Phagocytic Activity of Bovine Leukocytes During Pregnancy F. H. S. Newbould*
ABSTRACT
RESUMA
The phagocytic competence, measured as the total number of polymorphonuclear leukocytes per mm3 which phagocytosed Staphylococcus aureus, strain 321, in vitro, was determined in eight cows during complete pregnancies. Such leukocytes are referred to as "Active PMNs". There was a gradual decline in the number of these cells from conception to a minimum between the 16th and 20th weeks of pregnancy, followed by a steady increase to the cessation of lactation when a marked drop occurred, after which there was an increase to a maximum during the second week prepartum. From this maximum there was a rapid decrease to an absolute minimum during the first week after parturition. From the second week postpartum there was a gradual increase to conception. The correlation coefficient (r) of numbers of active PMNs with time before conception was -0.474 (p=0.01). There were significant differences (p=0.01) in numbers of active PMNs among the eight cows. It was found that the cows fell into two groups, one whose members had, overall, significantly more active PMNs (p=0.001) than those in the second group. The between cow differences may have been due to 1) age, since the cows with the highest numbers of circulating active PMNs were younger than those in the other group or 2) the combined stress of pregnancy and lactation, as those cows which were both pregnant and milking had the lowest numbers of active PMNs.
Cette experience visait 'a determiner la capacite phagocytaire des neutrophiles de huit vaches, tout au long de leur gestation. L'auteur adopta comme standard le nombre de neutrophiles/mm3 qui phagocytierent, in vitro, la souche 321 de Staphylococcus aureus et donna 'a ces leucocytes le nom de neutrophiles actifs. II decela une diminution graduelle de ces cellules, de la conception jusqu'a un minimum de 16 'a 20 semaines de gestation; elles augmentierent ensuite progressivement jusqu'au tarissement, alors qu'elles subirent une baisse drastique. Une autre augmentation survint par la suite et elle atteignit un sommet au cours de la deuxieme semaine avant la parturition. II se produisit ensuite une diminution rapide, jusqu'a un minimum absolu au cours de la semaine qui suivit le v&lage. A compter de la deuxieme semaine apres la parturition, se dessina une augmentation progressive qui persista jusqu'a la conception. Le coefficient de correlation (r) entre le nombre de neutrophiles actifs et la periode precedant la conception s'etablit 'a -0.474 (p=0.01). Les huit vaches presenterent des differences appreciables (p=0.01) dans le nombre de leurs neutrophiles actifs. L'auteur realisa qu'elles formaient deux groupes distincts; celles du premier poss6daient un nombre sensiblement plus eleve de ces cellules (p=0.001) que celles du second. Les differences enregistrees d'une vache it l'autre resultaient probablement 1) de l'age, puisque celles dont le sang recelait le plus de neutrophiles actifs etaient plus jeunes que les autres, ou 2) de l'agression combinee de la gestation et de la lactation, puisque les vaches 'a la fois gestantes et en lactation possedaient le nombre le moins eleve de neutro-
of Veterinary Microbiology and Immuno*Department logy, University of Guelph, Guelph, Ontario.
Submitted March 19, 1975.
Volume 40
-
January, 1976
philes actifs.
ill
INTRODUCTION In a series of papers from this laboratory (3, 7, 10, 11, 12) we have reported results of investigations into the efficacy in vitro of bovine leukocytes, both from blood and from milk. We have found that among the cells collected from the secretion from artificially irritated mammary glands there was a smaller proportion of efficient phagocytes than among those from blood, due at least partly to the lack of an energy source in the gland secretion. Although at a lower level, the proportion of phagocytosing PMNs in milk reflected that in blood of the same cow, i.e. where one was higher or lower than average, so was the other. Since, over short periods, among cows, significant differences in proportion of phagocytosing PMNs were found, it seemed advisable to monitor the activity of leukocytes from a group of cows over a long period which necessitated using blood leukocytes rather than those from milk. It has been reported (4, 5, 6) that the lenkocytes in pregnant women showed enhanced phagocytic and bactericidal activity. Since lactating cows are pregnant during most of their adult life, we have examined the phagocytic activity against Staphylococcus aureus of the blood leukocytes of eight cows throughout complete pregnancies and in some cases during early lactation.
tended to one and one half hours. Staph.
aureus, O.V.C. strain 321 (10) was used throughout. At the beginning of the work, nine cows were assigned to this investigation, of which two were unbred heifers, two were bred heifers within two to three weeks of calving, one was in her third lactation, one in her fourth and one in her fifth. The cow in third lactation was removed later because of failure to conceive. Thus data were collected from eight cows from some time prior to conception to and until shortly after parturition. All cows were free of overt disease. As far as possible the cells of each cow were tested every three weeks, although occasionally this schedule could not be maintained. During the last three to four weeks before the estimated calving date, cows were bled and observations made at weekly intervals and in a few cases even more frequently. At each bleeding the total white blood cell (WBC) count was estimated, the percentage of polymorphonuclear neutrophils (PMNs) determined and the percentage of the PMNs which phagocytosed staphylococci (effective PMNs) calculated from the examination of at least 200 cells in smears stained by Leishman's Stain. For convenience in analyzing and presenting the data the observations were grouped into four-week periods from conception to calving. All observations obtained within each period were pooled and means determined. Where needed, between period data was subjected to an analysis of variance (14), between animal comparisons compared by determining the significance between two means (1) and correlation coefficients were determined (14).
