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The Use of Fibroma Virus (Shope) for the Protection of Rabbits Against Myxomatosis
J.
COMPo
PATH.
1956. VOL. 66.
THE USE OF FIBROMA VIRUS (SHOPE) FOR THE PROTECTION OF RABBITS AGAINST MYXOMATOSIS By
J.
BARBARA ROWE, W. MANSI AND R. HUDSON Veterinary Laboratory, Ministry cif Agriculture, W'!Ybridge INTRODUCTION
Shope (1936, 1938) showed that the inoculation of fibroma virus conferred a high degree of protection to rabbits against experimental inoculation with myxoma virus. He reported that fifty-nine out of sixty-two of his fibroma-treated rabbits resisted challenge by inoculation with the latter. The early results of Shope were confirmed by Berry and Litchty (1936), Martin (1936), Hurst (1938), Hyde (I939),Jacotot and Vallee (1953), and Fenner and Woodroofe (1954), and a brief summary of early work performed at Weybridge was given by Ritchie, Hudson and Thompson (1954). The percentage of rabbits that have been found to have acquired a serviceable immunity after inoculation with fibroma virus has varied from one worker to another. The present paper gives a detailed account of our investigation of the keeping properties of the vaccine, the duration of immunity, the safety of the vaccine for young rabbits, the effect of immunity of the dam on the result of vaccinating suckling rabbits and the relative value of the two sub-strains of the Boerlage strain of fibroma virus isolated by Shope in 1947, given subcutaneously and intradermally. MATERIALS AND METHODS
Rabbits. Seven hundred and ninety-nine rabbits were used in these experiments. Some were purchased in the open market, some were bred, but none had been immunized before being put on an experiment. They varied in age and breed. Fibroma virus (Shope). Two sub-strains of the Boerlage strain of this virus were used. The first, obtained from Dr. Jacotot in September, 1953, is referred to as "P", and the second, from Professor Fenner, is referred to as "F". Vaccine was normally prepared by the method used at the Pasteur Institute, Paris. Male rabbits were inoculated into each testicle and subcutaneously at four or more sites with 0·5 cc. doses of a ten per cent suspension of material harvested previously. The seed virus came from a bank, stored frozen at -40°C., and, in order to minimise the possibility of changes in the virus, no unnecessary passages were made. On the day before the maximum development of the tumours was expected, usually on the eighth day, the rabbits were killed and the testicles and subcutaneous, oedematous pulp were collected, weighed and homogenized in a "Magimix" household pulping machine in five volumes of five per cent whole-egg buffer of pH 7·4. This concentrated suspension was titrated on rabbits and stored frozen until required. The vaccine itself was then prepared by diluting the concentrate so that each 0·5 cc. dose contained 1,000 m.i.d. Usually, this meant diluting IO to 100 fold.
BARBARA ROWE, W. MANS[ AND
J.
R. HUDSON
Myxoma virus. A fully virulent strain, isolated from a rabbit from Cornwall in April, [954, was used. The material was kept frozen during the early part of the work, but in September, [954, freeze-dried material was prepared and, since then, challenge has been mainly with this material. Inoculation of this strain produced typical myxomatosis in susceptible rabbits, with death in I I to 15 days. The preparation before freeze-drying contained between 10-6 and 10-10 m.i.d. per cc. The challenge dose was at least 1,000 m.i.d. and was inoculated intradermally or subcutaneously. RESULTS
Keeping Properties of the Vaccine Concentrate A number of rabbits were inoculated with the original material from Paris and the virus thus obtained has been used to infect all the vaccine producers required in our work. Retention of the concentrated vaccine for several months made it necessary to determine the period during which it could be kept without loss of activity. Table I shows that when stored frozen at -400e. the TABLE
I
KEEPING PROPERTIES OF THE VACCINE CONCENTRATE ( 1/5)
Period Temperature in weeks -40 °C.
+4°C .
I
2 9 15 32 36 46 52 I
2 39 58 +4°C .
69
88 1= Immune.
Numbers Fibroma inoculated reaction
Diluent Buffer saline pH 7'4
"
"
" " "
"
" " " "
" " " " " " " "
" " "
"
" " " "
" "
13 3 5 43 3 5 3 3
Beef-heart infusion broth pH 7 ' 4 and freeze-dried
"
"
"
"
Buffer saline pH 7'4 and freeze-dried
12 3 5
II
2 2
3
0
Myxoma challenge
- - - -- D. I. R. -II
0
2
I
I
I
2 29 I
2 2 3
4
3 3 3
3 3
2
2
I
3
3
2
R = Recovered.
I
5
2 2
0
I
0
0 0
0
0
0 0
I
- - - -- -
4
2
2 9
4
3 3 2
I
0 0 0
I
0
0
I
- - ---
D=Died.
concentrate can be held for a year. After freeze-drying it may be kept at -4°e. for a longer period, both in buffer saline or in beefheart infusion broth. Tests after longer periods of storage have still to be made.
