Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk production

Veterinary Immunology and Immunopathology 44(1995)251-261

Veterinary immunology and immunopathology

Study of immunological dysfunction in periparturient Holstein cattle selected for high and average milk productiont J.C. Detilleux”, M.E. Kehrli, Jr.b-*, J.R. Stabel”, A.E. Freemad, D.H. Kelley” “Animal Science Department, Iowa State University, Ames, IA 5001 I, USA bUS Department OfAgriculture, Agricultural Research Service, National Animal Disease Center, Metabolic Diseases and Immunology Research Unit, 2300 Dayton Avenue, Ames, IA 50010, USA ‘US Department OfAgriculture, Agricultural Research Service, National Animal Disease Center, LeptospirosisMycobacteriosis Research Unit, Ames, IA 50010, USA Accepted 4 February 1994

Abstract Data from twenty assays of traits associated with innate and adaptive immunity were evaluated from 137 periparturient Holstein cows. These cows had been selected through planned matings for four different levels of milk production (high and average pounds of milk, and high and average pounds of milk fat plus protein). For up to seven generations, the genetic lines were produced by mating females of each line to sires of corresponding merit. With the exceptions of neutrophil ingestion of Staphylococcus aureus and directed migration, all assays measuring neutrophil functions were depressed beginning 2 to 3 weeks before calving through 3 weeks after calving. Serum concentrations of immunoglobulin G, decreased while those of immunoglobulin G2 increased around calving time. Serum complement and conglutinin concentrations decreased before calving and reached a minimum around calving time. Cows selected for high milk production (pounds of milk and pounds of milk fat plus proteins) had significantly higher (PC 0.10) numbers of circulating neutrophils and mononuclear cells, had higher (PC 0.10) neutrophil resting chemiluminescence and higher (PC 0.10) neutrophil directed migration than cows with average production potentials. There were significant (P < 0.00 1) sire progeny group differences for most traits associated with the immune system that we tested. These results can be considered * Corresponding author. ’ Journal Paper No. J- 15360 of th Iowa Agriculture and Home Economics Experiment Station, Ames, IA. Project No. 3 146. All programs and services of the U.S. Department of Agriculture are offered on a nondiscriminatory basis without regard to race, color, national origin, religion, sex, age, marital status, or handicap. 0165-2427/95/$09.50 0 1995 Elsevier Science B.V. All rights reserved SSDIO165-2427(94)05302-9

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encouraging, in that selection for high milk yield did not produce unfavorable correlated responses in the functional capacity of immune function traits, and that there is sufftcient genetic variation in these immunological traits among sires of high genetic merit for milk production to potentially improve the immunocompetence of periparturient cows through planned mating experiments. Abbreviations

ACTH, adrenocorticotrophic hormone; ADNC, antibody-dependent neutrophil cytotoxicity; ConA, concanavalin A, cpm, counts per min; Ig, immunoglobulin; PHAP, phytohemagglutinin P; PBS, phosphate buffered saline; PBMC, peripheral blood mononuclear cells; PDM, predicted difference for milk; PTA-FP, predicted transmitting ability for combined pounds of milk fat plus protein yields; PWM, pokeweed mitogen.

1. Intmduction

Infectious disease in livestock is a significant factor in reducing the efficiency of cheap and quality food production. Annual losses associated with mastitis alone have been estimated at $140 to $300 per cow (Gill et al., 1990). Mastitis and other diseases may result when the immune system is unable to respond effectively to the presence of infectious agents. Increased prevalence of clinical mastitis has been shown in cows whose neutrophils had a low capacity for phagocy-

