Periparturient Hypocalcemia in Cows: Effects
on Peripheral Blood Neutrophil and Lymphocyte Function
MARCUS E. KEHRLI, JR., and JESSE P. GOFF
Mineral Metabolism and Mastitis Research Laboratory
National Animal Disease Center, USDA-ARS
Ames, lA 50010
ABSTRACT
Adverse changes in immune cell function during the periparturient period have been reported, which may contribute to the suscepubility
of the bovine mammary gland to infection and
clinicai mastitis (10, 12, 14, 15, 16, 21, 34).
Ahhough a causal relationship is not established, epidemiologicai studies indicate that
cows with parturient hypocaicemia (PH) have a
five to eight times greater chance of having
mastitis than if PH is absent and a nine times
greater chance of having coliform mastitis (5).
Parturient hypocalcemia is primarily a disorder
of Ca homeostasis associated with the onset of
lactation in dairy cows. We hypothesized that
changes in plasma Ca concentrations during PH
and the associated changes in concentrations of
hormones involved with Ca homeostasis might
contribute to changes in neutrophil (PMN) and
lymphocyte (Lc) function in periparturient
cows. If this were true, then prepartum parathyroid hormone (PTH) administration to increase
bone resorption and 1,25-dihydroxyvitamin D
[1,25-(OH)2D ] concentrations (which would increase intestinal absorption of Ca) would prevent a decline in plasma Ca concentration at
calving (8) and might alter the adverse periparturient changes in immune cell function. The
objective of the present study was to evaluate
the ability of PTH treatment and the associated
normocaicemia to restore normal peripherai
blood PMN and Lc function during the periparturient period.
Periparturient dairy cows are quite
susceptible to intramammary infections
and clinical mastitis, epidemiologic evidence indicates that parturient paresis
(milk fever) greatly increases the risk of
mastitis, although a causai relationship
has not been established. In the present
experiment the effects of hypocaicemia at
parturition on the immune status of dairy
cows were investigated. Ten heaithy,
multiparous Holstein cows were fed a
high Ca diet prepartum to induce hypocalcemia at parturiuon. Five of these
cows received intramuscular parathyroid
horrnone (crude synthefic N-terminus 134) to prevent hypocaicemia at parturition. Effects of hypocaicemia on various
neutrophil and lymphocyte functions
were determined during the periparturient
period, ranging from 6 wk prepartum to 5
wk postpartum. All cows exhibited severe
loss of immune cell function in the weeks
surrounding parturition. Hypocaicemia or
the development of parturient paresis did
not exacerbate the immune cell dysfunction, This implies that the degree of
hypocaicemia observed did not have a
large or irreversible influence on neutrophil and lymphocyte function in periparturient cows.
INTRODUCTION
MATERIALS AND METHODS
The bovine mammary gland during the periparturient period is more susceptible to infection and clinical disease than at other times
during lactation or the dry period (19, 22, 31).
Received August 1, 1988.
Accepted November 21, 1988.
1989 J Dairy Sci 72:1188-1196
Animals and Expedmental Design
Ten healthy, multiparous Holstein cows
were evaluated during the periparturient period
ranging from 6 wk prepartum to 5 wk postpartum. All gestation periods were synchronized to
minimize the duration of the calving period to
be studied. As described by Goff et ai. (8), all
1188
HYPOCALCEMIA AND PERIPARTURIENTIMMUNITY
cows received a high Ca diet (about 150 g Ca
and 80 g P/d) prepartum to predispose them to
hypocalcemia and milk fever alter calving. One
group of five cows was given crude synthetic
bovine PTH (N-terminal 1-34 fragment, Peninsula Laboratories, Inc., San Carlos, CA) beginning approximately 6.4 d prepartum with a 20
mg dose every 8 h for the first six treatments as
a loading dose. Maintenance doses of 10 mg of
PTH every 8 h were continued until parturition
at which time 20 mg of PTH was administered
three times at 8-h intervals. Cows were then
gradually withdrawn from PTH therapy by 50%
reduction in dose per day down to 2.5 mg/8 h.
Withdrawal from PTH was completed after 48
h of treatment at the 2.5 mg dose/8 h (on
average by 6.25 d postpartum). Control cows
were treated with the carrier alone following
this same schedule.
Because normal leukocytes cannot be stored
for use as laboratory standards in our function
assays, leukocytes isolated daily from five Holstein steers were used as internal laboratory
standards to determine the daily mean for each
assay (15, 16). This mean was then used to
block out the day-to-day variability typically
seen with immune cell function assays by converting the daily individual cow results to a
percent of the steers' dally standard mean. Initially, sampling was once a week beginning
about 4 wk berøre the expected calving time.
