- Email: [email protected]
Antigenic Relationship Between Mastitis Streptococci Belonging to Group-C
J.
COMPo PATH.
1964.
VOL.
74.
ANTIGENIC RELATIONSHIP BETWEEN MASTITIS STREPTOCOCCI BELONGING TO GROUP-C
II.
PATHOGENICITY AND RECIPROCAL MOUSE PROTECTION POWER
By S.
N.
BAKSHI AND
C.
M. SINGH
Department ofPathology and Bacteriology, U.P. College of Veterinary Science and Animal Husbandry, Mathura, India INTRODUCTION
In addition to the group specific polysaccharide Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis possess type and species specific antigens as well as antigens shared with each other (Bakshi and Singh, 1964). Trials were conducted to assess if the property of having shared antigens could form a basis of reciprocal mouse protection against cross challenge with the three organisms under study. MATERIALS AND METHODS
Cultures. Type cultures No. 4669, 6176 and 6181 of Str. dysgalactiae, SIr. zooepidemicus and Str. equisimilis respectively were imported from the National Collection of Type Cultures, England. Pathogenicity for mice. An 18 hours growth of culture in IO CC. of the medium recommended by Plastridge, Banefield and William (1940) was centrifuged and washed twice with phosphate buffer pH 7'0, containing 0·85 per cent. saline solution. The sediment was then resuspended in 20 times its volume of buffered solution and 0'5 cC. of the suspension was inoculated intra-peritoneally into each mouse. In the case of Str. dysgalactiae I CC. was required to produce a mortality pattern comparable to that given by 0'5 cC. of Str. zooepidemicus and Str. equisimilis. The inoculated mice were kept under observation for 15 days and the mortality was recorded. Spleens of the dead mice were triturated separately with sterile sand and I CC. of sterile normal saline solution. Of this suspension o· I CC. was streaked on blood agar plates. The surviving mice were destroyed on the 15th day and their spleens were collected for bacteriological examination. Active immunisation qf mice. The mice were actively immunised by the technique reported by Pattison (1948). To an 18 hours culture in the medium of Plastridge et al. (1940) formalin was added to give a final concentration of 0'35 per cent. The culture was then incubated for one hour and kept in a refrigerator overnight. The growth was centrifuged, washed thrice with sterile normal saline and resuspended in saline to give a final concentration of approximately 300 million organisms ml. Three groups of 21 mice each were inoculated with 0'5 cC. of Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis vaccines intraperitoneally on three consecutive days, and after an interval of four days, on two more consecutive days. Eight days after the last immunising dose each group was divided into three equal batches of seven mice which were then challenged with Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis cultures separately. The controls were challenged simultaneously. The mice were kept under observation for 15 days and the survivors were sacrificed. The spleens were collected, triturated with sterile sand and saline, and plated on blood agar.
410
GROUP-C MASTITIS STREPTOCOCCI: PATHOGENICITY RESULTS
A preliminary study was conducted to assess an approximately L.D. 50 of the organisms. The comparative pathogenicity of the organisms is given in Table 1. It is clear that 1·0 cc. of a 24 hours culture of Str. dysgalactiae injected intra peritoneally gave a mortality pattern comparable with that 0·5 cc. of Str. zooepidemicus or Str. equisimilis culture given by the same route. Str. equisimilis was the only organism which could be isolated from the spleen of mice surviving for 15 days. The cultures of this organism were tested biochemically and serologically with Wellcome Research Laboratories streptococcus grouping sera and with locally prepared Str. equisimilis antiserum, and were found to be similar to the N.C.T.C. strain of Str. equisimilis in reaction. Reciprocal Mouse Protection Test Three groups of 21 mice each were vaccinated according to the technique of Pattison (1948), with formalinised suspensions Group A with Str. dysgalactiae, Group B with Str. zooepidemicus and group C with Str. equisimilis. Eight days after the last inoculation each group was divided into three sub-groups of seven mice each and each sub-group was challenged with the approximate L.D. 50 of the homologous or heterologous organism (Table 2). Group A. All the mice challenged with Str. dysgalactiae, two out of6 challenged with Str. zooepidemicus and 5 out of six challenged with Str. equisimilis, survived. Pure cultures of the organisms were isolated from the spleens of all the mice challenged with Str. zooepidemicus and which died on third and fourth day of the challenge. A pure culture of Str. equisimilis was isolated from the spleen of the mouse which died within 24 hours of the challenge with the same organism. No organism could be isolated from the spleen of surviving mice sacrificed on 16th day of the challenge. Group B. As in group A, all the mice challenged with Str. dysgalactiae survived. In the sub-group challenged with Str. zooepidemicus five out of the six died and pure cultures of the organism were isolated from their spleens. Three mice challenged with Str. equisimilis died and pure cultures were isolated. No organisms could be isolated from the spleens of survivors challenged with Str. dysgalactiae and Str. zooepidemicus, and sacrificed on the 16th day. Pure cultures were isolated from the spleens of survivors inoculated with Str. equisimilis culture. Group C. In the sub-group challenged with Str. dysgalactiae there was only one death. All the seven mice challenged with Str. zooepidemicus died within six days of challenge. Four of the seven mice challenged with Str. equisimilis died within three days .Pure cultures were isolated from all the mice which succumbed within six days. No organism could be isolated from mice which were challenged with Str. equisimilis and died on the eighth day. No organism could be isolated from spleens of survivors of this group on the 16th day.
