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Mycoplasma mycoides var mycoides in mice
.1.
COMPo PATH.
1962.
MYCOPLASMA COURSE
OF
THE
VOL. 72.
MYCOIDES
VAR
LESION,
THE
AND
MYCOIDES PRIMARY
AND
IN
MICE
SECONDARY
SEROLOGICAL RESPONSE IN AN INBRED LINE OF MICE
By
J. Department of Federal
G. Ross
Veterina~y
Research, Vom, Nigeria*
INTRODUCTION
The history of attempts to utilise small laboratory animals in researches with Mycoplasma mycoides has been described by Hyslop (1958). Gerlach and Heikkila (1956) reported the successful culture of the organism in mice and this work was further developed by Hyslop (1958). Gerlach and Heikkila (1956) have already suggested the use of a mouse-passaged strain as a vaccine, while Lindley (1959) has compared strains of varying virulence for cattle in mice. Lindley (1959) recorded varying virulence for mice between 2 cattle virulent strains and an attenuated strain. The investigations described here were done to obtain additional information concerning the infection in mice, in particular the s'erological response, for the purpose of genetical studies. MATERIALS AND METHODS
Mice. The mice used were an inbred line of Swiss Albino which had originated from the Rockefeller Institute, U.S.A., and had subsequently been inbred in Africa for a considerable number of years. Inoculum. An avianised strain of Mycoplasma mycoides, designated ASS, of partial virulence for cattle was used throughout the investigation. The parent strain, S /3 was obtained from the East African Veterinary Research Organisation. The organism was grown in 50 m!. of serum broth at 37°C. After 48 hours, when the culture exhibited "hanging threads" indicative of the mycelial growth phase, it was thoroughly shaken and dispensed in 1 m!. amounts into ampoules. The ampoules were then freeze-dried in an Edwards' centrifugal freeze drier and stored in a deep freeze at .- 10 to .- IS °c. Five ampoules were reconstituted to their original volume with serum broth, mixed and counts of the number of organisms made following the method of Miles and Misra (1938). The plate count for the ampoules used was 2·75 ± 0·55 X 107• This was diluted one in ten with sterile normal saline and the resulting suspension of organisms was mixed with an equal quantity of a sterile suspension of I per cent agar gel in saline (B.D.H. agar). The final organism suspension in 0·5 per cent agar gel constituted the inoculum. Inoculation oj mice. The mice were inoculated with 0·5 ml. of the inoculum subcutaneously over the sacral region. The injection was made from before backwards and the hypodermic needle was inserted not less than 1·5 cm. before inoculating. Serum collection. The mice were anaesthetised with ether, decapitated and the blood from a group of mice collected in a centrifuge tube. The
* Present address: Glasgow University Veterinary School.
2
Mycoplasma mycoides
IN MICE
blood was allowed to clot, knocked free from the side of the tube and then centrifuged so that the serum could be drawn off with a pipette. Sera from five mice was normally mixed to constitute one bulked serum sample for serological tests as sufficient serum was rarely obtained from a single mouse. Measurement qf lesions. Lesions were measured in mm. using calipers, and two measurements of each lesion were taken, one parallel to the spine and one at right angles to the spine. The summation of these two measurements was recorded as the lesion size. Complement fixation test. The tests were carried out on perspex plates according to the technique of Fulton and Dumbell (1949). Dropping pipettes delivering drops of 0·04 m!. were used for the five test components. The diluent used was 0·35 per cent saline buffered with veronal at pH. 7.2 (Mayer, Osler, Bier and Heidelberger, 1946). Guinea pig serum was freeze-dried and stored at - 10 to - 15°C for use as complement in the test. The complement was used at 0·2 log. IO mm 3 dilutions and titrated at each test. Washed sheep red cells were used at one in eighty dilution, and haemolysin (Burroughs Wellcome and Co.) at a one in two hundred dilution. The antigens used, in this and the agglutination test, were prepared by the method of Campbell (1938). Sera were tested at 0, t, t to 1/32 dilutions after heating for half an hour at 56°C. Any serum dilution fixing 0·2 log IO mm 3 • dilution of complement greater than the unit end point of complement was scored as positive at that titre. Serum and antigen controls were used throughout and suitable corrections for anticomplementary activity made when necessary. Agglutination test. Tube tests were employed using 0·2 m!. of each reagent. Normal saline was used as the diluent. Positive and negative bovine sera controls were employed throughout. Electrophoresis analysis of serum proteins. Paper electrophoresis analysis of the serum proteins was done using a horizontal Shandon tank, Whatman 3 MM. paper and a barbiturate buffer of pH 8·6 and ionic strength o· I. The separated proteins were fixed by heat coagulation and stained with bromphenol blue. The dyed strip was screened in a photo-electric densitomer and quantitative estimates of the protein fractions calculated from total nitrogen estimates obtained by micro-Kjeldahl digestions (Flynn and De Mayo, 1951; Jenks, Jetton and Durrum, 1955). RESULTS
Preliminary investigations showed that peak antibody response was invariably on the 16th day post-inoculation. Throughout the present investigations, therefore, all sera for analysis w ere collected on that day.
