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Rinderpest Interference With Caprinized Rinderpest Virus
J. COMPo PATH.
222
Ig61. VOL. 71
RINDERPEST INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS By
J.
K. H.
WILDE*
Department of Veterinary Services, Tanganyika
and
G. R.
SCOTT
East African Veterinary Research Organization, Muguga, Kef!JIa INTRODUCTION
The so-called interference phenomenon has been well documented in recent years but in the late thirties interference between animal viruses was still a novel concept. In 1938 Pfaff ( I 940) vaccinated cattle with caprinized rinderpest virus. Two animals were inoculated with virulent rinderpest virus 24 hours later and although both developed rinderpest they recovered. Two other cattle resisted the effects of virulent virus given 48 hours after vaccination. Pfaff attributed the protection to the rapid development of immunity which he considered was serviceable at 24 hours and absolute in 48 hours. In the light of present knowledge a sounder hypothesis attributes the protection to interference of the virulent virus by the attenuated caprinized virus. Many field veterinarians in Africa hold the entrenched belief that caprinized rinderpest virus has a therapeutic action in animals incubating or suffering from rinderpest. Edwards (1950) concurred and attributed the action to interference, but he failed to support his opinions experimentally. The confirmation that caprinized rinderpest virus interferes with virulent rinderpest virus and that it lacks therapeutic action are herein reported. MATERIALS AND METHODS
Caprinized rinderpest virus. The Kabete attenuated (KAG) strain of caprinized rinderpest virus was used. It has been employed widely and successfully in East Africa as a live virus vaccine. The virus was packed in vacuum-sealed ampoules in the form of powdered freeze-dried infected goat spleens. The ampoules were kept at refrigerator temperatures until required and the contents were reconstituted with saline just prior to inoculation. Virulent rinderpest virus. The Mpwapwa (M) strain of virulent rinderpest virus in the form of suspensions of infected bovine spleen was used. Unexpectedly the strain proved to be relatively avirulent and the case mortality rate was only 10 per cent. Cattle. Tanganyika Shorthorn Zebu steers were used. They had never been in contact with rinderpest and had never been vaccinated. *Present address: Wellcome Research Laboratory, Kabete, Kenya.
J.
223
K. H. WILDE AND G. R. SCOTT
Experimental design. The cattle were divided into eleven groups of four animals. Group A were given M virus alone, Groups B, C and D were given M virus followed by KAG 48, 24 and 13 hours later respectively. Group E were given M and KAG simultaneously. Groups F, G, H, I and J were given KAG followed by M virus I I, 24, 33, 48 and 57 hours later respectively and group K were given KAG alone. The dose of KAG was the routine field dose. The dose of M virus was equivalent to one gramme of wet spleen. Aliquots of the same bovine spleen were used throughout. At the end of the experiment the remaining spleen was titrated in cattle and proved to have a titre in excess of 10-6 ID50 per gramme. The cattle were kept under conditions as nearly identical as possible. Rectal temperatures were taken every morning and the cattle were carefully examined at short intervals. Particular attention was paid to clinical signs which were assessed for severity by a point system (Table I). The system was quite arbitrary but was based on the experiences of the authors. All surviving cattle were challenged three weeks later with virulent rinderpest virus. Statistical analysis. The data were subjected to the Analysis of Variance and significant differences among groups were determined by the Multiple Range Test (Duncan, 1955). RESULTS
(;ase lkfortalitv ~ate Only four (g. I per cent) cattle died. Two deaths occurred in cattle given M virus before KAG and two deaths occurred in cattle given KAG before M virus. TABLE I ASSESSMENT SYSTEM
System
Signs
Points
Ocular
points 4
Injected conjunctivae Slight injection Conjunctival catarrh Ocular discharge Slight ocular discharge
2
Nasal discharge Slight discharge
2
Erosions Erythema of mucous membrane Slight erythema of mucous membrane Inflammation of gums Erythema of gums "Rinderpest smell" Slight smell
3
Dysentery Diarrhoea Slight diarrhoea
3
1
2 2 1
Nasal
2 1
Oral
5 I
0'5 2 1
2 I
Intestinal
Death
Maximum
3 2 1 14
224
INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS TABLE
2
ASSESSMENT OF CLINICAL SIGNS
Group
A
Treatment
Cattle
Malone
4 10 411 412 4 13
Clinical assessment 8°0 °0 8°0 8 °5
II
B
M 48 hours before KAG
414 4 15 4 16 4 17
II
C
M 24 hours before KAG
4 18 4 19 4 20 421
II
D
M 13 hours before KAG
422 423 424 4 25
10°0 8°0 7°5 14° 0 *
E
M and KAG simultaneously
4 26 427 428 4 29
F
KAG 11 hours before M
43 0 43 1 43 2 433
°0 10°0 14° 0 * 5° 0 °0 8°0 13° 0 12°0
5° 0 I I °0
8 °5 12°0 II
°0
I I °0
14°0 *
I I °0
G
KAG 24 hours before M
434 435 43 6 437
8°0 7° 0 6 °5 8°0
H
KAG 33 hours before M
43 8 439 440 441
9° 0 5"5 4° 0 14°0 *
I
KAG 48 hours before M
442 443 444 445
7° 0 4°5 1°5 4°5
J
KAG 57 hours before M
446 447 448 449
7° 0 3° 0 3° 0 2°0
K
KAG alone
450 45 1 45 2 453
2 °5 4°5 2 °5 1°0
* Diedo
J.