The frequency of sampling was increased to a
Monday, Wednesday, and Friday schedule
about 2 wk before expected parturition and
continued at that frequency for at least 2 wk
postpartum. The animals were then sampled
once a week for the next 2 wk.
Leukocytø Preparation
Neutrophils were separated by hypotonic lysis from packed erythrocytes as previously described (26). Remaining cells, usually >95%
granulocytes [PMN + eosinophils (PME)], were
resuspended to 5.0 x 107 granulocytes/ml in
.015 M phosphate-buffered saline solution for
functional analysis. Lymphocytes were isolated
as previously described (17).
Neutrophil Function Assays. The procedures
for evaluating cytochrome C reduction, ingestjon of 125I-labeled Staphylococcus aureus, iodination, native (nonluminol dependent) chemi-
1189
luminescence,
antibody-dependent
cellmediated cytotoxicity (ADCC), antibody-independent neutrophil-mediated cytotoxicity, directed migration, and random migration under
agarose by PMN were done as described (15).
Lymphocyte Blastogenesis. Lymphocyte
blastogenesis was done as previously described
with 2.0 x 105 lymphocytes per well (17).
Tissue culture media (.2 ml of RPMI 1640,
Gibco Laboratories, Grand Island, NY. #3802400) with 25 mM HEPES buffer and 2 mM Lglutamine was supplemented with the following: heat-inactivated fetal bovine serum (Al ll5-L, Hyclone Laboratories, Inc., Logan,
UT) to a 10% final concentration and an antibiotic/antimycotic solution (A-9909, Lot 76F6832, Sigma Chemical Company, St. Louis,
MO) resulting in 100 units of penicillin G, .1
mg of dihydrostreptomycin, and 250 ng of amphotericin B/ml of medium. Media for mitogenic stimulation contained one of the following amounts of mitogen: 25, 12.5, or 6.25 I,tg of
phytohemagglutinin-P (PHAP, L-9132, Sigma
Chemical Company, St. Louis, MO), 15.6, 7.8,
or 3.9 ktg of Concanavalin A (Con A, C-2010,
Sigma Chemical Company, St. Louis, MO), or
20, 2, or .2 ktg of pokeweed mitogen (PWM, L9379, Sigma Chemical Company, St. Louis,
MO)/ml of medium. Data o b t t n e d from all
three concentrations of each mitogen were averaged for an overall mitogen mean which was
used in statistical analysis.
Data Analysis
Data from each cow were converted to a
percent of the standard steers' values for each
sampling day and then coded relative to their
actual calving dates. Standardized values for
individual cows were averaged within each
week relative to calving. To evaluate the effect
of PTH administrafion on each parameter, wk
- 2 was chosen from Figures 1 and 2, as the
pretreatment baseline value for comparison.
Changes for each cow from wk - 2 to each of
the following 3 wk were determined. Average
differences between the control cows and the
PTH-treated cows were calculated from the individual weekly changes. Any difference different from zero (P<.05) was considered a significant effect of PTH treatment.
Journal of Dalry Science Vol. 72, Nø. 5, 1989
1190
KEHRLI, JR., AND GOFF
PTH A D M I N I S T R A T I O N TO P E R I P A R T U R I E N T C O W S
S. sureus Ingestion
Controls
Controls
PTH
PTH
.w--..
3oDifference
2 o D i f f e r e n c e f r o m c o n t r o l s (%)
f r o m c o n t r o l s (%)
B
S. aureus Ingestion
/'4,
0
20
-2O
-40
-60
-1' - 4
5
-3
-2
-1
1
2
-80
3
Difference from controls (%)
-3
4oDifference
b
-2
1
2
3
f r o m c o n t r o l s (%)
e
Cytochrome C Røduction
-1
Antibody-Døt>endønt CelI-Medhltød Cytotoxicit
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20
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-10
-15
-20
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-40
-25
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-2
-1
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2
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T I M E ( w e e k s r e l a t i v e to c a l v i n g )
20 D i f f e r e n c e f r o m c o n t r o l s (%)
c
Nstive Chemiluminescence
10
,pø,o a"
/-
0
s s ,~'ø
-10
-20
-304
-3
-2
-I
I
2
3
T I M E ( w e e k s r e l a t i v e to c a l v i n g )
Figure 1. Effect of intramuscular parathyroid hormone (PTH) administration on neutrophil functions in periparturient
Holstein cows compared with control cows. Data are the difference of the standardized weekly means (n = 5 cows/group)
from assay controls derived from 5 Holstein steers. Time of parturition was assigned a zero time value and the weeks
represent 168 h time periods before or after parturition.