MATERIALS AND METHODS Methods used were essentially as described previously (3) with the following modifications. Blood was collected from the jugular vein instead of the tail vein and heparinized at the rate of 10 U.S.P. units/ml. The buffy coat was resuspended in fresh autologous plasma. A sufficient volume of bacterial culture, the number of bacteria in which was estimated by a Coleman nephelocolorimeter, was added directly to the leukocyte suspension. The incubation time to allow ingestion of the bacteria was ex-
112
RESULTS The period means for each parameter are shown in Fig. 1. Examination of the three curves shows that there was a good deal of variation in each during pregnancy. There was a steady decline in both the total numbers of WBC and the percentage of PMNs up to the period 16 to 20 weeks after conception. The percent of PMNs phagocytosing increased up to ten to 12
Can. J. camp. Med.
I
\
P.MNs 75.
phagocyto8a&g
30/ 20 wsc
12F
n 103/mm3
101_
_ _
_
_
8
_ _
_
_
16 Weeks
_
jl
__ _
_
_
_
24
_
32
_
_
40
Preg nant
Fig. 1. Mean total WBC counts per mm3, % PM!Ns and % of PMNs phagocytosing Staph. aureus. Each point plotted represents the mean of all values obtained in a four week period from six to eight pregnant cows.
weeks, then declined to a low level also at 16 to 20 weeks after conception. The number of circulating phagocytes capable of phagocytosing was determined by multiplying together the three sets of data from Fig. 1 and the total thus obtained termed "Active PMNs". For the cows studied, the figures thus arrived at are shown in Table I. It can be seen that there was a gradual decline in the number of these cells from conception to a minimum during the fifth period, i.e. between the 16th and 20th week, followed by an increase to the eighth period, a drop during the ninth during which the cows were dried off and reaching a peak during the last period, although an analysis of variance showed no overall significance between periods in numbers of active PMNs. However, the mean difference between the fifth period which was the lowest and the tenth period which was the highest is significant (p = 0.01), although when no overall significant difference exists such significances should be regarded with some caution. In Table II are shown the mean weekly activities for six cows for which sufficient figures were available during the period three weeks before parturition to two weeks after. These data indicate that the number of active PMNs reached a maximum during the second week before parturition, followed by a decline to a minimum during the first week postpartum, after which there was a return towards former numbers. An analysis of variance showed the between period differences to be significant at the five per cent level. Also, all
Volume 40
-
January, 1976
three parameters individually showed a significant decline at or immediately after calving. Since, as shown in Table I, the numbers of circulating active PMNs were high immediately following conception and, as seen in Table II, were lowest immediately after parturition, an attempt was made to anal-
yze the trend in active numbers between these two events. There were insufficient figures available for any cow or group of cows as there was considerable variation in the length of time before breeding at which individual cows were introduced to the investigation. Thirty individual figures, which were obtained during 13 weeks preconception, were plotted and are shown in Fig. 2, together with the calculated regression line of activity on time. The correlation coefficient (r) of activity with time before conception was -0.474 which is significant at p=0.01. TABLE I. Mean Number of Active Circulating PMNs in Blood of Cows in Ten 28-day Periods
Following Conception
Period 1...........
2...........
3....... 4.......
5........... 6........... 7........... 8........... 9......
10......
No. of Cows 6 7 7 7 8 8 8 7 7 7
Active PMNs i S.D./mm3 (x 103) 2.31 :4: 1.29 2.11 1.02
2,09 1 0.99 1.83 : 0.86 4
1.56 1.89 1.91 2.44 1.93 2.51
0.63 + 0.95
:1: 1 + :1
0.55 1.37 0.75 0.52
5
E E 0 x
3
z
0
S
-
--v
2-
's 1
12
10 8 6 4 Weeks before conception
2
Fig. 2. The numbers of circulating "Active PMNs" per mm3 in cows at various times between parturition and conception. Correlation coefficient (r) = -0.474, p = 0.01, y = -0.llx + 2.79.
113
14 E E 0
C o
6
6
~ ~ L a c t at i o n
4 ~~2
6
Ag e
Figr. 3. The relationship betwreen lactation age and mean leetation WBC counst in eight cowrs. Correlation coefficient (r) = -0.761, p = >0.05, y = -1.23 + 14.6.