225
K. H. WILDE AND G. R. SCOTT
Reaction Assessments The cattle given M virus alone had a point average of8·875 with a standard deviation of I '44 whereas the group given KAG alone had a point average of 2.625 with a standard deviation of I '44. The difference was highly significant (t = 6'158, P <0'001). Dual Infections Data are recorded in Table 2 and the Analysis of Variance is shown in Table 3. The Variance Ratio between treatments was 5.18 (d.f. 10, 30) which was highly significant (P< 0'01). The Variance Ratio between replicates was not significant. TABLE 3 ANALYSIS OF VARIANCE OF THE CLINICAL ASSESSMENTS
Source
Degrees of freedom
Sum of squares
.
Mean square
Between treatments
..
..
10
37 1 '98
Between replicates
..
..
3
5"79
1'93 0
"
..
..
30
21 5'5 2
7' 184
..
..
..
43
593'29
..
Error
Total
37'198 **
The Multiple Range Test revealed that the group means fell into four series which differed significantly from each other (Table 4). The clinical assessments of the groups in the first series were indistinTABLE 4 GROUPS WITH SIGNIFICANTLY DIFFERENT CLINICAL ASSESSMENT MEANS
Series
I
Series
2
Series 3
Series -I
A
G
G
I
B
H
I
J
c
I
J
K
D
E F G
H
9'5 16
6.625
5" 16 7
3'58 3
226
INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS
guishable from the assessment made when M virus alone was inoculated. The groups included all those in which M virus was given before KAG, the group given M and KAG simultaneously and the groups in which KAG was given up to 33 hours before M virus. The clinical assessments of the groups in the fourth series were indistinguishable from the assessment made when KAG alone was inoculated. The groups were those in which KAG was given 48 and 57 hours before M virus. The second and third series had assessments which fell between that of M virus alone and KAG alone. The groups were all those given KAG virus 24 to 57 hours before M virus. In other words, the second series overlapped the first and the third series overlapped the fourth.
Immunity The surviving cattle proved to be Immune when challenged three weeks later. DISCUSSION
Despite the weakness occasioned by the virulent virus proving to be relatively mild which forced us to use an arbitrary assessment system for clinical severity, Pfaff's findings were confirmed, namely, that if caprinized rinderpest virus was administered 48 hours or more before virulent rinderpest virus the cattle were protected and underwent a caprinized virus reaction only. Neutralizing or complementfixing antibodies have never been detected in cattle within 48 hours of vaccination (Plowright and Ferris, 1958) and consequently the protection can only be attributed to interference. Our findings failed to support the belief that caprinized rinderpest virus possesses a therapeutic action in cattle already infected with virulent rinderpest virus. In fact, the simultaneous inoculation of both viruses and the inoculation of caprinized less than 33 hours before the virulent virus produced virulent rinderpest reactions. Unfortunately the virulent virus used in these experiments killed only g'l per cent of the cattle inoculated or 12'5 per cent of the cattle at risk and consequently the assessment of any therapeutic action could only be subjective and as such is open to criticism. Nevertheless the system adopted revealed highly significant differences between cattle given virulent virus only and cattle given caprinized virus only. CONCLUSIONS
Cattle inoculated with virulent rinderpest virus before, simultaneously or up to 33 hours after caprinized rinderpest virus underwent a typical virulent rinderpest reaction. Cattle given virulent virus 48 and 57 hours after vaccination with caprinized rinderpest virus did not contract virulent rinderpest but underwent a typical vaccine virus reaction.
J.
K. H. WILDE AND G. R. SCOTT
227
REFERENCES
Duncan, D. B. (1955). Biometrics, 11, I. Edwards, J. T. (1950). Vet. Rec., 52, 833. Pfaff, G. (1938). Onderstepoort J., ll, 261. Plowright, W., and Ferris, R. D. (1958). Rep. E. Afr. vet. res. Org. 1956-57, p.28. [Received for publication, December 19th, 1960]
222
Ig61. VOL. 71
RINDERPEST INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS By
J.
