IMMUNOLOGY
Immunity to Pasteurella
multocida in Protein-Deficient Chickens
C. J. PAYNE, T. R. SCOTT,1 J. W. DICK, and B. GLICK
Department of Poultry Science, Clemson University,
Clemson, South Carolina 29634-0379
(Received for publication January 25, 1990)
1990 Poultry Science 69:2134-2142
INTRODUCTION
It has long been observed that undernutrition predisposes the host to the risk of acquired
infection, and the severity of the illness is
augmented under these circumstances. Thus,
research on protein deficiencies, among other
nutritional deficiencies, has been conducted for
many years. Both humoral and cell-mediated
immune functions have been studied in protein-deprived animals.
Malave and Layrisse (1976) observed no
effect or an increase in serum hemagglutinin
titers of protein-deficient mice during a primary humoral response. During the secondary
response, there was marked depression in
titers. Also, synthesis of IgG hemagglutinins
was depressed during the primary and secondary responses. Later researchers found mice
on protein-restricted (PR) diets had significantly lower plaque-forming cell responses at
all ages tested, when compared with controls
(Stoltzner and Dorsey, 1980). Birds placed on
PR diets until 5 wk of age and injected with
To whom correspondence should be addressed.
5% SRBC showed a reduced primary IgG
antibody response (Glick et al., 1981).
Cooper et al. (1974) observed a significantly enhanced response to phytohemagglutinin (PHA) of lymphocytes from proteindeprived mice. Utilizing a graft-versus-host
(GvH) reaction, a functional test, Bell and
Hazell (1975) found that protein deficiency did
not negatively affect T-cell activity.
During the first weeks of protein restriction
of mice, Malave et al. (1980) found enhanced
responses of spleen and thymus cells to PHA
and concanavalin A (Con A). Beyond the 4th
wk, the proliferative activity and mitogenic
response ratio of protein-deprived (8% protein)
animals decreased to values close to those of
the 27% protein group. A highly significant
decrease in response to mitogens, PHA, Con
A, lipopolysaccharide (LPS), and staphylococcal enterotoxin B, occurred in PR mice (Mann,
1978). The PR mice also exhibited a depressed
mixed leukocyte culture response. However,
mice maintained on the PR diets until 7 mo of
age demonstrated normal responses (Mann,
1978). Chickens fed a 1% PR diet were not
compromised in their ability to produce T cells
capable of a GvH response (Glick et al., 1983).
2134
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ABSTRACT Studies were conducted to determine the effects of dietary protein restriction on the humoral
immunity (HI) and cell-mediated immunity (CMT) of chickens. New Hampshire chickens were separated into
two dietaiy treatment groups: basal, containing 3,200 kcal/kg and 21% protein; or protein restricted (PR),
containing 3,200 kcal/kg and 7% protein. In studies involving HI, half of the birds in each dietary treatment
were vaccinated against fowl cholera at 4 and 8 wk of age. Blood samples were collected weekly beginning at
4 wk of age. Overall, unvaccinated birds had lower titers than vaccinated birds and PR groups generally
showed lower titers than basal groups. All birds were challenged by palatine cleft inoculation of live, virulent
Strain X-73 of Pasteurella multocida. The vaccinated PR group survived live challenge as well as the
vaccinated basal group, but all unvaccinated birds died as a result of the challenge, regardless of antibody titer.
In studies involving CMI, half of the birds in each dietary treatment were vaccinated at 5 wk of age. At 2 to
3 wk postvaccination, representative birds from each treatment were bled for total and differential blood
counts. Also, birds were sacrificed and spleen cells collected. Cells were cultured in Roswell Park Memorial
Institute (RPMI) medium with phytohemagglutinin-M (PHA-M), sonicated P. multocida (X-73), or RPMI
only. The PR birds had significantly decreased numbers of lymphocytes, as well as an overall decrease in total
white blood cell counts. Within dietary treatments, vaccinated birds tended to have higher numbers of white
blood cells, but (he differences were only significant for the PR groups in Experiment 4. The PR diet
suppressed proliferation of splenic cells stimulated with PHA-M, and vaccination significantly increased
lymphocyte proliferation in response to sonicated P. multocida. Strain X-73.
