can

positively influence a broiler's immune state. For example, dietary vitamin E (c_- °

tocopherol) supplementation at levels 5 to 10 times above standard levels was found to

significantly increase the CD4:CD8 ratios in thymus, blood, and spleen in broilers raised

on litter (Erf and Bottje, 1996). The enhanced CD4:CD8 ratio in the blood was associated

with increases in the ability of lymphocytes to respond to T cell mitogen and in the

antibody response to a T-independent antigen (S. pullonvn) (Eft and Bottje, 1996).

More information is needed regarding immune development and function in

broilers. This information will be valuable in identifying selection criteria at the pedigree

level to improve future commercial broiler performance. Until the genetic immune system

potential has increased, better immune system development and function in broiler chicks

can be achieved through optimizing nutritional and environmental conditions.

Acknowledgments

The excellent technical help provided by Tina Bersi, Chris Fritts, and Belinda Floyd is

very much appreciated. This manuscript is published with the approval of the Director of

the Arkansas Agriculture Experiment Station, University of Arkansas. This work was

supported, in part, by a grant from the US Poultry and Egg Association (Tucker, GA).

References

Bucy, R.P., Chert, C.-L.H. and Cooper, M.D., 1991. Analysis ofy_ T cells in the

chicken. Sem. Immunol., 3: 109-117.

Chen, C.-L.H., Pickel, J.M., Lahti, J.M. and Cooper, M.D., 1991. Surface markers on

avian immune cells. In: J.M. Sharma (Editor), Avian Cellular Immunology, CRC

Press, Boca Raton, FL, pp. 1-22.

Cihak, J., Hoffmann-Fezer, G., Ziegler-Heibrock, H.W.L., Stein, H., Kaspers, B., Chen,

C.-L.H., Cooper, M.D. and Ltsch, U., 1991. T cells expressing the .VB1T-cell

116

receptor are required for IgA production in chicken. Proc. Natl. Acad. Sci., 88:

10951-10955.

Davidson, N.J. and Boyd, R.L., 1992. Delineation of chicken thymocytes by CD3-TCR

complex, CD4 and CD8 antigen expression reveals phylogenicaUy conserved and

novel thymocyte subsets. Int. Immunol., 4:1175-1182.

" Davison, T.F., Morris, T.R. and Payne, L.N., 1996. Poultry Immunology. Poultry

Science Symposium Series, Vol.24, Carfax Publishing Company, Abingdon,

England.

Erf, G.F. and Bottje, W.G., 1996. Nutrition and immune function in chickens: Benefits

of dietary vitamin E supplementation. Proceedings of the Arkansas Nutrition

Conference, pp. 113-130.

Err, G.F., Bottje, W.G. and Bersi, T.K., 1997. CD4, CD8, and TCR defined T cell

subsets in thymus and spleen of 2- and 7-week-old commercial broiler chickens.

Vet. Immunol. Immunopathol. (submitted).

G6bel, T.W.F., 1996. The T-dependent immune system. In: T.F. Davison, T.R. Morris

and L.N. Payne (Editors), Poultry Immunology, Poultry Science Symposium Series

Vol.24, Carfax Publishing Company, Abingdon, England, pp. 31-45.

Han, P.F.-S. and Smyth, J.R., Jr., 1972. The influence of growth rate on the

development of Marek's disease in chickens. Poultry Sci., 51: 975-985.

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.

Higgins, D.A., 1996. Comparative immunology in avian species. In: T.F. Davison, T.R.

Morris and L.N. Payne (Editors), Poultry Immunology, Poultry Science

Symposium Series, Vol.24, Carfax Publishing Company, Abingdon, England, pp.

149-205.

Luhtala, M., Lassila, O., Toivanen, P. and Vainio, O., 1997. A novel CD4+CD8 + T

cell population: inheritance of CDSot expression on CD4 + T cells. Eur. J.

Immunol., 27: 189-193.

. McCorkle, F.M. and Glick, B., 1980. The effect of aging on immune competence in the

chicken: antibody mediated immunity. Poultry Sci., 59: 669-672.