Keeping Properties of the Diluted Vaccine A series of preparations were made from one batch of concentrate (sub-strain P) by diluting with the following:- (a) phosphate
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
buffer saline pH 7'4; (b) phosphate buffer saline pH 7'4 containing five per cent whole egg; (c) phosphate buffer with I: 100,000 thiomersalate. Part of the preparation containing egg, but without thiomersalate was freeze-dried. Samples of the liquid preparations were stored at room temperature and at +4 ce. Freeze-dried material was stored at +4 ce. and reconstituted to the original volume immediately before use. The results of tests of these samples are given in Table 2. It will be observed that neither the addition TABLE
2
KEEPING PROPERTIES OF FIBROMA VIRUS SUSPENSIONS OF VACCI:--iE STRE:--iGTH
Periodqf storage in weeks
Preparation and temperature of storage
Number qf rabbits ----,---,-----_._---1 Results of challenge Inoculated Reacted Immune Recovered Died
--1--2--
---I-B-u-ffe-r-s-a-li-n-e-p-H-7-·4-+-4-oC-. Egg buffer +4°C. Eggbuffer+thiomersalate +4°C.1
--2--
--3--1-~--~
2
1
1
-
3
2
2
-
Buffer saline pH 7'4 +4°C. Egg buffer +4°C. Egg buffer+thiomersalate + 4°C.
7 3 3
6
5 3
Buffer saline pH 7'4 +4°C. Egg buffer +4° C . Egg buffer+thiomersalate +4°C.
4 4 4
4 4 4
Egg buffer Room temperature Egg buffer+thiomersalate
2
2
4
4
20
Egg buffer, freeze-dried +4°C.
3
3
2
32
-HOC.
3
3
3
41
+4°C .
3
3
94
+4°C .
3
2
20
2 2
1
-
2
I
3
of egg proteins as protective colloids nor thiomersalate as a bacteriostatic agent improved the keeping qualities of the vaccine so that it gave any protection after storage for five months in the refrigerator. Freeze-drying enabled the vaccine to be kept for periods up to twenty-two months, but the extra cost entailed in producing a freeze-d:ied vaccine would perhaps render such a product uneconomIC. Duration of Immunity The rabbits included in Table 3 were inoculated with different batches of vaccine at various times, but the vaccine was always standardized to contain about 1,000 minimum infective doses of fibroma virus and it was always used fresh. It is felt that this table gives a general picture of what may be expected of the vaccine and it is probably a better guide to the value of the vaccine in the field
BARBARA ROWE, W. MANSI AND
J.
293
R. HUDSON
than an experiment designed with one batch of vaccine on a single large group of rabbits. It will be observed that about three quarters of the rabbits vaccinated had a serviceable immunity for about a year. TABLE 3 DURATION OF IMMUNITY
Period between vaccination and challenge
Number of rabbits vaccinated
Result of challenge Immune
Recovered
Died
0(%)
..
·.
34
29 (86%)
5 (14%)
Between 60-120 days
·.
41
24 (60%)
11(28%)
6 (12%)
Between 120-200 days
67
24 (3 6 %)
26 (39%)
17 (25%)
Between 200-300 days
.. ..
36
26 (73%)
4 (II%)
6 (16%)
Between 300-400 days
..
12
6 (50%)
3 (25%)
3 (25%)
Between 400-500 days
..
14
3 (22%)
1(7%)
·.
4
0(0%)
1(25%)
Under 60 days
Over 500 days
..
10
(71%)
3 (75%)
Safety and Value of the Vaccine in Baby Rabbits The following experiments were designed to find out how young, suckling rabbits could be vaccinated safely and whether the immunity of the dam interfered with the result of vaccination. Many litters of rabbits were used in these experiments and several different batches of freshly-prepared vaccine. The results are given in Table 4 in which are also included the results of tests made with myxoma virus. It will be observed that during the first three weeks of life the offspring of fibroma-immune rabbits are less likely to react to a dose of fibroma virus than are the offspring of a susceptible rabbit. Contrary to what is found with fibroma virus, it is clear that no protection is afforded to the young of myxomaimmune does against inoculation with myxoma virus. This result confirms those reported by Jacotot, Vallce and Virat (I 954b). Unfortunately, a proportion of the young rabbits was lost before the second part of this experiment, an attempt to study the possibility that the young of a vaccinated dam might not respond as well as those of a susceptible dam to vaccination, could be completed. Nevertheless, it was possible to challenge 81 offspring from vaccinated does, themselves vaccinated when two to seven weeks old, and 14 offspring from normal does, vaccinated when four to seven weeks old. The results (Table 5) suggest that the immunity of the doe does not affect the development of immunity in the young. Effect of Vaccination on Pregnant Does Pregnant does did not abort as a result of the vaccination, but we observed some abortions in pregnant rabbits which were in-
I
8
-12
20
5
6 -
9
8
5 5
4
3
2
I1
i
,- -;--
·.
8
I
3
·. ·.
7
6
5
3
4
..
Number inoculated
0
0 20
5 20
3
8
8
0
5
5
5
5
0
0
Died
0
5
3
3
Reacted
5
I I
I
0
0
0
0
No reaction
I
Offspring of myxoma-immune rabbits
· .
I
I
ciffibroma-immune
I
4
21
5
10
12
II
8
0
0
0
0
8
I
20
5
5
18
0
0
0
0
20
5
5
18
1
20
5
3
18
- - -- - - -- - - - -- - - -
..