(Key words: protein deficiency, fowl cholera, antibodies, white blood cells, lymphocyte proliferation)
PROTEIN DEFICIENCY AND IMMUNITY IN CHICKENS
2135
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TABLE 1. Basal and protein-restricted (PR) diet
Total white blood cells and relative numbers of
formulations for all experiments
lymphocytes and heterophils were also determined, and the protein restriction did not alter
Ingredient
PR
Basal
these numbers significantly at 1, 2, or 3 wk of
79.54
Com
57.71
age.
32.82
Soybean meal (48.5% CP)
In those previous studies, uninfectious Fat
5.50
6.08
agents (e.g., BSA and SRBC) and mitogens Dicalcium phosphate
1.82
2.57
were used in tests to assess immunocompe- Calcium carbonate
1.34
1.13
.26
.58
tence of protein-deficient animals. It has been Methionine
.30
.30
established that both humoral and cell-medi- Salt
1
.25
Vitamin and mineral premix
.25
ated immunities were involved in the protec- Celufil
9.55
tion of poultry against Pasteurella multocida Calculated content
21.00
Dietary protein, %
7.00
(Maheswaran et al., 1976; Schlink and Olson,
3,200
Metabolizable energy, kcal/kg 3,200
1979; Baba, 1984; Briggs and Skeels, 1984;
Avakian and Dick, 1986). However, antibody
Vitamin and mineral premix added to diet to meet or
production and cell-mediated immune activity exceed National Research Council (1984) requirements.
toward P. multocida have not been examined
in chickens subjected to a dietary deficiency
such as inadequate protein content. The objectives of the present study were to determine synthesis of IgG antibodies (Tsiagbe et al.,
whether a severe protein restriction would 1987); therefore, methionine was included in the
affect immunocompetence of chickens to P. diet at the NRC (1984) recommended level. The
multocida and to relate their immune respon- diet formulation used in all experiments is
siveness with survival to a live pathogenic shown in Table 1. Feed samples were analyzed;
challenge.
PR feed was shown to have 8.1% crude protein
on a dry matter basis, and the basal feed
contained 23.7% crude protein.2 At 5 wk of age,
MATERIALS AND METHODS
the low-protein feed was removed from the PR
groups and birds were placed on basal feed for
the remainder of the experiments. Body weight
Chickens
and feed consumption data were collected
Chickens from a closed flock of the Lester J. weekly and feed conversion values calculated.
Dreesen (LTD) strain of New Hampshires were
grown in battery brooders until 5 wk of age at
which time birds on basal diets were moved to Vaccinations
grower batteries. At 8 wk of age, the PR birds
At 4 and 8 wk of age in Experiments 1 and 2,
were moved into grower batteries. Feed and birds received primary and secondary vaccinawater were supplied for ad libitum access tions with a commercial bacterin of P. multocida
throughout the experiments.
(.5 mL, PaBac, P. multocida)? At 5 wk of age in
Experiments 3 and 4, birds were vaccinated with
the same bacterin. The injections were subDiets
cutaneous in the dorsal side of the neck for all
Two dietary treatments were used. The basal birds.
diet contained 21% protein and the PR diet
The vaccination scheme employed resulted in
contained 7% protein. Both diets contained four treatment groups in Experiments 1, 2, and
3,200 kcal/kg and met National Research 4; basal vaccinated, basal unvaccinated, PR
Council (NRC, 1984) requirements for other vaccinated, and PR unvaccinated. The PR
nutrients. Previous work involving methionine unvaccinated group was not included in Experisupplementation and immune status has sug- ment 3. Experiment 1 contained 8 basal
gested that methionine is important in the unvaccinated birds, 16 basal vaccinated birds, 8
PR unvaccinated birds, and 13 PR vaccinated
birds. Experiment 2 contained 10 birds in each
of the basal unvaccinated, basal vaccinated, and
^Department of Agricultural Chemical Services, ClemPR vaccinated groups, and 9 PR unvaccinated
son University, Clemson, SC 29634.
3
Salsbury Labs, Inc., Charles City, IA 50616-9989. birds. For determination of white blood cell
2136
PAYNE ET AL.
counts in Experiments 3 and 4,6 birds from each
treatment group were used. Four birds from each
treatment group were used for Experiments 3
and 4 lymphocyte proliferation assays.
