Experimental streptococcal mastitis

J.

64·

COMPo PATH. 1954 VOL.

35 1

EXPERIMENTAL STREPTOCOCCAL MASTITIS X.

THE IMPORTANCE OF A SERUM STAPHYLOCOCCAL

HYALURONIDASE INHIBITOR IN INFECTION OF THE COW

By

D. G.

HOWELL,

I.

H. PATTISON, H. H. HOLMAN AND

I.

M. SMITH

Agricultural Research Council, Field Station, Compton, Berks

INTRODUCTION

The experimental introduction of Streptococcus agalactiae into the bovine udder has frequently been associated with a low infection rate (Plastridge, 1953J' Howell, Pattison, Holman, and Smith (1954) concluded that certain cows possessed some inherent resistance to Str. agalactiae and considered that the nature of such resistance should be the subject offurther investigation. At the time these studies were being pursued, experiments were also being carried out on the streptococcus itself, with special reference to hyaluronidase production. Examination of the sera of experimentally i.nfected cows had revealed the presence of an inhibitor to the hyaluronidase of Str. agalactiae. A further examination showed that certain of these cows also possessed a serum inhibitor to the hyaluronidase of Staphylococcus aureus, and the presence of this inhibitor seemed to be related to the type of reaction seen following the introduction of Str. agalactiae into the udder. Thus cows which possessed the inhibitor gave a transient reaction whereas cows which lacked the inhibitor gave a crisis-type reaction with the establishment of infection (Howell, et al). It was accordingly decided to investigate this relationship more closely and cows were selected as being respectively inhibitor-positive and inhibitor-negative to staphylococcal hyaluronidase and subsequently inoculated with Str. agalactiae into the teat sinus. MATERIALS AND METHODS

The materials and methods for producing infection and for studying the ensuing mastitis were as described by Howell, et al (1954). Two strains of Str. agalactiae were used viz. strain SI3 and strain AIOO; 21 cows were inoculated with the former and 19 with the latter. The test for the presence of hyaluronidase inhibitor was that of Burnet (1948) carried out in the manner described by Howell (1954), In assessing whether or not the disease was produced, the establishment of the inoculated strain of Str. agalactiae within the udder has been taken as the criterion of infection. For this reason transient reactions were not included as valid infections and crises were especially looked for in view of the importance attached to them as a manifestation of the chronic syndrome (Howell, et al).

352

SERUM STAPHYLOCOCCAL HYALURONIDASE INHIBITOR RESULTS

Observations using Strain SI3

The results for 2 I cows that received an intramammary inoculation of 10 9 viable organisms are recorded in Table I. These include six cows which gave rise to the original observation. TABLE I INFECTION OF

Cows

WITH 10· VIABLE

Titre of Staph. hyaluronidase inhibitor

Cow No. I 2

1:32 1:64 1:3 2 1:256 0 1:3 2 1:128 1:3 2 1:64 1:32 0 0 0 0 0 0 1:3 2 1:64 1:3 2 1:128 1:64

3 4 5 6 7 8 9 10 II 12 13 14 15 16 17 18 19 20 21

Note:

-

=

Str. Agalactiae STRAIN SI3 No. of crzses recorded

Whether infection established

No No No Yes Yes No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes

Duration of experiment (days)

o

5

2

3 4 2 I

o o o o

no crises as infection not established.

Establishment of infection. In 13 infected cows inhibitor was present in six and absent in seven while in eight non-infected cows, inhibitor was present in every case. This suggests that where the inhibitor is absent there is 100 per cent infection. By the "exact method" p = 0.015 for a chance result as favourable to this hypothesis. Occurrence of crisis in infected cows. In eight cows showing crises the inhibitor was present in one and absent in seven while in five cows which failed to show a crisis the inhibitor was present in every case. These figures strongly suggest an association between the presence of an inhibitor and the absence of a crisis. By the "exact method" p = 0.0047 for a chance result as favourable to the hypothesis. Observations using Strain AIOa

Table 2 records the results for inoculation with strain AIOO.

19

cows given an intramammary

D. G. HOWELL, 1. H. PATTISON, H. H. HOLMAN AND 1. M. SMITH

353

TABLE 2 INFECTION OF COWS WITH

Cow No. I 2 3 4 5 6 7 8 9 10 II

12 13 14 15 16 17 18 19

Titre of Staph. hyaluronidase inhibitor 0 0 0 0 0 0 1:128 1:128 1:3 2 1:128 1:64 0 1:64 0 1:64 0 1:128 0 1:32

Note:

-

Approx. No. of viable organisms inoculated 5 5 5 5

X 10

5

X IU

5 5 5 5 5 5 5 5 5 5

X

5 5 5

5

X 10

8 8 8

X 10 8

X 10

x x x x x x x x x x x x

8 8

10 8 10 8 10 8 10 8 10 8 10 10' 10' 10· 10· 5

10 105 10'

x 10 4

Str .. Agalactiae Whether infection established

STRAIN AIOO

No. of crzses recorded

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Yes No Yes No

I 0 0 0 0 0 0 0 0 0 0

Duration qf experiment (days) 21 46 46 46 46 46 46 46 46 46 46 10 10 10 10 10 10 10 10

no crises as infection not established.

