A Mechanism of Interaction between Dietary Protein and coccidiosis

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A Mechanism of Interaction between Dietary Protein
Levels and Coccidiosis in Chicks'
W. M. BRITTON, C. H. HILL ANDC. W. BARBER
Department of Poultry Science, North Carolina State of the University of
North Carolina at Raleigh, North Carolina
ABSTRACT Chicks were used to study a mechanism of interaction between dietary
protein levels and coccidiosis. The results of the study indicate mortality from coc-
cidiosis is less among groups of chicks fed a protein-free diet for 48 hours, or a 5%
protein diet for 14 days before inoculation with coccidia, than among chicks fed a
20% protein diet. Starvation had the same effect as the protein-free diet. Observation
of gross cecal lesions also indicates less infection in chicks fed a protein-free diet than
in chicks fed a 20% protein diet. Low protein diets also reduced intestinal trypsin
activity. A positive correlation was shown between dietary protein level and intestinal
trypsin activity and between intestinal trypsin activity and degree of infection of chicks
with coccidiosis. Addition of trypsin to the inoculum of the chicks fed a protein-free
diet resulted in an infection as severe as when the chicks were fed a 20% protein
diet. Since trypsin has been found to be essential for the establishment of coccidiosis
infection, it is proposed that the effect of low protein diets in decreasing the severity
of coccidiosis is due to the effect of these diets on intestinal trypsin activity.
The effect of dietary protein levels on
resistance or susceptibility to bacterial in
fection has been found to be unpredictable.
In some instances increasing protein levels
increased resistance (1); in others, in
creased susceptibility (2); and in at least
one instance (3) was found to have both
effects or neither depending upon the pres
ence of other dietary factors.
Coccidiosis has been studied in relation
to dietary protein levels by Allen (4) and
Jones (5) who reported no marked effect
on coccidiosis of chickens. In preliminary
experiments in this laboratory, it was ob
served that chicks fed a protein-free diet
for 2 weeks were much less susceptible to
coccidiosis than were chicks fed a diet
containing 20% protein.2 The purpose of
the work presented in this report was to
seek an explanation of this observation.
EXPERIMENTAL
The chicks used in these studies were
White Plymouth Rocks obtained from a
commercial hatchery. They were housed
in a conventional battery brooder and fed
a commercial diet until they were given
the experimental diets, at which time they
were placed in broiler batteries with raised
wire floors. The composition of the experi
mental diets is presented in table 1. The
protein content of the various diets was
adjusted by changing the soybean meal
level. The methionine level was changed
in proportion to the protein level. A con
stant energy level was maintained by ad
justment with fat and fiber.
Uninfected chicks were maintained on
the same regimen as the infected. Since
there was no mortality in the uninfected
animals, all mortality must be ascribed to
the infection.
Trypsin activity in the intestinal tract
was determined by a modified spectropho-
tometric method described by Kunitz (6).
A section of the lower small intestine just
above the cecum was taken from each
chick. The section was from 5 to 10 cm
long so as to make the sections approxi
mately the same size from each chick. The
section was slit open and swirled in 100 ml
of 0.0025 M HC1. This solution was filtered
through filter paper and 2 ml of the filtrate
was incubated with 2 ml of a 1% solution
of casein in Sorensen phosphate buffer at
pH 7.6 in a water bath at 37°Cfor 20
minutes. Six milliliters of 5% trichloro-
acetic acid were mixed with each sample
at this time and allowed to stand for one
hour. The sample was filtered and the
Received for publication October 23, 1963.
1Published with the approval of the Director of the
Experiment Station as Paper no. 1705 of the Journal
Series.
2Unpublished data, F. R. Craig and W. L. Payne.
306
J. NUTRITION,82: '64
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DIETARY PROTEIN LEVELS AND COCCIDIOSIS 307
TABLE 1
Composition of basal diets
Soybean meal (50%protein)Glucose
'
Defluorinated rockphosphateNaClVegetable
oil2Fiber
3MnSO,Vitamin
mix 4
DL-Methionine0%
083.2
3.20.51.011.9mg/kg
2201980
05%
1077.7
3.20.51.07.4mg/kg
220198075Protein10%
2072.3
3.20.51.02.7mg/kg
2201980
15020%
4053.3
3.20
53.00mg/kg
2201980
30030%
6029.6
3.20
5600mg/kg
2201980
450
1Cerelose, Com Products Refining Company, New York
2 Wesson Oil, The Wesson Oil Company, New Orleans, Louisiana
3 Solka Floe, Brown Company, Berlin, New Hampshire.
"Supplies per kilogram of diet: (in milligrams) thiamine, 3.5: riboflavin, 5.7; folie acid 11-
a-tocopheryl acetate, 22.0; menaditrae sodium bisulfite, 0.79; biotin. 0.18; pà ntothenic acid 183:
iitTmin D i S^ICU1116' cyanocobalamin, 8.8 „g; vitam n A, 11,000 USP 5ni?s; and
optical density at 280 mu was determined
on a Gary recording spectrophotometer.