Antibody Determinations
(Experiments 1 and 2)
Sigma Chemical Company, St. Louis, MO
63178-9916.
5
Pel-Freez, Rogers, AR 72757.
^Aldrich Chemical Co., Milwaukee, WI 53233.
'Dynatech Laboratories Inc., Chantilly, VA 22021.
8
American Scientific Products, McGaw Park, IL 60085.
S/P =
SQD
-
NCOD/PCOD
- NC0D
where:
SOD = raw absorbance value of test sera;
NCOD = raw absorbance value of known
negative control; and
P Q ) D = r a w absorbance value of a known
positive control.
Virulent Challenge
At 10 wk (Experiment 2) or at 12 wk
(Experiment 1), all birds received a palatine cleft
inoculation (Derieux and Dick, 1980) of the
virulent Strain X-73 of P. multocida, grown in
brain-heart infusion broth for 20 h at 37 C.
Mortality was monitored for 2 wk following the
challenge. The P. multocida was isolated from
liver, lungs, and heart of dead birds, grown at 37
C for 20 h, and Gram stained.
White Blood Cell Determinations
At 7 wk of age in Experiments 3 and 4,
peripheral blood was taken from the cutanea
ulnaris of six birds from each treatment. From
each bird, two blood smears were prepared and
stained with MacNeal's stain (Experiment 3) or
with a modified Wright's stain8 (Experiment 4)
for differential blood counts. A .1-mL sample of
blood was drawn into blood pipets and diluted to
1.0 mL with Natt-Herrick stain (Natt and
Herrick, 1952) for total counts. Total counts
were made by microscopic examination of cells
on a hemocytometer.
Lymphocyte Proliferation Assay
Preliminary assays were run to determine a
suitable level of sonicated X-73 strain of P.
multocida to add to cultures. Varying levels (i.e.,
2.5,5,10,20,40, and 80 ug/mL were incubated
with lymphocytes from the LJD strain of New
Hampshires, and an optimal response was
obtained when 20 ug/mL of antigen was used.
At 7 wk of age in Experiments 3 and 4, four
birds from each treatment were killed by
cervical dislocation and spleens were aseptically
removed. Spleens from PR diet birds were
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All birds were bled weekly beginning at 4 wk
of age. The PR birds were bled by the filter paper
method (Avakian and Dick, 1985) until they
were large enough to obtain 1 mL of blood
intravenously via syringe and needle. All birds
were bled from the cutanea ulnaris vein. The
indirect ELISA was performed as previously
described (Dick and Johnson, 1985) with some
modification. The ELISA plate wells were
coated with 250 uL of sonicated CU strain of P.
multocida (6 ug protein/mL) and incubated at 4
C overnight. Following incubation, excess
antigen was removed and plates washed once
with phosphate-buffered saline-.05% polyoxyethylenesorbitan (Tween) 80 (PBS-T80)4 pH
7.4. Plates were blocked with 250 |iL of PBST80 containing .5% gelatin for 30 min at 37 C.
Sera were diluted 1:500 in PBS-T80 containing. 1 % gelatin, and 100-uL samples were added
to plate wells and incubated 30 min at 37 C.
Samples were run in triplicate, and all plates
contained positive and negative controls.
Plates were washed and 100-uL rabbit
antichicken IgG (heavy- and light-chain specific) horseradish peroxidase conjugate,5 diluted 1:
20,000 with PBS-T80-.1% gelatin, or rabbit
antichicken IgG (heavy chain specific) horseradish peroxidase conjugate,5 diluted 1:100,000
with PBS-T80-.1% gelatin, was added to each
well. Plates were incubated 30 min at 37 C and
washed three times with PBS-T80. One hundred
microliters of orthophenylenediamine (OPD)6
was added as substrate at 40 u.g/100 mL of .05 M
sodium phosphate-citric acid buffer, pH 5.0,
containing 40 uL/100 mL of 30% hydrogen
peroxide, and incubated 20 min at 37 C. The
reaction was stopped with the addition of 50 uL
of 5 N sulfuric acid. Absorbance readings were
determined with a MicroELIS A Auto Reader7 at
490 nm. Antibody titers were expressed as test
serum to positive ratio to minimize variation
among plates. This value was calculated by the
following formula:
2137
PROTEIN DEFICIENCY AND IMMUNITY IN CHICKENS
•vaccinated or mvaccinated chickens from
Experiments 1 and 2 fed either a basal or
protein-restricted (PR) diet
Statistical Analysis
All experiments had completely randomized
designs. In Experiments 1 and 2, there were
split-plot arrangements of treatments with diet
and vaccination as the whole-plot effects and
time as the split-plot effect. In Experiment 3,
there was a completely random arrangement of
9
GIBCO Laboratories, Grand Island, NY 14072-0068.