tosis (Heyneman et al., 1990). Several authors have observed impairment in bovine blood neutrophil and lymphocyte functions at parturition (Newbould, 1976; Guidry et al., 1976; Nagahata et al., 1988; Kehrli and Goff, 1989; Kehrli et al., 1989a,b; Saad et al., 1989; Daniel et al., 1991; Hogan et al., 1992). This immunosuppression may compromise the ability of the cow to combat subclinical infections occurring around parturition and lead to the development of clinical disease. The bovine mammary gland has been reported to be more susceptible to infection and clinical disease during the periparturient period than during the remainder of lactation or during the dry period (Smith et al., 1985). Estimates of genetic correlation between milk yield and mastitis incidence range from -0.30 to +0.66, with most estimates being positive (Emanuelson et al., 1988 ) . This positive genetic correlation indicates that genetic improvement for milk yield is associated with increased mastitis incidence. Because of the role of the innate immune response in protecting cows against infectious agents, one might speculate that intensive breeding programs for increased milk production in cattle may have adversely altered disease defense mechanisms. We have previously reported immunological dysfunction in 39 periparturient dairy cows in our own research herd (Kehrli and Goff, 1989; Kehrli et al., 1989a,b, 1990b, 199 1a; Stabel et al., 199 1) . Our primary purpose was to see whether any immunological dysfunction is observed under more commercially typical management and husbandry conditions in a dairy herd distinct from the herd used for our earlier reports of periparturient immunosuppression. A second objective

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was to determine if genetic differences in immune function could be found between milk production lines or sire progeny groups. Reported here are alterations in functional activities of bovine neutrophib and mononuclear cells, as well as changes in serum protein levels (immunoglobulin, complement, and conglutinin) during the periparturient period. We also present preliminary results of the overall effect of selection for four different levels of milk production and sire progeny groups on innate immunity factors.

2. Materials and methods 2.1. Animals and experimental design Periparturient Holstein cows (n = 137 ) from the I-O-State long-term selection experiment were used in this study. Initially, two selection lines were maintained. For up to seven generations ( 1968-l 986), cows were selected for milk production by using sires, from artificial insemination organizations in the US, selected for highest and average predicted difference for milk (PDM) (Shanks et al., 1978). Starting in 1986, sires were then selected for highest and average predicted transmitting ability for combined pounds milk fat plus protein (PTA-FP ) . Daughters from the high milk line were randomly bred to high and average PTAFP sires, as were daughters from the average milk line. Thus, four lines of cows resulted being present in the current experiment, two from the original high and average PDM lines and two from the recently initiated selection experiments for PTA-FP. Matings have been at random within line. In the experiment reported here, 84 cows were from the PTA-FP study initiated in 1986 and 53 were from the PDM study initiated in 1968. All cows are managed the same and fed for high production (Bertrand et al., 1985 ). The difference between high and average PDM lines is 1500 kg mature equivalent milk per lactation and 16 kg of milk fat plus protein. Cows included in this study were chosen randomly using tables of random numbers. Each sire had, on average, three daughters (range 1- 13 ) included in the study. Blood samples were collected once a week, beginning an expected 5 weeks before the expected calving date until 5 weeks after calving. Leukocytes were isolated from five Holstein steers on the same day that the cows were bled. Leukocytes from these steers were used as laboratory standards to reduce the day-today variability typically seen with immune cell function assays (Kehrli et al., 1989b ). If parturition was delayed in a particular cow and additional prepartum data was collected, only data from the immediate 5 weeks prepartum was used. 2.2. Leukocyte preparation Granulocytes were separated by hypotonic lysis from packed red blood cells (Kehrli et al., 1989b). Remaining cells, usually more than 95% granulocytes (neutrophils and eosinophils ), were resuspended to 5 x 10’ mL- ’ in physiologic

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phosphate buffered saline (PBS). Peripheral blood mononuclear cells (PBMC) were isolated from whole blood buffy coats by density gradient centrifugation and were resuspended to a concentration of 2 x 1O6cells mL- ’ in supplemented culture medium, as previously described (Kehrli and Goff, 1989). 2.3. Hematological variables Total leukocyte counts (cells mL- ’ ) in blood were obtained by electronic counting (CellTrack, Angel Engineering Corp., Trumbull, CT) of blood obtained by jugular venipuncture into tubes containing EDTA. Cytocentrifuge films were stained (StatStain, Volu-Sol Corp., Henderson, NV) and 200 cells or more were differentiated into neutrophils, neutrophil bands, eosinophils, or mononuclear cells, and counted. Relative proportions (percentages) and numbers of cells per microliter of blood were calculated for each cell type. 2.4. Neutrophil functional assays