I
6
cc.
1·0
6
0·5 cc.
Dose tif culture
**21**11**1
1-6
·2
Mortality in 15 days No. tif mice 1~81~~-11I-;-;I~~I---;;inoculated 1 - ' 2 13-r;-]-5
*2
*4
Survival
..
0·5 cc.
12 *"'21"''''21''''''1
- '··1
- '''''''I
*6
0·5 cc. (Sterile culture medium).
6
12 **31 **3' -
"'. Pure culture of the infecting organism isolated from spleen.
Controls
SIr. equisimilis
• No organism isolated from the spleen.
"'6
"'' '5
----------------1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1---
Str. 1;ooepidemicus
-------------1----1-1-1-1--1-1-1-1-1-1-1-1-1-1---1-1---
SIT. dysgalactiae
["fecling organism
TABLE
RESULTS OF INOCULATION OF STR. nrSGALACTlAE, STR. ZOOEPIDEMICUS AND STR. EQUISIMILIS CULTURE
.. ~
~ o :.:
~
p
~ o
~
b:I
:<:
!"
rtr. dysgalactiae Str. zooepilkmicus Str. equisimilis
rtr. dysgalactiae Str. zooepilkmicus Str. equisimilis
Group B. Sir. zooepilkmicus
Group C. Str. equisimilis
7 7 7
6 6 6
7 6 6
No. of mice challenged
•• Challenge organism isolated from the spleen.
rtr. dysgalactiae Str. zooepidemicus Str. equisimilis
Group A. Sir. dysgalactiae
Vaccine
Challenge organism
TABLE 2
-
-
-
-
-
-
··2 ··3 ··1 ··3 - ··1 -
··3
-
I -I -
-
- - - ··1 - -
-
8
9
-
-
-
, -I -I -
7
10
II
-
-
-
·1 -
-
··1
-
--
-
- - --
-
-
-
-
- --
-
14
-
IS
-
-
1-
.
-
-
-
-
-
I
--I - - - - - - - -
-
-
-
I-
13
·S ·0 ·2
··3
·1
·6
·2 ·S
·7
Survival
• Challenge organism not isolated from the spleen.
--~-
-
-
··1 -
12
1-'----,-
- - - I -I - -- - - - - - - - ··1 - - - - ··1 - - **2 - -
-
4
- - - ··3 ··1 ··1 - - - -
3
6
2 S
1
1-
MMtality in 15 days
MORTALITY PATTERN AMONG THE VACCINATED MICE
...