To evaluate immunosuppression in all 10
cows across treatments, the individual cow's
standardized values within each week relative
to the day of calving (d 0) were averaged and
analyzed by fitUng the general linear model: y
Journal of Dalry Science Vol. 72, No. 5, 1989
= mean + week + cow + error. In this model,
data were blocked by week (a 7-d period before
or after the time of calving) and by cow (representing animal differences), and error represented the residual animal variation after fitting
HYPOCALCEMIA AND PERIPARTURIENT IMMUNITY
PTH A D M I N I S T R A T I O N TO P E R I P A R T U R I E N T C O W S
Controls
PTH
80 D i f f e r e n c e from c o n t r o l s (%)
1191
this model. Significant differences between wk
- 2 and each of the 3 successive wk were
judged by F-tests of the week effect. Probabilifles were considered significant if P<.05.
Lymphocyte Blastogenesis to CON A
RESULTS
60
40
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20
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Lyrnphocyte Blastogenesis to PWM
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1
General Observatlons at Parturition
One control cow developed parturient paresis and required medical intervention; another
cow bad difficulty standing but recovered without intravenous calcium borogluconate administration. All controls had retained placentas,
metritis, and were clinically ill. Two cows receiving PTH had twins and both cows developed retained placentas; all other PTH cows
cleaned normally. One cow receiving PTH became clinically weak and febrile alter calving,
was treated symptomatically, and ultimately euthanafized 2 d postpartum. Nø renal damage
nor hepatic damage was evident on postmortem
which might have explained her clinical illness.
Data from this cow prior to her clinical illness
was retained in the data set because the immune cell function results were not disparate
from the other cows.
Two cows (one in each group) bad experimentally induced Staphylococcus aureus
intramammary infections. Both cows developed
clinical mastitis during the first 2 d of lactation.
Neither cow was treated for mastitis and both
cows were clinically normal by 5 d after calving. Throughout the experiment, data obtained
from these cows were not disparate from cows
free of intramammary infections and were
therefore kept in the data set for analysis.
Periparturient Plasma Calcium
and 1,25-Dihydroxyvitamin D Concentrations
-3
-2
1
1
2
3
T I M E ( w e e k s r e l a t i v e to c a l v i n g )
Figure 2. Effect of intramuscular parathyroid hormone
(PTH) administration on lymphocyte blastogenic responses
to mitogens in peripartudent Holstein cows compared with
control cows. Data are the difference of the standardized
weekly means (n = 5 cows/group) from assay controls
derived from five Holstein steers. Time of parturition was
assigned a zero time value and the weeks represent 168 h
time periods before or alter parturition. Con A = Concanavalin A; PHAP = phytohemagglutinin P; PWM = pokeweed
mitogen.
Blood Ca concentrafions are shown by Goff
et al. (9). In brief, in control cows (prone to
milk fever), blood Ca concentrations decreased
within 24 h after parturition to below 7 mg/100
ml, whereas blood Ca of cows receiving exogenous PTH was maintained abøve 9 mg/100 ml.
Blood concentrations of 1,25-(OH)ED rose significantly in both groups of cows with peak
concentrafions occurring approximately 7 d earlier in the cows receiving exogenous PTH.
Journal of Dairy Science Vol. 72, Nø. 5, 1989
1192
KEHRLI, JR., AND GOFF
Periparturient Changøs in
Nøutrophil FuncUon
Changes in PMN in vitro functions, consistent with the concept of immunosuppression in
vivo, were observed in all cows around parturidon. In viu-o test data of PMN from control
cows and PTH-treated cows were virtually
identical or parallel throughout the periparturient period despite significant differences in
plasma Ca concentration (see companion paper)
around parturition (Figure 1).
Data from both treatment groups were then
analyzed as one group to determine significant
changes in PMN function around calving (Table 1). The burst of oxidative metabolism associated with phagocytosis (measured collectively
by cytochrome c reduction, native chemiluminescence, and iodination assays) in stimulated
PMN was significantly impaired during the first
2 wk postpartum. Production of superoxide anion by phagocytically active PMN, as measured
by cytochrome c reduction was 23 percentage
points below the wk -2 standardized value for
all cows. The iodination reaction measures
myeloperoxidase (MPO) catalyzed halogenation of tyrosine residues present on ingested
proteins, in the presence of H202. This reaction
is dependent on adequate production of H202
(which is formed by spontaneous dismutation
of superoxide anion), degranulation of PMN
primary granules that contain MPO, and the
presence of adequate quantities of funcUonal
MPO in phagolysosomes. Iodination by PMN
was significantly impaired for the first 3 wk
postpartum. Compared with the standardized
value of the cows at wk -2, iodination values
were 43, 37, and 20 percentage points less the 3
wk postpartum, respectively. Nauve chemiluminescence, which is a general measure of
oxidative metabolic capacity of PMN, was impaired by 28 and 29 percentage points, respectively, the first 2 wk postpartum when compared with the standardized values for wk -2.