Procedures for evaluating the following blood neutrophil functions were performed as previously described (Roth and Kaeberle, 198 1b,c; Canning et al., 1986; Kehrli et al., 1989a): random (mm*) and directed (mm) migration under agarose, opsonized zymosan phagocytosis-associated native (nonluminol-dependent) chemiluminescence activity (log,0 photons detected per 74 min assay), cytochrome C reduction (nmoles O2 reduced per 10’ neutrophil per h ), iodination (nmoles NaI per 10’ neutrophil per h ), ingestion (percentage of 60 Staphylococcus aureus ingested per neutrophil), and antibody-dependent neutrophil cytotoxicity ( ADNC; percentage release of 51Cr ). 2.5. Peripheral blood mononuclear cells response to mitogen stimulation

Mitogen-induced and unstimulated blastogenic transformation (log,, counts per min (cpm) ) of PBMC was measured as described (Kehrli et al., 1990a). Culture medium for mitogenic stimulation contained either 5 mg of concanavalin A (ConA, C-20 10; Sigma Chemical Company, St Louis, MO), 5 mg of phytohemagglutinin P (PHAP, L-9 132; Sigma Chemical Company, St Louis, MO) or 5 mg of pokeweed mitogen (PWM, L-9379; Sigma Chemical Company, St Louis, MO) per milliliter. 2.6. In vitro antibody production Pokeweed mitogen-driven production of polyclonal immunoglobulin M by B lymphocytes was determined, as previously described (Stabel et al., 1991), by harvesting 12-day PBMC culture supematants and assaying for IgM concentration (ng mL- ’ ) by ELISA.

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2.7. Serum protein assays Concentrations of immunoglobulin isotypes IgM, IgGI, and IgGz were determined by radial immunodiffusion with commercially available kits (mg dL- ‘; VET-RID, Bethyl Labs, Inc., Montgomery, TX). Serum complement activity (hemolysis area in mm’) was determined by a hemolysis in gel assay, in which hemolysis of guinea pig erythrocytes opsonized with specific bovine antisera was induced by the tested sera (Thurston et al., 1986). Serum conglutinin activity was determined by agglutination of Escherichia coli (Thurston et al., 1989)) and was expressed as a ratio to a pooled serum sample used to standardize all results to a common baseline. 2.8. Statistical analyses For each cow, neutrophil and PBMC assay values were adjusted for laboratory variability by dividing them by the mean value of the live control steers (Kehrli et al., 1989b). These neutrophil ratios were computed for each week a cow was sampled, and expressed in relation to each cow’s calving date. For subsequent statistical analyses, a logarithmic (log,,) transformation was applied to each neutrophil ratio, and a square root transformation was applied to the number of white blood cells to stabilize the variance and to approximate a normal distribution of these data. It is known that eosinophils, which are co-isolated with neutrophils, alter results from neutrophil assays (Roth and Kaeberle, 198 1c). Therefore, the effect of the percentage of eosinophils on the neutrophil assays (ratio values) was estimated by linear regression. For each individual neutrophil assay, ordinary least squares estimation was applied with model [ 1 ] : y=p+/3x+e

(1)

where y is log,, of neutrophil ratio, x is log,, of the percentage of eosinophil contamination in the neutrophil preparation, p is the regression coefficient of y on x, and e is the random error assumed N (0, a: ). Least squares estimate of the regression coefficient (/?) was used to adjust the values (y) obtained for each neutrophil assay to a common level of eosinophil contamination (y-px) and used for subsequent analyses. To study the overall effect of genetic line on our indicators of innate immunity, Eq. (2 ) was applied to the adjusted values of each immune response assay separately. Eq. (2 ) was used to relate immune assay values to genetic lines, adjusted to a common season of calving, parity, and week with respect to calving (as reflected by the variables season, parity, and week, included in Eq. (2 ) ). Yijklmn

=m+l,+s,+rk+W~+a,+tijklmn

(2)

is the adjusted values for one assay on ijum,th cow, m is the overall where Yiiklmn mean, Li is the genetic line (i= 1,2,3,4), sil is the sire effect u= 1,2,...n,; n, is the number of sires per genetic line), ck is the season of calving (k= I,2), yt is the

