The role of white cells and frequency of milking in the control of staphylococcal mastitis

J. COMP.PATH.

1967.

VOL.

77.

143

THE ROLE OF WHITE CELLS MILKING IN THE CONTROL MASTITIS

AND FREQUENCY OF STAPHYLOCOCCAL

OF

RY

F. WHITE

and

Row&t

E. A. S. RATTRAY

Research Institute,

Aberdeen

INTRODUCTION

The dairy cow can carry low levels of coagulase positive staphylococci in the udder for long periods without clinical signs of mastitis. It is assumed that the cow has some means of control over the staphylococci present since the number found in freshly drawn milk of a carrier cow is far less than the maximum population attained in milk in vitro. Mastitis can be looked upon as a failure of the controlling mechanism resulting in a rapid growth of staphylococci, damage to the udder tissue and the appearance of clinical symptoms. The results reported here which demonstrate some of the factors involved in the control of the numbers of staphylococci in the cow’s udder, arose from observations on the diurnal variation of the number of white cells in milk (White and Rattray, 1965). Milk obtained at the end of milking had a higher content of fat and solids-not-fat than that obtained just before milking. The effect of this change in quality of milk on the growth of staphylococci has been investigated. MATERIALS

AND

METHODS

Animals. The animals used were lactating Ayrshires and Friesians in the dairy herd of the Duthie Experimental Stock Farm. Milk samples. Samples for white cell counts were collected as previously described (White and Rattray, 1965). Where larger samples for in vitro growth curves were required it was occasionally necessary to stop just short of complete milking out to ensure that sufficient milk was available for the after-milking sample. Bacterial strains. Where a coagulase positive staphylococcus was already present in the quarter supplying milk for experiments, it was isolated and used as the inoculating organism. In other cases a type A or type C staphylococcus was used. These strains were isolated from the herd and serotyped by a method previously described (White, Rattray and Davidson, 1962). Media and methods. Plates were prepared with Oxoid blood agar base No. 2, with the addition of 5 per cent. washed sheep red blood cells. The red cells were washed because the plasma of certain sheep had been shown to contain anti Q and p haemolysins, which were probably antibodies and made identification of staphylococci on plates more difficult. Identical volumes of milk varying from 50 to 100 ml. depending on the volume available, were used for comparative growth curves. The inoculum was prepared from an overnight culture in a 10 per cent. papain digest broth which was centrifuged and the bacteria resuspended in 10 per cent. broth saline. The suspension was adjusted to correspond in density to Brown’s opacity tube No. 1, and added at the rate of 1 ml./100 ml. milk The milk was incubated at 37OC. For plate counts, 1 ml. of milk culture was suitably diluted in 10 per cent. broth saline. Three dilutions at lo-fold intervals were plated out from each sample. A platinum wire loop of 22 S.W.G. adjusted in size to deliver l/100 ml. of milk was used and

144

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WHITE

CELLS

IN

STAPHYLOCOCCAL

MASTITIS

5 counts carried out per plate..The bacterial count/ml. was calculated by the method of Finney (1952). Centrifugation to reduce the white cell content of milk. Although milk samples were centrifuged at 6,200 g. for 30 minutes, it was found impossible to remove white cells completely, possibly due to their being trapped in fat and rising to the surface. Prolonged centrifugation had little further effect on white cell count. Homogenization of milk culture samples. Fresh milk contains a factor causing the clumping of staphylococci and plate counts of staphylococci in milk can be increased by shaking (Smith\ 1957). Braude and Fettes (1953) showed that the apparent reduction, observed by the plate method, in numbers of coagulase-positive staphylococci after incubation in human blood, may result from clumping of living bacteria in leucocytes and in plasma, or from clumping of phagocytes containing living bacteria. Experiments were carried out on milk cultures of coagulase positive staphylococci in which samples were homogenized in an M.S.E. homogenizer for various periods prior to counting. Homogenization at 10,000 r.p.m. for 8 minutes gave the maximum increase in count of staphylococci. Preparation of smears and white cell counting procedure. Smears were prepared as previously described (White and Rattray, 1965). F rom a statistical analysis of the results in this publication it. was shown that the variability increased as white cell count decreased. In an attempt to stardardize errors over the range of counts, a procedure was adopted to give the number of microscope fields to be counted in relation to the number of white ceils present (Table 1). RESULTS

