A quatre reprises, l'auteur a recolte des. et teignit apres quatre heures de contact. rien de semblable. L'addition de 20 mg% de

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1 The Effect of Addled Serum and Glucose, and Some Inherent Factors, on Phagocytosis in vitro by Milk Leukocytes from Several Cows F. H. S. Newbould* ABSTRACT On four occasions leukocytes were obtained from mammary secretions of four cows following irritation by sterile distilled water. The effects of three levels of bovine immune serum, three levels of glucose, and combinations of these on the ability of the leukocytes to ingest staphylococci in vitro were studied. Phagocytosis was estimated by examination of stained slides prepared after four hours contact at 37 C. While 1% immune serum increased the percentage of milk leukocytes ingesting staphylococci, no improvement resulted from the addition of 3%, or 5%. Addition of 2mg% glucose produced a larger increase than did 1% serum, and 4 and 6mg% gave further increases. Little advantage was obtained by combining serum and glucose at any of the levels studied. Overall significant differences were found in the ability to ingest staphylococci by milk leukocytes from the four cows, and the previous observation that cells from some cows in vitro do not respond fully to added serum and glucose was confirmed. Neither of these phenomena was related to glycogen levels in blood or milk leukocytes as determined by the anthrone method, blood glucose levels, leukocyte viability, relative proportions of PMN's in circulating blood white cells, nor the amount of homologous blood serum in the mammary secretions. In two cows whose milk cells did not respond fully, very few blood PMN's were active. *Department of Veterinary Microbiology & Immunology, University of Guelph, Ontario, Canada. Presented in part at VI International Conference on Cattle Diseases, Philadelphia, 197. Submitted June 9, RESUME A quatre reprises, l'auteur a recolte des leucocytes dans les secretions mammaires de quatre vaches dont il avait irrite la glande avec de l'eau distillee. II etudia l'effet de trois concentrations d'immun-serum bovin, de glucose, ou d'une combinaison de ces deux substances sur le pouvoir phagocytaire des leucocytes a l'endroit des staphylocoques, in vitro. Il evalua le degre de phagocytose par 1'examen de lames qu'il prepara et teignit apres quatre heures de contact it 37 C. Tandis qu'une concentration de 1% d'immunserum augmenta le pourcentage de leucocytes du lait ingerant des staphylocoques, des concentrations de 3% ou de 5% ne produisirent rien de semblable. L'addition de 2 mg% de glucose donna une plus grande augmentation que ne le fit 1% d'immun-serum; l'addition de 4 ou 6 mg% de glucose intensifia ce phenomene. Le fait de combiner de l'immunserum et du glucose, i l'une ou l'autre des concentrations utilisees, ne produisit pas d'avantage appreciable. L'auteur trouva des differences appreciables dans le pouvoir que possedaient les leucocytes du lait de quatre vaches d'ingerer des staphylocoques; il confirma aussi une observation anterieure selon laquelle les leucocytes de certaines vaches ne repondent pas parfaitement i l'addition d'immun-serum ou de glucose, in vitro. Aucun de ces phenomenes ne semblait relie 'a la teneur en glycogene du sang ou des leucocytes du lait, comme le demontrerent la methode d'anthrone, la teneur du sang en glucose, la viabilite des leucocytes ou la proportion relative de neutropiiles parmi les leucocytes du sang circulant; ils n'etaient pas relies non plus a la quantiti de Vol April,

2 serum sanguin homologue que contenaient les secretions mammaires. Le sang de deux vaches dont les leucocytes du lait ne repondirent pas parfaitement ne comptait que tres peu de neutrophiles actifs. INTRODUCTION It has been reported that in vitro the numbers of bovine milk leukocytes which phagocytosed live staphylococci were significantly increased by the addition to the leukocyte-bacteria mixture of either.4% glucose or 5 % of bovine anti-staphylococcal serum (5). It is not known what effect other concentrations of glucose and serum or combinations of them might have, and the present work was undertaken to elucidate these. In addition, the effect of these augmentations on viability of the phagocytes during the experimental period were studied. In an attempt to elucidate between-cow difference in competence of phagocytes in vitro, limited investigations were made of stored energy sources in bovine milk leukocytes and in blood polymorphonuclear leukocytes (PMN's) in each cow. A comparison was made between the efficiencies of the leukocytes from milk and blood, particularly from those cows whose milk leukocytes have appeared not to respond fully to the addition of serum or glucose (5). Blood glucose levels in the cows were compared, and the amount of blood serum in the milk of each was estimated. The proportion of polymorphonuclear leukocytes (PMN's) in a minimum of 2 white blood cells (WBC) in the blood of each cow was determined at each test period. MATERIALS AND METHODS With the exception of methods for estimation of blood