TABLE II. Mean Nunmber of Active CAirculating PMNs in Blood of Six Cows Immediately Before and After Parturition Active PMNs ± S.D./mm3 Days from Parturition (X 103) - 14 to - 18 ............... 2.23 :1: 0.53 - 7 to-13 ............... 2.82 4 1.28 - 6 to 2.07 i 0.90 0............... 1.27 i0.53 + lto+ 7 ............... ......... + 8to + 15 . 1.73 0.65 Differences of 0.96 x 103/mm3 or more are significant at p = 0.05
Analysis of the data summarized in Fig. 1 and Table I showed that there were highly significant difference (p=0.&1) in numbers of circulating active PMNs between the eight individual cows. The mean number for each cow throughout pregnancy is given in Table III. From these figures it is apparent that the cows fell into two distinct groups, group 1 composed of cows 1, 3 and 4, while the other five constitute group 2. The means for the two groups are shown in Table IV and the difference between these is highly significant (p =0.001). This finding led to a critical examination of the individual cow records to see if any obvious differences between the two groups could be found. Of the cows in Group 1 it was determined that two were in their first pregnancy, i.e. had never lactated, while the third was in her second pregnancy, but, because of having been bred unusually late in her first lactation, was dry for seven months of the nine. In group 2, all cows of which lactated for seven of the nine months
14
of pregnancy, two were in second and one in each of third, fourth and fifth lactation. Thus, with the minor exception of twomonths of one pregnancy the cows in group, 1 were dry during their pregnancies while those in group 2 were lactating during the first seven months. Since the figures for active PMNs were calculated from three separate measurements, these were reexamined to see which of the three or combination of any two might have produced the between group, difference. Originally, phagocytosing PMNs were expressed as a percentage of those examined and PMNs in the blood as a percentage of the WBC count. These multiplied together produced a figure representing the percentage of WBC which were effective phagocytes and when this resulting figure was applied to the total WBC count the number of circulating active PMNs per mm3 was found. From the data in Fig. 1 it has already been suggested that a balance was maintained between percentage of PMNs phagocytosing and percentage of PMNs among the WBC. The mean percent of all WBC which were effective phagocytes was calculated for each of the two groups of cows and these figures compared. Likewise the mean total WBC/mm3 for the two groups were determined and compared. These figures are shown in Table V. From this it is apparent that the difference between the groups resulted exclusively from difference in the total WBC count. The relationship of total WBC count to lactation age is shown in Fig. 3 and the regression line, which was calculated from the points, shown. This shows that there was a drop in total WBC count with increasing lactation age.
DISCUSSION The reservoir of phagocytes potentially available to attack an invader of the body is formed largely by the PMNs present in the circulating blood. Thus at any one time the total number of circulating cells capable of phagocytosis may well represent the primary ability of a host to combat a developing infection and variations in that number may affect the host's resistance to disease.
Can. J. comp. Med.
In this paper, data are presented to show that during pregnancy, cows' phagocytes (PMNs) showed marked variation in the three parameters studied. It has been shown previously that there were significant differences between cows in the percentage of effective phagocytes circulating in the blood over short periods (12), but no long term data have been published, nor have there been reports concerning the total numbers of effective phagocytes in bovine circulating blood. In view of the findings (4, 5, 6) that there are increases in both phagocytic and bactericidal activities of PMNs in women during pregnancy, the data presented here were compiled and evaluated throughout complete pregnancies, i.e. from the day of conception, accepted as the day of final breeding, to the day of parturition and in some cases for a limited time before breeding and immediately after TABLE III. Mean Number of Active Circulating PMNs in Blood of Eight Cows During Pregnancy
Cow 1................ 2................ 3................ 4................ 5................ 6................ 7................ 8................
No. of
Trials 10 7 8 9 10 9 10 9
Active PMNs 4
S.D./mm3
(x 103)
3.12 i 1.03 1.61 + 0.71 2.53 ± 0.63 2.69± 0.77 1.47 ± 0.53 1.78± 0.50 1.59 ± 0.41 1.48 4 0.79
TABLE IV. Mean Number of Active Circulating PMNs in Blood of Two Groups of Cows During Pregnancy Active PMNs ± S.D./mm3
Group
1.~~ 2
.
-
(X 103) 2.80 ± 0.85 1.58 i 0.58
t = 6.6
p = > 0.001''
parturition. It is hard to assess the significance of the changes found in the individual parameters during pregnancy. The most notable feature was that all three reached minimum levels during the period 16 to 20 weeks after conception. The total WBC count and the percentage of PMNs showed an almost steady decline during that interval. The phagocytosing PMNs at first increased rapidly up to eight to 12 weeks and then dropped precipitously to a minimum at 16 to 20 weeks. The reasons for these changes are obscure. There is some indication of a balance being maintained between the percentage of phagocytosing PMNs and the percentage of PMNs in the circulating WBC, since these two parameters, to some extent, varied inversely. The values for the three parameters were combined to develop a single figure for the total number of circulating PMNs capable of phagocytosis, a value which has been called "Active PMNs", and the minimum again occurred during the period of 16 to 2& weeks after conception. Whether this is a time of extreme stress in pregnant cows is not known. A peak was reached at between 28 and 32 weeks, just before drying off which was followed by a noticeable drop. Active PMNs then increased to a maximum during the second week before parturition, followed by a decrease to an absolute minimum in the first week post partum. This pattern is different from that found in women, in whom both leukocyte numbers and phagocyte activity were at a consistently high level during the three trimesters of pregnancy (5). The data presented agree in regard to numbers of WBC and percentage of PMNs with those previously published (16) showing that both these parameters peaked on the day of parturition and then dropped off rapidly thereafter. From shortly after parturition it appeared that the circulating active PMNs increased up to the time of conception (Fig. 2). It is pos-