9
31
16
II
22 28
0
8
I I
20
0
5
5
I
I
1 - -- - -1
10
8
3
15
36
14
24
4
0
5
0
0
3
5
I
12
24
I
0
0
!
I
I
20
5
5
5
7
3
2
3
12
13
24
4
0
0
0
0
0
0
6
4
Die~
20
5
5
5
---
I
Reacte~
Offipring of normal rabbits
Number No I Die~ ~noculate~ ~tion
rabbits
Number No inoculated :..,eaction -.!!eacted
--- --
Offspring
Offspring of myxoma immune, fibroma immune and normal rabbits.
3
2
I
Age in weeks when inoculated
MY"om· 1
Fibroma ..
Virus inoculated
TABLE 4-
SUSC EPTIBILITy TO EXPERIMENTAL INFECTION WITH FIBROMA AND MYXOMA VIRUSES
<
til
VJ
o
..,
-< >: o :::;J>
:::
~ ~..,
;J>
~
i:Ij i:Ij
;J>
'"
o"1
Z
(3
..,;J>
Z
lJ lJ
;J>
~
to .....
BARBARA ROWE, W. MANSI AND
J.
295
R. HUDSON
advertently provided as experimental subjects, frequently handled and unsuitably housed. These observations were restricted to a very :small number of does. TABLE 5 IMMUNITY TESTS ON RABBITS, THE PROGENY OF VACC Il'ATED AND NORMAL DOES
Vaccinated when
I
Age at ____ va_cc_in_a__ti_on_I ____D_o_e_ _ _ 2
! Vaccinated
2 -
7 weeks of age.
I,. _ _ Result Number chalienged__ I__ .!~
__ _R_.__
5
3
4 4 5
5
6
7
Normal 1= Immune.
o
o
4
o
5
o
13
8
4
19
12
5
34
7
4
o
, Normal Vaccinated
o
13 R = Recovered.
_ I._D_-_ __
2
4
8
4
D=Died.
'Comparison of the Protection against Myxomatosis Afforded by Two ,Substrains of the Boerlage Strain of Fibroma Virus and Using Two Routes ,of Inoculation It was desirable to make a comparison between the two substrains, "P" and "F," and the different routes of inoculation. Accordingly, a vaccine-concentrate was prepared from substrain "F" and suitable dilutions were used on three occasions. On two ·of these, batches of strain "P" were also prepared by dilution and used on some of the rabbits. The dose was 0·5 cc., given subcutaneously to some, intradermally to others. The reactions to the vaccinating dose and the results of challenge 30 to 195 days later with virulent myxoma virus were noted. The results, detailed in Table 6, show that 50 out of 70, or 71.4 per cent, of rabbits inoculated with the "F" substrain reacted to the fibroma virus, as compared with 45 out of 60, or 75 per cent, -of those which received the "P" substrain. This difference is probably of no significance. On the other hand, 49 of the 60, or 81·7 per cent, of the rabbits inoculated intradermally reacted, as compared with 46 of the 70, or 65·7 per cent, of the rabbits inoculated :subcutaneously, a difference which suggests that the intradermal route provokes more severe reactions. Unfortunately, a number of the rabbits, kept for challenge from .the looth day onwards, died. However, results were available from
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
50 rabbits that had received the "F" substrain and from 38 that had received the "P" substrain. They suggest that the "F" subs train produced a better immunity since 14 per cent succumbed to challenge, as compared with 26 per cent vaccinated with the "P" subs train, and that the intradermal route gave a better protection (93 per cent) than the subcutaneous (78 per cent). TABLE 6 SUMMARY OF THE RESULTS OF THE COMPARATIVE TESTS ON THE SUBSTRAINS OF SHOPE'S FIBROMA VIRUS
Fibroma strain
Australian 'F'
Period between vaccination and NonNo . specific No. challenge Route reacted mortality challenged (days )
Numbers of rabbits inoculated
Date
10
19·5·54
i/d
17.6,54
sic
20
-9 15
I
8
7 2
I
101 195
0
6
I
I
I
0
5
I
I
I
5
I
0
I
4
5
4
6
104
15
29· I 2·54
sic
7
5
10
169
15
29. 12 .54
i/d
9
2
13
50
D.
0 0 0
10
70
R.
3
i/d
- - -
I.
- - -- -
30 104 166
17.6,54
--
- - -- -
6 4 2
10
-- -
Result
16 9
I
I
I
7 1 5
I
- -j - 23
7
20
. . ... .. . . . .. .. .. . . . . . .... .. . . . . ... . .. .. . . . " . . .. . . . . . . . .. . .. . ... .. . . . . .. French
, P'
20
sic
20
8
6 6
30 104
10
17.6,54
ild
10
8
2
104
15
29· I 2.54
sic
4
3
12
16 9
29. 12.54
i/d
II
3
12
15
- - --
-
17.6,54
60
-_._ \- - ~;-I-- -
Total number of rabbits inoculated with each strain Non specific deaths . . N umber challenged in each group Total immune Total recovered Total mortality Intradermal inoculation : Total Immune Recovered Died Subcutaneous inoculation : Total Immune Recovered Died . .