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time the sample was taken (week with respect to calving date), 7t, is the parity number ( m = I,2 ), and eiiklm,,is the random error assumed N (0, rrz ). Sources of variation in the data were assessed by least-squares by using the General Linear Models procedure on SAS (Statistical Analysis Systems Institute, Inc., 1988). The sires-within-line mean square was used to test for line effects. Genetic lines were defined as high PDM, average PDM, high PTA-FP, and average PTA-FP. In the data set, two summer seasons of calving were used. The summer seasons included cows calving from 1 May 1990 to 1 October 1990, and from 1 May 199 1 until the end of the study in September 199 1. Cows calving outside the summer season were included in the winter season effect. Cows were also classified as first parity cows, and second or greater parity cows. For data presented graphically, the immune assay values were averaged across cows and week relative to parturition, when the samples were collected. There were no interactions in Eq. (2) because errors between repeated measurements were negatively auto correlated and therefore, could cause increased type I errors (Diggle, 1990) when testing the null hypothesis of no interaction effect of week with other main effects. 3. Results 3. I. Periparturient changes in leukograms and immune assays Marked suppression of most of the immune system assays was observed around parturition. The number of circulating neutrophils increased from 5 weeks prepartum until parturition when there was a sharp decline at one week postpartum to values nearer to those at 5 weeks prepartum (Fig. 1). This decline in neutrophi1 numbers was followed by an increase in the number of circulating immature neutrophils (band cells) at 2 weeks postpartum. A second increase and peak in circulating neutrophil numbers was observed during the second and third weeks postpartum. The number of circulating eosinophils had a tendency to decrease from 5 weeks prepartum through 2 weeks postpartum (Fig. 1). Random migration (Fig. 2) by neutrophils increased until two weeks before parturition, and decreased rapidly by the first week after calving. No periparturient changes were observed for neutrophil directed migration. Neutrophil ingestion of bacteria was enhanced at calving time, reaching a plateau 20% higher than initial prepartum levels. Antibody-dependent neutrophil cytotoxicity was also altered around parturition (Fig. 2 ), as values increased gradually from 67% 5 weeks before calving, to a maximum at 72% three weeks before calving, and then decreased again the week following calving. The burst of oxidative metabolism associated with phagocytosis was clearly impaired one week after calving (Fig. 2). For example, compared with values obtained before calving, values during the first week postpartum were decreased by 25%, 596, and 10% for iodination, cytochrome C, and stimulated chemiluminescence assays, respectively. Compared with values at 5 weeks prepartum, the proliferative responses of

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Km

1

Time Relative

257

Neuuophds

to Calving

(weeks)

Fig. 1. Total number of leukocytes per miililter of blood. Data presented are the mean ( +_SEM ) values from 137 Holstein cows repeatedly sampled once a week, 5 weeks prepartum to 5 weeks postpartum.

PBMC stimulated by mitogens did not appear to be reduced around calving time (Fig. 3 ) . The general lymphoproliferative response induced by all three mitogens was maximal 3 weeks before calving, and then decreased gradually to reach a minimum 1 week after calving. One week after calving, PBMC responses to PWM, ConA, and PHAP were 24%, 48%, and 42%, respectively, lower than responses

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110

1

I

104

1

IW 75

Random

Migration

Directed

Migration

“1

Iodination

I

I 1

65’

Andbody-dependent

-5

-4

-3

-2

Neutrophil

~1

I

*

Cyrotoxicity

3

4

Time Relative to Calving (weeks)

5

,

851 .5

-4

-3

-2

-I

Chemihxninescence

I

2

3

4

5

Time Relative to Calving (weeks)

Fig. 2. Neutrophil random migration and directed migration under agarose toward chemotactic fac‘251-labeled Staphylococcusautor. Neutrophil Fc receptor-mediated ingestion of antibody-opsonized reu.s. Antibody-dependent neutrophil-mediated cytotoxicity toward 51Cr-labeled chicken erythrocyte target cells. Neutrophil myeloperoxidase-catalyzed iodination reaction initiated by phagocytosed C3bcoated particles. Neutrophil superoxide anion production initiated by phagocytosed C3b-coated zymosan particles measured by cytochrome C reduction. Neutrophil stimulated chemiluminescence assay using zymosan particles preopsonized with serum C3-derived ligands. Data presented are the mean ( f SEM) values from 137 Holstein cows repeatedly sampled once a week, 5 weeks prepartum to 5 weeks postpartum.

