The Effects of Cold Stress on Neonatal Calves
II. Absorption of Colostral Immunoglobulins
D. P. Olson, C. J. Papasian and R. C. Ritter*
ABSTRACT
Newborn calves were subjected to cold
stress and made hypothermic by immersion in water at 15 to 170C. Cold stress delayed the onset and significantly decreased
(P < 0.05) the rate of absorption of immunoglobulins (IgM, IgG1 and IgG2) up to
15 hours after first feeding of pooled
colostrum. However, the net absorption of
colostral immunoglobulins was not affected. The possible deleterious effect of
cold stress on absorption of colostral immunoglobulins by newborn calves under
range conditions is discussed.
RCSUMC
Les auteurs ont provoque le stress du
froid, chez des veaux nouveau-nes, et les
ont rendus hypothermiques, en les immergeant dans un bassin d'eau dont la temperature variait de 15 a 170C. Ce stress retarda le debut de l'absorption des immunoglobulines IgM, IgGi et IgG2; il en diminua aussi de faqon appreciable (P <
0.05) le taux d'absorption, jusqu'a 15
heures apres le premier repas d'un me
lange de colostrum. L'absorption nette des
*University of Idaho Agricultural Experiment
Station, Department of Veterinary Science,
Moscow, Idaho 83843.
Published with the approval of the Director
of the Idaho Agricultural Experiment Station,
as Research Paper No. 7986.
Supported by funding from the Idaho Agricultural Experiment Station.
Based on a thesis submitted by the second
author to the Graduate School of the University of Idaho, in partial fulfillment of the requirements of the MS degree.
Submitted May 24, 1979.
immunoglobulines colostrales ne s'en
trouva cependant pas affectee. Les auteurs
commentent les effets dommageables possibles du stress du froid sur l'absorption
des immunoglobulines colostrales par les
veaux nouveau-nes, dans les conditions
naturelles du velage au champ.
INTRODUCTION
Newborn calves are usually born either
hypogammaglobulinemic (12, 13) or agammaglobulinemic (20, 22) due to the absence
of transplacental transfer of antibodies
from the maternal to the fetal circulation
during normal gestation (2). The calf, like
many other neonates, is passively immunized by the ingestion and absorption of
colostral immunoglobulins during the early
postnatal period. Nonselective absorption
of immunoglobulins and other macromolecules in colostrum by the intestine of the
suckled neonate normally begins within
three hours after ingestion and continues
for only 24 to 36 hours after birth (8, 11).
Consequently, suckled calves may die from
a variety of infectious diseases due to failure of absorption and the resulting persistent hypogammaglobulinemia or agammaglobulinemia (3, 5).
Range calves, born during late winter
and early spring, are often cold stressed
and may become hypothermic due to exposure to harsh environmental conditions
(15, 16, 23, 24). Work with dogs has shown
that hypothermia is responsible for decreased venous outflow from the small intestine, decreased intestinal motility and a
net reduction in transport of substances
from intestinal lumen into the blood (10).
Therefore, it seemed possible that hypothermia might impair absorption of colostral immunoglobulins by calves. This work
was undertaken to determine the effect of
cold stress on the ability of neonatal calves
to absorb colostral immunoglobulins.
Can. J. comp. Med. 44: 19-23 (January 1980)
19
MATERIALS AND METHODS
GENERAL PROCEDURES
Procedures for cold stress treatment of
26 Holstein-Friesian calves have been described (18). Briefly, calves in groups 1
and 2 were cold stressed by immersion in
water at 15 to 17°C until the core body
temperature (CBT) was lowered 10°C.
Calves in group 1 were euthanatized two
hours after cold stress. Calves in group 2
were allowed to recover (after cold stress)
for 72 to 96 hours in a cold (4°C) wet environment and then euthanatized. Calves in
group 3 remained normothermic during immersion in water at thermoneutral temperature (35 to 37°C) for two hours. They
were then maintained in a 250 C dry environment for 72 to 96 hours and then
euthanatized. Calves in groups 1 and 2
were fed one liter of pooled colostrum by
stomach tube when the CBT was lowered
2°C or 5°C, respectively. Calves in group 3
were fed after one hour of immersion.
column (2.5 by 90 cm) using 0.1M tris (hydroxymethyl aminomethane) buffer (pH
8.18) to which sodium azide (400 mg/mL)
had been added as a preservative (6).
Ion exchange chromatography was performed on diethylaminoethyl cellulose2
columns (1.5 by 30 cm) at 25°C. Immunoglobulin G2 was semipurified by single step
elution with phosphate buffer solution
(PBS) (0.O1M, pH 7.4) (6). Immunoglobulin G1 from gel filtration chromatography
was further purified by stepwise elution
with PBS. Buffer concentrations of phosphate and pH value used were 0.O1M, 0.02M
and 0.05M at pH 6.3 (6).