TABLE V. Mean WBC Counts and Percent of Total WBC which were Effective PMNs, for Two Groups of Cows During 280 Days of Pregnancy
Effective PMNS as % of WBC Total WBC x 103/Mm3.......... -Difference not significant at p = 0.05 bDifference significant at p = > 0.001
Volume 40 - January, 1976
Five Milking Three Dry Cows Cows Means ± S.D. 17.23 ± 5.24 18.97 ± 5.22 9.13 ± 1.86 14.71 ± 2.01
S. E. of Difference 1.27&
0.47b
115
sible that this increase is related to the presence of high estrogen concentrations during this time, as estrogens have been shown to be strong stimulants of the reticuloendothelial system and increased phagocytic activity (2, 8, 9). The reasons for the significant differences between cows and the two groups of cows are not clear, although the data do suggest several hypotheses. A balance appeared to have been maintained in the proportion of effective PMNs among the WBC, since pregnancy means for this combined parameter were essentially the same for all cows. Thus the primary difference was in the total WBC count both between cows and except at the very end of pregnancy possibly between periods. While the number of cows used in these experiments was small, the correlation coefficient (r) between mean total WBC count and age was -0.761 (p=0.05), which is in agreement with earlier observations that with advancing age both the total WBC count and the absolute number of neutrophils decrease (15). Thus the differences found may have been due to age since the one group of cows was much younger than the other. On the other hand the cows in group 1 which were pregnant but nonlactating had a significantly higher total count of WBC than those in group 2 which were not only pregnant but also were lactating for seven of the nine months. This suggests two other possible explanations. Either the combined stresses of pregnancy and lactation result in a lower total production of WBC including effective PMNs by the hematopoietic system, or in lactating cows there is a steady drain of leukocytes into the milk even in uninfected glands which the hemopoietic system is unable to replace. That the latter may have significance is indicated by the fact that the total WBC count is at a low level at the time when milk yield is at its height. There are two times at which the cow is considered to be more than normally susceptible to infection of the mammary gland. These are during the first two to three weeks of the dry period and at and immediately following parturition (13). Whether the fact that these are times when the active PMN population was found to be at a low point is related to the incidence of mastitis is open to speculation as indeed is its relation to the incidence of any other infection in the cow. That the phagocytic and bactericidal activity of PMNs is
116
related to the incidence of disease in women has been shown, since in those suffering from a bacteriuria, the result of gram negative organisms, there was no increase. in PMN activity during pregnancy. The lack of activity was most marked against the particular organism causing the disease (4, 5).
ACKNOWLEDGMENTS Financial support was received from the National Research Council of Canada, the Ontario Milk Marketing Board and the Ontario Ministry of Agriculture and Food. The technical help of R. Johnston and Kathy Schumley is gratefully acknowledged. REFERENCES 1. ARKIN, H. and R. R. COLTON. Statistical Methods. 4th Ed. revised. p. 121. New York: Barnes and Noble Inc. 1968. 2. HARTVEIT, F. and K. ANDERSEN. Reticuloendothelial activity related to age and sex in mice. Acta. path microbiol. scand. 76: 161-163. 1969. 3. KENT, G. M. and F. H. S. (NEWBOULD. Phagocytosis and related phenomena in polymorphonuclear leukocytes from cow's milk. Can. J. comp. Med. 33: 214-219. 1969. 4. MITCHELL, G. W., Jr. and A. J. SBARRA. The role of the phagocyte in host-parasite interaction II. The phagocytic capabilities of leukocytes in pregnant women. Am. J. Obstet. Gynec. 91: 755-762. 1965. 5. MITCHELL, G. W., Jr., R. J. McRIPLEY, R. J. SELVARAJ and A. J. SBARRA. The role of the phagocyte in host-parasite interactions IV. The phagocytic activity of leukocytes in pregnancy and its relationship to urinary tract infections. Am. J. Obstet. Gynec. 96: 687.697. 1966. 6. MITCHE'LL, G. W., A. A. JACOBS, V. HADDAD, B. B. PAUL, R. R. STRAUSS and A. J. SBARRA. The role of the phagocyte in host-parasite interactions XXV. Metabolic and bactericidal activities of leukocytes from pregnant women. Am. J. Obstet Gynec. 108: 805-813. 1970. 7. NAIDU, T. G. and F. H. S. NEWBOULD. GlYcogen in leukocytes from bovine blood and milk. Can. J. comp. Med. 37: 47-55. 1973. 8. NICOL, T.. D. L. J. BILBEY, L. M. N. CHARLES, J. L. CORDINGLEY and B. VERNON-ROBERTS. Oestrogen: The natural stimulant of body defence. J. Endocr. 30: 277-291. 1964. 9. NICOL, T., B. VERNON-ROBERTS and D. QUANTOCK. The influence of various hormones on the reticulo-endothelial system: endocrine control or body defence. J. Endocr. 33: 365-383. 1965. 10. NEWBOULD, F. H. S. Some effects of the source of bovine milk leukocytes and strain of Staphylococcus aureus on their interaction in vitro. Can. J. comp. Med. 31: 303-308. 1967. 11. NEWBOULD, F. H. S. Enhancement of phagocytosis in bovine milk leukocytes in vitro. Can. J. comp. Med. 34: 261-264. 1970. 12. NEWBOULD, F. H. S. The effect of added serum and glucose, and some inherent factors, on phagocytosis in vitro by milk leukocytes from several cows. Can. J. comp. Med. 37: 189-194. 1973. 13. NEWBOULD, F. H. S. Microbial diseases of the mammary gland. In Lactation, a Comprehensive Treatise. Vol. II. B. L. Larson and V. R. Smith, Eds. p. 277. New York and London: Academic Press, Inc. 1974. 14. QUENOUILLE, M. H. Rapid Statistical Calculations. pp. 16, 23. London: Griffin. 1959. 15. SCHALM, 0. W. Veterinary Hematology. 2nd Ed. p. 231. Philadelphia: Lea and Febiger. 1965. 16. STRAUB, 0. C.. 0. W. SCHALM, J. P. HUGHES and G. H. THEILEN. Bovine hematology TI. Effect of parturition and retention of fetal membranes on blood morphology. J. Am. vet. med. Ass. 135: 618622. 1959.