,
5 4
I
0
I
0
I
I
0
5
6
I 3
3
I
II
-1- --'I-169
6
i
, F'
70 20 50 23 (46 % ) 20 (40%) 7 (14% ) 28 13(46 '5%) 13(46 '5%) 2(7 % ) 22 10(45% ) 7(33%) 5(22%)
17
I I
i
i
9
' P'
60 22 38
17 (45% ) (29% ) 10 (26% ) 14 7(5°% ) 4(28 ·6% ) 3(21'4%) 24 10(42.6%) 7(28 ' 7% ) 7(28 '7%) II
BARBARA ROWE, W. MANSI AND
J.
R. HUDSON
297
DISCUSSION
Fibroma virus, in the form of a concentrate vaccine, was found to be viable, when kept either frozen at -40DC. or freeze-dried, for over a year. Although, by freeze-drying, the keeping properties 0' the vaccine can be greatly extended, the increased cost of producing such a preparation would probably make its price uneconomic. The fresh vaccine has, if properly handled, a life of fourteen days, and this should be ample for use in a country with good communications. I t is clear from the work of Andrewes (1936) that the virus of Shope's fibroma is liable to mutate into a strain giving an atypical reaction; thus, it was necessary to passage the original "P" strain as little as possible. It may be stated that in all the rabbits inoculated at Weybridge during two and a half years, no lesions have been detected suggesting a mutation of the virus in the direction of the inflammatory type of the original fibroma virus reported by Andrewes (1936). It is generally recognised that some rabbits which have been inoculated with Shope's fibroma virus are completely 'resistant to inoculation of myxoma virus, others react to the inoculation of myxoma virus, often quite severely, but eventually recover, while a third group behave like fully-susceptible rabbits dying within the normal period after challenge. Most of those that are protected probably remain resistant for twelve months (Fenner and Woodroofe, 1954). Revaccination every six months is recommended if myxomatosis continues as a threat to tame rabbits in any area. Comparison between the protection against myxomatosis afforded by the "P" and "F" subs trains of Shope's fibroma, indicated the superiority of the latter. The results recorded in Table 6 suggest that the "F" substrain produced a better immunity since 14 per cent succumbed to challenge as compared with 26 per cent vaccinated with the "P" substrain. Fenner and Woodroofe (1954) reported that, using 1,000 ID/50 of their "F" substrain of the Boerlage strain given intradermally, they had immunized seventeen rabbits; representative animals drawn for challenge 8 to 52 weeks afterwards invariably survived. Seven had no local lesion and most of the others developed no secondary manifestations. These results were considerably better than those reported in the present paper. Intradermal inoculation gave 14.2 per cent more protection than subcutaneous. In some virus diseases the offspring of an immune dam receive antibodies in the colostrum or through the placenta. While such antibodies are circulating in their blood, the young animals will not react to the inoculation of a vaccinating dose of virus contained in a living virus vaccine. Results recorded in Table 4 indicated that no protection is afforded to the young of myxoma-immune does against inoculation of either fibroma or myxoma viruses. During the first
298
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
week of life it is unsafe to vaccinate any young rabbits. During the second week, although the young, born to a vaccinated doe, can be inoculated without risk, the offspring of a susceptible doe may still die following treatment. Hyde and Gardner (1939) have reported deaths in rabbits inoculated before they were more than three days old. In very young rabbits which die, the fibroma virus generalizes and a clinical picture is produced similar to that recorded as· developing very rarely in older, vaccinated domesticated rabbits, by Jacotot, Vallee and Virat (1954a). CONCLUSIONS
The paper describes in detail experiments made to answer practical questions that arise in regard to vaccination of tame rabbits against myxomatosis with Shope's fibroma virus. ACKNOWLEDGMENT
We are grateful to Dr. H. jacotot of the Institut Pasteur, Paris, and Professor F. Fenner, Australian National University, for the provision of strains of virus, and to the former for much useful information on the routine preparation of vaccine. REFERENCES
Andrewes, C. H. (1936).]. expo Med., 63, 157. Berry, G. P., and Litchty, j. A. (1936).]. Bact., 31,49. Fenner, F., and Woodroofe, Gwendolyn, M. (1954). Aust. ]. expo Bioi. 32,653· Hurst, E. W. (1938). Ibid., 16,265. Hyde, R. R. (1939). Amer.]. Hyg., 30,47. Hyde, R. R., and Gardner, R. E. (1939). Ibid., 57. jacotot, H., and Vallee, A. (1953). Ann. Inst. Past. Paris., 85, 133. jacotot, H., Vallee, A., and Virat, B. (1954a). Bull. Acad. vet. Fr., 27, 105; (1954b). Ibid., 465. Martin, C. j. (1936). Bull. Coun. sci. indust. Res. Aust. No. 96. Ritchie,j. N., Hudson,j. R., and Thompson, H. V. (1954). Vet. Rec., 66, 79 6 . Shope, R. E. (1936).]. expo Med., 63,33; Ibid., 43; (1938). Proc. Soc. expo Biol. N.Y., 38,86. [Received for publication, February 15th, 1956]
COMPo
PATH.