Establishment oj injection. In 16 infected cows inhibitor was present in six and absent in ten while in three non-infected cows inhibitor was present in each case. These figures support the result that there is 100 per cent infection in cows lacking the inhibitor but it can be seen from Table 2 that all cows were infected where a large infecting dose was used and the proportion of high infecting doses hides any significance these figures can show by themselves. Occurrence oj crisis in infected cows. In six cows showing crises the inhibitor was present in one and absent in five while in ten cows which failed to show a crisis the inhibitor was present in five and absent in five. This also gives support to the observation made on strain SI3 that there is an association between the presence of the inhibitor and the absence of crises. The only cow contradicting this hypothesis is one that received a high infecting dose. Again the numbers are too few for a useful test of significance. D{fJerence oj appearance oj milk after injection. Fig 1 shows the macroscopic appearance of the milk from the infected quarters of two cows, one of which possessed the inhibitor and the other which did not. The dose was 5 x 10 6 viable organisms. The clots present in the milk of the cow in which the inhibitor was absent are clearly seen. A similar difference was noted in the pair of cows receiving 5 x 10 5 and 5 x 10 4 viable organisms.

354

SERUM STAPHYLOCOCCAL HYALURONIDASE INHIBITOR

Fig.

I.

Macroscopic appearance of milk three days after inoculation with 5 x ro S viable Str. agalactiae strain Aroo. The sample on the left was from cow No. 14 (ear mark 119) and on the right from cow No. 15 (ear mark 508). The gross abnormality of the milk of cow No. 14, which was inhibitor-negative, as compared with cow No. 15, which was inhibitor-positive, can be clearly seen.

Comparison of Results obtained with Strain SI3 and Strain AIOa

It will be seen that the results obtained with the two strains are similar although it would appear that strain Aloo was better able to overcome any resistance in the inhibitor positive cows than was strain S13. As the results are similar for both strains and as the significance of the results in each case depends on a four cell value which in some cells has a theoretical value of less than five, it is convenient to combine the two results. Establishment if infection. Using either of the two strains a total ot 29 cows were successfully infected. Of these inhibitor was present in 12 and absent in 17. In 1 I non-infected cows inhibitor was present in every case. From these figures X2 = 8.g and p = 0.003 and it would seem reasonable to suppose there is an association between successful infection and the absence of the inhibitor. Occurrence of crises. Using either of the two strains 14 cows showed a crisis; inhibitor was present in two and absent in 12 of these cows. In 15 cows which failed to show a crisis the inhibitor was present in ten and absent in five. From these figures X2 = 6.17 and p = 0.013 and it would seem reasonable to assume that there is an association between the presence of the inhibitor and the absence of crises.

D. G. HOWELL, I. H. PATTISON, H. H. HOLMAN AND I. M. SMITH

355

DISCUSSION

Howell, et al. confirmed the findings of previous workers in demonstrating that certain cows were able to resist experimental infection with Str. agalactiae. The observations recorded in this paper though statistically significant raise considerable speculation. One of us (Howell, 1954) has brought forward evidence suggesting that this inhibitor to staphylococcal hyaluronidase is a true antibody and it is unlikely that it could have any influence on the infectivity of Str. agalactiae. The findings of McClean (1943) Friou (1949) and Caprio, Rantz and Randall (1952) indicate that a marked antigenic specificity exists between the hyaluronidases of the various cocci. While the nature of resistance to Str. agalactiae, which certain cows possess, still remains obscure it appears that it does arise in parallel with the production of an inhibitor to the hyaluronidase of Staph. aureus. CONCLUSIONS

The results of an investigation into the relationship of a serum staphylococcal hyaluronidase inhibitor to the infectivity of Str. agalactiae in cows is described. The experiments involved a total of 40 cows, the serum of 23 of which possessed the inhibitor and that of 17 of which did not. Each cow was inoculated into the teat sinus with Str agalactiae. A significant difference was obtained in the ability of the inhibitor-postive cows to resist infection as compared with the inhibitor-negative cows. There was also a negative association between cows possessing the inhibitor and the occurrence of crises. It was suggested that it was unlikely that such an inhibitor was directly concerned with the resistance of certain cows to Str. agalactiae, but that it occurred in parallel with some other and more important protective factor. ACKNOWLEDGMENT

vVe are grateful to Mr. F. H. Summerfield for preparing the photograph. REFERENCES

Burnet, F. M. (1948). Aus.]. expo Biol. Med. Sci.) 26) 71. Caprio, J. M., Rantz, L. A., and Randall, E. (1952). A.M.A. Arch. Int. Med.) 89, 374. Friou, G. J. (1949).]. info Dis.) 84,240 .. Howell, D. G. (1954). ]. Path. Bact.) In press. Howell, D. G., Pattison, I. H., Holman, H. H., and Smith, I. M. (1954). ]. compo Path.) 64) 335. McClean, D. (1943). Biochem. ].) 37) 169. Plastridge, W. N. (1953). In Brandly & Jungherr "Advance' in Veterinary Science" Vol. I. Academic Press Inc., New York. [Received for publication February 12th 1954]