Samples of fractions of IgM, IgG, and
IgG2 from column chromatography were
tested by spectrophotometry, immunoelectrophoresis (21) and double diffusion in
agar gel (19) to determine purity.
Guinea pig antiwhole bovine serum and
monospecific antiglobulins against bovine
IgM, IgG, and IgG2 were prepared as previously described (17). Antiglobulins were
stored (-20°C) until used.
QUANTITATION OF IMMUNOGLOBULINS
COLLECTION
OF SPECIMENS
A polyethylene catheter (PE 90) was implanted into the jugular vein of each calf
before immersion. Blood was collected
before immersion, hourly for nine hours
after the start of immersion and approximately every 12 hours thereafter. Serum
was separated from clotted blood and stored
(-20°C) until analyzed for immunoglobulin.
Rennin precipitated and defatted whey was
also prepared from the pooled colostrum
fed to calves and stored (-20°C) until
analyzed for immunoglobulins.
IMMUNOLOGICAL PROCEDURES
Bovine IgM, IgG1 and IgG2 were isolated and purified by minor modifications of
previously described procedures (6). Immunoglobulin G2 was prepared from whole
blood serum whereas IgM and IgGi were
prepared from colostral whey.
Gel filtration chromatography' was performed for semipurification of IgM by
gravity flow elution at 4°C on a single
'Sephadex G-200, Pharmacia Fine Chemicals,
Piscataway, New Jersey.
20
Concentrations of IgM, IgG1 and IgG2
in pooled colostral whey and calf serum
were determined by single radial immunodiffusion (14). A stock solution of 2%
(w/v) agar gel3 was prepared in 0.05M barbital buffer (pH 8.6) to which sodium azide
(400 mg/mL) had been added as a preservative. Antiserum was diluted with 0.05M
PBS (pH 7.4), heated to 55°C, and thoroughly mixed with an equal volume of
heated (60°C) agar solution. The final dilutions of antiglobulins for bovine IgM, IgG,
and IgG2 were 1:25, 1:15 and 1:20, respectively. Preliminary tests were conducted to
determine the optimum final dilution of
the sample to be used in the analysis.
Calibration curves were obtained by testing fractions of bovine IgM, IgG, and IgG2
of known concentrations.4 All pooled colostral whey and calf serum samples were
tested in duplicate and incubated at 250C
in a moist chamber for 48 hours. Ring dia2Whatman DE 52, Reeve Angel, Clifton, New
Jersey.
3Noble Agar, Difco. Laboratories, Detroit, Michigan.
4Miles Laboratories, Inc., Elkhart, Indiana.
0.9
GROUP 3
0.8I
_
4.0
z
30
II
GROUJP 3
I
E 07
IIX
I
I
z
0
06
II
11
GROUP 2
11
0.5-
11
1
F
z
w
0.4
E-
0.3-
11
11
II
II
20
II
_,
.
.
.
6
9
II
II
0.
II
_
II
1.0
II
0I-
II
II
II
3
6
9
12
5
18
21
r
v-j
24
3
TIME (hours)
Fig. 1. Mean concentrations (mg/mL) of IgM
in serum of cold stressed (group 2) and noncold stressed (group 3) calves at selected times
after ingestion of colostrum.
meters were read in two dimensions on a
calibration viewer5 and the mean diameter
calculated. An equation for the calibration
curve for each immunoglobulin class (concentration vs. the square of the mean ring
diameters) was determined by linear regression. The concentrations of IgM, IgG,
and IgG2 in pooled colostral whey and in
calf serum were calculated using the appropriate regression equations.
12
es
24
21
TME (hours)
Fig. 2. Mean concentrations (mg/mL) of IgG,
in serum of cold stressed (group 2) and noncold stressed (group 3) calves at selected times
after ingestion of colostrum.
_
GROUP 3
OA-
# ,GROP
0.3
2
/
i-I
Z
0.2-
/
w
i0
I/
~0.Ii
RESULTS
The concentration of immunoglobulins
(mg/mL) in whey from pooled colostrum
fed to the calves was: IgM=9.10, IgG,=
60.73 and IgG2=7.87. Mean concentrations
of IgM, IgG, and IgG2 in serum of calves
in groups 2 and 3 are summarized (Table
I, Figs. 1-3). Data from calves in group 1
are not included since the mean concentrations of immunoglobulins measured did not
differ significantly (P > 0.05) from those
of calves in group 2 by three hours after
first feeding and euthanasia at an earlier
age prevented a full length comparison
with the noncold stressed calves.
There was a delay of from one and onehalf to three and one-half hours in first
appearance of immunoglobulins in serum of
5Transdyne General Corporation, Ann Arbor,
Michigan.