1956. VOL. 66.
THE USE OF FIBROMA VIRUS (SHOPE) FOR THE PROTECTION OF RABBITS AGAINST MYXOMATOSIS By
J.
BARBARA ROWE, W. MANSI AND R. HUDSON Veterinary Laboratory, Ministry cif Agriculture, W'!Ybridge INTRODUCTION
Shope (1936, 1938) showed that the inoculation of fibroma virus conferred a high degree of protection to rabbits against experimental inoculation with myxoma virus. He reported that fifty-nine out of sixty-two of his fibroma-treated rabbits resisted challenge by inoculation with the latter. The early results of Shope were confirmed by Berry and Litchty (1936), Martin (1936), Hurst (1938), Hyde (I939),Jacotot and Vallee (1953), and Fenner and Woodroofe (1954), and a brief summary of early work performed at Weybridge was given by Ritchie, Hudson and Thompson (1954). The percentage of rabbits that have been found to have acquired a serviceable immunity after inoculation with fibroma virus has varied from one worker to another. The present paper gives a detailed account of our investigation of the keeping properties of the vaccine, the duration of immunity, the safety of the vaccine for young rabbits, the effect of immunity of the dam on the result of vaccinating suckling rabbits and the relative value of the two sub-strains of the Boerlage strain of fibroma virus isolated by Shope in 1947, given subcutaneously and intradermally. MATERIALS AND METHODS
Rabbits. Seven hundred and ninety-nine rabbits were used in these experiments. Some were purchased in the open market, some were bred, but none had been immunized before being put on an experiment. They varied in age and breed. Fibroma virus (Shope). Two sub-strains of the Boerlage strain of this virus were used. The first, obtained from Dr. Jacotot in September, 1953, is referred to as "P", and the second, from Professor Fenner, is referred to as "F". Vaccine was normally prepared by the method used at the Pasteur Institute, Paris. Male rabbits were inoculated into each testicle and subcutaneously at four or more sites with 0·5 cc. doses of a ten per cent suspension of material harvested previously. The seed virus came from a bank, stored frozen at -40°C., and, in order to minimise the possibility of changes in the virus, no unnecessary passages were made. On the day before the maximum development of the tumours was expected, usually on the eighth day, the rabbits were killed and the testicles and subcutaneous, oedematous pulp were collected, weighed and homogenized in a "Magimix" household pulping machine in five volumes of five per cent whole-egg buffer of pH 7·4. This concentrated suspension was titrated on rabbits and stored frozen until required. The vaccine itself was then prepared by diluting the concentrate so that each 0·5 cc. dose contained 1,000 m.i.d. Usually, this meant diluting IO to 100 fold.
BARBARA ROWE, W. MANS[ AND
J.
R. HUDSON
Myxoma virus. A fully virulent strain, isolated from a rabbit from Cornwall in April, [954, was used. The material was kept frozen during the early part of the work, but in September, [954, freeze-dried material was prepared and, since then, challenge has been mainly with this material. Inoculation of this strain produced typical myxomatosis in susceptible rabbits, with death in I I to 15 days. The preparation before freeze-drying contained between 10-6 and 10-10 m.i.d. per cc. The challenge dose was at least 1,000 m.i.d. and was inoculated intradermally or subcutaneously. RESULTS
Keeping Properties of the Vaccine Concentrate A number of rabbits were inoculated with the original material from Paris and the virus thus obtained has been used to infect all the vaccine producers required in our work. Retention of the concentrated vaccine for several months made it necessary to determine the period during which it could be kept without loss of activity. Table I shows that when stored frozen at -400e. the TABLE
I
KEEPING PROPERTIES OF THE VACCINE CONCENTRATE ( 1/5)
Period Temperature in weeks -40 °C.
+4°C .
I
2 9 15 32 36 46 52 I
2 39 58 +4°C .
69
88 1= Immune.
Numbers Fibroma inoculated reaction
Diluent Buffer saline pH 7'4
"
"
" " "
"
" " " "
" " " " " " " "
" " "
"
" " " "
" "
13 3 5 43 3 5 3 3
Beef-heart infusion broth pH 7 ' 4 and freeze-dried
"
"
"
"
Buffer saline pH 7'4 and freeze-dried
12 3 5
II
2 2
3
0
Myxoma challenge
- - - -- D. I. R. -II
0
2
I
I
I
2 29 I
2 2 3
4
3 3 3
3 3
2
2
I
3
3
2
R = Recovered.
I
5
2 2
0
I
0
0 0
0
0
0 0
I
- - - -- -
4
2
2 9
4
3 3 2
I
0 0 0
I
0
0
I
- - ---
D=Died.
concentrate can be held for a year. After freeze-drying it may be kept at -4°e. for a longer period, both in buffer saline or in beefheart infusion broth. Tests after longer periods of storage have still to be made.