of Age and Sex of Mice A comparison was made of the lesion in mice, of both sexes, of two, three and four months of age. Hyslop (1958) recorded that mice of all ages were susceptible to subcutaneous inoculation and this was largely substantiated by the r esults shown in Table I. No significant differences were seen between age or sex, either in the lesion size or serological response. The large lesion recorded in the 2 month female group on the 14th day was due to a lesion which subsequently Effects
4
M
4
3
4
F
3
4
3
3
----~~-
4
4
F
3
3
M
4
M
2
3
--
4
--
F
2
3
0
0
0
0
0
0
II
II
II
10
10
9
-
t
4
0
--
1
0
0
1'4
1'4
5
0
4
0
1'4
1'4
1'4
~----
13
II
9
8
12
12
Complement fixation test.
14
12
II
II
2 ,8 0
13
17
2·8
1'4
-----
13
14
2,8 2·4
12
II
10
13
0
1'4
2·8
0
deviation,
10
10
14
II
10
10
* Standard
3
1'4
1'4
1'4
2·8
0
-~-
+
- -
+ + -- ---
+ - - - --
----~-
-
--i-
+
+
+
+ - -+
-
+
Agglutination test
Titres
+
C.F.T·t
6th 10th 12th I,ph 16th 8th Mean Alean Mean Mean Mean Mean lesion S.D. * lesion S.D . lesion S.D. lesion S.D. Ie.non S.D. lesion S.D.
Sex
Age months
Number of mice
Titres
Lesion measurement in mm. and standard dtviation: days post inoculation
TABLE I COMPARISON OF AGE AND SEX EFFECT ON THE LOCAL LESION AND SEROLOGICAL RESPONSE FOLLOWING SUBCUTANEOUS INFECTION WITH STRAIN OF MrCOPLASMA MrCOIDES WITH AGAR GEL
:<:I
""
~
o
P
'-
4
Mycoplasma mycoides
IN MICE
ruptured on the 15th day and may well have been contaminated. It was found that in the younger mice of 2 months that the inoculum was liable to diffuse owing to the looseness of the skin and that a local lesion was technically more difficult to produce. Fink and Quinn (I953) have reported that a ntibody response is low in young mice and that the response increases from 2 months of age. In the light of these findings, male mice of 3 to 4 months of age were used exclusively in the following investigations.
Course of Lesion The initial inoculation of either agar gel only or agar gel/organism mixture produced an immediate swelling of a pproximately 26 mm. This swelling ra pidly disappeared and was normally gone 24 to 48 hours following inoculation. Around the 5th day post inoculation an agar gel/ M. mycoides inoculum produced a palpable lesion, which increased gradually until the I2th to 14th day. This took the form of a subcutaneous swelling and, unless it became diffuse, it could be measured by calipers. Lesions which were palpable but which were too small to be measured were given the arbitrary reading of 4 mm. In a small proportion of the mice the lesion ruptured; this occurred in controls as well as in those inoculated with Mycoplasma mycoides. The lesion in the majority of these cases was sterile but a fe w yielded extraneous organisms. Lesions which ruptured were considered as contaminated and were not included in the analysis of the results. The course of the lesion to the twenty-fourth day following inoculation with o· 5 m!. of the agar/ Mycoplasma mycoides suspension is shown in Table 2. The lesion regressed slowly from the twenty-fourth day onwards and was usually negligible by the sixth week after inoculation. Macroscopically the lesion was a n abscess and the contents purulent, of a greenish or yellow colour. A haemorrhagic zone normally existed round the abscess. In the agar gel controls, although no lesion was palpable, a greyish amorphous sterile deposit was still evident at post-mortem on the twelfth day after inoculation . Primary Serological Response Four groups of mice were inoculated subcutaneously with agar gel only, agar gel plus sterile culture broth, lvIycoplasma mycoides broth culture only, and agar gel plus Mycoplasma mycoides broth culture, respectively. The course of the lesion was followed and sera collected on the sixteenth day post-inoculation. In addition two groups were inoculated with agar gel plus Mycoplasma mycoides broth culture, and sera collected on the twentieth a nd twenty-fourth day post inoculation. Except in the secondary serological response group which will be described next, complement fixation titres of over! and agglutination titres of over 11, were not found. Titres of such low value have been recorded in the Tables as +. The results of the serological response are shown in T a ble 3.