Can. J. comp. Med.
ABSTRACT
RESUMA
The phagocytic competence, measured as the total number of polymorphonuclear leukocytes per mm3 which phagocytosed Staphylococcus aureus, strain 321, in vitro, was determined in eight cows during complete pregnancies. Such leukocytes are referred to as "Active PMNs". There was a gradual decline in the number of these cells from conception to a minimum between the 16th and 20th weeks of pregnancy, followed by a steady increase to the cessation of lactation when a marked drop occurred, after which there was an increase to a maximum during the second week prepartum. From this maximum there was a rapid decrease to an absolute minimum during the first week after parturition. From the second week postpartum there was a gradual increase to conception. The correlation coefficient (r) of numbers of active PMNs with time before conception was -0.474 (p=0.01). There were significant differences (p=0.01) in numbers of active PMNs among the eight cows. It was found that the cows fell into two groups, one whose members had, overall, significantly more active PMNs (p=0.001) than those in the second group. The between cow differences may have been due to 1) age, since the cows with the highest numbers of circulating active PMNs were younger than those in the other group or 2) the combined stress of pregnancy and lactation, as those cows which were both pregnant and milking had the lowest numbers of active PMNs.
Cette experience visait 'a determiner la capacite phagocytaire des neutrophiles de huit vaches, tout au long de leur gestation. L'auteur adopta comme standard le nombre de neutrophiles/mm3 qui phagocytierent, in vitro, la souche 321 de Staphylococcus aureus et donna 'a ces leucocytes le nom de neutrophiles actifs. II decela une diminution graduelle de ces cellules, de la conception jusqu'a un minimum de 16 'a 20 semaines de gestation; elles augmentierent ensuite progressivement jusqu'au tarissement, alors qu'elles subirent une baisse drastique. Une autre augmentation survint par la suite et elle atteignit un sommet au cours de la deuxieme semaine avant la parturition. II se produisit ensuite une diminution rapide, jusqu'a un minimum absolu au cours de la semaine qui suivit le v&lage. A compter de la deuxieme semaine apres la parturition, se dessina une augmentation progressive qui persista jusqu'a la conception. Le coefficient de correlation (r) entre le nombre de neutrophiles actifs et la periode precedant la conception s'etablit 'a -0.474 (p=0.01). Les huit vaches presenterent des differences appreciables (p=0.01) dans le nombre de leurs neutrophiles actifs. L'auteur realisa qu'elles formaient deux groupes distincts; celles du premier poss6daient un nombre sensiblement plus eleve de ces cellules (p=0.001) que celles du second. Les differences enregistrees d'une vache it l'autre resultaient probablement 1) de l'age, puisque celles dont le sang recelait le plus de neutrophiles actifs etaient plus jeunes que les autres, ou 2) de l'agression combinee de la gestation et de la lactation, puisque les vaches 'a la fois gestantes et en lactation possedaient le nombre le moins eleve de neutro-
of Veterinary Microbiology and Immuno*Department logy, University of Guelph, Guelph, Ontario.
Submitted March 19, 1975.
Volume 40
-
January, 1976
philes actifs.
ill
INTRODUCTION In a series of papers from this laboratory (3, 7, 10, 11, 12) we have reported results of investigations into the efficacy in vitro of bovine leukocytes, both from blood and from milk. We have found that among the cells collected from the secretion from artificially irritated mammary glands there was a smaller proportion of efficient phagocytes than among those from blood, due at least partly to the lack of an energy source in the gland secretion. Although at a lower level, the proportion of phagocytosing PMNs in milk reflected that in blood of the same cow, i.e. where one was higher or lower than average, so was the other. Since, over short periods, among cows, significant differences in proportion of phagocytosing PMNs were found, it seemed advisable to monitor the activity of leukocytes from a group of cows over a long period which necessitated using blood leukocytes rather than those from milk. It has been reported (4, 5, 6) that the lenkocytes in pregnant women showed enhanced phagocytic and bactericidal activity. Since lactating cows are pregnant during most of their adult life, we have examined the phagocytic activity against Staphylococcus aureus of the blood leukocytes of eight cows throughout complete pregnancies and in some cases during early lactation.
tended to one and one half hours. Staph.
aureus, O.V.C. strain 321 (10) was used throughout. At the beginning of the work, nine cows were assigned to this investigation, of which two were unbred heifers, two were bred heifers within two to three weeks of calving, one was in her third lactation, one in her fourth and one in her fifth. The cow in third lactation was removed later because of failure to conceive. Thus data were collected from eight cows from some time prior to conception to and until shortly after parturition. All cows were free of overt disease. As far as possible the cells of each cow were tested every three weeks, although occasionally this schedule could not be maintained. During the last three to four weeks before the estimated calving date, cows were bled and observations made at weekly intervals and in a few cases even more frequently. At each bleeding the total white blood cell (WBC) count was estimated, the percentage of polymorphonuclear neutrophils (PMNs) determined and the percentage of the PMNs which phagocytosed staphylococci (effective PMNs) calculated from the examination of at least 200 cells in smears stained by Leishman's Stain. For convenience in analyzing and presenting the data the observations were grouped into four-week periods from conception to calving. All observations obtained within each period were pooled and means determined. Where needed, between period data was subjected to an analysis of variance (14), between animal comparisons compared by determining the significance between two means (1) and correlation coefficients were determined (14).