^ecton-Dickinson, Oxnard, CA 93030.
n
ICN Biochemicals, Irvine, CA 92713.
12
Cambridge Technology, Inc., watertown, MA 02172.
13
Beckman Instruments, Inc., Fullerton, CA
92634-3100.
Dietary treatment
Age (wk)
Basal
PR
1
2
3
4
5
6
7
8
9
10
11
12
54"
118"
233"
376"
530"
743"
977"
1,200"
1,457"
1,682"
1,910"
2,288*
39"
44 b
44 b
46 b
49 b
60 b
138b
258 b
402 b
592 b
832 b
l,136 b
"•''Values within rows with no common superscripts are
significantly different (P<05).
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placed on sterile lens paper resting on a treatments (basal vaccinated, basal unvaccinatMaximov chamber, minced, and cells were ed, and PR vaccinated), and Experiment 4 had a
expressed into Roswell Park Memorial Institute factorial arrangement of treatments with diet and
(RPMI) 1640 medium9 by pressing with a blunt vaccination as the main effects. All data were
instrument (Glick and Schwartz, 1975). Spleens subjected to least squares analysis of variance.
from basal diet birds were placed in sterile petri The least squares means were further separated
dishes, minced in RPMI 1640 medium, clumps by least significant difference (LSD).
allowed to settle, and medium containing cells
pipetted into centrifuge tubes. Cell suspensions
RESULTS
were washed two times (5 min, 500 x g) in
RPMI 1640 medium containing 100 units/mL
penicillin, 100 u,g/mL streptomycin, .25 |Jg/mL Growth and Feed Conversion
fungizone, 1 mM L-glutamine, and .2% sodium
Table 2 shows weekly bird weights from
bicarbonate. Viable cells were counted and the
mononuclear cell concentration was adjusted to Experiments 1 and 2. Figure 1 shows feed
1 x 107 cells/mL. One hundred microliters of conversion data from Experiments 1 and 2,
each suspension (106 cells) was added to each of calculated by dividing body weight gain into
12 flat-bottom microtiter wells (96-well overall feed consumption. The PR diet signifiMicrotest m tissue culture plate with lid). 10 One cantly suppressed growth as early as 2 wk of age.
hundred uL of either 20 ug/mL sonicated X-73 Feed conversion values for the PR birds were
Strain of P. multocida, 250 u.g/mL phytohemag- significantly higher (P<.05) than those for basal
glutinin-M (PHA-M), or RPMI 1640 medium birds. Following the change in feeding regimen
was then added to three wells per bird. Cells for PR birds, their body weights increased but
were incubated for 48 h (40 C, 5% C0 2 ). One remained below the basal bird weights. Feed
microcurie of pHlthymidine11 (specific activity conversion values decreased to values compa6.7 Ci/mmol) was added and cultures were rable to those of basal birds during the control
incubated another 18 h. Cells were harvested feed period (i.e., 6 to 12 wk). Half of the birds in
onto filters using a PHD Cell Harvester.12 Four each dietary treatment were vaccinated twice
milliliters of scintillation fluid were added to with a commercial bacterin at 4 and 8 wk of age
vials holding the disks, and vials were counted in each experiment. Weight gain and feed
on a liquid scintillation counter.13 Lymphocyte conversion values were not influenced by
proliferation was measured based on vaccination.
[3H]thymidine uptake, and stimulation indices
(SI) reported were obtained by dividing the cpm
of test cultures containing PHA-M or sonicated
P. multocida by the cpm of cultures containing
TABLE 2. Combined average weights (g) of
only RPMI.
2138
PAYNE ET AL.