27
26'5
Lesion mean in agar controls
0
Lesion mean in infected mice
Days after inoculation
4
4
1st
0
0
2nd
0
4
4th
0
4
6th
0
5
8th
0
12 ' 7
I---
10th 14th
16th
18th
0
14
--0
11'2
0
11'5
0
g'6
- - - - - - - - -I - - -
12th
TABLE 2 COURSE OF TYPICAL LESION IN 25 INFECTED AND CONTROL MICE
0
8·g
20th
8·6
24th
0
0
0
0
---
35 th to 12nd
---------
8·6
22nd
u-.
P i':l o
';-<
1% Agar only
45
16
20
24
Agar gel plus A55 culture 1/ 10
Agar gel plus A55 culture 1/ 10
Agar gel plus A55 culture 1/10
30
25
25
16
Pure A55 culture only
10
16
Agar plus sterile broth
5"98
5"9
5"93
5"7 82
6- 178
2- 87
2-54
2-61
~-
2-97
2-35
• Complement fixation test
0-94
0-75
f----
0-7 8
1-08
0-61
3-5 2
1-1 5
6-52
16
- - - - - -1----
Albumin g/roo mI_
Albumin; globulin ratio
Total protein g/lOo ml_
10
- --
Inoculum
Number of mice
Days post inoculation serum collected 1-43
a and f3' globulin g/roo mI_
1-25
1-12
1-13
1-42
1-890 1----
I
1-79
2-18
2- 19
1-3 12
1-93 8
I-56
and Y globulin g/roo ml. f32
-
+
+
+
-
+
+
+
-
-
-
-
Agglutination titre
C_F_ T.* titre
- - --
TABLE S SEROLOGICAL RESPONSE FOLLOWING SUBCUTANEOUS INOCULATION OF AGAR GEL, MrCOPLASMA MrCOIDES A55 CULTURE
-
[;l
~
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~
~
~
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~
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~ %
0">
J.
7
G. ROSS
Due to changes occurring in the [3 globulin fractions in the mice inoculated subcutaneously with Mycoplasma mycoides plus agar gel, the [31 fraction became difficult to distinguish from the a globulin fraction and the (32 difficult to distinguish from the y globulin fraction; consequently, in the results of the electrophoresis analysis, the a and (31 globulins have been grouped together, and the y and the (32 globulin similarly grouped.
Secondary Serological Responses Ten male mice of three to four months of age were given o· 5 ml. of the agar gel, Mycoplasma mycoides inoculum subcutaneously behind the left shoulder. Six control mice, designated "37 day controls", were given a similar inoculum in the sacral region. Five controls were given agar gel only subcutaneously. On the 2 1st day the ten test mice were given 0'5 ml. of the agar gel, Mycoplasma mycoides inoculum in the sacral region. A third fresh control group of five mice were given the same inoculum in the sacral region on the same day. This group was designated the r6 day controls. A comparison of the secondary lesion, at the peak period on the 8th and roth days following the secondary challenge, with the three control groups is shown in Table 4.
TABLE 4 COMPARISON OF SECONDARY LESION ON 8TH AND 10TH DAY AND 3 CONTROLS
No. lif mice
Group
5
Agar only
6
37
day control
5
16
day control
10
Secondary response
Lesion
8th day
S.D.·
Lesion
10th day
S.D.
0
0
0
0
0
0
0
0
I1'7
9'5
1'7 3
10
8·8
4'4 1'4
• S.D.-Standard deviation
The secondary lesion appears smaller than the primary lesion of the 16 day controls, but this difference is not significant. A comparison of the serological response of the secondary challenge mice and the three control groups is shown in Table 5. A significantly greater rise in the antibody titres is evident in the secondary response group. The 16 day control group, and the secondary response group both showed a fall in the serum albumen level, and a rise in the (32 and y globulin fractions.
-----
--
---
5.91
Secondary response
--
5"93
16 day response
- -
5"94
G~
37 day control
Agar only
Total protein g/lOO mi.
0·73
0· 78
1·03
1·15
Albumin globulin ntio
---
2.48
2·61
3.0 3
3.52
Albumin g/lOO mi.