Parmentier, H.K., Kreukniet, M.B., Goeree, B., Davison, T.F., Jeurissen, S.H.M.,

Harmsen, E.G.M. and Nieuwland, M.G.B., 1995. Differences in distribution of

lymphocyte antigens in chicken lines divergently selected for antibody responses to

sheep red blood cells. Vet. Immunol. Immunopathol., 48: 155-168.

t17

Parmentier, H.K., Nieuwland, M.G.B., Rijke, E., De Vries Reilingh, G. and Schrama,

J.W., 1996. Divergent antibody responses to vaccines and divergent body weights

of chicken lines selected for high and low humoral responsiveness to sheep red

blood cells. Avian Dis., 40: 634-644.

Peleg, B.A., Heller, D.E. and Pitcovski, J., 1985. The ontogeny of the humoral response

to Escherichia coli vaccine and to sheep red blood cells in young chicks. Avian

Pathol., 14: 471-481.

Pitcovski, J., Heller, D.E., 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.

Praharaj, N.K., Dunnington, E.A. and Siegel, P.B., 1995. Growth,

immunoresponsiveness, and disease resistance of diverse stocks of chickens reared

under two nutritional regimens. Poultry Sci., 74: 1721-1729.

Qureshi, M.A. and Havenstein, G.B., 1994. A comparison of the immune performance of

a 1991 commercial broiler with a 1957 randombred strain when fed typical 1957

and 1991 broiler diets. Poultry Sci., 73: 1805-1812.

Ratcliffe, M.J.H., Paramithiotis, E., Coumidis, A., Sayegh, C., Demaries, S., Martinez,

O. and Jacobsen, K.A., 1996. The bursa of Fabricius and its role in avian B

lymphocyte development. In: T.F. Davison, T.R. Morris and L.N. Payne

(Editors), Poultry Immunology, Poultry Science Symposium Series, Vol.24, Carfax

Publishing Company, Abingdon, England, pp. 11-30.

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Table 1. CD4 and CD8 Defined LymphocyteSubsets (%) in Thymus from SCWL and

Broiler Chicks Reared in Isolation

Strain Age CD4 CD8 CD48

SCWL 1 wk 8.0 4- 1.1 5.9 4- 0.7 92.8 4- 1.0Broiler 1 wk 5.7 4- 0.9 4.5 4- 0.4 89.6 + 2.2

SCWL 2 wk 9.2 4- 1.4 7.3 4- 1.5 81.4 4- 5.4Broiler 2 wk 9.9 4- 1.4 13.2 4-3.1 70.9 4- 6.3

SCWL 4 wk 6.7 4- 0.7 5.6 4- 0.7b 80.1 4-2.3aBroiler 4 wk 7.9 4- 1.0 7.5 4- 0.6a 72.4 4- 2.5b

Data shown are mean % + SEM.

See legend Fig. 2 for experimental detail.a,bWithin a cell type and an age group, means with differentsuperscripts are significantly different (P < 0.05).

Table 2. CD4 and CD8 Defined LymphocyteSubsets (%) in Spleen from SCWL and

Broiler Chicks Reared in Isolation

Strain Age CD4 CD8 CD48

SCWL 1 wk 20.0 4-0.9a 23.2 4- 1.5 2.3 4- 0.3Broiler 1 wk 14.5 4- 1.5b 26.2 4- 1.8 2.1 4- 0.4

SCWL 2 wk 15.5 4- 1.5a 29.3 4- 4.6 3.9 4- 0.7Broiler 2 wk 11.2 4-0.5b 29.0 4-3.3 2.6 4- 0.5

- SCWL 4wk 14.3 4- 1.0a 32.1 4- 1.7 5.2 4- 0.8Broiler 4wk 10.9 4- 0.9b 36.4 4- 2.5 3.7 4- 0.6

Data shown are mean % + SEM;

See legend Fig. 2 for experimental detail.a,bwithin a cell type and an age group, means with different

superscripts are significantly different (P < 0.05).