The ability of PMN to perform ADCC was also
impaired each of the ftrst 2 wk alter calving by
21 and 39 percentage points below the standardized values during wk -2.
Chemokinesis and chemotaxis were not affected in this group of 10 cows during the
periparturient period. Neutrophil bacterial ingestion mediated by Fc receptors was enhanced
the week before and after calving by 12 and 10
Journal of Dairy Science Vol. 72, No. 5, 1989
percentage points, respectively, compared with
standardized values dufing wk -2.
It was also evident that some PMN functions
were altered prior to calving (Figure 1). Average iodination and ADCC values had decreased
by wk -2 and -1, respectively, by 14 percentage points below the wk -3 va.lue. Neutrophil
ingestion capacity had also increased 22 percentage points by wk -1 from the wk -3 value.
Contamination of the PMN preparation by
PME affects the results of some PMN assays.
Pearson correlation coefficients were calculated
between all PMN function parameters and the
percent of PME in the PMN preparations. Significant correlation coefficients for PMN contamination were found with PMN ADCC (r =
.33; P<.002), iodination (r = .80; P<.0001), and
PMN directed migration (r = .21; P<.05). Some
of the variation in PMN function can therefore
be explained by PME contamination of the
PMN preparations used in the function assays.
The percentage of PME contamination ranged
from a high of 13% during the 5th wk
prepartum down to 2% during the 2nd wk
postpartum, and back up to 10% by the 4th wk
postpartum. The overall average contamination
of PME in the PMN preparations was 8%.
Pøriparturiønt Chøngøs in Lymphocyte Function
Nø significant differences in Lc blastogenic
responses to mitogens were observed between
PTH-treated cows and control cows during or
alter PTH administration. As shown in Figure
2, data from control cows and PTH-treated
cows were virtually paraUel throughout the
periparturient period in spite of significant differences in plasma Ca concentrations.
Data from both treatment groups were then
analyzed as one group to determine significant
changes in Lc function around calving time.
Significant decreases in mitogen-induced blastogenesis of peripheral blood Lc were detected
during the periparturient period in all cows.
Compared with standardized wk -2 values, Lc
mitogenic responses to Con A and PWM were
reduced by 22 and 25 percentage points, œspectively, the week before calving, and responses to PHAP were reduced by 26 and 41
percentage points the weeks before and after
calving, respectively (Table 1).
HYPOCALCEMIA AND PERIPARTURIENT IMMUNITY
1193
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Journal of Dairy Science Vol. 72, No. 5, 1989
1194
KErmLI. JR., AND GOFF
DISCUSSION
The data suggest that prevention of hypocalcemia by administratjon of PTH does not prevent the development of periparturient immunosuppression. In vitro immune cell function
changes (consistent with immunosuppression
existing in vivo) were evident before calving,
indicating that plasma cortisol and 1,25(OH)2D concentration increases at calving are
not the primary causes of periparturient immunosuppression. This was surprising, especially
since plasma concentrations of both hormones
rose significantly near the time of parturition
(9) and since high concentrations of cortisol in
vitro (20) and in vivo (23, 28) and of 1,25(OH)2D (18, 24, 25) are known to impair PMN
function and Lc blastogenesis, It is possible
that increased cortisol and 1,25(OH)2D concentrations may have contributed to the magnitude
or duration of postpartum immunosuppression.
In other species, 1,25-(OH)2D has a variety
of effects on immune cell function, including
enhanced H202 production by phagocytes (3)
and inhibition of interferon-y (24), DNA (18),
and interleukin-2 (25) synthesis in lymphocytes. Many functional impairments of PMN
and Lc in our study were detected simultaneously, in spite of a 7-d difference in peak
1,25-(OH)2D plasma concentrations between
treated and control cows. Therefore, we think
the primary cause(s) of immune cell function
defects reported here is, most likely, not directly related to hormone fluxes associated with
Ca homeostasis.
The data reported here must be considered in
the following context: the PMN and Lc in our
studies were isolated from normocalcemic and
hypocalcemic cows and evaluated under normocalcemic conditions in vitro. Although the
cells were kept in Ca-free media until each
assay was conducted, there would be adequate
time for extracellular Ca to restore intracellular
Ca concentrations (if intracellular pools were
decreased in vivo) during the assay. These assay conditions should be considered when interpreting the severity of immunosuppression
that existed in vivo.