---
~.
a
-
1·30
- - - - - - - - -- -
2·1 3
-
1/8 -
1/30
+ 2·1 9
+ 1·13
a
a
1.42
a
Agglutination titre
1.48
-
Complement fixation titre 0
I 1. 56
--
globulin g/ IOO mi.
f32 and y
1·43
and f31 globulin g/lOO mi.
TABLE 5 COMPARISON OF SECONDARY SEROLOGICAL RES PONSE AND CONTROLS
~
52
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~
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~
00
J.
G. ROSS
9
DISCUSSION
The lesion which developed in mice following the inoculation of
Mycoplasma rnycoides was similar to that described by Gerlach and
Heikkila (1956) and in no instance was a progressive or generalised reaction seen. The lesion was surprisingly uniform throughout the experiments and this fact, coupled with the differences in virulence between strains of Mycoplasma mycoides in mice reported by Lindley (1959), make the use of the mouse as an experimental animal in investigations with this organism worthy of further consideration. The serological response as measured by the complement fixation and agglutination tests was extremely low, so much so, that further development of this approach is not encouraging. The secondary serological response following challenge of previously infected mice, although not high, was significantly greater than the primary response. Between the 16th and the 20th day following inoculation of the agar gel/Mycoplasma mycoides inoculum, certain deviations from the normal serum protein levels were evident. There was a fall in the serum al bumin level and in the a and {3I glo bulins, and a rise in the [32 and y globulins. The results show that a drop in serum albumin occurred with primary and secondary inoculations with agar/Mycoplasma mycoides inocula, and with agar/sterile broth inoculum and also to a lesser extent with Mycoplasma mycoides broth culture only. No fall in serum albumin followed the inoculation of agar gel alone. This fall is presumably a reflection of the systemic reaction which occurs, and suggests that the agar adjuvant may be necessary to hold the inoculum under the skin for a sufficient time to produce this systemic response. This is substantiated by the globulin rise following the agar/sterile broth and agar/Mycoplasma mycoides inocula, and by the relatively small serum albumin drop following the Mycoplasma mycoides culture only inoculum and the lack of rise in the globulin fractions following this inoculum. Following the agar/sterile broth inoculum a rise occurred in all the globulin fractions, while following the agar/Mycoplasma mycoides inoculum a fall occurred in the [32 and y fractions. The proportionally larger [32 and y fractions rise following the agar/Mycoplasma mycoides inoculum points to the antibody response to the organism as being contained in these fractions. That this is not the complete explanation is illustrated by the failure of the [32 and y globulin fractions in the secondary response group to reach a higher level than the single inoculum 16 day control group, despite the higher complement fixation and agglutinating titres recorded in the secondary response group. These investigations do not answer the question of the relationship between the antibody response and the resistance of the host to the development of the lesions, but it is hoped that future studies will clarify this important aspect. CONCLUSIONS
A study of subcutaneous infections of mIce with agar gel/
10
Mycoplasma mycoides
IN MICE
Mycoplasma mycoides inocula is described. The primary and secondary serological responses as measured by complement fixation and agglutination tests and electrophoretic analysis of the sera are reported and discussed. ACKNOWLEDGMENTS
The author is grateful to Dr. J. I. Taylor, Director of Veterinary Research, for assistance and criticism during the preparation of this paper and to Mr. E. P. Lindley and the staff of the Bacteriology Division, Vom, who supplied the culture and antigen materials. REFERENCES
Campbell, A. D. (1938). ]. Counc. Sci. Ind. Res. Aust., n, 103. Flynn, F. U., and De Mayo, P. (1951). Lancet, ii, 235. Fink, M. A., and Quinn, V. A. (1953). ]. Immunol, 70,61. Fulton, F., and Dumbell, K. R. (1949). ]. gen. Microbiol,5, 7. Gerlach, F., and Heikkila I. (1956). Off. Internat. Epiz. Dis., 26th Session, No. 417. Hyslop, M. St. G. (1958). ]. Path. Bact., 75, 189. Jencks, W. P., Jetton, M. R., and Durrum, E. L. (1955). Biochem. ].,60, 20 5. Lindley, E. P. (1959). Bull. epiz. Dis. Afr., 1,238. Mayer, M. M., Osler, A. G., Bier, O. G., and Heidelberger, M. (1946). ]. expo Med., 84, 535. Miles, A. A., and Misra, S. S. (1938).]. Hyg. Camb., 38,732. [Received for publication, May 18th, 196 I]
COMPo PATH.