serum in milk, the procedures used were as described previously (2, 3, 4, 5). Cows For the main part of the experiment, four cows (A-D) were selected from the Ontario Veterinary College mastitis research herd. This work was carried out in the Spring, just prior to the cows being turned out for 19 the Summer. They were fed a normal diet of corn silage, hay and 16% dairy concentrate. One cow was in her first lactation, one in her second, and two in their third. No apparent infection of any kind had occurred in these cows prior to these experiments, with the exception that cow D had an experimental Staphylococcus aureus infection of one week's duration in one quarter, two years previously. Cow A was included, as she was one whose cells had been found previously not to respond fully (5). A fifth cow, whose cells were also reported previously not to respond fully, was included in later experiments, as were six other cows regarded as normal. The latter work was carried out during the Summer and Autumn. STAPHYLOCOCCI Staphylococcus aureus, strain 321 (4), was used throughout these experiments. ESTIMATION OF BLOOD SERUM IN MILK On each test day, from the same secretion from which milk leukocytes were obtained, non-acid whey was prepared by the method of Hall and Learmouth (1). Duplicate samples of each serum were separated on Sepraphore III' cellulose polyacetate strips, in a Gelman Electrophoresis Chamber2. Strips were stained with Ponceau S Stain3 and scanned in an automatic recording and integrating scanner, and the percentage of blood albumin in the milk serum was estimated. Blood serum in milk was estimated by comparing the mean blood albumin content in the test samples with that in similar samples to which known amounts of blood serum had been added. STATISTICAL ANALYSIS Each set of data was subjected to an analysis of variance from which the significance of any resulting differences was estimated (7) and least significant differences calculated. PLAN OF EXPERIMENTS Following irritation by infusion of sterile water, milk cells from each of the four 1,2,3Gelman Instrument Co., Ann Arbor, Michigan, U.S.A. Can. J. comp. Med.

3 RESU LTS.=41 4 -J -2 A B C D Cows Fig. 1. Percentage3 of milk leukocytes from each of four cows, ingesting Staphylococci in vitro. Means of all treatments. Cl I > bacteria/leukocyte; 8 > bacteria/leukocyte. cows were studied on four occasions. Each time leukocytes and staphylococci were reacted in fat-free homologous secretion alone, and secretion to which was added 1%, 3% or 5% (V/V) bovine immune serum alone; 2, 4 or 6mg% of glucose alone; and all combinations of these concentrations of serum and glucose. Thus, in all, 16 determinations were made on each test day. Phagocytosis in each reaction mixture was estimated by examining Gram stained slides prepared after four hours reaction time at 37 C. (5). At least 2 leukocytes were examined, and the percentage of all cells ingesting one or more (1>) staphylococci and of those ingesting eight or more (8>) staphylococci determined. Viability of the leukocytes in each reaction mixture was estimated after four hours. On each test day a sample of milk leukocytes was analysed for glycogen content. On the day preceding each test day, a blood sample was obtained. From this leukocytes were obtained for estimation of their glycogen content, and the blood glucose content estimated enzymatically. In the final series of experiments, blood was drawn from two cows (A and E) on four occasions and on six cows on one or more occasions each, and the percentage of PMN's ingesting staphylococci after one and a half hours reaction time determined. PHAGOCYTOSIS The overall activity of cells from each of the four cows is shown in Fig. 1. The mean percentage of cells phagocytosing both 1 > and 8 > staphylococci in all 16 treatments on four test days is shown for each cow. Each then represents the mean of 64 determinations. Standard deviations were - 6.1, ± 8.4, - 1.5, ± 9.5 for 1> and - 7.3, ±8.5, ± 1., ± 1.7 for 8> staphylococci in cells from cows A, B, C and D respectively. For either criterion these differences were highly significant (p = >.1), Cow A having the least efficient cells, although relatively more of these cells ingested 8> bacteria than did those of the apparently more efficient cells of cows C and D. Figure 2 shows the results of the 16 variations of serum and glucose concentrations, alone and in combination. Each bar represents the mean of 16 determinations. On the whole, it can be seen that after the initial increase resulting from the addition of 1 % serum, no significant improvement occurred with increasing concentrations. In contrast, the efficiency of the leukocytes improved with each glucose increment. TABLE I. Significance of Increases in Percentage of Cells Ingesting, Resulting from Additions of Blood Serum and Glucose in vitro Treatment Increastes in Percentage of Cells Ingesting 1 > 8> Staphylococci Staphylococci Serum alone 1% vs 17.7b 15.8b Glucose alone 2 vs mg% 19.9b 16.lb 6 vs 4 mg% 6.3b 6.Ob 6 vs 2 mg%,r 6.b 9.5b Serum + Glucose 1% 2 vs mg% 4.3a N. 6 vs 2 mg N.S. 8.4b 3% 4 vs 2 mg% 4.2a N.S. 6 vs 4 mg%c 4.5a N.S. 6 vs 2 mg%, 8.7b 8.3b 5c 2vs Omg% N.S. 5.7a 6 vs 4 mg%, 5.2b N.S. 6 vs 2 mg% 8.b 8.4b N.S. = Not significant a = p =.5 b = p = >.1 Vol April,