~
~
z
o
o ~
:z:
~
Q
o
oo
~
~
§
I
o
~
"0
~
~
S. N. BAKSHI AND C. M. SINGH DISCUSSION
Holman, Smith and Pattison (1953) reported that a dose of cc. of Sir. dysgalaciiae culture of strain 419 inoculated intraperitoneally killed 36 per cent of inoculated mice, 0'5 cc. 56 per cent, and 1 cc. 93 per cent. Davis (1957) claimed that Sir. zooepidemicus was very fatal for mice. Evans (1944) reported that 47'1 per cent. inoculated intraperitoneally with Sir. equisimilis died. In the present study it was noted that Sir. dysgalaciiae was comparatively less pathogenic than Sir. zooepidemicus or Sir. equisimilis: neither Sir. dysgalaciiae nor Sir. zooepidemicus could be isolated from the spleen of surviving mice after fifteen days. Sir. equisimilis could, however, be regularly isolated from the spleens of surviving mice 15 days after inoculation. The results of cross-protection test suggested that immunisation with Sir. dysgalaciiae vaccine could protect against challenge with Str. dysgalaciiae and Sir. equisimilis. Although one of the mice of group A challenged with Sir. equisimilis died after one day, five mice survived and no organisms could be isolated from their spleens. Sir. zooepidemicus vaccine does not seem to give any protection against Sir. zooepidemicus or Sir. equisimilis. Similarly Sir. equisimilis proved to be a poor antigen and there was little protective antibody response. The cross-protection tests in this study were conducted on only a limited number of mice and can be considered exploratory in nature, but the results suggest the need for further work on the protecting capacity of Str. dysgalaciiae vaccine against Sir. dysgalaciiae and Str. equisimilis infection. 0'25
CONCLUSIONS
Pathogenicity trials with the three mastitis organisms belonging to group C revealed that Str. dysgalaciiae was less pathogenic for mice than Str. zooepidemicus and Str. equisimilis. Str. equisimilis could be regularly isolated from the spleens of infected mice 15 days after inoculation. Str. dysgalactiae and Str. zooepidemicus could not be isolated from surviving mice after 15 days. The results of crossprotection test indicated that mice hyperimmunised with Str. dysgalactiae vaccine could develop resistance against challenge with Str. dysgalactiae and Str. equisimilis. Str. zooepidemicus and Str. equisimilis were poor protecting antigens. ACKNOWLEDGMENT
The authors are grateful to Dr. S. T. Cowan, Director, National Collection of Type Culture, Collindale, London for the supply of type cultures and to Sri C. V. G. Choudary, Principal, U.P. College of Veterinary Science and Animal Husbandry, Mathura for providing the facilities for the work.
414
GROUP-C MASTITIS STREPTOCOCCI: PATHOGENICITY REFERENCES
Bakshi, S. N., and Singh, C. M. (1964). J. camp. Path., 74, 398. Davis, D. H. S. (1957). Nature, London, 179, 869. Evans, A. C. (1944). J. Bact, 48, 267. Holman, H. H., Smith, I. M., and Pattison, I. H. (1953). J. camp. Path., 63,199· Pattison, I. H. (1948). J. Path. Bact., 60, 217. Plastridge, W. N., Banefield, L. F., and William, L. F. (1940). J. inf. Dis., 66, 202. [Received for publication, January 25th, 1964]
COMPo PATH.
1964.
VOL.
74.
ANTIGENIC RELATIONSHIP BETWEEN MASTITIS STREPTOCOCCI BELONGING TO GROUP-C
II.
PATHOGENICITY AND RECIPROCAL MOUSE PROTECTION POWER
By S.
N.
BAKSHI AND
C.
M. SINGH
Department ofPathology and Bacteriology, U.P. College of Veterinary Science and Animal Husbandry, Mathura, India INTRODUCTION
In addition to the group specific polysaccharide Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis possess type and species specific antigens as well as antigens shared with each other (Bakshi and Singh, 1964). Trials were conducted to assess if the property of having shared antigens could form a basis of reciprocal mouse protection against cross challenge with the three organisms under study. MATERIALS AND METHODS
Cultures. Type cultures No. 4669, 6176 and 6181 of Str. dysgalactiae, SIr. zooepidemicus and Str. equisimilis respectively were imported from the National Collection of Type Cultures, England. Pathogenicity for mice. An 18 hours growth of culture in IO CC. of the medium recommended by Plastridge, Banefield and William (1940) was centrifuged and washed twice with phosphate buffer pH 7'0, containing 0·85 per cent. saline solution. The sediment was then resuspended in 20 times its volume of buffered solution and 0'5 cC. of the suspension was inoculated intra-peritoneally into each mouse. In the case of Str. dysgalactiae I CC. was required to produce a mortality pattern comparable to that given by 0'5 cC. of Str. zooepidemicus and Str. equisimilis. The inoculated mice were kept under observation for 15 days and the mortality was recorded. Spleens of the dead mice were triturated separately with sterile sand and I CC. of sterile normal saline solution. Of this suspension o· I CC. was streaked on blood agar plates. The surviving mice were destroyed on the 15th day and their spleens were collected for bacteriological examination. Active immunisation qf mice. The mice were actively immunised by the technique reported by Pattison (1948). To an 18 hours culture in the medium of Plastridge et al. (1940) formalin was added to give a final concentration of 0'35 per cent. The culture was then incubated for one hour and kept in a refrigerator overnight. The growth was centrifuged, washed thrice with sterile normal saline and resuspended in saline to give a final concentration of approximately 300 million organisms ml. Three groups of 21 mice each were inoculated with 0'5 cC. of Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis vaccines intraperitoneally on three consecutive days, and after an interval of four days, on two more consecutive days. Eight days after the last immunising dose each group was divided into three equal batches of seven mice which were then challenged with Str. dysgalactiae, Str. zooepidemicus and Str. equisimilis cultures separately. The controls were challenged simultaneously. The mice were kept under observation for 15 days and the survivors were sacrificed. The spleens were collected, triturated with sterile sand and saline, and plated on blood agar.