MATERIALS AND METHODS Methods used were essentially as described previously (3) with the following modifications. Blood was collected from the jugular vein instead of the tail vein and heparinized at the rate of 10 U.S.P. units/ml. The buffy coat was resuspended in fresh autologous plasma. A sufficient volume of bacterial culture, the number of bacteria in which was estimated by a Coleman nephelocolorimeter, was added directly to the leukocyte suspension. The incubation time to allow ingestion of the bacteria was ex-
112
RESULTS The period means for each parameter are shown in Fig. 1. Examination of the three curves shows that there was a good deal of variation in each during pregnancy. There was a steady decline in both the total numbers of WBC and the percentage of PMNs up to the period 16 to 20 weeks after conception. The percent of PMNs phagocytosing increased up to ten to 12
Can. J. camp. Med.
I
\
P.MNs 75.
phagocyto8a&g
30/ 20 wsc
12F
n 103/mm3
101_
_ _
_
_
8
_ _
_
_
16 Weeks
_
jl
__ _
_
_
_
24
_
32
_
_
40
Preg nant
Fig. 1. Mean total WBC counts per mm3, % PM!Ns and % of PMNs phagocytosing Staph. aureus. Each point plotted represents the mean of all values obtained in a four week period from six to eight pregnant cows.
weeks, then declined to a low level also at 16 to 20 weeks after conception. The number of circulating phagocytes capable of phagocytosing was determined by multiplying together the three sets of data from Fig. 1 and the total thus obtained termed "Active PMNs". For the cows studied, the figures thus arrived at are shown in Table I. It can be seen that there was a gradual decline in the number of these cells from conception to a minimum during the fifth period, i.e. between the 16th and 20th week, followed by an increase to the eighth period, a drop during the ninth during which the cows were dried off and reaching a peak during the last period, although an analysis of variance showed no overall significance between periods in numbers of active PMNs. However, the mean difference between the fifth period which was the lowest and the tenth period which was the highest is significant (p = 0.01), although when no overall significant difference exists such significances should be regarded with some caution. In Table II are shown the mean weekly activities for six cows for which sufficient figures were available during the period three weeks before parturition to two weeks after. These data indicate that the number of active PMNs reached a maximum during the second week before parturition, followed by a decline to a minimum during the first week postpartum, after which there was a return towards former numbers. An analysis of variance showed the between period differences to be significant at the five per cent level. Also, all
Volume 40
-
January, 1976
three parameters individually showed a significant decline at or immediately after calving. Since, as shown in Table I, the numbers of circulating active PMNs were high immediately following conception and, as seen in Table II, were lowest immediately after parturition, an attempt was made to anal-
yze the trend in active numbers between these two events. There were insufficient figures available for any cow or group of cows as there was considerable variation in the length of time before breeding at which individual cows were introduced to the investigation. Thirty individual figures, which were obtained during 13 weeks preconception, were plotted and are shown in Fig. 2, together with the calculated regression line of activity on time. The correlation coefficient (r) of activity with time before conception was -0.474 which is significant at p=0.01. TABLE I. Mean Number of Active Circulating PMNs in Blood of Cows in Ten 28-day Periods
Following Conception
Period 1...........
2...........
3....... 4.......
5........... 6........... 7........... 8........... 9......
10......
No. of Cows 6 7 7 7 8 8 8 7 7 7
Active PMNs i S.D./mm3 (x 103) 2.31 :4: 1.29 2.11 1.02
2,09 1 0.99 1.83 : 0.86 4
1.56 1.89 1.91 2.44 1.93 2.51
0.63 + 0.95
:1: 1 + :1
0.55 1.37 0.75 0.52
5
E E 0 x
3
z
0
S
-
--v
2-
's 1
12
10 8 6 4 Weeks before conception
2
Fig. 2. The numbers of circulating "Active PMNs" per mm3 in cows at various times between parturition and conception. Correlation coefficient (r) = -0.474, p = 0.01, y = -0.llx + 2.79.
113
14 E E 0
C o
6
6
~ ~ L a c t at i o n
4 ~~2
6
Ag e
Figr. 3. The relationship betwreen lactation age and mean leetation WBC counst in eight cowrs. Correlation coefficient (r) = -0.761, p = >0.05, y = -1.23 + 14.6.