Antibody Titers
14
12
Diet, as well as vaccination, had significant
effects on titers, hi Experiment 1, the basal
10vaccinated birds had significantly higher titers
-Q- Basal Diet
than all other groups at 1.5,2.0, and 2.5 wk after J «
-•- PR Diet
the primary injection and 2.5 and 3.5 wk after
the second injection and significantly higher
titers than unvaccinated birds at all bleeding
times except 4.0 to 5.0 and 8.5 wk of age (Table
3). The PR-vaccinated birds had significantly
higher titers than unvaccinated birds between 2
4
6
10
12
and 4 wk after the primary injection and between
Age in Weeks
1 and 3 wk after the second injection. At 11.5 wk
FIGURE 1. Combined Experiment 1 and 2 feed
of age, the PR vaccinated titers fell significantly conversion values (grams of feed per gram of body weight)
lower than both unvaccinated and basal vacci- for chickens fed either a basal or protein restricted (PR) diet.
nated groups. In Experiment 2, diet, as well as The asterisk denotes the age when the PR diet was changed
to the basal diet.
vaccination, had significant effects on titers
(Table 4). Basal vaccinated birds expressed
significantly higher titers than PR vaccinated
birds at 5.5., 6.0, and 7.0 wk of age, and significantly higher titers than all unvaccinated
significantly higher titers than all unvaccinated birds.
birds at 5.0 to 7.0 wk of age. Beginning 2 wk
The birds in Experiments 1 and 2 showed
after vaccination, the PR vaccinated birds had gradual increases in IgG antibody titers over the
course of the experiments. In Experiment 1, the
basal vaccinated birds had significantly higher
IgG titers than PR vaccinated birds 1 to 4 wk
after primary injection; the two groups showed
TABLE 3. Antibody titers (test serum to positive
no significant difference after the second injecratio) of chickens fed basal or protein-restricted
tion (Table 5). The PR vaccinated birds had
(PR) diet and unvaccinated or vaccinated (Vac) at
significantly higher IgG titers than all unvacci4 and 8 wk of age with a polyvalent
nated
birds at 2 and 3 wk postprimary injection
commercial bacterin (Experiment 1)
and 1, 2, and 3 wk postsecondary injection. In
I
Age
Basal
Basal
Vac
PR
PR Vac
(wk)
4.0
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
8.5
9.0
9.5
10.0
10.5
11.0
11.5
.109a
.224"
.210*b
.228b
.277°
.320°
.372°
.290b
.462b
.461 b
.452b
.820°
.538b
.629°
.596b
.790b
.022 a
.086b
.404"
.900"
1.198a
.974a
.981 ab
.873a
1.059"
1.010"
1.052a
1.489"
1.353"
1.524"
1.337"
1.111"
.009"
.005b
.014b
.026b
.006dd
.029
.016d
.154b
.230°
.966"
.219°
.281 d
.330°
.218d
.197°
.689b
.021"
.014"b
.092b
21SP°
.584bb
.580
.746b
.897"
.824"
.856*
.917"
1.183b
1522"
1252 b
1.223"
.401c
""^Values within rows with no common superscripts are
significantly different (P<05).
'Basal birds fed a diet containing 21% protein; PR birds
fed a diet containing 7% protein.
TABLE 4. Antibody titers (test serum to positive
ratio) of chickens fed basal or protein-restricted
(PR) diet and unvaccinated or vaccinated (Vac) at
4 and 8 wk of age with a polyvalent
commercial bacterin (Experiment 2)
Dietary and vaccination treatment1
Age (wk)
Basal
Basal
Vac
PR
PR Vac
4
5
6
7
8
9
10
.083"
.206"
.267b
.300"=
.246c
.434b
.705c
.088"
.220"
.850"
.935"
.762"
.915*
1.335"
.026*
.036b
.0184b
218°
.198c
.318b
AIT6
.036"
sm^
.758*
.513 b
.531 b
.925*
.922b
Values within rows with no common superscripts are
significantly different (P<.05).
'Basal birds fed a diet containing 21% protein; PR birds
fed a diet containing 7% protein.