4 6, 4 -I C 2 IL rmn FEWL Per cent of Serum Glucose mg / Fig. 2. The effect from the addition of serum, glucose or combinations of both in vitro on the phagocytic activity of milk leukocytes from four cows. I percentage of leukocytes ingesting 1> bacteria/leukocyte; percentage of leukocytes ingesting 8> bacteria/leukocyte. little decrease in viability during the four hours contact with staphylococci. Increasing amounts of serum alone had little or no effect. The addition of 2 and 4mg% glucose improved the viability slightly, that shown for 4mg% being significantly (p = >.1) better than when no glucose was added. This effect, however, appeared to be partially reversed by 6mg% of glucose. Combinations of serum and glucose appeared to offer no advantage over glucose alone. BLOOD GLUCOSE The mean blood glucose content of each cow is shown in Fig. 5. The differences between cows were not significant. e 6o J e 4 2 -j e 2 a. rmlf 4 a a c2 c Percent of Se r u m 2 4 GI uc o se mg.-'. Fig. 3. The effect from the addition of serum, glucose or combinations of both in vitro on the phagocytic activity of milk leukocytes from Cow A. L I percentage of leukocytes ingesting 1> bacteria/leukocyte: percentage of leukocyte ingesting 8> bacteria/leukocyte. The significance of increases associated with individual treatments is shown in Table I. Only those treatments which resulted in a significant increase in at least one of the two criteria are included. The results obtained with cells from Cow A, which previously appeared not to repond fully, are shown separately in Fig. 3. It can be seen that, while the percentages of active phagocytes is lower in all cases and the increases smaller, the pattern of increasing activity from the additions of serum and glucose is essentially the same as that shown in Fig. 2. In the presence of 6mg% glucose a very high proportion of the cells ingested 8> bacteria. LEUKOCYTE VIABILITY The decreases in viability, as indicated by absorption of Trypan blue, are indicated in Fig. 4. It can be seen that there was very O7 Percent of Serum 2 4 GI ucose mg /o Fig. 4. The decrease in percentage of viable leukocytes during four hours contact with staphylococci in homologous milk with the addition of three levels of glucose, serum and combinations of both. COWS Fig. 5. Between-cow differences in three factors possibly affecting phagocytosis. Blood Glucose; Glycogen in Blood PMN's; VGlycogen in Milk Leukocytes. Can. J. 6 comp. Med.

5 TABLE II. Blood Serum in Milk from Irritated Mammary Glands of Four Cows Blood Albumin Estimated Blood % of Whey Serum in Milk Cow Proteina A B 8.37 i C i D i ameans of two determinations on each of four test days, i S.D. TABLE III. Polymorphonuclear Leukocytes in Blood from Four Cows Cow Percent Polymorphonuclear Cellsa A ± 5.83 B ± 2.42 C ± 4.92 D ameans of two determinations at each of four test days, i S.D. GLYCOGEN IN MILK AND BLOOD LEUKOCYTES The glycogen content of both milk cells and blood PMN's for each cow are shown in Fig. 5. Because of a laboratory accident blood PMN values are the means of three test days only. Between cows there was no significant difference in the amount of glycogen in milk cells, but a significant (P -.5) difference occurred in that of blood PMN's mainly as the result of the much lower value in the cells of Cow C. PHAGOCYTOSIS BY BLOOD PMN's In Cows E and A, both of which have been reported not to respond fully, the mean percentage of blood PMN's phagocytosing staphylococci was and respectively. From six other cows, during the same period, the mean percentage was BLOOD SERUM IN INFLAMMATORY SECRETIONS For each cow the estimates from samples taken on each of the four test days were available for comparison. The estimated mean for each cow is found in Table II, which shows mean serum albumin and estimated total blood serum contents. These differences are highly significant (p = >.1). POLYMORPHONUCLEAR LEUKOCYTES IN BLOOD The mean percentage of PMN's derived from differential white cell counts (minimum of 2 cells on each strip) made on the blood of each cow on each test day, are shown in Table III. DISCUSSIO'N The present work has demonstrated significant differences in the phagocytic competence of milk leukocytes obtained from mildly irritated glands of several cows, confirming previous observations (6). Overall, the difference in this respect between the least and most competent cells, from Cows A and D respectively, amounted to 23 percentage points; a difference which was largely mediated by the addition of serum and/or glucose to the leukocyte-staphylococci reaction medium since in milk alone the difference was just over seven percentage points. The data presented confirm previous observations (4) of the effects of the addition of immune serum and/or glucose to the reaction medium, and, further, show that no advantage was gained by adding more than 1% immune serum, whereas increasing concentrations of