Keeping Properties of the Diluted Vaccine A series of preparations were made from one batch of concentrate (sub-strain P) by diluting with the following:- (a) phosphate
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
buffer saline pH 7'4; (b) phosphate buffer saline pH 7'4 containing five per cent whole egg; (c) phosphate buffer with I: 100,000 thiomersalate. Part of the preparation containing egg, but without thiomersalate was freeze-dried. Samples of the liquid preparations were stored at room temperature and at +4 ce. Freeze-dried material was stored at +4 ce. and reconstituted to the original volume immediately before use. The results of tests of these samples are given in Table 2. It will be observed that neither the addition TABLE
2
KEEPING PROPERTIES OF FIBROMA VIRUS SUSPENSIONS OF VACCI:--iE STRE:--iGTH
Periodqf storage in weeks
Preparation and temperature of storage
Number qf rabbits ----,---,-----_._---1 Results of challenge Inoculated Reacted Immune Recovered Died
--1--2--
---I-B-u-ffe-r-s-a-li-n-e-p-H-7-·4-+-4-oC-. Egg buffer +4°C. Eggbuffer+thiomersalate +4°C.1
--2--
--3--1-~--~
2
1
1
-
3
2
2
-
Buffer saline pH 7'4 +4°C. Egg buffer +4°C. Egg buffer+thiomersalate + 4°C.
7 3 3
6
5 3
Buffer saline pH 7'4 +4°C. Egg buffer +4° C . Egg buffer+thiomersalate +4°C.
4 4 4
4 4 4
Egg buffer Room temperature Egg buffer+thiomersalate
2
2
4
4
20
Egg buffer, freeze-dried +4°C.
3
3
2
32
-HOC.
3
3
3
41
+4°C .
3
3
94
+4°C .
3
2
20
2 2
1
-
2
I
3
of egg proteins as protective colloids nor thiomersalate as a bacteriostatic agent improved the keeping qualities of the vaccine so that it gave any protection after storage for five months in the refrigerator. Freeze-drying enabled the vaccine to be kept for periods up to twenty-two months, but the extra cost entailed in producing a freeze-d:ied vaccine would perhaps render such a product uneconomIC. Duration of Immunity The rabbits included in Table 3 were inoculated with different batches of vaccine at various times, but the vaccine was always standardized to contain about 1,000 minimum infective doses of fibroma virus and it was always used fresh. It is felt that this table gives a general picture of what may be expected of the vaccine and it is probably a better guide to the value of the vaccine in the field
BARBARA ROWE, W. MANSI AND
J.
293
R. HUDSON
than an experiment designed with one batch of vaccine on a single large group of rabbits. It will be observed that about three quarters of the rabbits vaccinated had a serviceable immunity for about a year. TABLE 3 DURATION OF IMMUNITY
Period between vaccination and challenge
Number of rabbits vaccinated
Result of challenge Immune
Recovered
Died
0(%)
..
·.
34
29 (86%)
5 (14%)
Between 60-120 days
·.
41
24 (60%)
11(28%)
6 (12%)
Between 120-200 days
67
24 (3 6 %)
26 (39%)
17 (25%)
Between 200-300 days
.. ..
36
26 (73%)
4 (II%)
6 (16%)
Between 300-400 days
..
12
6 (50%)
3 (25%)
3 (25%)
Between 400-500 days
..
14
3 (22%)
1(7%)
·.
4
0(0%)
1(25%)
Under 60 days
Over 500 days
..
10
(71%)
3 (75%)
Safety and Value of the Vaccine in Baby Rabbits The following experiments were designed to find out how young, suckling rabbits could be vaccinated safely and whether the immunity of the dam interfered with the result of vaccination. Many litters of rabbits were used in these experiments and several different batches of freshly-prepared vaccine. The results are given in Table 4 in which are also included the results of tests made with myxoma virus. It will be observed that during the first three weeks of life the offspring of fibroma-immune rabbits are less likely to react to a dose of fibroma virus than are the offspring of a susceptible rabbit. Contrary to what is found with fibroma virus, it is clear that no protection is afforded to the young of myxomaimmune does against inoculation with myxoma virus. This result confirms those reported by Jacotot, Vallce and Virat (I 954b). Unfortunately, a proportion of the young rabbits was lost before the second part of this experiment, an attempt to study the possibility that the young of a vaccinated dam might not respond as well as those of a susceptible dam to vaccination, could be completed. Nevertheless, it was possible to challenge 81 offspring from vaccinated does, themselves vaccinated when two to seven weeks old, and 14 offspring from normal does, vaccinated when four to seven weeks old. The results (Table 5) suggest that the immunity of the doe does not affect the development of immunity in the young. Effect of Vaccination on Pregnant Does Pregnant does did not abort as a result of the vaccination, but we observed some abortions in pregnant rabbits which were in-
I
8
-12
20
5
6 -
9
8
5 5
4
3
2
I1
i
,- -;--
·.
8
I
3
·. ·.
7
6
5
3
4
..
Number inoculated
0
0 20
5 20
3
8
8
0
5
5
5
5
0
0
Died
0
5
3
3
Reacted
5
I I
I
0
0
0
0
No reaction
I
Offspring of myxoma-immune rabbits
· .
I
I
ciffibroma-immune
I
4
21
5
10
12
II
8
0
0
0
0
8
I
20
5
5
18
0
0
0
0
20
5
5
18
1
20
5
3
18
- - -- - - -- - - - -- - - -
..