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Dietary and vaccination treatment
2139
PROTEIN DEFICIENCY AND IMMUNITY IN CHICKENS
TABLE 6. Antibody titers (IgG, test serum to
positive ratio) of chickens fed a basal or
protein-restricted (PR) diet and unvaccinated or
vaccinated (Vac) at 4 and 8 wk of age with a
polyvalent commercial bacterin (Experiment 2)
TABLE 5. Antibody titers (IgG, test serum to
positive ratio) of chickens fed a basal or
protein-restricted (PR) diet and unvaccinated or
vaccinated (Vac) at 4 and 8 wk of age with a
polyvalent commercial bacterin (Experiment 1)
Dietary and vaccination treatment1
Dietary and vaccination treatment1
Basal
Basal
Vac
PR
PR Vac
Age (wk)
Basal
Basal
Vac
PR
PR Vac
4
5
6
7
8
9
10
11
.177°
.224°
.370°
.445°
.605b
.486b
.366b
.540b
.065d
.281°
.787*
.762*
.871*
.930*
1.016*
1.024*
.411*
.492*
.020d
.088d
.161°
.565b
.258°
.378°
.295b
.390b
.579b
.653b
.696b
.872*
.952*
.952*
4
5
6
7
8
9
10
.082*
.206*
.267b
.300°
3A&
.423b
.705c
.088*
.220*
.853*
.935*
.762*
.916*
1.318*
.027*
.033 b
.199°
.154°
.212°
.332b
.409d
.035*
.097*
.740*
.48 l b
.531 b
.925*
.934 b
*-dtValues within rows with no common superscripts are
significantly different (P<05).
'Basal birds fed a diet containing 21 % protein; PR birds
fed a diet containing 7% protein.
Experiment 2, basal vaccinated birds had
significantly higher IgG titers than PR vaccinated birds at 3 and 4 wk postprimary and 2 wk
postsecondary injection. The PR vaccinated
birds had significantly higher IgG titers than all
unvaccinated birds at all ages except 4 and 5 wk
(Table 6).
Virulent Challenges
Table 7 shows survival data from Experiments 1 and 2 following a live pathogenic
challenge with the virulent Strain X-73 of P.
multocida. Survival percentages for vaccinated
birds of either dietary treatment in Experiments
1 and 2 were 90% or greater, while survival
percentages of unvaccinated birds were zero.
"^Values ^vithin rows wiithnoconimonsup erscrintsare
significantly different (P<05).
'Basal birds fed a diet containing 21% protein; PR birds
fed a diet containing 7% protein.
cells than PR vaccinated birds, although only
monocyte and total counts were significantly
lower than the vaccinated groups.
Lymphocyte Proliferation
In Experiment 3, diet significantly effected
lymphocyte proliferation, as shown by the
decreased response to PHA-M in PR birds and
high SI in basal birds (Table 9). Also, the PR
vaccinated group did not show increased lymphocyte proliferation when cultured with sonicated P. multocida, Strain X-73. However, in
Experiment 4, vaccination was shown to have
significant effects within both dietary groups.
White Blood Cell Counts
In Experiment 3, the PR vaccinated birds had
significantly decreased numbers of lymphocytes
and significantly increased numbers of heterophils and basophils over both basal groups
(Table 8). The basal vaccinated and PR vaccinated birds had significantly higher numbers of
monocytes than birds in the basal groups. In
Experiment 4, the PR birds, both vaccinated and
unvaccinated, had significantly lower numbers
of peripheral lymphocytes and eosinophils. The
total white cell counts were significantly lower
in the PR groups compared to the basal
vaccinated birds. The PR unvaccinated birds
tended to have lower numbers of all types of
TABLE 7. Results of live pathogenic challenge to
Pasteurella multocida, Strain X-73, of 12-wk-old
(Experiment 1) or 10-wk-old (Experiment 2) chickens
fed a basal or protein-restricted (PR) diet and
unvaccinated or vaccinated (Vac) at 4 and 8 wk of
age with a polyvalent commercial bacterin
Experiment 1
Experiment 2
Treatment
Death
ratio1
Percentage
survival
Death
ratio
Percentage
survival
Basal
Basal Vac
PR
PR Vac
8/8
1/16
8/8
0/13
0
93.75
0
100
10/10
0/9
9/9
1/10
0
100
0
90
Number dead out of total number in treatment
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Age (wk)
2140
PAYNE ET AL.