TIME (hours)
Fig. 3. Mean concentrations (mg/mL) of
IgG2 in serum of cold stressed (group 2) and
noncold stressed (group 3) calves at selected
times after ingestion of colostrum.
calves in group 2 when compared to calves
in group 3. Furthermore, the mean concentrations and thus the rate of absorption of
IgM and IgG1 in the serum of calves in
group 2 were significantly lower (P
0.05) than the respective concentrations in
the calves in group 3 during the period
three to 18 hours after feeding of colostrum. Similarly, the mean concentration of
IgG2 in the serum of calves in group 2 was
significantly lower
(P
-
0.05)
than
that
found in calves in group 3 during the
period of three to 15 hours after feeding of
colostrum. In all cases, the rate of absorption of immunoglobulins by noncold
stressed calves was exponential and nearmaximal concentrations were attained by
21
TABLE I. Mean Concentrations (mg/mL) of IgM, IgGi and IgG2 in the Serum of Cold Stressed (group 2) and
Noncold Stressed (group 3) Calves at Selected Times After Ingestion of Colostrum
Immunoglobulin Concentrations (Mean
IgGi
IgM
Groups
Groups
3
2
3
2
0.00
lg99a
0.00
0.16a
Time (hours)
after Ingestion of Colostrum
3
0.06
9
0.29
+0.25
0.46
+0.27
0.53
+0.25
0.59
+0.27
0.79d
2.16
+1.50
2.42
-41.42
2.70
+0.23
+ 0.26
+
12
15
18
21
0.62
+ 0.21
0.63a
0.82c
0.77e
+ 0.26
4.09a
2.08
4.25a
0.55
1.07
+0.24
0.79a
+0.27
0.83b
+0.29
IgG2
Groups
0.00
2
3
0.20a
0.06
+0.12
=I:0. 1
0.36a
0.13
+ 1.29
dz 0.14
6
0.12
i SD)
1.38
a1.43
1.37
3.08
-4-1.44
+2.06
4.30b
+2.03
4.39c
+2.06
4.50d
2.10
4.16e
+ 1.55
=i
0.15
+0.16
0.25
h 0.14
0.41a
+0.13
0.42b
+0.14
i0.13
0.43C
+0.14
0.33
+0.12
0.36
+0. 12
0.43e
+0.12
0.42e
+ 0.14
0.29
ap<50.00
bp <
0.o1
cP <0.025
dp < 0.05
eNot significant
six to nine hours after first feeding.
The net absorption of colostral immunoglobulins by calves was not affected by
cold stress. Data show that the mean concentrations of IgM and IgG1 in the serum
of calves in groups 2 and 3 did not significantly differ (P 2 0.05) by 21 hours after
feeding of colostrum. Also, there was no
significant difference (P 2 0.05) between
calves in groups 2 and 3 in concentration of
serum IgG, by 18 hours after feeding.
DISCUSSION
Data from the present investigation show
that cold stress of newborn calves caused
a delay in onset and a significant decrease
in the rate of absorption of colostral immunoglobulins up to 15 hours after the
first feeding. The reasons for the delay in
onset and the decrease in rate of absorption of colostral immunoglobulins were
not determined, although they may have
been the same as those previously described in cold stressed dogs (10).
Resistance of newborn calves against diseases such as colibacillosis is dependent
in part on ingestion and absorption of sufficient quantities of protective colostral
antibodies (1, 4, 7, 9). Factors which cause
a delay in onset and a decrease in rate of
22
absorption of colostral antibodies could
have a deleterious effect on calf survival,
particularly when natural exposure to infectious organisms occurs within the first
few hours of life. Data from the present
investigation suggest that severe cold stress
of newborn calves under range conditions
could cause a delay in onset and a decrease
in rate of absorption of colostral antibodies
and enhance the susceptibility of these
animals to infectious organisms.
The apparent absence of an effect on net
absorption of colostral immunoglobulins by
calves may have been due to the short
duration of the cold stress. A previous report (18) showed that the core body temperature of these calves had returned to
normal by eight hours after the first feeding of colostrum, whereas the present
report indicates that absorption of immunoglobulins continued for 18 to 21 hours.
Consequently, cold stressed calves were
hypothermic for less than half the time
period during which immunoglobulin absorption occurred. Prolonged hypothermia
would probably have decreased net absorption.
Muscular weakness and reluctance to
stand and nurse are behavioral changes
which were observed in the cold stressed
calves (18). Similar alterations in clinical
behavior by cold stressed calves under range
conditions could cause a delay in onset of
nursing activity and thus cause a decrease
in the total amount of colostrum ingested
and absorbed.
12.
ACKNOWLEDGMENTS
13.
The authors acknowledge the technical
assistance of Ms. L.M. Evenson and K.M.
Hendrix and the graphic art of Ms. T.
Peek.
14.
15.
REFERENCES
1. BOYD, J. W. The relationship between
serum immune globulin deficiency and disease in calves: A farm survey. Vet. Rec.
