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Immunogenic Stability of Heat-Killed Mycoplasma Mycoides Subsp. Mycoides at 4°C
Br. vet.]. (1980). 136, 614
SHORT COMMUNICATION
IMMUNOGENIC STABILITY OF HEAT-KILLED MYCOPLASMA MYCOIDES SUBSP. MYCOIDES AT 4°C
BY
J.
M . HOOKER ,
G. R. SMITH AND R. A. MILLIGAN
Nuffield Laboratories of Comparative Medicine, Institute of Zoology, The Zoological Society of London, Regent's Park, London NWJ 4R Y INTRODUCTION
The disadvantages associated with the living vaccines currently used for the control of contagious bovine pleuropneumonia (CBPP) have been discussed by Hooker, Smith & Milligan (1980). These authors reported experiments in mice, rabbits and cattle on the development of a heat-killed vaccine; different adjuvants were compared, antigen preparations were titrated for immunizing potency, and the duration of the immune response was measured . A killed CBPP vaccine would be required to retain its ability to protect for a year or more in the refrigerator. This point is the subject of the present report.
MATERIALS AND METHODS
Killed mycoplasma suspension The method of preparation was that of Hooker et al. (1980). Cells of Mycoplasma mycoides subsp. mycoides (strain 'Blenheim'), harvested from a four-day broth culture, were washed, resuspended in phosphate buffered saline (PBS) to the opacity of Brown's tube 10, and killed by heating at 56°C for 30 min. The sterility of the heated material was confirmed by culture of samples. The suspension was stored at approximately 4°C for 18 months; samples were withdrawn at intervals to test their protective potency . Tests for protective potency in mice The method was that of Smith (1969). Each sample of killed mycoplasma suspension was treated as follows. Six five-fold dilutions in PBS were prepared, ranging from 1 in 25 to 1 in 78 125 . Groups of 12 mice were immunized intravenously (0 · 25 ml per mouse) with each dilution. A control group of 20 mice received PBS alone intravenously . Three weeks later all mice were challenged intraperitoneally with strain Blenheim . The test was assessed on the presence or absence of mycoplasmaemia 24 h after challenge. as judged by a selective blood-culture technique.
..,
615
OF HEAT-KILLED MYCOPLASMA
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>
b:l
615
STABILITY OF HEAT-KILLED MYCOPLASMA TABLE I
r'
POSITIVE BLOOD CULTURES, 24 HAFTER CHALLENGE. IN GROUPS OF MICE IMMUNIZED THREE WEEKS EARLIER WITH HEAT-KILLED MYCOPLASMAS STORED AT4°C
~
0
"'1
:t Heat-killed mycoplaJma suspension (Brown's tube JO) diluted I in
~
Storage-time-months (challenge doJe-millions of M. Mycoides subsp. mycoides)
':1
0 (45)
0·75 (66)
4 (84)
6 ()J)
8 (20)
10 (40)
(12)
18 (12)
0/12 0/12 1/12 6/11 7/12t 9/12* 19/20
0/12 0/12 l/12 3/12 7/12 11/12* 20/20
0/12 0/12 0/12 1/12 4/12 9/12t 20/20
1/12 1/12 0/12 4/12 10/12* 12/12* 21/21
0/12 0/12 1/12 6/12t 8/12* 10/12* 18/20
0/12 0/12 l/12 3/12 11/12* 11/12* 20/20
1/12 2/12 5/12 8/12 8/12 8/12 20/20
0/12 0/12 3/12 5/12* 4/12t 9/12* 15/20
15
cr' r'
tT1
25 125 625 3125 15625 78125 Control- PBS
• Not significantly different from controls (P>0·05}; t significantly different (P<0·025); all other results significantly different (P<0·0l).
t:,
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n
0
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616
BRITISH VETERINARY JOURNAL, 136, 6 RESULTS
The heat-killed mycoplasma suspension remained protective for mice throughout 18 months' storage at 4°C (Table I). If any loss of potency occurred it was too slight to be detected by the method used. The suspension was protective at the following dilutions: 25, 125 and 625 (invariably); 5125 (almost invariably); 15 625 (usually); 78 125 (occasionally). If protection for mice can be equated with that for cattle - a point that requires confirmation-the keeping qualities of the heat -killed mycoplasmas were consistent with those expected of a satisfactory killed vaccine.
ACKNOWLEDGEMENTS
The experiment formed part of a project financed by Merck Sharp and Dohme Ltd. REFERENCES
j. M., SMITH, G. R. & MILLIGAN, R . A. (1980). journal of Comparative Pathology 90. In press. SMITH , G. R. (1969) . joumal of Comparative Pathology 79,255. HOOKER ,
(Accept ed for publication 15 May 1980)
SHORT COMMUNICATION
IMMUNOGENIC STABILITY OF HEAT-KILLED MYCOPLASMA MYCOIDES SUBSP. MYCOIDES AT 4°C
BY
J.