9
31
16
II
22 28
0
8
I I
20
0
5
5
I
I
1 - -- - -1
10
8
3
15
36
14
24
4
0
5
0
0
3
5
I
12
24
I
0
0
!
I
I
20
5
5
5
7
3
2
3
12
13
24
4
0
0
0
0
0
0
6
4
Die~
20
5
5
5
---
I
Reacte~
Offipring of normal rabbits
Number No I Die~ ~noculate~ ~tion
rabbits
Number No inoculated :..,eaction -.!!eacted
--- --
Offspring
Offspring of myxoma immune, fibroma immune and normal rabbits.
3
2
I
Age in weeks when inoculated
MY"om· 1
Fibroma ..
Virus inoculated
TABLE 4-
SUSC EPTIBILITy TO EXPERIMENTAL INFECTION WITH FIBROMA AND MYXOMA VIRUSES
<
til
VJ
o
..,
-< >: o :::;J>
:::
~ ~..,
;J>
~
i:Ij i:Ij
;J>
'"
o"1
Z
(3
..,;J>
Z
lJ lJ
;J>
~
to .....
BARBARA ROWE, W. MANSI AND
J.
295
R. HUDSON
advertently provided as experimental subjects, frequently handled and unsuitably housed. These observations were restricted to a very :small number of does. TABLE 5 IMMUNITY TESTS ON RABBITS, THE PROGENY OF VACC Il'ATED AND NORMAL DOES
Vaccinated when
I
Age at ____ va_cc_in_a__ti_on_I ____D_o_e_ _ _ 2
! Vaccinated
2 -
7 weeks of age.
I,. _ _ Result Number chalienged__ I__ .!~
__ _R_.__
5
3
4 4 5
5
6
7
Normal 1= Immune.
o
o
4
o
5
o
13
8
4
19
12
5
34
7
4
o
, Normal Vaccinated
o
13 R = Recovered.
_ I._D_-_ __
2
4
8
4
D=Died.
'Comparison of the Protection against Myxomatosis Afforded by Two ,Substrains of the Boerlage Strain of Fibroma Virus and Using Two Routes ,of Inoculation It was desirable to make a comparison between the two substrains, "P" and "F," and the different routes of inoculation. Accordingly, a vaccine-concentrate was prepared from substrain "F" and suitable dilutions were used on three occasions. On two ·of these, batches of strain "P" were also prepared by dilution and used on some of the rabbits. The dose was 0·5 cc., given subcutaneously to some, intradermally to others. The reactions to the vaccinating dose and the results of challenge 30 to 195 days later with virulent myxoma virus were noted. The results, detailed in Table 6, show that 50 out of 70, or 71.4 per cent, of rabbits inoculated with the "F" substrain reacted to the fibroma virus, as compared with 45 out of 60, or 75 per cent, -of those which received the "P" substrain. This difference is probably of no significance. On the other hand, 49 of the 60, or 81·7 per cent, of the rabbits inoculated intradermally reacted, as compared with 46 of the 70, or 65·7 per cent, of the rabbits inoculated :subcutaneously, a difference which suggests that the intradermal route provokes more severe reactions. Unfortunately, a number of the rabbits, kept for challenge from .the looth day onwards, died. However, results were available from
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
50 rabbits that had received the "F" substrain and from 38 that had received the "P" substrain. They suggest that the "F" subs train produced a better immunity since 14 per cent succumbed to challenge, as compared with 26 per cent vaccinated with the "P" subs train, and that the intradermal route gave a better protection (93 per cent) than the subcutaneous (78 per cent). TABLE 6 SUMMARY OF THE RESULTS OF THE COMPARATIVE TESTS ON THE SUBSTRAINS OF SHOPE'S FIBROMA VIRUS
Fibroma strain
Australian 'F'
Period between vaccination and NonNo . specific No. challenge Route reacted mortality challenged (days )
Numbers of rabbits inoculated
Date
10
19·5·54
i/d
17.6,54
sic
20
-9 15
I
8
7 2
I
101 195
0
6
I
I
I
0
5
I
I
I
5
I
0
I
4
5
4
6
104
15
29· I 2·54
sic
7
5
10
169
15
29. 12 .54
i/d
9
2
13
50
D.
0 0 0
10
70
R.
3
i/d
- - -
I.
- - -- -
30 104 166
17.6,54
--
- - -- -
6 4 2
10
-- -
Result
16 9
I
I
I
7 1 5
I
- -j - 23
7
20
. . ... .. . . . .. .. .. . . . . . .... .. . . . . ... . .. .. . . . " . . .. . . . . . . . .. . .. . ... .. . . . . .. French
, P'
20
sic
20
8
6 6
30 104
10
17.6,54
ild
10
8
2
104
15
29· I 2.54
sic
4
3
12
16 9
29. 12.54
i/d
II
3
12
15
- - --
-
17.6,54
60
-_._ \- - ~;-I-- -
Total number of rabbits inoculated with each strain Non specific deaths . . N umber challenged in each group Total immune Total recovered Total mortality Intradermal inoculation : Total Immune Recovered Died Subcutaneous inoculation : Total Immune Recovered Died . .