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259

Pokeweed mitogen I

s cl

m:-

Phytohemagglutinm

I

130

1

P

I

In vitro IgM production

.

I

~5

-4

3

-2

Time Relative

1

I

2

to Calving

3

4

5

(weeks)

Fig. 3. Lymphocyte blastogenesis assays: ConA-stimulated lymphocyte blastogenesis, PHAP-stimulated lymphocyte blastogenesis, PWM-stimulated lymphocyte blastogenesis. In vitro IgM production by B cells was determined in vitro by measuring IgM concentrations in culture supematants of mixed mononuclear cell cultures stimulated with pokeweed mitogen. Data presented are the mean ( +_SEM) values from 137 Holstein cows repeatedly sampled once a week, 5 weeks prepartum to 5 weeks postpartum.

observed three weeks before calving. Five weeks after calving, PBMC responses to ConA and PWM were higher than responses observed three weeks before calving. Responses to PHAP returned to initial prepartum levels three to four weeks

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J.C. Detilkux et al. / Veterinary Immunology and Immunopathology 44 (1995) 251-267 lmmunoglobulin GZ

Immunoglobulin G,

Hemolytic Complement

E 2

220~

-5

4

3

-2

-I

/

z

3

I

Time Relative to Calving (weeks)

5

‘5.5

4 -3 -2 ~1 1 2 3 4 5 Time Relative to Calving (weeks)

Fig. 4. Serum immunoglobulin concentration: IgG,, IgG2, IgM isotypes. Conglutinin activity determined by agglutination of Escherichia cob. Serum complement activity measured by hemolysis in gel of guinea pig erythrocytes opsonized with specific bovine antisera. Data presented are the mean ( + SEM) values from 137 Holstein cows repeatedly sampled once a week, 5 weeks prepartum through 5 weeks postpartum.

after calving. In vitro IgM production decreased as calving approached, reached a minimum at calving time ( 18.5% lower than 4 weeks before calving), and increased after parturition (Fig. 3 ) . For serum IgM, IgG, and conglutinin values there was an overall decline as calving time approached (Fig. 4). Compared with levels observed 5 weeks before calving, serum IgG, concentration was 62% lower 1 week before calving and then recovered by 3 weeks after calving to values exceeding the 5 week prepartum value (Fig. 4). Compared with values observed 5 weeks before calving, serum IgM concentration was 7% lower 4 weeks after calving (Fig. 4). Serum conglutinin levels reached a minimum one week after calving and returned to initial prepartum levels in the second week after calving (Fig. 4 ) . Serum IgGz and he-

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26 1

Table 1 Least-squares means for leukograms and neutrophil functions by production lines Immune assays

R2

P value’

Least-squares means by line PDM”

PTA-I? High (n=42)

Average (n=42)

High (n=25)

Average (n=26)

Leukogram (cellspL - ‘) Neutrophils Eosinophils Mononuclear cells

0.21 0.35 0.34

0.05 0.29 0.06

2997 332 4806

2958 264 4761

2668 274 3722

2205 222 3152

Neutrophilassays (% controls) Random Migration Directed Migration

0.16 0.05

0.83 0.05

91 102

94 96

95 99

95 101

Resting chemiluminescence Stimulated chemiluminescence Cytochrome C reduction Iodination

0.24 0.19 0.07 0.20

0.06 0.11 0.65 0.93

68 97 104 62

64 91 104 61

84 101 100 63

78 95 104 62

Ingestion

0.13

0.72

116

116

115

121

ADNCd

0.10

0.18

63

60

73

72

“PTA-FP, predicted transmitting ability for pounds of milk fat plus protein; bPDM, predicted difference for milk, “probability of F test for significance of line effect; dantibody-dependent neutrophil cytotoxicity.