1962.
MYCOPLASMA COURSE
OF
THE
VOL. 72.
MYCOIDES
VAR
LESION,
THE
AND
MYCOIDES PRIMARY
AND
IN
MICE
SECONDARY
SEROLOGICAL RESPONSE IN AN INBRED LINE OF MICE
By
J. Department of Federal
G. Ross
Veterina~y
Research, Vom, Nigeria*
INTRODUCTION
The history of attempts to utilise small laboratory animals in researches with Mycoplasma mycoides has been described by Hyslop (1958). Gerlach and Heikkila (1956) reported the successful culture of the organism in mice and this work was further developed by Hyslop (1958). Gerlach and Heikkila (1956) have already suggested the use of a mouse-passaged strain as a vaccine, while Lindley (1959) has compared strains of varying virulence for cattle in mice. Lindley (1959) recorded varying virulence for mice between 2 cattle virulent strains and an attenuated strain. The investigations described here were done to obtain additional information concerning the infection in mice, in particular the s'erological response, for the purpose of genetical studies. MATERIALS AND METHODS
Mice. The mice used were an inbred line of Swiss Albino which had originated from the Rockefeller Institute, U.S.A., and had subsequently been inbred in Africa for a considerable number of years. Inoculum. An avianised strain of Mycoplasma mycoides, designated ASS, of partial virulence for cattle was used throughout the investigation. The parent strain, S /3 was obtained from the East African Veterinary Research Organisation. The organism was grown in 50 m!. of serum broth at 37°C. After 48 hours, when the culture exhibited "hanging threads" indicative of the mycelial growth phase, it was thoroughly shaken and dispensed in 1 m!. amounts into ampoules. The ampoules were then freeze-dried in an Edwards' centrifugal freeze drier and stored in a deep freeze at .- 10 to .- IS °c. Five ampoules were reconstituted to their original volume with serum broth, mixed and counts of the number of organisms made following the method of Miles and Misra (1938). The plate count for the ampoules used was 2·75 ± 0·55 X 107• This was diluted one in ten with sterile normal saline and the resulting suspension of organisms was mixed with an equal quantity of a sterile suspension of I per cent agar gel in saline (B.D.H. agar). The final organism suspension in 0·5 per cent agar gel constituted the inoculum. Inoculation oj mice. The mice were inoculated with 0·5 ml. of the inoculum subcutaneously over the sacral region. The injection was made from before backwards and the hypodermic needle was inserted not less than 1·5 cm. before inoculating. Serum collection. The mice were anaesthetised with ether, decapitated and the blood from a group of mice collected in a centrifuge tube. The
* Present address: Glasgow University Veterinary School.
2
Mycoplasma mycoides
IN MICE
blood was allowed to clot, knocked free from the side of the tube and then centrifuged so that the serum could be drawn off with a pipette. Sera from five mice was normally mixed to constitute one bulked serum sample for serological tests as sufficient serum was rarely obtained from a single mouse. Measurement qf lesions. Lesions were measured in mm. using calipers, and two measurements of each lesion were taken, one parallel to the spine and one at right angles to the spine. The summation of these two measurements was recorded as the lesion size. Complement fixation test. The tests were carried out on perspex plates according to the technique of Fulton and Dumbell (1949). Dropping pipettes delivering drops of 0·04 m!. were used for the five test components. The diluent used was 0·35 per cent saline buffered with veronal at pH. 7.2 (Mayer, Osler, Bier and Heidelberger, 1946). Guinea pig serum was freeze-dried and stored at - 10 to - 15°C for use as complement in the test. The complement was used at 0·2 log. IO mm 3 dilutions and titrated at each test. Washed sheep red cells were used at one in eighty dilution, and haemolysin (Burroughs Wellcome and Co.) at a one in two hundred dilution. The antigens used, in this and the agglutination test, were prepared by the method of Campbell (1938). Sera were tested at 0, t, t to 1/32 dilutions after heating for half an hour at 56°C. Any serum dilution fixing 0·2 log IO mm 3 • dilution of complement greater than the unit end point of complement was scored as positive at that titre. Serum and antigen controls were used throughout and suitable corrections for anticomplementary activity made when necessary. Agglutination test. Tube tests were employed using 0·2 m!. of each reagent. Normal saline was used as the diluent. Positive and negative bovine sera controls were employed throughout. Electrophoresis analysis of serum proteins. Paper electrophoresis analysis of the serum proteins was done using a horizontal Shandon tank, Whatman 3 MM. paper and a barbiturate buffer of pH 8·6 and ionic strength o· I. The separated proteins were fixed by heat coagulation and stained with bromphenol blue. The dyed strip was screened in a photo-electric densitomer and quantitative estimates of the protein fractions calculated from total nitrogen estimates obtained by micro-Kjeldahl digestions (Flynn and De Mayo, 1951; Jenks, Jetton and Durrum, 1955). RESULTS
Preliminary investigations showed that peak antibody response was invariably on the 16th day post-inoculation. Throughout the present investigations, therefore, all sera for analysis w ere collected on that day.