410
GROUP-C MASTITIS STREPTOCOCCI: PATHOGENICITY RESULTS
A preliminary study was conducted to assess an approximately L.D. 50 of the organisms. The comparative pathogenicity of the organisms is given in Table 1. It is clear that 1·0 cc. of a 24 hours culture of Str. dysgalactiae injected intra peritoneally gave a mortality pattern comparable with that 0·5 cc. of Str. zooepidemicus or Str. equisimilis culture given by the same route. Str. equisimilis was the only organism which could be isolated from the spleen of mice surviving for 15 days. The cultures of this organism were tested biochemically and serologically with Wellcome Research Laboratories streptococcus grouping sera and with locally prepared Str. equisimilis antiserum, and were found to be similar to the N.C.T.C. strain of Str. equisimilis in reaction. Reciprocal Mouse Protection Test Three groups of 21 mice each were vaccinated according to the technique of Pattison (1948), with formalinised suspensions Group A with Str. dysgalactiae, Group B with Str. zooepidemicus and group C with Str. equisimilis. Eight days after the last inoculation each group was divided into three sub-groups of seven mice each and each sub-group was challenged with the approximate L.D. 50 of the homologous or heterologous organism (Table 2). Group A. All the mice challenged with Str. dysgalactiae, two out of6 challenged with Str. zooepidemicus and 5 out of six challenged with Str. equisimilis, survived. Pure cultures of the organisms were isolated from the spleens of all the mice challenged with Str. zooepidemicus and which died on third and fourth day of the challenge. A pure culture of Str. equisimilis was isolated from the spleen of the mouse which died within 24 hours of the challenge with the same organism. No organism could be isolated from the spleen of surviving mice sacrificed on 16th day of the challenge. Group B. As in group A, all the mice challenged with Str. dysgalactiae survived. In the sub-group challenged with Str. zooepidemicus five out of the six died and pure cultures of the organism were isolated from their spleens. Three mice challenged with Str. equisimilis died and pure cultures were isolated. No organisms could be isolated from the spleens of survivors challenged with Str. dysgalactiae and Str. zooepidemicus, and sacrificed on the 16th day. Pure cultures were isolated from the spleens of survivors inoculated with Str. equisimilis culture. Group C. In the sub-group challenged with Str. dysgalactiae there was only one death. All the seven mice challenged with Str. zooepidemicus died within six days of challenge. Four of the seven mice challenged with Str. equisimilis died within three days .Pure cultures were isolated from all the mice which succumbed within six days. No organism could be isolated from mice which were challenged with Str. equisimilis and died on the eighth day. No organism could be isolated from spleens of survivors of this group on the 16th day.
I
6
cc.
1·0
6
0·5 cc.
Dose tif culture
**21**11**1
1-6
·2
Mortality in 15 days No. tif mice 1~81~~-11I-;-;I~~I---;;inoculated 1 - ' 2 13-r;-]-5
*2
*4
Survival
..
0·5 cc.
12 *"'21"''''21''''''1
- '··1
- '''''''I
*6
0·5 cc. (Sterile culture medium).
6
12 **31 **3' -
"'. Pure culture of the infecting organism isolated from spleen.
Controls
SIr. equisimilis
• No organism isolated from the spleen.
"'6
"'' '5
----------------1-1-1-1-1-1-1-1-1-1-1-1-1-1-1-1---
Str. 1;ooepidemicus
-------------1----1-1-1-1--1-1-1-1-1-1-1-1-1-1---1-1---
SIT. dysgalactiae
["fecling organism
TABLE
RESULTS OF INOCULATION OF STR. nrSGALACTlAE, STR. ZOOEPIDEMICUS AND STR. EQUISIMILIS CULTURE
.. ~
~ o :.:
~
p
~ o
~
b:I
:<:
!"
rtr. dysgalactiae Str. zooepilkmicus Str. equisimilis
rtr. dysgalactiae Str. zooepilkmicus Str. equisimilis
Group B. Sir. zooepilkmicus
Group C. Str. equisimilis
7 7 7
6 6 6
7 6 6
No. of mice challenged
•• Challenge organism isolated from the spleen.
rtr. dysgalactiae Str. zooepidemicus Str. equisimilis
Group A. Sir. dysgalactiae
Vaccine
Challenge organism
TABLE 2
-
-
-
-
-
-
··2 ··3 ··1 ··3 - ··1 -
··3
-
I -I -
-
- - - ··1 - -
-
8
9
-
-
-
, -I -I -
7
10
II
-
-
-
·1 -
-
··1
-
--
-
- - --
-
-
-
-
- --
-
14
-
IS
-
-
1-
.