Table 2 Least-squares means for lymphocyte assay and serum protein levels by production lines Immune assays

R2

Pvalue’

Least-squares means by line PTA-FP”

PDMb

High (n=42)

Average (n=42)

High (n=25)

Average (n=26)

Lymphocyte assays Concanavalin A (% controls) Phytohemagglutinin P (96 controls) Pokeweed mitogen (% controls) In vitro IgM (ng dL_’ )

0.18 0.20 0.18 0.18

0.27 0.75 0.42 0.52

105 78 98 99

133 81 128 111

73 49 73 75

70 49 85 73

Protein assays IgG, (mgdL-i) IgGz (mg dL- ’ ) IgM (mg dL- ’ ) Complement (mm of hemolysis) Conglutinin (reciprocal titer)

0.56 0.37 0.39 0.28 0.14

0.23 0.23 0.20 0.42 0.27

411 1597 238 8.6 7.5

458 1335 282 8.6 7.2

459 1607 233 8.3 6.8

548 1584 209 8.1 6.9

“PTA-FP, predicted transmitting ability for pounds of milk fat plus protein; ‘PDM, predicted difference for milk, ‘Pvalue for significance of line effect.

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molytic complement levels were not altered much before calving but showed increases postpartum. Complement levels were 6% higher 2 weeks postpartum and IgGz concentrations were 20% higher at 3 weeks postpartum than observed values at 5 weeks prepartum. 3.2. Eflects of genetic lines on immune response indicator values Statistically significant effects of milk production lines (P-C 0.10) were found for resting chemiluminescence, directed migration, number of circulating neutrophils and number of circulating mononuclear cells (Table 1). Directed migration was highest (Pc.05) in cows selected for high PTA-FP. Although not always statistically significant, cows selected for high PTA-FP or high PDM had higher numbers of circulating leukocytes, higher values for four neutrophil assays (native resting and stimulated chemiluminescence, iodination, and ADNC), lower or equal values for S. aureus ingestion (Table 1)) lower serum IgG, concentration, and lower PBMC response to PHAP and PWM (Table 2) than cows with average PTA-FP or PDM, respectively. With the only exception of neutrophil directed migration (P> 0. lo), significant differences (P-C 0.00 1) in sire progeny groups were detected for all assays shown in Tables 1 and 2.

4. Discussion 4.1. Periparturient changes in leukograms and immune assays Impaired immune cell function at parturition has been shown in Holsteins (Kashiwazaki, 1984; Kashiwazaki et al., 1985; Ishikawa, 1987; Nagahata et al., 1988; Kehrli and Goff, 1989; Kehrli et al., 1989a,b; Hogan et al., 1992; Gilbert et al., 1993). It is probable that periparturient hormonal changes contribute to impaired immune function. Indeed, among other hormones, serum levels of progesterone, estrogen and cortisol change dramatically at calving (Wetteman, 1980). Increased concentrations of progesterone have been associated with depressed iodination, enhanced ADNC, and increased neutrophil random migration (Roth et al., 1982a). Also, administration of adrenocorticotrophic hormone ( ACTH) to yearling steers has been shown to cause neutrophilia (with a left shift), eosinopenia, a decrease in lymphocyte blastogenic responses to mitogens, a decrease in neutrophil iodination, and an increase in the ability of neutrophils to migrate under agarose (Roth et al., 1982b). The synthetic glucocorticoid, dexamethasone, inhibits ingestion of S. aureus, nitroblue tetrazolium reduction, iodination, chemiluminescence, and ADNC by bovine neutrophils (Roth and Kaeberle, 1981a). As shown in previous studies (Nagahata et al., 1988; Kehrli et al., 1989b; Saad et al., 1989; Gilbert et al., 1993), all cows experienced a transient leukopenia after calving (Fig. 1). It is thought that the extensive influx of neutrophils into