of Age and Sex of Mice A comparison was made of the lesion in mice, of both sexes, of two, three and four months of age. Hyslop (1958) recorded that mice of all ages were susceptible to subcutaneous inoculation and this was largely substantiated by the r esults shown in Table I. No significant differences were seen between age or sex, either in the lesion size or serological response. The large lesion recorded in the 2 month female group on the 14th day was due to a lesion which subsequently Effects
4
M
4
3
4
F
3
4
3
3
----~~-
4
4
F
3
3
M
4
M
2
3
--
4
--
F
2
3
0
0
0
0
0
0
II
II
II
10
10
9
-
t
4
0
--
1
0
0
1'4
1'4
5
0
4
0
1'4
1'4
1'4
~----
13
II
9
8
12
12
Complement fixation test.
14
12
II
II
2 ,8 0
13
17
2·8
1'4
-----
13
14
2,8 2·4
12
II
10
13
0
1'4
2·8
0
deviation,
10
10
14
II
10
10
* Standard
3
1'4
1'4
1'4
2·8
0
-~-
+
- -
+ + -- ---
+ - - - --
----~-
-
--i-
+
+
+
+ - -+
-
+
Agglutination test
Titres
+
C.F.T·t
6th 10th 12th I,ph 16th 8th Mean Alean Mean Mean Mean Mean lesion S.D. * lesion S.D . lesion S.D. lesion S.D. Ie.non S.D. lesion S.D.
Sex
Age months
Number of mice
Titres
Lesion measurement in mm. and standard dtviation: days post inoculation
TABLE I COMPARISON OF AGE AND SEX EFFECT ON THE LOCAL LESION AND SEROLOGICAL RESPONSE FOLLOWING SUBCUTANEOUS INFECTION WITH STRAIN OF MrCOPLASMA MrCOIDES WITH AGAR GEL
:<:I
""
~
o
P
'-
4
Mycoplasma mycoides
IN MICE
ruptured on the 15th day and may well have been contaminated. It was found that in the younger mice of 2 months that the inoculum was liable to diffuse owing to the looseness of the skin and that a local lesion was technically more difficult to produce. Fink and Quinn (I953) have reported that a ntibody response is low in young mice and that the response increases from 2 months of age. In the light of these findings, male mice of 3 to 4 months of age were used exclusively in the following investigations.
Course of Lesion The initial inoculation of either agar gel only or agar gel/organism mixture produced an immediate swelling of a pproximately 26 mm. This swelling ra pidly disappeared and was normally gone 24 to 48 hours following inoculation. Around the 5th day post inoculation an agar gel/ M. mycoides inoculum produced a palpable lesion, which increased gradually until the I2th to 14th day. This took the form of a subcutaneous swelling and, unless it became diffuse, it could be measured by calipers. Lesions which were palpable but which were too small to be measured were given the arbitrary reading of 4 mm. In a small proportion of the mice the lesion ruptured; this occurred in controls as well as in those inoculated with Mycoplasma mycoides. The lesion in the majority of these cases was sterile but a fe w yielded extraneous organisms. Lesions which ruptured were considered as contaminated and were not included in the analysis of the results. The course of the lesion to the twenty-fourth day following inoculation with o· 5 m!. of the agar/ Mycoplasma mycoides suspension is shown in Table 2. The lesion regressed slowly from the twenty-fourth day onwards and was usually negligible by the sixth week after inoculation. Macroscopically the lesion was a n abscess and the contents purulent, of a greenish or yellow colour. A haemorrhagic zone normally existed round the abscess. In the agar gel controls, although no lesion was palpable, a greyish amorphous sterile deposit was still evident at post-mortem on the twelfth day after inoculation . Primary Serological Response Four groups of mice were inoculated subcutaneously with agar gel only, agar gel plus sterile culture broth, lvIycoplasma mycoides broth culture only, and agar gel plus Mycoplasma mycoides broth culture, respectively. The course of the lesion was followed and sera collected on the sixteenth day post-inoculation. In addition two groups were inoculated with agar gel plus Mycoplasma mycoides broth culture, and sera collected on the twentieth a nd twenty-fourth day post inoculation. Except in the secondary serological response group which will be described next, complement fixation titres of over! and agglutination titres of over 11, were not found. Titres of such low value have been recorded in the Tables as +. The results of the serological response are shown in T a ble 3.