Growth

Curves

of Staphylococci

Milk with a low cell count. A typical result is given in Fig. 1. Essentially the same result was obtained with milk from several different cows and several

2

3

4

5

6

HOURS AFTER INOCULATION Growth curves in milk with a low white cell count of a coagulase positive staphyloccocus isolated from Cow 793 quarter RH: Type C. Milk taken from Cow 793 quarter LF. (A) one hour after morning milking, white count 120,OOO/ml.; (B) just before morning milking, white cell count 2,50O/ml.

cell

F.

WHITE

AND

E.

A.

S.

145

RATTRAY

different quarters. The range of white cell count was nil to 2 - 3 x 105. In milk taken after milking, the generation time was on average 70 per cent. and the maximum population 2 to 4 times of that in milk taken before milking. There were no consistent differences in the lag phase, which was generally between 20 minutes and 1 hour.

FIGURE

2

23 HOURS

AFTER

25

26

curves in milk with a high celi count of coagulase positive staphylococcus 462: Type A. taken from Cow 554 quarter LF. (A) one hour after morning milking, white cell 38,400,00O/ml.; (B) just before morning milking, white cell count 10,560,000/m1.

Growth

Milk coun

TABLE

Four

smears

Sequential depending

referred

procedure on total

Total

+ SlF2

SlFl

to SlF4

SlFl

to SlF8

SlFl

to SlF16

SlFl

to SlF32

count

of white

fields within

Action

to completion

each smear

denoted

of count

count

SlFl

SlFl

1

to as: Sl, S2, S3, S4. Microscope SlFl, SlF2, etc.

of counts from

Total

24

INOCULATION

Over 50 to 25 to Under 25 or Under

100 100 50 25 more 25

25 or more

Under 25 25 or more Under 25 25 or more Under 25 25 or more Under 25 cells/ml.

of milk

No more counts needed. Count 1 field on S2. Count 1 field on S&S3 and S4. Count SlF2, then see next step. Count 2 fields on S2, S3 and S4. Count SlF3 and SlF4, then see next step. Count 4 fields on S2, S3, and S4. Count SlF5 to SlF8, then see next step. Count 8 fields on S2, S3 and S4. Count SlF9 to SlF16, then see next step. Count 16 fields on S2, S3 and S4. Count SlF17 to SlF32, then see next step. Count 32 fields on S2, S3 and S4. Count 64 fields on Sl, S2, S3, 54. was obtained

from

the mean

count/field

x 32 x 10,000.

Milk with a high cell count. A typical result is given in Fig. 2. Consistent results were obtained with milk from several different cows and different quarkm. The range of white cell count was 2 - 3 x 10g to 30 x 106.

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MASTITIS

Comparing the growth curves in milk with a high cell count with those in milk with a low cell count there was a marked increase in lag phase and with the higher white cell counts an apparent mortality (Fig. 2), counts after 4 or 5 hours being well below inoculum level. There was considerable variability from cow to cow and quarter to quarter. This could be explained by differences in the ratio of white cells to staphylococci in the different experiments, but much more experimental evidence will be required to prove this point. In all the samples with a high cell count which were examined the staphylococci eventually entered the logarithmic phase of growth and the maximum population was of the same order as that reached in milk with a low cell count.