TABLE 8. Total and absolute white blood cell counts (x lCplmrn3) of 7-wk-old chickens fed a basal or
protein-restricted (PR) diet and vaccinated (Vac, at 5 wk of age) or unvaccinated
Treatment
Basal
Basal Vac
PR Vac
6.01 b
9.64 b
14.35*
7.24*
5.75*b
4.34 b
6.84*b
Lymphocytes Eosinophils
26.96a
23.82*
12.88b
14.37b
17.73*
10.14c
10.44°
Monocytes
Experiment 3
.78a
3.46b
.86*
10.12*
.68*
8.94*
Experiment 4
1.47*
6.28*
1.01*
6.13*b
.42°
3.66b
.60 bc
6.40*
Basophils
Total
.19 b
.35 b
1.64*
37.50*
44.67*
39.17*
.31*
.05*
.07*
.24*
29.38*1
30.87*
18.50°
24.63 b
^Values within columns and experiments with no common superscripts are significantly different (P<05).
Both vaccinated groups had significantly higher
SI when cultured with sonicated X-73 than
unvaccinated groups. Also, diet had no significant effect on lymphocyte proliferation in
response to PHA-M.
DISCUSSION
As shown in previous work (Bell et al.,
1976; Glick et al, 1981; Hambor et al, 1983;
Carsia et al, 1988; Sanchez-Munez et al,
1988), protein deprivation results in reduced
weight gain and increased feed conversion
values. The body weight and feed conversion
data presented from Experiments 1 and 2
clearly showed drastic growth inhibition in
birds fed the low-protein diet. However, PR
birds responded well when placed on the basal
feed at 5 wk of age. An experiment was
conducted to determine the effects of continued feeding of the low-protein diet, but due to
a very high mortality rate by 8 wk of age,
further information could not be obtained.
Humoral immune function of RP animals
has been assessed in the past, with varying
results (Cooper et al, 1974; Glick et al.,
1981). In the present studies, IgG antibody
titers in PR birds were lower than those of
basal birds, indicating a reduction in Ig
production. This concurs with results from the
work of Glick et al. (1981), showing reduction
in serum IgG concentrations in PR birds.
These results are also in agreement with work
by Cooper et al. (1974) and with work by Bell
and Hazell (1975) that indicated that a
moderately severe protein deficiency may
cause suppression in humoral immunity. Further, the protein restriction not only reduced
the titers following the first vaccination but
also had a suppressing effect on the titers
following the second vaccination, which occurred 3 to 4 wk after PR birds were placed on
basal feed. This shows a long-term effect from
a protein restriction early in life, or during
bursal development, which may explain the
reduction in antibody titer, even after several
weeks of feeding a diet containing 21%
protein. However, the PR diet did not compromise the vaccinated birds' abilities to
survive P. multocida infection.
The results of the challenge also indicated
that the polyvalent commercial bacterin
TABLE 9. Lymphocyte proliferation (incorporation of
[3H] thymidine (stimulation indices) of 7-wk-old basal
or protein-restricted (PR) vaccinated (Vac, at S wk
of age) or unvaccinated birds from
Experiment 3 and 4
Treatment
Phytohemagglutinin-M Sonicated X-73
SI*
SI 2
Basal
Basal Vac
PR Vac
204.10*
184.75*
22.36 b
Basal
Basal Vac
PR
PR Vac
439.21*
295.70*
207.01*
295.87*
Experiment 3
4.19 b
14.06*
1.36b
Experiment 4
3.51 b
7.85*
2.81 b
7.31*
"•"Values within column and experiments with no
common superscripts are significantly different (P<05).
'Stimulation index (SI) = phytohemagglutinin-M
(counts per minute) + Roswell Park Memorial Institute
medium (RPMI) (counts per minute).
2
SI = Sonicated X-73 (counts per minute) x RPMI
(counts per minute).
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 12, 2016
Basal
Basal Vac
PR
PR Vac
Heterophils
PROTEIN DEFICIENCY AND IMMUNITY IN CHICKENS
tion in Experiment 3, the same treatment group
of birds responded quite well in Experiment 4,
particularly to the sonicated X-73 stimulation.
Based on white blood cell counts (i.e.,
increased heterophils), this significant decrease
in Experiment 3 may be explained by undefined environmental stressors.