27
26'5
Lesion mean in agar controls
0
Lesion mean in infected mice
Days after inoculation
4
4
1st
0
0
2nd
0
4
4th
0
4
6th
0
5
8th
0
12 ' 7
I---
10th 14th
16th
18th
0
14
--0
11'2
0
11'5
0
g'6
- - - - - - - - -I - - -
12th
TABLE 2 COURSE OF TYPICAL LESION IN 25 INFECTED AND CONTROL MICE
0
8·g
20th
8·6
24th
0
0
0
0
---
35 th to 12nd
---------
8·6
22nd
u-.
P i':l o
';-<
1% Agar only
45
16
20
24
Agar gel plus A55 culture 1/ 10
Agar gel plus A55 culture 1/ 10
Agar gel plus A55 culture 1/10
30
25
25
16
Pure A55 culture only
10
16
Agar plus sterile broth
5"98
5"9
5"93
5"7 82
6- 178
2- 87
2-54
2-61
~-
2-97
2-35
• Complement fixation test
0-94
0-75
f----
0-7 8
1-08
0-61
3-5 2
1-1 5
6-52
16
- - - - - -1----
Albumin g/roo mI_
Albumin; globulin ratio
Total protein g/lOo ml_
10
- --
Inoculum
Number of mice
Days post inoculation serum collected 1-43
a and f3' globulin g/roo mI_
1-25
1-12
1-13
1-42
1-890 1----
I
1-79
2-18
2- 19
1-3 12
1-93 8
I-56
and Y globulin g/roo ml. f32
-
+
+
+
-
+
+
+
-
-
-
-
Agglutination titre
C_F_ T.* titre
- - --
TABLE S SEROLOGICAL RESPONSE FOLLOWING SUBCUTANEOUS INOCULATION OF AGAR GEL, MrCOPLASMA MrCOIDES A55 CULTURE
-
[;l
~
'"Z
~
~
~
'"
~
§"
<">
~ %
0">
J.
7
G. ROSS
Due to changes occurring in the [3 globulin fractions in the mice inoculated subcutaneously with Mycoplasma mycoides plus agar gel, the [31 fraction became difficult to distinguish from the a globulin fraction and the (32 difficult to distinguish from the y globulin fraction; consequently, in the results of the electrophoresis analysis, the a and (31 globulins have been grouped together, and the y and the (32 globulin similarly grouped.
Secondary Serological Responses Ten male mice of three to four months of age were given o· 5 ml. of the agar gel, Mycoplasma mycoides inoculum subcutaneously behind the left shoulder. Six control mice, designated "37 day controls", were given a similar inoculum in the sacral region. Five controls were given agar gel only subcutaneously. On the 2 1st day the ten test mice were given 0'5 ml. of the agar gel, Mycoplasma mycoides inoculum in the sacral region. A third fresh control group of five mice were given the same inoculum in the sacral region on the same day. This group was designated the r6 day controls. A comparison of the secondary lesion, at the peak period on the 8th and roth days following the secondary challenge, with the three control groups is shown in Table 4.
TABLE 4 COMPARISON OF SECONDARY LESION ON 8TH AND 10TH DAY AND 3 CONTROLS
No. lif mice
Group
5
Agar only
6
37
day control
5
16
day control
10
Secondary response
Lesion
8th day
S.D.·
Lesion
10th day
S.D.
0
0
0
0
0
0
0
0
I1'7
9'5
1'7 3
10
8·8
4'4 1'4
• S.D.-Standard deviation
The secondary lesion appears smaller than the primary lesion of the 16 day controls, but this difference is not significant. A comparison of the serological response of the secondary challenge mice and the three control groups is shown in Table 5. A significantly greater rise in the antibody titres is evident in the secondary response group. The 16 day control group, and the secondary response group both showed a fall in the serum albumen level, and a rise in the (32 and y globulin fractions.
-----
--
---
5.91
Secondary response
--
5"93
16 day response
- -
5"94
G~
37 day control
Agar only
Total protein g/lOO mi.
0·73
0· 78
1·03
1·15
Albumin globulin ntio
---
2.48
2·61
3.0 3
3.52
Albumin g/lOO mi.