119

Table 3. CD4 and CD8 Defined Lymphocyte

Subsets (%) in Blood from SCWL and BroilerChicks Reared in Isolation

Strain Age CD4 CD8 CD48

SCWL 1 wk 43.9± 3.4 15.0± 1.0 1.4 4-0.2Broiler 1 wk 42.9 4- 2.6 15.8 4, 1.8 2.0 4, 0.3

SCWL 2 wk 51.2 4-2.4a 17.54- 1.1 1.64, 0.2aBroiler 2 wk 27.6 4- 2.9b 17.4 4- 2.5 0.3 4- 0.1b

SCWL 4 wk 21.6 4- 2.8 9.6 4- 1.7 1.54- 0.2aBroiler 4 wk 19.7 4, 1.9 8.8 4- 0.8 0.7 4- 0.2b

Data shown are mean % + SEM;

See legend Fig.2 for experimental detail.a,bwithin a ceil type and an age group, means with

different superscripts are different (P < 0.05).

120

O. r

SCWL Broiler

Figure 1.Bursa (a) and spleen (b) weights in specific pathogen freeSingle Comb White Leghorn (SCWL) and commercialbroiler chicks raised in isolation (biocontainment level 2).At 1, 2, and 4 weeks of age, the bursa and spleen were

removed from 12 to 15 birds per strain. Bursa and spleenweights were determined and adjusted for body weight (BW)differences.Ig

Indicates significant strain differences within an age-group(P < 0.05).

121

3.5

3 - Q1 wk

2.52 _ m2 wk

1 114 wk

0.5

0

S B S B S B

Thymus Spleen Blood

Figure 2.Proportions between CD4 + CD8" and CD4-CD8 + T cells(CD4:CD8 ratio) in thymus, spleen, and blood from specificpathogen free Single Comb White Leghorn (SCWL) andcommercial broiler chicks raised in isolation (biocontainment

level 2). At 1, 2, and 4 weeks of age, the thymus, spleen,and blood were obtained from 12 to 15 birds per strain.

Cell suspensions were immunofluorescently stained usingmonoclonal antibody specific for chicken CD4 (FITC-labeled) and CD8 (PE-labeled) and a two-color, direct

staining procedure. The percentage of cells expressing CD4and/or CD8 was determined by flow cytometry.*Indicates significant strain differences within a tissue type

and an age group (P < 0.05).

122

Questions

Question: S. J. Lamont

Many past studies have compared distinct populations, and have found a negative

• relationship of body weight and immune function. Have you examined the

" relationship of body weight and immune parameters within the population that you

have studied?

Response: G. F. Erf

No I have not. Healthy commercial broilers examined in our laboratory had

relatively uniform body weights. Hence, it will be difficult to establish a

relationship between body weight and immune function within a population of

healthy broilers. Another approach used to determine the relationship between

body weight and immune function was to divergently select a population of

chickens for high or low immunocompetence (e.g." H. K. Parmentier and co-

workers; P. B. Siegel and co-workers). Using this approach, it was demonstrated

that selection of chickens for high or low antibody titers tOsheep red blood cells

resulted in lower and higher body weights, respectively, as compared to random-

bred controls.

Question: M. Boyle

Can you envision using a flow cytometer on an industrial level (i.e. large numbers

of samples)? How much does the machine cost?

Response: G. F. Erf

Yes I can. A basic flow cytometer, equipped with a single argon laser (488 nm)

and three fluorescence detectors, can simultaneously provide information about

size, internal complexity, and fluorescence (up to three different fluorescent labels)

of a single particle (e.g. cell). It can perform these tasks at high speed (10,000

particles in less than 5 seconds). Hence, a large number of samples can be

processed in a day. Various diagnostic kits are currently being developed for

, poultry species requiring the use of a flow cytometer. A basic flow cytometer, with

• the capabilities described above will cost between $80,000 to $100,000. The

service contract will be between $10,000 to $15,000 a year.

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