If returning the cells to a normocalcemic
media influenced the results of these assays,
then it is possible that the cows that became
hypocalcemic were more immunosuppressed
than we were able to demonstrate. Therefore,
Journal of Dairy Science Vol. 72, Nø, 5, 1989
we cannot conclude with certainty that the in
vivo functjons of immune cells from hypocalcemic cows were the same as those from normocalcemic cows. Free Ca released into the
cytosol of acUvated cells is derived from intracellular compartments and can occur in the
absence of extracellular free Ca (13). Extracellular Ca probably serves to replete the intracellular pools of Ca, thus maintaining cell activatjon
(13).
However,
extracellular
Ca
concentrations may not be critical for proliferation of T cells (33) or for PMN random migratjon (6). In stimulated hepatocytes, 180 nM extracellular free Ca was sufficient to maintain
the release of Ca (2). Free Ca concentration in
dairy cows experiencing PH should not fall
below .3 mM; therefore, it is unlikely that the
degree of hypocalcemia in PH dairy cows is
low enough to disrupt cell activation events. If
cells from hypocalcemic cows are different in
vivo, the differences are readily reversed by
normal in vitro extracellular Ca concentrations.
The appearance of significant immunosuppression at least 1 wk prepartum suggests that
physiological changes in late gestation adversely influence immune cell function in dairy
cows. Estrogen increases 1 to 2 wk prepartum,
coincident with our observed development of
immunosuppression (4, 11). Estrogens have
been associated with altered bovine PMN and
Lc function (27, 29). The type of estrogen
present may also be important since plasma
estrone concentrations are higher than 171]-estradiol concentratjons prior to parturition; this
contrasts to mainly 1713-estradiol during estrus.
Total plasma estrogen in cows at the end of
gestation is significantly higher (>100 times)
than during esmis and is elevated along with
progesterone, which is four times greater than
progesterone during the luteal phase of the
estrous cycle (11).
The impaired immune cell functions observed in multiparous dairy cows at parturiUon
agrees with our earlier findings in primiparous
dairy cows (15, 16). The increase in bacterial
ingestion by PMN reported here was also observed in our previous study (15). We think this
increase in PMN ingestion is associated with
defective oxidative killing of ingested bacteria.
Because less energy is being consumed by oxidative microbicidal reactjons, more energy is
available in the PMN to perform ingestion.
HYPOCALCEMIA AND PERIPARTURIENT IMMUNITY
We are unaware of reports in other species
that describe a parmrition-associated immunosuppression that is similar in magnitude and
duration to the findings in this and other studies
of dairy cows and heifers. Giesecke (7) suggested that lactating dairy cows are unique in
their response to stress, because ruminant metabolism is dependent on glycogenesis and glycogenolysis as well as lipogenesis and lipolysis
for energy efficient and glucose-sparing feed
conversion. The lactational ability of dairy
cows, combined with ruminant metabolism,
may be a metabolically demanding phenomenon unique to dalry cows. The ensuing negative
energy and protein balances in early lactation
may limit the immune system of cows.
In conclusion, some of the highest physiological plasma concentrations of estrogens, progesterone, prolactin, and somatotropin occur in
dairy cows during the periparturient period (1,
4). It is unlikely that reduced immune cell
function is the result of a change in concentration of a single entity; more likely, it will be
several entities acting in concert with profound
effects on the function of many organ systems
of the dairy cow. Therefore, it may be very
difficult to discern which hormone(s) might
contribute to suppression of immune ceU function.
The most evident effect of periparturient
stressors on dairy cows may be immunosuppression, thus explaining the high incidence of
clinical mastitis. It is evident that PMN are
critical in the control of infectious bovine mastitis (30). The effect of impalred Lc function on
udder health is not well defined, although bovine Lc are known to activate PMN function
(32). Because hypocalcemic cows are not
detectably immunosuppressed more than normocalcemic cows at parturition, we think the
increased risk of mastitis in parturient paretic
cows is the combined result of impaired immune cell functions, increased exposure of the
teat end to environmental bacteria due to prolonged recumbency of paretic cows, and loss of
teat sphincter muscle tone.
ACKNOWLEDGMENTS
The authors thank Arien Anderson, Travis
Stills, and Kim Driftmier for excellent technical
assistance; Chong Hong for statistical analyses;
1195
Gene Hedberg for illustrafions; and Norm
Tjelmeland, Don Robinson, and Jack Moore for
animal care.
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KEHRLI, JR., AND GOFF
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