FtGURE

3

4 HOURS

AFTEK

INOClJLATlON

Growth curves in milk of a coagulase positive staphylococcus isolated from Cow LF; Type C. Milk taken from 592 LF. one hour after morning milking. Growth aliquot centrifuged before inoculation. Cell count remaining 2,76tY$OOO/ml. homogenized before plating out for counting; (B) samples not homogemzed before for counting. Growth curves in aliquot not centrifuged before inoculation. 20,500,000/m1. (C) samples homogenized before plating out for counting; (D) homogenized before plating out for counting.

592 quarter curves in (A) samples plating out Cell count samples not

Although the white cells were clearly implicated in the extended lag phase and apparent mortality, a possible explanation was an increase in clumping of staphylococci leading to a reduction in plate counts. To check this possibility and to establish the role of the white cells, milk with a high white cell count taken from one quarter 1 hour after milking was divided into two samples, one of which was centrifuged to remove as many white cells as possible. Both samples were inoculated with the same number of staphylococci. Two growth curves constructed from each culture, one from counts obtained by homogenizing the samples before diluting and plating out, and the other from the counts obtained when samples

F.

WHITE

AND

E.

A.

FIGURE

HOURS

AFTER

S.

4

INOCULATION

Growth curves in milk of a coagulase positive staphylococcus isolated from LF; Type C. Milk taken from Cow 592 LF. 1 hour after morning milking. (A) centrifuged remaining white cell count 4,520,00O/mI; (B) centrifuged deposit resuspended 9,480,00O/ml.

FIGURE

I

2

3 HOURS

4

5 AFTER

6

147

RATTRAY

Cow

592 quarter

deposit removed white cell count

5

?

8

9

INOCULATION

Growth curves in milk of a coagulase positive staphylococcus isolated from Cow 592 quarter LF; Type C. Milk taken from Cow 592 LF. one hour after morning milking. (A) homogenized before inoculation, no intact white cells; (B) not homogenized before inoculation, white cell count 2o,ooo,ooo/ml. C

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CELLS

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MASTITIS

were directly diluted and plated out. The results (Fig. 3) show that the decrease in the counts of staphylococci in the presence of the higher white cell count, is real and not due to additional clumping. In further experiments a sample of milk with high white cell count was divided into two. Both samples were centrifuged and the deposited white cells were resuspended in one and removed from the other. Both were inoculated with the same numbers of staphylococci and growth curves constructed. A representative result is given in Fig. 4. As it was found impossible to remove the white cells completely by centrifugation, homogenization was used to disintegrate the white cells of milk prior to inoculation with staphylococci. After homogenization for 15 to 20 minutes intact nuclei could still be found on microscopic examination but the cytoplasm had disappeared. After homogenizing for 25 minutes no intact nuclei were found and this time was used in the experiments. The result of a typical experiment is given in Fig. 5. Since the white cells were implicated in lowering the numbers of staphylococci in milk with a high cell count, smears were prepared from in vitro cultures in milk with a high cell count at various times after inoculation and incubation. All smears prepared after 2 hours incubation showed considerable phagocytosis. If the white cells restrict the numbers of staphylococci in the udder, then the pattern of white cell output in relation to milking is of great importance (White and Rattray, 1965). In the in vitro cultures, depression of the staphylococcal numbers in the milk with a high cell count ceases if incubation is continued long enough (Fig. 5). Thus the interval of time between successive milkings may well be of importance in determining how high the staphylococcal count in the udder will rise before the next milking. It was postulated that the total bacterial count in the udder should be lower just before the afternoon milking after an interval of 9 hours from the last milking than before the morning milking, which takes place 15 hours from the last milking. Comparison of the Total Bacterial and Staphylococcat same Quarter before Morning and Afternoon Milking