---
~.
a
-
1·30
- - - - - - - - -- -
2·1 3
-
1/8 -
1/30
+ 2·1 9
+ 1·13
a
a
1.42
a
Agglutination titre
1.48
-
Complement fixation titre 0
I 1. 56
--
globulin g/ IOO mi.
f32 and y
1·43
and f31 globulin g/lOO mi.
TABLE 5 COMPARISON OF SECONDARY SEROLOGICAL RES PONSE AND CONTROLS
~
52
~
c ~
"
~
§" ~
" ~
~
00
J.
G. ROSS
9
DISCUSSION
The lesion which developed in mice following the inoculation of
Mycoplasma rnycoides was similar to that described by Gerlach and
Heikkila (1956) and in no instance was a progressive or generalised reaction seen. The lesion was surprisingly uniform throughout the experiments and this fact, coupled with the differences in virulence between strains of Mycoplasma mycoides in mice reported by Lindley (1959), make the use of the mouse as an experimental animal in investigations with this organism worthy of further consideration. The serological response as measured by the complement fixation and agglutination tests was extremely low, so much so, that further development of this approach is not encouraging. The secondary serological response following challenge of previously infected mice, although not high, was significantly greater than the primary response. Between the 16th and the 20th day following inoculation of the agar gel/Mycoplasma mycoides inoculum, certain deviations from the normal serum protein levels were evident. There was a fall in the serum al bumin level and in the a and {3I glo bulins, and a rise in the [32 and y globulins. The results show that a drop in serum albumin occurred with primary and secondary inoculations with agar/Mycoplasma mycoides inocula, and with agar/sterile broth inoculum and also to a lesser extent with Mycoplasma mycoides broth culture only. No fall in serum albumin followed the inoculation of agar gel alone. This fall is presumably a reflection of the systemic reaction which occurs, and suggests that the agar adjuvant may be necessary to hold the inoculum under the skin for a sufficient time to produce this systemic response. This is substantiated by the globulin rise following the agar/sterile broth and agar/Mycoplasma mycoides inocula, and by the relatively small serum albumin drop following the Mycoplasma mycoides culture only inoculum and the lack of rise in the globulin fractions following this inoculum. Following the agar/sterile broth inoculum a rise occurred in all the globulin fractions, while following the agar/Mycoplasma mycoides inoculum a fall occurred in the [32 and y fractions. The proportionally larger [32 and y fractions rise following the agar/Mycoplasma mycoides inoculum points to the antibody response to the organism as being contained in these fractions. That this is not the complete explanation is illustrated by the failure of the [32 and y globulin fractions in the secondary response group to reach a higher level than the single inoculum 16 day control group, despite the higher complement fixation and agglutinating titres recorded in the secondary response group. These investigations do not answer the question of the relationship between the antibody response and the resistance of the host to the development of the lesions, but it is hoped that future studies will clarify this important aspect. CONCLUSIONS
A study of subcutaneous infections of mIce with agar gel/
10
Mycoplasma mycoides
IN MICE
Mycoplasma mycoides inocula is described. The primary and secondary serological responses as measured by complement fixation and agglutination tests and electrophoretic analysis of the sera are reported and discussed. ACKNOWLEDGMENTS
The author is grateful to Dr. J. I. Taylor, Director of Veterinary Research, for assistance and criticism during the preparation of this paper and to Mr. E. P. Lindley and the staff of the Bacteriology Division, Vom, who supplied the culture and antigen materials. REFERENCES
Campbell, A. D. (1938). ]. Counc. Sci. Ind. Res. Aust., n, 103. Flynn, F. U., and De Mayo, P. (1951). Lancet, ii, 235. Fink, M. A., and Quinn, V. A. (1953). ]. Immunol, 70,61. Fulton, F., and Dumbell, K. R. (1949). ]. gen. Microbiol,5, 7. Gerlach, F., and Heikkila I. (1956). Off. Internat. Epiz. Dis., 26th Session, No. 417. Hyslop, M. St. G. (1958). ]. Path. Bact., 75, 189. Jencks, W. P., Jetton, M. R., and Durrum, E. L. (1955). Biochem. ].,60, 20 5. Lindley, E. P. (1959). Bull. epiz. Dis. Afr., 1,238. Mayer, M. M., Osler, A. G., Bier, O. G., and Heidelberger, M. (1946). ]. expo Med., 84, 535. Miles, A. A., and Misra, S. S. (1938).]. Hyg. Camb., 38,732. [Received for publication, May 18th, 196 I]