-
-
-
-
-
I
--I - - - - - - - -
-
-
-
I-
13
·S ·0 ·2
··3
·1
·6
·2 ·S
·7
Survival
• Challenge organism not isolated from the spleen.
--~-
-
-
··1 -
12
1-'----,-
- - - I -I - -- - - - - - - - ··1 - - - - ··1 - - **2 - -
-
4
- - - ··3 ··1 ··1 - - - -
3
6
2 S
1
1-
MMtality in 15 days
MORTALITY PATTERN AMONG THE VACCINATED MICE
...
~
~
z
o
o ~
:z:
~
Q
o
oo
~
~
§
I
o
~
"0
~
~
S. N. BAKSHI AND C. M. SINGH DISCUSSION
Holman, Smith and Pattison (1953) reported that a dose of cc. of Sir. dysgalaciiae culture of strain 419 inoculated intraperitoneally killed 36 per cent of inoculated mice, 0'5 cc. 56 per cent, and 1 cc. 93 per cent. Davis (1957) claimed that Sir. zooepidemicus was very fatal for mice. Evans (1944) reported that 47'1 per cent. inoculated intraperitoneally with Sir. equisimilis died. In the present study it was noted that Sir. dysgalaciiae was comparatively less pathogenic than Sir. zooepidemicus or Sir. equisimilis: neither Sir. dysgalaciiae nor Sir. zooepidemicus could be isolated from the spleen of surviving mice after fifteen days. Sir. equisimilis could, however, be regularly isolated from the spleens of surviving mice 15 days after inoculation. The results of cross-protection test suggested that immunisation with Sir. dysgalaciiae vaccine could protect against challenge with Str. dysgalaciiae and Sir. equisimilis. Although one of the mice of group A challenged with Sir. equisimilis died after one day, five mice survived and no organisms could be isolated from their spleens. Sir. zooepidemicus vaccine does not seem to give any protection against Sir. zooepidemicus or Sir. equisimilis. Similarly Sir. equisimilis proved to be a poor antigen and there was little protective antibody response. The cross-protection tests in this study were conducted on only a limited number of mice and can be considered exploratory in nature, but the results suggest the need for further work on the protecting capacity of Str. dysgalaciiae vaccine against Sir. dysgalaciiae and Str. equisimilis infection. 0'25
CONCLUSIONS
Pathogenicity trials with the three mastitis organisms belonging to group C revealed that Str. dysgalaciiae was less pathogenic for mice than Str. zooepidemicus and Str. equisimilis. Str. equisimilis could be regularly isolated from the spleens of infected mice 15 days after inoculation. Str. dysgalactiae and Str. zooepidemicus could not be isolated from surviving mice after 15 days. The results of crossprotection test indicated that mice hyperimmunised with Str. dysgalactiae vaccine could develop resistance against challenge with Str. dysgalactiae and Str. equisimilis. Str. zooepidemicus and Str. equisimilis were poor protecting antigens. ACKNOWLEDGMENT
The authors are grateful to Dr. S. T. Cowan, Director, National Collection of Type Culture, Collindale, London for the supply of type cultures and to Sri C. V. G. Choudary, Principal, U.P. College of Veterinary Science and Animal Husbandry, Mathura for providing the facilities for the work.
414
GROUP-C MASTITIS STREPTOCOCCI: PATHOGENICITY REFERENCES
Bakshi, S. N., and Singh, C. M. (1964). J. camp. Path., 74, 398. Davis, D. H. S. (1957). Nature, London, 179, 869. Evans, A. C. (1944). J. Bact, 48, 267. Holman, H. H., Smith, I. M., and Pattison, I. H. (1953). J. camp. Path., 63,199· Pattison, I. H. (1948). J. Path. Bact., 60, 217. Plastridge, W. N., Banefield, L. F., and William, L. F. (1940). J. inf. Dis., 66, 202. [Received for publication, January 25th, 1964]