Counts

in Milk

from the

Milk samples were taken from each quarter before each milking for two, three or four successive days. A total of 194 samples were collected from 44 quarters. A total bacterial count and a staphylococcal count were made on each sample. The total count was defined as that obtained by culture of milk aerobically on the medium used. Statistical analysis was carried out on the 97 differences between total bacterial count of milk taken before morning (log, a.m. count/ml.) and before afternoon (log, p.m. count/ml.) milkings. There was considerable variability among the differences between the two log counts, but in 84 out of the 97 instances the morning count was the higher of the two. The mean difference of the logs was estimated as 0.70 with a standard error of f0.10, based on a residual variance of O-529 plus an additional variance (significant at P < 0.05 level) of O-177 from quarter to quarter. The mean difference of 0.70 is equivalent to the afternoon count being 20 per cent. of the morning count. Confidence limits (95 per cent.) for the mean difference were approximately 0.50 to 0.90 that is the afternoon count is on average between 12 per cent. and 31 per cent.

F.

WHITE

AND

E.

A.

S.

149

RATTRAY

of the morning count. There were no apparent differences of total bacterial counts and staphylococcal counts. Decline in White Cell Count between

between

the pattern

Milking

In the work on the diurnal variation of white cell output, white cell counts/ml. appeared to fall lower before morning than before afternoon milking. Cell counts were carried out on a total of 200 samples of milk from 44 quarters taken before each milking for two, three or four successive days. Statistical analysis was carried out on the 100 differences between milk taken before morning (log, a.m. count/ml.) and afternoon (log, p.m. count/ml.) milking: 75 were negative and 23 were positive. The mean difference was estimated as -0.226 with a standard error of &O-O32 based on a residual variance of 0.104. There was no significant additional variance from quarter to quarter. The mean difference of -0.226 is equivalent to the afternoon count being 168 per cent. of the morning count. Confidence limits (95 per cent.) for the mean difference are approximately -0.161 to -0.291 corresponding to 145 per cent. and 196 per cent.

of Clinical

Occurrence

Mastitis

If there is any connection between the level of bacterial numbers in the udder and the incidence of clinical mastitis then it follows from the results of comparison

FIGURE COW MILKING

6 762

MILKING

7 I//

MILKING

I I

I I P-.

-- ---

L

-.. ---

. \ \

-\

I I /

\y----

.J

---_

---

-‘.

I I I I

f

. ‘*

.

’ ---_

I

Ic

e e AM

IO

12 12

2

\ . I . \ / I

I

\

\

I

I II II

I II II 4

6

e

IO

PM

NOON

TIME Relation

K.t.

I A,

v,

I I

4

I I

_

i

\

MILKING

I I

between

milking

IN

HOURS

and white

cell count/ml.

of milk.

150

ROLE

OF

WHITE

CELLS

IN

STAPHYLOCOCCAL

MASTITIS

of bacterial counts before morning milking with those before afternoon milking that clinical mastitis should be more commonly detected before the former. Over the period October ZOth, 1965, to March 29th, 1966, 36 cases of clinical mastitis were detected in the Rowett Dairy herd by use of the strip cup : 31 before the morning and 5 before the afternoon milking. E8ect of Milking Three Times a Day on the White Cell Output as compared with Milking Twice a Day It has been shown that a rise in white cell count occurs during and after milking, the inference being that it is associatedwith the act of milking. To confirm this, four cows were milked three times a day, the extra milking being carried out at 10 p.m. Milk samples were taken just prior to milking and one hour after milking. A count of white cells/ml. was carried out on the samples and the log10 of white cell count plotted against time. All four cows showed a higher white cell count in the milk from each quarter after milking, including the additional one. A typical result is given in Fig. 6. DISCUSSION