K. H.
WILDE*
Department of Veterinary Services, Tanganyika
and
G. R.
SCOTT
East African Veterinary Research Organization, Muguga, Kef!JIa INTRODUCTION
The so-called interference phenomenon has been well documented in recent years but in the late thirties interference between animal viruses was still a novel concept. In 1938 Pfaff ( I 940) vaccinated cattle with caprinized rinderpest virus. Two animals were inoculated with virulent rinderpest virus 24 hours later and although both developed rinderpest they recovered. Two other cattle resisted the effects of virulent virus given 48 hours after vaccination. Pfaff attributed the protection to the rapid development of immunity which he considered was serviceable at 24 hours and absolute in 48 hours. In the light of present knowledge a sounder hypothesis attributes the protection to interference of the virulent virus by the attenuated caprinized virus. Many field veterinarians in Africa hold the entrenched belief that caprinized rinderpest virus has a therapeutic action in animals incubating or suffering from rinderpest. Edwards (1950) concurred and attributed the action to interference, but he failed to support his opinions experimentally. The confirmation that caprinized rinderpest virus interferes with virulent rinderpest virus and that it lacks therapeutic action are herein reported. MATERIALS AND METHODS
Caprinized rinderpest virus. The Kabete attenuated (KAG) strain of caprinized rinderpest virus was used. It has been employed widely and successfully in East Africa as a live virus vaccine. The virus was packed in vacuum-sealed ampoules in the form of powdered freeze-dried infected goat spleens. The ampoules were kept at refrigerator temperatures until required and the contents were reconstituted with saline just prior to inoculation. Virulent rinderpest virus. The Mpwapwa (M) strain of virulent rinderpest virus in the form of suspensions of infected bovine spleen was used. Unexpectedly the strain proved to be relatively avirulent and the case mortality rate was only 10 per cent. Cattle. Tanganyika Shorthorn Zebu steers were used. They had never been in contact with rinderpest and had never been vaccinated. *Present address: Wellcome Research Laboratory, Kabete, Kenya.
J.
223
K. H. WILDE AND G. R. SCOTT
Experimental design. The cattle were divided into eleven groups of four animals. Group A were given M virus alone, Groups B, C and D were given M virus followed by KAG 48, 24 and 13 hours later respectively. Group E were given M and KAG simultaneously. Groups F, G, H, I and J were given KAG followed by M virus I I, 24, 33, 48 and 57 hours later respectively and group K were given KAG alone. The dose of KAG was the routine field dose. The dose of M virus was equivalent to one gramme of wet spleen. Aliquots of the same bovine spleen were used throughout. At the end of the experiment the remaining spleen was titrated in cattle and proved to have a titre in excess of 10-6 ID50 per gramme. The cattle were kept under conditions as nearly identical as possible. Rectal temperatures were taken every morning and the cattle were carefully examined at short intervals. Particular attention was paid to clinical signs which were assessed for severity by a point system (Table I). The system was quite arbitrary but was based on the experiences of the authors. All surviving cattle were challenged three weeks later with virulent rinderpest virus. Statistical analysis. The data were subjected to the Analysis of Variance and significant differences among groups were determined by the Multiple Range Test (Duncan, 1955). RESULTS
(;ase lkfortalitv ~ate Only four (g. I per cent) cattle died. Two deaths occurred in cattle given M virus before KAG and two deaths occurred in cattle given KAG before M virus. TABLE I ASSESSMENT SYSTEM
System
Signs
Points
Ocular
points 4
Injected conjunctivae Slight injection Conjunctival catarrh Ocular discharge Slight ocular discharge
2
Nasal discharge Slight discharge
2
Erosions Erythema of mucous membrane Slight erythema of mucous membrane Inflammation of gums Erythema of gums "Rinderpest smell" Slight smell
3
Dysentery Diarrhoea Slight diarrhoea
3
1
2 2 1
Nasal
2 1
Oral
5 I
0'5 2 1
2 I
Intestinal
Death
Maximum
3 2 1 14
224
INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS TABLE
2
ASSESSMENT OF CLINICAL SIGNS
Group
A
Treatment
Cattle
Malone
4 10 411 412 4 13
Clinical assessment 8°0 °0 8°0 8 °5
II
B
M 48 hours before KAG
414 4 15 4 16 4 17
II
C
M 24 hours before KAG
4 18 4 19 4 20 421
II
D
M 13 hours before KAG
422 423 424 4 25
10°0 8°0 7°5 14° 0 *
E
M and KAG simultaneously
4 26 427 428 4 29
F
KAG 11 hours before M
43 0 43 1 43 2 433
°0 10°0 14° 0 * 5° 0 °0 8°0 13° 0 12°0
5° 0 I I °0
8 °5 12°0 II
°0
I I °0
14°0 *
I I °0
G
KAG 24 hours before M
434 435 43 6 437
8°0 7° 0 6 °5 8°0
H
KAG 33 hours before M
43 8 439 440 441
9° 0 5"5 4° 0 14°0 *
I
KAG 48 hours before M
442 443 444 445
7° 0 4°5 1°5 4°5
J
KAG 57 hours before M
446 447 448 449
7° 0 3° 0 3° 0 2°0
K
KAG alone
450 45 1 45 2 453
2 °5 4°5 2 °5 1°0
* Diedo
J.