,
5 4
I
0
I
0
I
I
0
5
6
I 3
3
I
II
-1- --'I-169
6
i
, F'
70 20 50 23 (46 % ) 20 (40%) 7 (14% ) 28 13(46 '5%) 13(46 '5%) 2(7 % ) 22 10(45% ) 7(33%) 5(22%)
17
I I
i
i
9
' P'
60 22 38
17 (45% ) (29% ) 10 (26% ) 14 7(5°% ) 4(28 ·6% ) 3(21'4%) 24 10(42.6%) 7(28 ' 7% ) 7(28 '7%) II
BARBARA ROWE, W. MANSI AND
J.
R. HUDSON
297
DISCUSSION
Fibroma virus, in the form of a concentrate vaccine, was found to be viable, when kept either frozen at -40DC. or freeze-dried, for over a year. Although, by freeze-drying, the keeping properties 0' the vaccine can be greatly extended, the increased cost of producing such a preparation would probably make its price uneconomic. The fresh vaccine has, if properly handled, a life of fourteen days, and this should be ample for use in a country with good communications. I t is clear from the work of Andrewes (1936) that the virus of Shope's fibroma is liable to mutate into a strain giving an atypical reaction; thus, it was necessary to passage the original "P" strain as little as possible. It may be stated that in all the rabbits inoculated at Weybridge during two and a half years, no lesions have been detected suggesting a mutation of the virus in the direction of the inflammatory type of the original fibroma virus reported by Andrewes (1936). It is generally recognised that some rabbits which have been inoculated with Shope's fibroma virus are completely 'resistant to inoculation of myxoma virus, others react to the inoculation of myxoma virus, often quite severely, but eventually recover, while a third group behave like fully-susceptible rabbits dying within the normal period after challenge. Most of those that are protected probably remain resistant for twelve months (Fenner and Woodroofe, 1954). Revaccination every six months is recommended if myxomatosis continues as a threat to tame rabbits in any area. Comparison between the protection against myxomatosis afforded by the "P" and "F" subs trains of Shope's fibroma, indicated the superiority of the latter. The results recorded in Table 6 suggest that the "F" substrain produced a better immunity since 14 per cent succumbed to challenge as compared with 26 per cent vaccinated with the "P" substrain. Fenner and Woodroofe (1954) reported that, using 1,000 ID/50 of their "F" substrain of the Boerlage strain given intradermally, they had immunized seventeen rabbits; representative animals drawn for challenge 8 to 52 weeks afterwards invariably survived. Seven had no local lesion and most of the others developed no secondary manifestations. These results were considerably better than those reported in the present paper. Intradermal inoculation gave 14.2 per cent more protection than subcutaneous. In some virus diseases the offspring of an immune dam receive antibodies in the colostrum or through the placenta. While such antibodies are circulating in their blood, the young animals will not react to the inoculation of a vaccinating dose of virus contained in a living virus vaccine. Results recorded in Table 4 indicated that no protection is afforded to the young of myxoma-immune does against inoculation of either fibroma or myxoma viruses. During the first
298
VACCINATION OF RABBITS AGAINST MYXOMATOSIS
week of life it is unsafe to vaccinate any young rabbits. During the second week, although the young, born to a vaccinated doe, can be inoculated without risk, the offspring of a susceptible doe may still die following treatment. Hyde and Gardner (1939) have reported deaths in rabbits inoculated before they were more than three days old. In very young rabbits which die, the fibroma virus generalizes and a clinical picture is produced similar to that recorded as· developing very rarely in older, vaccinated domesticated rabbits, by Jacotot, Vallee and Virat (1954a). CONCLUSIONS
The paper describes in detail experiments made to answer practical questions that arise in regard to vaccination of tame rabbits against myxomatosis with Shope's fibroma virus. ACKNOWLEDGMENT
We are grateful to Dr. H. jacotot of the Institut Pasteur, Paris, and Professor F. Fenner, Australian National University, for the provision of strains of virus, and to the former for much useful information on the routine preparation of vaccine. REFERENCES
Andrewes, C. H. (1936).]. expo Med., 63, 157. Berry, G. P., and Litchty, j. A. (1936).]. Bact., 31,49. Fenner, F., and Woodroofe, Gwendolyn, M. (1954). Aust. ]. expo Bioi. 32,653· Hurst, E. W. (1938). Ibid., 16,265. Hyde, R. R. (1939). Amer.]. Hyg., 30,47. Hyde, R. R., and Gardner, R. E. (1939). Ibid., 57. jacotot, H., and Vallee, A. (1953). Ann. Inst. Past. Paris., 85, 133. jacotot, H., Vallee, A., and Virat, B. (1954a). Bull. Acad. vet. Fr., 27, 105; (1954b). Ibid., 465. Martin, C. j. (1936). Bull. Coun. sci. indust. Res. Aust. No. 96. Ritchie,j. N., Hudson,j. R., and Thompson, H. V. (1954). Vet. Rec., 66, 79 6 . Shope, R. E. (1936).]. expo Med., 63,33; Ibid., 43; (1938). Proc. Soc. expo Biol. N.Y., 38,86. [Received for publication, February 15th, 1956]