A standard curve was obtained by using
trypsin (2 X crystallized) 50% MgSO43as
a standard with the activity of 1 ug being
defined as a trypsin unit. The protein con
tent of the filtrate of the intestinal contents
was determined by measuring the optical
density at 260 and 280 mu and the trypsin
activity was expressed in trypsin units per
milligram of protein.
The results of the experiments were
evaluated for statistical significance by
analysis of variance. Arcsin /percentage
transformation was used on all of the per
centage data analyzed (7).
RESULTS AND DISCUSSION
The first experiment was conducted to
test the effect of dietary protein levels on
the resistance of chickens to coccidiosis.
The protein levels varied from zero to 30%
in 5% intervals. These diets were fed to
4-week-old chicks for 2 weeks before and
one week after oral inoculation with
200,000 sporulated oocysts of Eimeria
tenella* One week after inoculation the
chicks were fed a 20% protein commercial
ration. Mortality and gross cecal lesions
were observed. Mortality occurred between
the fifth and ninth days post-inoculation.
The results of this study are presented in
table 2. Statistical analysis of the mortal
ity and lesion scores showed a significant
decrease in both at the zero and 5% pro-
TABLE 2
Mortality and gross cecal lesion scores of chicks
fed different levels of protein and infected
with Eimeria tenella
Protein
in diet>%
051015202530Mortality
2%
00101515520Gross
cecal
lesion scores31.51.73.02.73.33.13.3
. 'Diets fed for 2 weeks before and 1 week after
inoculation.
2 Twenty birds were fed each diet.
3 Mean of 10 birds scored zero for no lesions to
4 for most severe lesions.
tein levels. Furthermore, the chicks fed
the higher levels of protein (15 to 30% )
developed symptoms of coccidiosis, bloody
excreta and lethargy, earlier after inocula
tion.
In seeking an explanation of these re
sults, attention was directed to the life
cycle of the coccidia. It has been found
that excystation of the sporozoites from the
oocyst in dependent upon the presence of
trypsin (10). Levine (8) and Ikeda (9)
observed that chicks with ligated pancre
atic ducts did not become infected when
inoculated orally with sporulated oocysts.
When trypsin was added to the inoculum,
infection occurred (10).
' Nutritional Biochemical* Corporation, Cleveland.
«Donated by Merck and Company, Inc., Rahway,
New Jersey.
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308 W. M. BRITTON, C. H. HILL AND C. W. BARBER
Trypsin activity of the pancreas has been
found to be reduced by low levels of dietary
protein (11, 12). It was possible, there
fore, that the effect of dietary protein levels
on coccidiosis was a reflection of the effect
of these levels on intestinal trypsin activity.
To assess the effect of level of protein on
trypsin activity, an experiment was con
ducted in which intestinal trypsin activi
ties of chicksfed diets containing 5, 20,
and 30% soybean protein and a 20% pro
tein commercial diet were determined. The
results (table 3), show a significant reduc
tion in the trypsin activity of the 5% pro
tein fed group when compared with those
fed higher protein levels. These data sup
ported the possibility that the effect of the
low protein diet on trypsin activity was
causally related to the effect on mortality
from coccidiosis.
The effect of low dietary protein levels
on intestinal trypsin activity was most
probably a rapidly occurring phenomenon.
To determine how rapid, one group of
chicks receiving the commercial diet was
changed to a protein-free diet and another
group was starved. After 24, 48, and 72
hours, 4 chicks from each treatment, along
with four continuing to receive the com
mercial diet, were killed and the intestinal
trypsin activity determined. The results
are presented in table 4. Within 24 hours
the intestinal trypsin activity of those
chicks receiving the protein-free diet or
those chicks starved was much lower than
that of the 20% protein commercial ration.
These observations indicate that if intes
tinal trypsin activity and susceptibility to
coccidiosis were causally related a short
period of feeding a protein-free diet or
starvation should suffice to produce the
effect. Accordingly, an experiment was
conducted in which the chicks were either
TABLE 3
Trypsin activity of intestinal section of chicks
fed -various levels of protein in the diet
Diets
Time % Protein in diet
feddietsdays1416518411620trypsin
uniti4726443020
(com
mercial)ì/mg
protein1475456433395
TABLE 4
Trypsin activity of intestinal section of chicks
starved and of chicks fed a protein-free and
a 20% protein commercial ration
DietTime
feddiethours244872Starvedtrypi71518Tree¡in
a aih4181420%
i11' Protein
(commercial):/mg
protein'328813682
1Each value is a mean of 4 chicks tested.
TABLE 5
Mortality of chicks from coccidiosis when starved,
fed a protein-free diet, or a 20% protein diet
DietStarved
2
Protein-free 3
20% Protein3Days
post-inoculation56789Cumulative
% mortality l
3 10 27 27 27
0 17 29 29 29
32 45 50 55 55
1Mean value of 2 groups of 12 chicks each.
2Starved for 48 hours before and 96 hours after
inoculation.