Results obtained in milk with a low cell count show that post-milking samples of milk are a better media for the growth of staphylococci than pre-milking samples. The size of the inoculum of staphylococci was large enough to give a high ratio of staphylococci to white cells and, although it can be presumed that the white cells did exert an effect, the level attained was insignificant in both the pre- and post-milking samples, sufficient staphylococci remaining free in the medium to give logarithmic growth after a short lag phase. A comparison of the growth curve of staphylococci in milk containing large numbers of white cells with the curve in the same milk sample after the number of white cells had been reduced by centrifuging or disrupted by homogenizing, led to the conclusion that the white cells significantly reduced the number of organisms during the first 6 to 9 hours apparently by phagocytosis. In these experiments the ratio of staphylococci to white cells was low enough to allow the effect of phagocytosis to become apparent. The same mechanism can be expected to operate in the cow’s udder. From the relationship shown to exist between milking time and the white cell count of milk (White and Rattray, 19651, together with the results presented here of milking three times a day, it follows that the most favourable time for a reduction of the bacterial count in the udder is immediately after milking. The position will increasingly deteriorate the longer the cow is left unmilked. From the results presented, control during the 15-hour period between milkings deteriorates more than during the g-hour period. This conclusion was supported by the fact that a larger number of clinical mastitis cases was detected in the Rowett Dairy Herd before morning than before afternoon milking. Enquiries have shown that many dairymen are well aware that clinical symptoms of mastitis are more common before the morning milking. From the evidence presented, the milking of cows three times a day at 8-hour intervals should have a marked influence on the bacterial levels/ml. of milk in the cow’s udder and possibly on the incidence of mastitis in a dairy herd. No evidence has been found in the

F.

WHITE

AND

E.

A.

S.

151

RATTRAY

literature, however, to associate an increased frequency of milking with a reduced incidence of clinical mastitis, but frequent milking has often been suggested as a method of effecting a cure in active clinical cases of the disease (MunchPetersen, 1938). It is now suggested that a far better reason for more frequent milking would be to prevent clinical and subclinical mastitis. We have made one comparison between herds milked two and three times a day. Coagulase positive staphylococci were isolated from 401116 quarters from the herd milked twice a day and from 10/l 16 quarters from the herd milked three times a day. The only cases of clinical mastitis encountered in the herd milked three times a day occurred either at calving or during the drying-off period, drying-off being accomplished by reducing milking from three times a day to two, then once daily and eventually ceasing to milk. It is hoped to examine further herds milked three times a day. An experiment is in hand to compare the udder health and milk yields of two groups of heifers allocated at calving on a random basis, one group milked three times a day and the other twice, the management and feeding being identical. SUMMARY

It is suggested that the incidence of subclinical and clinical staphylococcal mastitis can be reduced by milking dairy cows three times a day at eight-hour intervals. This conclusion derives from experiments in which it was shown that the white cells suppressed the number of coagulase positive staphylococci in vitro in a milk culture in the early stages of incubation. In a herd milked twice a day at 9 and 15-hour intervals the bacterial count in udders was higher and clinical mastitis more common at the end of the long interval than a.t the end of the short interval. ACKNOWLEDGMENTS

Our grateful thanks are due to Mr. H. Denerley, the farm manager, Mr. J. Gunn and the Dairy Staff of the Duthie Stock Farm for their co-operation; to Mr. I. McDonald for statistical examinations of the results; to Messrs. J. W. and W. J. Argo, Farmers, Harvieston, Catterline, Stonehaven., Kincardineshire, for details of their dairy herd and for permission to examine mrlk samples; to Mr. D. J. G. Davidson for preparation of bacteriological media; Department for the photomicrographs.

and to Mr.

J. Robb

of the Photographic

REFERENCES

Braude, A. I., and Fettes, Joyce (1953). J. Lab. clin. Med., 42, 289. Finney, D. J. (1952). Statistical Method in Biological Assay, p. 574. Griffith & Co.; London. Munch-Petersen, E. (1938). Bovine Mastitis. Review Series No. 1. Imperial Bureau of Animal Health, Weybridge. Smith, H. W. (1957). Bull. Minist. Health, Lab. Service, 16, 39. White, F., Rattray, E. A. S., and Davidson, D. J. G. (1962). J. camp. Path., 72,

19; (1965). Ibid., 75, 253. T. D. (1931). Cir. 180. Dept.

Woodward,

[Received

for

Agric.

United

States.

publication, June 14th, 19661