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the reproductive tract is the major cause of the transient neutropenia after calving (Gunnink, 1984). As seen in previous studies (Kehrli and Goff, 1989; Kehrli et al., 1989a,b, 199 la), around calving time, the oxidative burst of metabolism by phagocyticalIy active neutrophils was reduced but the capacity of neutrophils to ingest antibody-opsonized bacteria was enhanced (Fig. 2). A possible interpretation for this inverse relationship, is that neutrophils possess a finite amount of energy, therefore, when less energy is consumed by oxidative reactions associated with phagocytosis, more energy is available to support the ingestion step of phagocytosis. An increased proportion of immature band neutrophils in the neutrophil isolates (Fig. 1) may have also contributed to this inverse relationship. Band cells have previously been shown to contribute little to oxidative burst activity but they have been shown to phagocytose E. coli (Silva et al., 1989). It was shown in previous studies, (Kashiwazaki et al., 1985; Ishikawa, 1987; Kehrli and Goff, 1989; Kehrli et al., 1989a; Saad et al., 1989) that proliferative responses of PBMC to mitogens are impaired at calving time. Our data on the proliferative responses of PBMC to mitogens around parturition (Fig. 3 ) are not entirely consistent with previous reports, it is possible that technical difficulties in our laboratory with this assay contributed to this difference. We believe the previous reports more accurately reflect the ability of lymphocytes to respond to stimuli since a lymphocyte assay (in vitro production of IgM) performed independently in our lab yielded data consistent with previous reports of impaired lymphocyte function in periparturient cows (Kashiwazaki et al., 1985; Ishikawa, 1987; Kehrli and Goff, 1989; Kehrli et al., 1989a; Saad et al., 1989; Stabel et al., 199 1). Periparturient impairment of lymphocyte activities has been suggested to be due to shifts in the ratio between lymphocyte subpopulations (helper versus suppresser/cytotoxic cells) (Saad et al., 1989 ) resulting in suppressed uptake of radiolabel in the blastogenesis assay. Direct measurement of CD4 and CD8 positive-staining cells in our previous studies did not support this hypothesis of shifts in subpopulations of lymphocytes however (Harp et al., 199 1) . The presence of inhibitory factors or suppressive cells in the assays, which may reduce cell metabolism or induce lymphotoxicity (Birkeland and Kristoffersen, 1980), has also been suggested, but cannot be proven or disproven with the current experiment. Consistent with our earlier report (Kehrli et al., 1990b), and contrary to a previous report (Ishikawa, 1987), we did not observe a decline in serum IgG2 concentration (Fig. 4). However, it is possible that IgG, concentration was already at a minimum when we started to sample our cows. The observed decline in serum IgG, could have been a result of compartmentalization of IgG, into lacteal secretions during colostrogenesis and/or due to impairment of plasma cell production of IgG, (Ishikawa, 1987). Impaired lymphocyte production of IgM was demonstrated in this study (Fig. 3 ) and in others (Stabel et al., 199 1). 4.2. Eflects of genetic lines on immune assays Our preliminary results (Tables 1 and 2 ) showed that high milk producers had high values for many immune assays. The findings that high PTA-FP and high

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PDM cows had a higher number of neutrophils and higher neutrophil ability to perform the metabolic burst is important because neutrophils provide first-line defenses that can be rapidly mobilized and activated against infectious or toxic agents (Van der Valk and Herman, 1987). The highly significant sire effects for all the immune assays confirmed that general immune functions have additive genetic variation (Kehrli et al., 1991b). Indeed, in dairy cattle, significant genetic influence has been found for the following innate immunologic traits: neutrophil phagocytosis and lymphocyte responses to mitogens (Kehrli et al., 199 1; Weigel et al., 1991b), serum immunoglobulin levels (Burton et al., 1989), and serum hemolytic complement levels (Lie et al., 1983 ). A previous study of the same herd reported no differences between immunological parameters tested during mid-lactation on daughters of high versus average PDM sires (Kehrli et al., 199 1b). It may be possible that the differences which exist between lines are greatest only among periparturient cows, when stress and disease incidence are greatest. Many of the bulls that produced progeny with either high or average milk production in this herd, had common sires in their pedigrees (Shanks et al., 1978). Although these common ancestors would reduce genetic differences between lines, we were still able to detect many sire progeny group differences in the immunological parameters tested. In theory, the phenotypic association between milk production level and immune response may be environmental or genetic in origin. Management and nutritional effects should not be considered here because all lines were managed and fed the same. Previously reported higher incidence of mastitis and other diseases in high versus average PDM lines (Bertrand et al., 1985), might be due to the observed association between differing immune function levels and genetic lines. The association between immune response assays and genetic lines may also be the result of linkage disequilibria between genes influencing the production traits and genes of the immune system. In this case, selection for improved immune function could be achieved without detriment to milk production. On the other hand, milk yield seems positively correlated with mastitis incidence (Emanuelson et al., 1988). Many factors undoubtedly contribute to this including a longer milking time, larger udders that tend to be closer to the ground and hence have more exposure to bacterial pathogens. It may even be that high producers have more competent defense mechanisms than low producers and they are merely overwhelmed by a greater pathogen challenge level due to the increased proximity to contaminated bedding and prolonged exposure time during milking. Another point here is that although higher producers have more mastitis, they are still more profitable to own for milk production (Bertrand et al., 1985). Clearly, additional work is needed to determine the genetic influence of the innate immune response upon mastitis resistance. Our approach is to use breeding (animal model) and statistical tools (non-linear, repeated measurement data analysis) to determine whether genetic selection for less severe periparturient immunosuppression also results in selection for better resistance to mastitis (Detilleux, 1993 ). In conclusion, we have demonstrated altered immunologic functions in 137