3Diets fed for 48 hours before and one week after
inoculation.
fed the protein-free diet or starved for 48
hours prior to inoculation with sporulated
oocysts of Eimeria tenella, Eimeria max
ima, Eimeria brunetti, and Eimeria acer-
vulina* Ninety-six hours after inoculation
the starved groups were fed the commer
cial diet. Seven days after inoculation the
groups fed a protein-free diet were fed the
commercial diet. The results of that study
are presented in table 5. A significant
reduction in mortality was noted in the
starved chicks and those fed the protein-
free diets even though this treatment was
started only 48 hours before inoculation.
A similar experiment in which the ef
fects of the protein-free diet fed for 48
hours before inoculation and the 20%
protein soybean meal basal diet were com
pared yielded similar results (table 6). In
this experiment the inoculum contained,
in addition to those mentioned above, spor
ulated oocysts of Eimeria hagani, Eimeria
necatrix, Eimeria praecox, and Eimeria
mivati."
The results of these 2 studies coupled
with those of a previous experiment (table
1Each value is a mean of 5 chicks tested.
5See footnote 4.
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DIETARY PROTEIN LEVELS AND COCCIDIOSIS 309
TABLE 6
Mortality of chicks from coccidiosis when fed a
protein-free or a 20% protein diet
Diet
Days post-inoculation
6 8 9
Cumulative % mortality 1
Protein-free2 0 22 55 65 75
20% Protein2 0 75 90 95 95
1Each value a mean of 4 groups of 10 birds each.
2Diets fed for 48 hours before and one week after
inoculation.
4) strengthened the theory that the dietary
protein effect on susceptibility to coccidio
sis was mediated by way of the intestinal
trypsin activity. Therefore, a more direct
test of the influence of protein levels and
trypsin activity on susceptibility to this in
fection was conducted. In this test chicks
were fed either a protein-free or a 20%
protein diet for 48 hours before inoculation
with the sporulated oocysts of the 8 species
of Eimeria shown above. The special in
oculum was prepared both with and with
out 1% added trypsin7 and given to 2
groups of 10 chicks/diet. The trypsin was
prepared as a 2% solution in Sorensen's
phosphate buffer at pH 7.6 and mixed with
equal volumes of the normal inoculum. Inthe control, Sorensen's phosphate buffer
was mixed with equal volumes of the nor
mal inoculum, but trypsin was not added.
The results of this study are presented
in table 7. The mortality was significantly
reduced in the chicks fed the protein-free
diet, and the addition of trypsin to the
inoculum increased the mortality to that of
the 20% protein-fed chicks. The addition
of trypsin to the inoculum of the 20%
protein-fed group did not result in a sig
nificant increase in mortality.
The results of this study establish more
clearly the relationship between dietary
protein levels, trypsin activity, and sus
ceptibility to coccidiosis. In this infectious
model, the effect of low dietary protein
levels was a definite reduction of the nor
mal trypsin activity of the intestinal tract
which is necessary for normal excystation
of the oocysts. Without excystation infec
tion does not occur. It should be noted that
even the chicks fed the low protein diets
still had some trypsin activity in the intes
tinal tract which may account for the in
fection that was observed in these groups.
LITERATURE CITED
1. Dubos, R. J., and R. W. Schaedler 1958
Effect of dietary proteins and amino acids on
the susceptibility of mice to bacterial infec
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2. Hill, C. H., and H. W. Garren 1961 Protein
levels and survival time of chicks infected
with Salmonella gallinarum. J. Nutrition,
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3. Hill, C. H., R. W. Colburn and H. A.
Schneider 1962 The relative roles of vita
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4. Allen, E. A. 1932 The influence of diet on
the development of experimental coccidiosis
of chickens kept under sanitary conditions.
Am. J. Hyg., 15: 163.
5. Jones, E. E. 1934 The effect of diet on the
course of experimental coccidiosis in the
chicken. J. A. V. M. A., 85: 193.
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'Trypsin 1:300,
tion, Cleveland.
Nutritional Biochemicals Corpora-
TABLE 7
Effect of protein-free and 20% protein diets on mortality from coccidiosis with
and without added trypsin in the inoculum
Diet i Inoculum
Days post-inoculation
Protein-freeProtein-free20%
Protein20%
Proteincontroltrypsin
addedcontroltrypsin
addedCumulative
'
010555756080957o
mortality
40708595z 45858595
1Diets fed for 48 hours before and one week after inoculation.
2Mean values of 2 groups of 10 chicks each.
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310 W. M. BRITTON, C. H. HILL AND C. W. BARBER
8. Levine, P. P. 1942 Excystation of coccidial
oocysts of the chicken. J. Parasitol., 28: 426.
9. Ikecla, M. 1955 Factors necessary for E.
tendÃ-ainfection of the chicken. I. Influence
of the digestive juices on infection. Japanese
J. Vet. Sci., 17: 197.
10. Ikeda, M. 1955 Factors necessary for E.
tenelÃ-ninfection of the chicken. II. Influence
of the pancreatic juice on infection. Japanese
J. Vet. Sci., 17: 225.
11. Grossman, M. I., H. Greengard and A. C. Ivy
1942 The effects of dietary composition on
pancreatic enzymes. Am. J. Physiol., 338:
676.
12. Sidransky, H., and M. Rechcigl, Jr. 1962
Chemical pathologyof acute amino acid de
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