225
K. H. WILDE AND G. R. SCOTT
Reaction Assessments The cattle given M virus alone had a point average of8·875 with a standard deviation of I '44 whereas the group given KAG alone had a point average of 2.625 with a standard deviation of I '44. The difference was highly significant (t = 6'158, P <0'001). Dual Infections Data are recorded in Table 2 and the Analysis of Variance is shown in Table 3. The Variance Ratio between treatments was 5.18 (d.f. 10, 30) which was highly significant (P< 0'01). The Variance Ratio between replicates was not significant. TABLE 3 ANALYSIS OF VARIANCE OF THE CLINICAL ASSESSMENTS
Source
Degrees of freedom
Sum of squares
.
Mean square
Between treatments
..
..
10
37 1 '98
Between replicates
..
..
3
5"79
1'93 0
"
..
..
30
21 5'5 2
7' 184
..
..
..
43
593'29
..
Error
Total
37'198 **
The Multiple Range Test revealed that the group means fell into four series which differed significantly from each other (Table 4). The clinical assessments of the groups in the first series were indistinTABLE 4 GROUPS WITH SIGNIFICANTLY DIFFERENT CLINICAL ASSESSMENT MEANS
Series
I
Series
2
Series 3
Series -I
A
G
G
I
B
H
I
J
c
I
J
K
D
E F G
H
9'5 16
6.625
5" 16 7
3'58 3
226
INTERFERENCE WITH CAPRINIZED RINDERPEST VIRUS
guishable from the assessment made when M virus alone was inoculated. The groups included all those in which M virus was given before KAG, the group given M and KAG simultaneously and the groups in which KAG was given up to 33 hours before M virus. The clinical assessments of the groups in the fourth series were indistinguishable from the assessment made when KAG alone was inoculated. The groups were those in which KAG was given 48 and 57 hours before M virus. The second and third series had assessments which fell between that of M virus alone and KAG alone. The groups were all those given KAG virus 24 to 57 hours before M virus. In other words, the second series overlapped the first and the third series overlapped the fourth.
Immunity The surviving cattle proved to be Immune when challenged three weeks later. DISCUSSION
Despite the weakness occasioned by the virulent virus proving to be relatively mild which forced us to use an arbitrary assessment system for clinical severity, Pfaff's findings were confirmed, namely, that if caprinized rinderpest virus was administered 48 hours or more before virulent rinderpest virus the cattle were protected and underwent a caprinized virus reaction only. Neutralizing or complementfixing antibodies have never been detected in cattle within 48 hours of vaccination (Plowright and Ferris, 1958) and consequently the protection can only be attributed to interference. Our findings failed to support the belief that caprinized rinderpest virus possesses a therapeutic action in cattle already infected with virulent rinderpest virus. In fact, the simultaneous inoculation of both viruses and the inoculation of caprinized less than 33 hours before the virulent virus produced virulent rinderpest reactions. Unfortunately the virulent virus used in these experiments killed only g'l per cent of the cattle inoculated or 12'5 per cent of the cattle at risk and consequently the assessment of any therapeutic action could only be subjective and as such is open to criticism. Nevertheless the system adopted revealed highly significant differences between cattle given virulent virus only and cattle given caprinized virus only. CONCLUSIONS
Cattle inoculated with virulent rinderpest virus before, simultaneously or up to 33 hours after caprinized rinderpest virus underwent a typical virulent rinderpest reaction. Cattle given virulent virus 48 and 57 hours after vaccination with caprinized rinderpest virus did not contract virulent rinderpest but underwent a typical vaccine virus reaction.
J.
K. H. WILDE AND G. R. SCOTT
227
REFERENCES
Duncan, D. B. (1955). Biometrics, 11, I. Edwards, J. T. (1950). Vet. Rec., 52, 833. Pfaff, G. (1938). Onderstepoort J., ll, 261. Plowright, W., and Ferris, R. D. (1958). Rep. E. Afr. vet. res. Org. 1956-57, p.28. [Received for publication, December 19th, 1960]