TABLE II. Mean Nunmber of Active CAirculating PMNs in Blood of Six Cows Immediately Before and After Parturition Active PMNs ± S.D./mm3 Days from Parturition (X 103) - 14 to - 18 ............... 2.23 :1: 0.53 - 7 to-13 ............... 2.82 4 1.28 - 6 to 2.07 i 0.90 0............... 1.27 i0.53 + lto+ 7 ............... ......... + 8to + 15 . 1.73 0.65 Differences of 0.96 x 103/mm3 or more are significant at p = 0.05
Analysis of the data summarized in Fig. 1 and Table I showed that there were highly significant difference (p=0.&1) in numbers of circulating active PMNs between the eight individual cows. The mean number for each cow throughout pregnancy is given in Table III. From these figures it is apparent that the cows fell into two distinct groups, group 1 composed of cows 1, 3 and 4, while the other five constitute group 2. The means for the two groups are shown in Table IV and the difference between these is highly significant (p =0.001). This finding led to a critical examination of the individual cow records to see if any obvious differences between the two groups could be found. Of the cows in Group 1 it was determined that two were in their first pregnancy, i.e. had never lactated, while the third was in her second pregnancy, but, because of having been bred unusually late in her first lactation, was dry for seven months of the nine. In group 2, all cows of which lactated for seven of the nine months
14
of pregnancy, two were in second and one in each of third, fourth and fifth lactation. Thus, with the minor exception of twomonths of one pregnancy the cows in group, 1 were dry during their pregnancies while those in group 2 were lactating during the first seven months. Since the figures for active PMNs were calculated from three separate measurements, these were reexamined to see which of the three or combination of any two might have produced the between group, difference. Originally, phagocytosing PMNs were expressed as a percentage of those examined and PMNs in the blood as a percentage of the WBC count. These multiplied together produced a figure representing the percentage of WBC which were effective phagocytes and when this resulting figure was applied to the total WBC count the number of circulating active PMNs per mm3 was found. From the data in Fig. 1 it has already been suggested that a balance was maintained between percentage of PMNs phagocytosing and percentage of PMNs among the WBC. The mean percent of all WBC which were effective phagocytes was calculated for each of the two groups of cows and these figures compared. Likewise the mean total WBC/mm3 for the two groups were determined and compared. These figures are shown in Table V. From this it is apparent that the difference between the groups resulted exclusively from difference in the total WBC count. The relationship of total WBC count to lactation age is shown in Fig. 3 and the regression line, which was calculated from the points, shown. This shows that there was a drop in total WBC count with increasing lactation age.
DISCUSSION The reservoir of phagocytes potentially available to attack an invader of the body is formed largely by the PMNs present in the circulating blood. Thus at any one time the total number of circulating cells capable of phagocytosis may well represent the primary ability of a host to combat a developing infection and variations in that number may affect the host's resistance to disease.
Can. J. comp. Med.
In this paper, data are presented to show that during pregnancy, cows' phagocytes (PMNs) showed marked variation in the three parameters studied. It has been shown previously that there were significant differences between cows in the percentage of effective phagocytes circulating in the blood over short periods (12), but no long term data have been published, nor have there been reports concerning the total numbers of effective phagocytes in bovine circulating blood. In view of the findings (4, 5, 6) that there are increases in both phagocytic and bactericidal activities of PMNs in women during pregnancy, the data presented here were compiled and evaluated throughout complete pregnancies, i.e. from the day of conception, accepted as the day of final breeding, to the day of parturition and in some cases for a limited time before breeding and immediately after TABLE III. Mean Number of Active Circulating PMNs in Blood of Eight Cows During Pregnancy
Cow 1................ 2................ 3................ 4................ 5................ 6................ 7................ 8................
No. of
Trials 10 7 8 9 10 9 10 9
Active PMNs 4
S.D./mm3
(x 103)
3.12 i 1.03 1.61 + 0.71 2.53 ± 0.63 2.69± 0.77 1.47 ± 0.53 1.78± 0.50 1.59 ± 0.41 1.48 4 0.79
TABLE IV. Mean Number of Active Circulating PMNs in Blood of Two Groups of Cows During Pregnancy Active PMNs ± S.D./mm3
Group
1.~~ 2
.
-
(X 103) 2.80 ± 0.85 1.58 i 0.58
t = 6.6
p = > 0.001''
parturition. It is hard to assess the significance of the changes found in the individual parameters during pregnancy. The most notable feature was that all three reached minimum levels during the period 16 to 20 weeks after conception. The total WBC count and the percentage of PMNs showed an almost steady decline during that interval. The phagocytosing PMNs at first increased rapidly up to eight to 12 weeks and then dropped precipitously to a minimum at 16 to 20 weeks. The reasons for these changes are obscure. There is some indication of a balance being maintained between the percentage of phagocytosing PMNs and the percentage of PMNs in the circulating WBC, since these two parameters, to some extent, varied inversely. The values for the three parameters were combined to develop a single figure for the total number of circulating PMNs capable of phagocytosis, a value which has been called "Active PMNs", and the minimum again occurred during the period of 16 to 2& weeks after conception. Whether this is a time of extreme stress in pregnant cows is not known. A peak was reached at between 28 and 32 weeks, just before drying off which was followed by a noticeable drop. Active PMNs then increased to a maximum during the second week before parturition, followed by a decrease to an absolute minimum in the first week post partum. This pattern is different from that found in women, in whom both leukocyte numbers and phagocyte activity were at a consistently high level during the three trimesters of pregnancy (5). The data presented agree in regard to numbers of WBC and percentage of PMNs with those previously published (16) showing that both these parameters peaked on the day of parturition and then dropped off rapidly thereafter. From shortly after parturition it appeared that the circulating active PMNs increased up to the time of conception (Fig. 2). It is pos-