glucose resulted in statistically significant increases in competence. This suggests that the presence of 1% serum provided all the opsonising factors previously shown to be helpful to the leukocyte (8). Even 2 mg% of glucose enhanced phagocytic activity more than the highest level of serum used, and larger amounts produced increasing enhancement, indicating that energy source for adhesion and engulfment had more importance than did opsonization. Microscopical observation of phagocytosis, by the technique used here, does not make possible a distinction between bacteria merely attached to or touching leukocytes and those actually ingested. It is probable that were such a distinction possible, the difference in activity by immune serum and glucose would be more apparent. In a previous publication (4), it was reported that the cells from two of 15 cows tested did not respond to the same degree Vol April,

6 as did those of the other 13. One of the two was included in the present study (Cow A), and the results obtained confirmed the earlier observations. Not only did fewer milk cells from Cow A ingest bacteria, but the maximum increase produced by the addition of serum and glucose amounted to only 17.97%, compared to a 35.26% increase in the case of cells from Cow D. This observation, together with the indication that a larger proportion of the cells from Cow A ingested 8> bacteria, led to the speculation that there was a maximum number of active milk leukocytes in Cow A which could utilize the added glucose. If this were so, either this cow's phagocytes may have suffered a significant loss of function during diapedesis, or possibly there were proportionately fewer active phagocytes in the circulating blood from which milk leukocytes were derived. Data presented show that while this cow's blood contained the highest percentage of PMN's among its leukocytes, there were significantly fewer active phagocytes among them than among those from the blood of other cows. Thus the phagocytic activity of milk leukocytes reflected that of blood PMN's in the same cow. There is little evidence among the data presented to indicate other reasons for the difference in competence between milk leukocytes from the four cows. There was no difference in the viability of their cells during the experiment, no significant difference in their glycogen content, and although significant differences occurred in the estimated amount of blood serum in milk of the four cows, there was no relationship between this parameter and phagocytic competence. Indeed, the cells from Cow A, whose milk contained the most serum, were the least competent and showed the least response to glucose addition. Further, that the amount of homologous serum present in the milk had little effect is evident since in all cases, even with cells from Cow A, the addition of 1% immune serum improved phagocytic activity significantly. While no apparent effect on phagocytic competence resulted, the glycogen content of the blood PMN's in Cow C was significantly lower than in those of the other three cows. There is no immediate explanation for this, nor for the fact that the mean levels of glycogen per 19 blood PMN's are considerably higher than reported previously (3). However in the latter case, subsequent work suggests that these values may vary with season and with certain physiological conditions in the cow. Thus it would appear that, in these experiments, the in vitro performance of milk leukocytes from the four cows in ingesting staphylococci was independent of the parameters studied and was probably related to some other factor inherent in the circulating PMN's before diapedesis. Much of the potential capability of these cells, once in the mammary secretion, could be reactivated in vitro by the addition of immune serum and glucose. ACKNOWLEDGMENTS Financial support from The National Research Council of Canada and the Ontario Department of Agriculture and Food is acknowledged. REFERENCES 1. HALL, I. C. and R. LEARMOUTH. A method for securing clear serums from the milk of cows and goats for agglutination tests. J. infect. Dis. 52: KEN'r, G. M. and F. H. S. NEWBOULD. Phagocytosis; and related phenomena in polymorphonuclear leukocytes from cow's milk. Can. J. comp. Med. 33: NAIDU, T. G. and F. H. S. NEWBOULD. Glycogen in leukocytes from bovine blood and milk. Can. J. comp. Med. 37: NEWBOULD, F. H. S. Some effects of the source of bovine milk leukocytes and strain of Staphylococcus or. their interaction in vitro. Can. J. comp. Med. 31: NEWBOULD, F. H. S. Enhancement of phagocytosis in bovine milk leukocytes in vitro. Can. J. Comrp. Med. 34: NEWBOULD, F. H. S. Some factors of potential importance in the pathogenesis of bovine staphylococcal mastitis. Proc. VI International Conference on Cattle Diseases, Philadelphia, Pa QUENOUILLE, M. H. Rapid Statistical Calculations. p. 16. London: Griffin WISNIOWSKI, J., K. ROMANIUKOWA and H. GRAJEWSKI. Phagocytosis phenomena in the mammary glands of cows. I. The opsonizing factor and the phagocytic activity of leukocytes in milk ani blood. Bull. vet. Inst. Pulawy Can. J. comp. Med.