Although decreased numbers of total white
blood cells and absolute lymphocytes do not
reflect the number of cells cultured in vitro,
effects of these lowered populations may be
occurring in vivo. However, if the assumption
is made that these birds would have performed
like birds in previous experiments (i.e., Experiments 1 and 2) when subjected to a live,
pathogenic challenge, it appears that the
decrease in lymphocyte or total cell population, or both, would not compromise the birds'
ability to survive. In Experiments 3 and 4,
significantly lower numbers of lymphocytes
were observed in PR vaccinated groups, but
based on previous challenge data, these birds
would have survived challenge. Also, the
results of the lymphocyte proliferation assay in
Experiment 4 demonstrate that PR vaccinated
birds were capable of responding to an
antigenic preparation from the sonicated X-73
strain. These data demonstrate that cells from
birds on PR diet have the ability to be
sensitized to P. multocida and thus may
contribute to protection against P. multocida.
The present studies, along with findings
from previous work (Maheswaran et al, 1976;
Schlink and Olson, 1979; Baba, 1984; Briggs
and Skeels, 1984; Avakian and Dick, 1986),
show that immunity to P. multocida in
chickens is dependent upon both humoral and
cell-mediated immunities. The current immunity data indicate that protein deficiency
results in reduced immunocompetence as assessed by antibody titers, white blood cell
counts, and T-cell activity. However, survival
following challenge of vaccinated birds with P.
multocida was essentially unaffected by this
dietary alteration. It was concluded mat protein
deficiency in chickens lowered their overall
immune status, but mere was a sufficient
sensitization of the immune system toward P.
multocida following vaccination to provide
specific protection.
ACKNOWLEDGMENTS
The authors wish to thank W. C. Bridges of
the Department of Experimental Statistics for
Downloaded from http://ps.oxfordjournals.org/ at Penn State University (Paterno Lib) on September 12, 2016
(PaBac) conferred protective immunity to birds
when they were challenged with virulent Strain
X-73 of P. multocida. Unvaccinated birds,
although exhibiting low titer levels, did not
survive challenge. Thus, the antibody titers
detected at that time in unvaccinated birds
were not protective antibodies to P. multocida.
In past work, antibodies against P. multocida
have been detected in unvaccinated birds;
however, protection was not ensured by the
presence of these antibodies (Avakian, 1985).
Furthermore, survival appeared to involve
more than just adequate levels of protective
antibodies in circulation. All of the PR
vaccinated birds survived pathogenic challenge
in Experiment 1, but their prechallenge antibody titers were the lowest of any of the
treatment groups (Table 3). Functional cellmediated immune mechanisms were apparently operative in vaccinated birds. The importance of thymus-dependent immunity in the
protection of chickens against fowl cholera has
been shown in the past (Yamaguchi and Baba,
1975; Baba et al, 1978; Baba, 1984); therefore, the role of cell-mediated immunity in the
protection of PR vaccinated birds was examined in Experiments 3 and 4.
A low-protein diet was not observed to
cause differences in total white blood cells,
absolute lymphocytes, or absolute heterophils
during the first 3 wk of life in a previous
experiment (Glick et al., 1983). The increased
numbers of heterophils in the present study
suggest that the birds may have been exposed
to additional environmental stressors other
than dietary restriction. However, the birds
used in the present studies were older than
those used by Glick et al. (1983), and age
difference may have contributed to the effects.
Data from Experiment 4 show a clear decrease
in total cell counts due to protein restriction as
well as an increase in total cell counts due to
vaccination.
The proliferation responses contrast somewhat with the results of Cooper et al. (1974),
which showed significant enhancement in the
ability of lymphocytes from protein-deprived
mice to respond to PHA stimulation. In view
of the increased stimulation to P. multocida,
these data are in agreement with those of Dua
and Maheswaran (1978), which showed immunized turkeys to have higher stimulation to
antigenic preparations of P. multocida than
unimmunized turkeys. Although the PR vaccinated birds had significantly reduced prolifera-
2141
2142
PAYNE ET AL.
his assistance with statistical analysis. Christy
Williams and Tim Meier provided technical
assistance, and Gloria Freeman typed the
manuscript. This is Technical Contribution
Number 3040 of the South Carolina Agricultural Experiment Station, Clemson University,
Clemson, SC.
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