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periparturient dairy cows, which confirms that innate immunity functions are altered in periparturient cows. This period coincides with increased incidence of subclinical and clinical infectious disease (Smith et al., 1985 ) . The unique aspect of this current study was that the cows were at a separate geographic location and under completely different housing conditions (free-stalls), fed a different ration (corn silage, haylage, and grain concentrate as a total mixed ration) and handled by a different herdsman and milkers different from those in our previous studies on periparturient immunosuppression (loose housing, grain concentrate mix with long-stem hay) (Kehrli and Goff, 1989; Kehrli et al., 1989a,b, 199 1a). Our previous studies also involved a milking schedule with 7 h and 17 h intervals. A more conventional, 12 h milking interval was followed in the current study. Clearly, as now evidenced by our series of studies, periparturient immunosuppression exists in dairy cattle under quite divergent management systems. So far, attempts to prevent immunosuppression by preventing hypocalcemia (Kehrli and Goff, 1989; Kehrli et al., 199Ob), by administrating cytokines (Kehrli et al., 199 1a; Stabel et al., 199 1) or by nutritional manipulation (Daniel et al., 1991; Hogan et al., 1992, 1993) have had limited success. In this study, we have shown genetic differences among sire progeny groups in the immune assays, therefore, a genetic approach to enhanced immunocompetence and disease resistance seems feasible and justified. If sufficient genetic variation and reasonable heritability estimates can be demonstrated for the immunocompetence of periparturient cows, genetic selection for dairy cows who experience less severe periparturient immunosuppression would appear to be both achievable and desirable in light of the research published over the past few years. 5. Acknowledgments

Appreciation is expressed to the Leopold Center for Sustainable Agriculture at Iowa State University, to Eastern Artificial Insemination Coop. Inc, and to 2 1st Century Genetics Coop., Inc. for partial support of this research. The authors thank Dr. Fletcher, Dr. Koehler, and Dr. Rothschild for their critical review and valuable discussions on this research. The authors thank A. Anderson, S. Bemick, B. Broekmeier, W. Buffington, K. Driftmier, L. Engelken, S. Howard, D. Valenciano, and R. Welper for excellent technical assistance. References Bertrand, J.A., Berger, P.J., Freeman, A.E. and Kelley, D.H., 1985. Profitability in daughters of high versus average Holstein sires selected for milk yield of daughters. J. Dairy Sci., 68: 2287-2294. Birkeland, S.A. and Kristoffersen, K., 1980. Lymphocyte transformation with mitogens and antigens during normal human pregnancy: a longitudinal study. &and. J. Immunol., 11: 321-325. Burton, J.L., Kennedy, B.W., Bumside, E.B., Wilkie, B.N. and Burton, J.H., 1989. Variation in serum concentrations of immunoglobulins G, A, and M in Canadian Holstein-Friesian calves. J. Dairy Sci., 72: 135-149.

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