TABLE V. Mean WBC Counts and Percent of Total WBC which were Effective PMNs, for Two Groups of Cows During 280 Days of Pregnancy
Effective PMNS as % of WBC Total WBC x 103/Mm3.......... -Difference not significant at p = 0.05 bDifference significant at p = > 0.001
Volume 40 - January, 1976
Five Milking Three Dry Cows Cows Means ± S.D. 17.23 ± 5.24 18.97 ± 5.22 9.13 ± 1.86 14.71 ± 2.01
S. E. of Difference 1.27&
0.47b
115
sible that this increase is related to the presence of high estrogen concentrations during this time, as estrogens have been shown to be strong stimulants of the reticuloendothelial system and increased phagocytic activity (2, 8, 9). The reasons for the significant differences between cows and the two groups of cows are not clear, although the data do suggest several hypotheses. A balance appeared to have been maintained in the proportion of effective PMNs among the WBC, since pregnancy means for this combined parameter were essentially the same for all cows. Thus the primary difference was in the total WBC count both between cows and except at the very end of pregnancy possibly between periods. While the number of cows used in these experiments was small, the correlation coefficient (r) between mean total WBC count and age was -0.761 (p=0.05), which is in agreement with earlier observations that with advancing age both the total WBC count and the absolute number of neutrophils decrease (15). Thus the differences found may have been due to age since the one group of cows was much younger than the other. On the other hand the cows in group 1 which were pregnant but nonlactating had a significantly higher total count of WBC than those in group 2 which were not only pregnant but also were lactating for seven of the nine months. This suggests two other possible explanations. Either the combined stresses of pregnancy and lactation result in a lower total production of WBC including effective PMNs by the hematopoietic system, or in lactating cows there is a steady drain of leukocytes into the milk even in uninfected glands which the hemopoietic system is unable to replace. That the latter may have significance is indicated by the fact that the total WBC count is at a low level at the time when milk yield is at its height. There are two times at which the cow is considered to be more than normally susceptible to infection of the mammary gland. These are during the first two to three weeks of the dry period and at and immediately following parturition (13). Whether the fact that these are times when the active PMN population was found to be at a low point is related to the incidence of mastitis is open to speculation as indeed is its relation to the incidence of any other infection in the cow. That the phagocytic and bactericidal activity of PMNs is
116
related to the incidence of disease in women has been shown, since in those suffering from a bacteriuria, the result of gram negative organisms, there was no increase. in PMN activity during pregnancy. The lack of activity was most marked against the particular organism causing the disease (4, 5).
ACKNOWLEDGMENTS Financial support was received from the National Research Council of Canada, the Ontario Milk Marketing Board and the Ontario Ministry of Agriculture and Food. The technical help of R. Johnston and Kathy Schumley is gratefully acknowledged. REFERENCES 1. ARKIN, H. and R. R. COLTON. Statistical Methods. 4th Ed. revised. p. 121. New York: Barnes and Noble Inc. 1968. 2. HARTVEIT, F. and K. ANDERSEN. Reticuloendothelial activity related to age and sex in mice. Acta. path microbiol. scand. 76: 161-163. 1969. 3. KENT, G. M. and F. H. S. (NEWBOULD. Phagocytosis and related phenomena in polymorphonuclear leukocytes from cow's milk. Can. J. comp. Med. 33: 214-219. 1969. 4. MITCHELL, G. W., Jr. and A. J. SBARRA. The role of the phagocyte in host-parasite interaction II. The phagocytic capabilities of leukocytes in pregnant women. Am. J. Obstet. Gynec. 91: 755-762. 1965. 5. MITCHELL, G. W., Jr., R. J. McRIPLEY, R. J. SELVARAJ and A. J. SBARRA. The role of the phagocyte in host-parasite interactions IV. The phagocytic activity of leukocytes in pregnancy and its relationship to urinary tract infections. Am. J. Obstet. Gynec. 96: 687.697. 1966. 6. MITCHE'LL, G. W., A. A. JACOBS, V. HADDAD, B. B. PAUL, R. R. STRAUSS and A. J. SBARRA. The role of the phagocyte in host-parasite interactions XXV. Metabolic and bactericidal activities of leukocytes from pregnant women. Am. J. Obstet Gynec. 108: 805-813. 1970. 7. NAIDU, T. G. and F. H. S. NEWBOULD. GlYcogen in leukocytes from bovine blood and milk. Can. J. comp. Med. 37: 47-55. 1973. 8. NICOL, T.. D. L. J. BILBEY, L. M. N. CHARLES, J. L. CORDINGLEY and B. VERNON-ROBERTS. Oestrogen: The natural stimulant of body defence. J. Endocr. 30: 277-291. 1964. 9. NICOL, T., B. VERNON-ROBERTS and D. QUANTOCK. The influence of various hormones on the reticulo-endothelial system: endocrine control or body defence. J. Endocr. 33: 365-383. 1965. 10. NEWBOULD, F. H. S. Some effects of the source of bovine milk leukocytes and strain of Staphylococcus aureus on their interaction in vitro. Can. J. comp. Med. 31: 303-308. 1967. 11. NEWBOULD, F. H. S. Enhancement of phagocytosis in bovine milk leukocytes in vitro. Can. J. comp. Med. 34: 261-264. 1970. 12. NEWBOULD, F. H. S. The effect of added serum and glucose, and some inherent factors, on phagocytosis in vitro by milk leukocytes from several cows. Can. J. comp. Med. 37: 189-194. 1973. 13. NEWBOULD, F. H. S. Microbial diseases of the mammary gland. In Lactation, a Comprehensive Treatise. Vol. II. B. L. Larson and V. R. Smith, Eds. p. 277. New York and London: Academic Press, Inc. 1974. 14. QUENOUILLE, M. H. Rapid Statistical Calculations. pp. 16, 23. London: Griffin. 1959. 15. SCHALM, 0. W. Veterinary Hematology. 2nd Ed. p. 231. Philadelphia: Lea and Febiger. 1965. 16. STRAUB, 0. C.. 0. W. SCHALM, J. P. HUGHES and G. H. THEILEN. Bovine hematology TI. Effect of parturition and retention of fetal membranes on blood morphology. J. Am. vet. med. Ass. 135: 618622. 1959.
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