90: 645. 1972.
2. BRAMBELL, F. W. R. The Transmission
of Passive Immunity from Mother to
Young. New York: American Elsevier.
1970.
3. FEY, H. Neuere untersuchungen uber die
colisepsis des kalbes. Schweizer Arch.
Tierheilk. 104: 1-12. 1962.
4. FEY, H. Colibacillosis in Calves. Bern:
Verlag Hans Huber. 1972.
5. FEY, H. und A. MARGADANT. Hypogammaglobulinamie bei der colisepsis des
kalbes. Path. Microbiol. 24: 970-976. 1961.
6. FEY, H., H. PFISTER, J. MESSERLI, N.
STURZENEGGER and F. GROLIMUND.
Methods of isolation, purification and
quantitation of bovine immunoglobulins.
Zentbl. VetMed. B 23: 269-300. 1976.
7. GAY, C. C., K. A. McKAY and D. A.
BARNUM. Studies on colibacillosis of
calves III. The experimental reproduction
of colibacillosis. Can. vet. J. 5: 314-325.
1964.
8. GAY, C. C., N. ANDERSON, E. W.
FISHER and A. D. McEWAN. Gammaglobulin levels and neonatal mortality in
market calves. Vet. Rec. 77: 148-149. 1965.
9. GLANTZ, P. J., D. C. KRADEL and J. F.
HOKANSON. Escherichia coli serogroup
115 isolated from animals: Experimental
infection of calves. Am. J. vet. Res. 27:
1205-1209. 1966.
10. GRIFFIN, W. 0., A. CASTANEDA, D. M.
NICOLOFF, N. H. STONE and 0. H.
WANGSTEEN. Influence of local hypothermia on absorption from isolated intestinal segments. Proc. Soc. exp. Biol.
Med. 103: 757-759. 1960.
11. HANSEN, R. G. and P. H. PHILIP. Studies
16.
17.
18.
19.
20.
21.
22.
23.
24.
on the proteins of bovine colostrum. II. The
absorption of g!obulins by the young calf.
J. Dairy Sci. 30: 560. 1947.
HANSEN, R. G. and P. H. PHILIP. Studies
on proteins from bovine colostrum. III.
The homologous and heterologous transfer
of ingested protein to the blood stream of
the young animal. J. biol. Chem. 179: 523527. 1949.
JOHNSON, P. and A. E. PIERCE. Ultracentrifugal and electrophoretic studies of
neonatal calf sera and maternal colostrum.
J. Hyg., Camb. 57: 309-320. 1959.
MANCINI, G., A. 0. CARBONERA and
J. F. HEREMANS. Immunochemical quantitation of antigens by single radial immunodiffusion. Immunochemistry 2: 235254. 1965.
MARTIN, S. W., C. W. SCHWABE and
C. E. FRANTI. Dairy cattle mortality rate:
Influence of meteorologic factors on calf
mortality rate in Tulare County, California. Am. J. vet. Res. 36: 1105-1109. 1975.
MOLL, T. The pathogenesis of diarrhea in
the newborn calf, with special reference to
physiologic function and environmental
conditions. J. Am. vet. med. Ass. 147: 13641366. 1965.
OLSON, D. P. and G. L. WAXLER. Immune responses of the bovine fetus and
neonate to Escherichia coli: Quantitation
and qualitation of the humoral immune
response. Am. J. vet. Res. 37: 639-647.
1976.
OLSON, D. P., C. J. PAPASIAN and R. C.
RITTER. The effects of cold stress on
neonatal calves I. Clinical condition and
pathological lesions. Can. J. comp. Med.
44: 11-18. 1980.
OUCHTERLONY, 0. Handbook of Immunodiffusion and Immunoelectrophoresis.
Ann Arbor: Ann Arbor Science Publishers.
1968.
PENHALE, W. J., G. CHRISTIE, A. D.
McEWAN, E. W. FISHER and I. E.
SELMAN. Quantitative studies on bovine
immunoglobulins. II. Plasma immunoglobulin levels in market calves and their
relationship to neonatal infection. Br. vet.
J. 126: 30-37. 1970.
SCHEIDEGGER, J. J. Une micro-methode
de l'immunoelectrophorese. Int. Archs
Allergy appl. Immun. 6: 103-110. 1955.
SMITH, E. L. and A. HOLM. Transference
of immunity to the newborn from colostrum. J. biol. Chem. 175: 349-357. 1948.
SPEICHER, J. A. and R. E. HEPP. Factors
associated with calf mortality in Michigan
dairy herds. J. Am. vet. med. Ass. 162:
463-466. 1973.
WITHERS, E. W. Mortality rates and
disease incidence in calves in relation to
feeding management and other environmental factors. Br. vet. J. 108: 382-405.
1952.
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