M . HOOKER ,
G. R. SMITH AND R. A. MILLIGAN
Nuffield Laboratories of Comparative Medicine, Institute of Zoology, The Zoological Society of London, Regent's Park, London NWJ 4R Y INTRODUCTION
The disadvantages associated with the living vaccines currently used for the control of contagious bovine pleuropneumonia (CBPP) have been discussed by Hooker, Smith & Milligan (1980). These authors reported experiments in mice, rabbits and cattle on the development of a heat-killed vaccine; different adjuvants were compared, antigen preparations were titrated for immunizing potency, and the duration of the immune response was measured . A killed CBPP vaccine would be required to retain its ability to protect for a year or more in the refrigerator. This point is the subject of the present report.
MATERIALS AND METHODS
Killed mycoplasma suspension The method of preparation was that of Hooker et al. (1980). Cells of Mycoplasma mycoides subsp. mycoides (strain 'Blenheim'), harvested from a four-day broth culture, were washed, resuspended in phosphate buffered saline (PBS) to the opacity of Brown's tube 10, and killed by heating at 56°C for 30 min. The sterility of the heated material was confirmed by culture of samples. The suspension was stored at approximately 4°C for 18 months; samples were withdrawn at intervals to test their protective potency . Tests for protective potency in mice The method was that of Smith (1969). Each sample of killed mycoplasma suspension was treated as follows. Six five-fold dilutions in PBS were prepared, ranging from 1 in 25 to 1 in 78 125 . Groups of 12 mice were immunized intravenously (0 · 25 ml per mouse) with each dilution. A control group of 20 mice received PBS alone intravenously . Three weeks later all mice were challenged intraperitoneally with strain Blenheim . The test was assessed on the presence or absence of mycoplasmaemia 24 h after challenge. as judged by a selective blood-culture technique.
..,
615
OF HEAT-KILLED MYCOPLASMA
U'>
>
b:l
615
STABILITY OF HEAT-KILLED MYCOPLASMA TABLE I
r'
POSITIVE BLOOD CULTURES, 24 HAFTER CHALLENGE. IN GROUPS OF MICE IMMUNIZED THREE WEEKS EARLIER WITH HEAT-KILLED MYCOPLASMAS STORED AT4°C
~
0
"'1
:t Heat-killed mycoplaJma suspension (Brown's tube JO) diluted I in
~
Storage-time-months (challenge doJe-millions of M. Mycoides subsp. mycoides)
':1
0 (45)
0·75 (66)
4 (84)
6 ()J)
8 (20)
10 (40)
(12)
18 (12)
0/12 0/12 1/12 6/11 7/12t 9/12* 19/20
0/12 0/12 l/12 3/12 7/12 11/12* 20/20
0/12 0/12 0/12 1/12 4/12 9/12t 20/20
1/12 1/12 0/12 4/12 10/12* 12/12* 21/21
0/12 0/12 1/12 6/12t 8/12* 10/12* 18/20
0/12 0/12 l/12 3/12 11/12* 11/12* 20/20
1/12 2/12 5/12 8/12 8/12 8/12 20/20
0/12 0/12 3/12 5/12* 4/12t 9/12* 15/20
15
cr' r'
tT1
25 125 625 3125 15625 78125 Control- PBS
• Not significantly different from controls (P>0·05}; t significantly different (P<0·025); all other results significantly different (P<0·0l).
t:,
s::><:
n
0
"'r'> U'>
s::
>
....
a, (jl
616
BRITISH VETERINARY JOURNAL, 136, 6 RESULTS
The heat-killed mycoplasma suspension remained protective for mice throughout 18 months' storage at 4°C (Table I). If any loss of potency occurred it was too slight to be detected by the method used. The suspension was protective at the following dilutions: 25, 125 and 625 (invariably); 5125 (almost invariably); 15 625 (usually); 78 125 (occasionally). If protection for mice can be equated with that for cattle - a point that requires confirmation-the keeping qualities of the heat -killed mycoplasmas were consistent with those expected of a satisfactory killed vaccine.
ACKNOWLEDGEMENTS
The experiment formed part of a project financed by Merck Sharp and Dohme Ltd. REFERENCES
j. M., SMITH, G. R. & MILLIGAN, R . A. (1980). journal of Comparative Pathology 90. In press. SMITH , G. R. (1969) . joumal of Comparative Pathology 79,255. HOOKER ,
(Accept ed for publication 15 May 1980)