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Rinderpest in Tissue Culture. III: Use of the Attenuated Strain as a Vaccine for Cattle
Brit. vet.
J . (1962),
ul, 141
RINDERPEST IN TISSUE CULTURE. III: USE OF THE ATTENUATED STRAIN AS A VACCINE FOR CATTLE By R. H.
JOHNSON
Federal Department of Veterinary Research, Vom, Nigeria
The two previous papers in this series have outlined the basic techniques and methods used in production and freeze drying of the rinderpest tissue culture virus and the use of qualitative and quantitative serum neutralization tests (Johnson, Ig62a, b). Plowright & Ferris (1959b) reported the use of the adapted and attenuated tissue culture rinderpest virus (TCRV) as a vaccine in East Mrican cattle, and the purpose of this report is to record the findings m West Mrican cattle vaccinated with t~e TORV strain. MATERIAL AND METHODS
The basic media and techniques have already been described (Johnson, 1962a, b). Virus for vaccine use is produced similarly to virus used for serum neutralization work. Bovine embryo kidney monolayers are used between the first and tenth passage levels, grown in a high sugar, high amino acid medium (HSH). The virus strain is that adapted and attenuated in tissue culture by Plowright & Ferris (I 959a, b) and is used between the 66th and 70th monolayer passages. Virus vaccine fluids are diluted 50 per cent in a modification of Mist. desiccans (Greaves, 1951; Johnson, Ig62b) and freeze dried in I ml. samples. SAFETY AND POTEN CY TESTING OF VACCINE BATCHES
Later in this paper it is shown that there is a very close relationship between the 50 per cent tissue culture infective dose (TCID so ) and the M1Dso for cattle. For this reason, cattle tests on vaccine batches concentrate on safety rather than potency. The concentration of virus in each batch of dried material is determined by titration in tissue culture (Johnson, 1962a). The following tests constitute the present checks on vaccine batches at Vom: A. In vitro tests Two I ml. ampoules of freeze-dried virus are titrated in tissue culture (Johnson, Ig62a) in 0'1 ml. samples over a twofold dilution range of 10 2 to 10 4 TCID 5o , being a corrected range per millilitre of 103 to 105 TCID so ' Undiluted virus is at the same time incubated with known positive and known negative rinderpest antisera in a serum neutrali7.ation (SN) test and the serumvirus mixtures are added to cell suspension.
BRITISH VETERINARY JOURNAL, lIB, 4
B. In vivo tests Two rinderpest-susceptible cattle checked by the SN test (Johnson, 1962b), are inoculated with the contents of one ampoule each of freeze-dried virus (50 doses). Two other susceptible bovines are inoculated with field dosage (1/50th of the contents of an ampoule). Morning temperatures and reactions ar{ recorded over a 14-day period. The four cattle, together with one control animal, are then challenged with 200 MID60 caprinized rinderpest virus (CRV) and temperatures taken for a further ten days. The criteria for passing a vaccine batch are: (a) An in vitro titre of not less than 103 . 6 TCID6o /ml. of the freeze-dried virus. The reason for this is associated with the dosage factor which is dealt with in the discussion. (b) No cytopathogenicity evident in the SN test containing positive serum. (c) No reactions other than mild hyperthermia in the four inoculated animals. (d) No reaction in the vaccinated cattle following challenge. RESULTS
A. Use of the strain as a vaccine in white Fulani (Zehu) cattle All initial work on TCRV was done on the humped White Fulani (Zebu) which is the only breed kept at these laboratories. Rinderpest-hypersusceptible breeds were later used in field trials. (i) Reactions foUowing vaccination Approximately 5,000 White Fulani (Zebu) cattle ranging in age from one day to three years have been vaccinated with doses of TCRV ranging from 1,000,000 to 10 TCID 60" Two hundred of these vaccinations were carried out at varying intervals using laboratory cattJe between 18 and 24 months old, which were kept under observation for periods up to 18 months after vaccination. The remaining 4,800 cattle were vaccinated in the field and the majority were calves varying from one day to nine months old. No adverse reactions have been reported from the field. In the 200 laboratory cattle there were no reactions other than a mild hyperthermia in occasional animals; 130 showed no observable rise in morning temperatures; 41 showed a rise of up to I ·5°F; i 9 a rise of between 1·5 and 3°F and loa rise of 3 to 4°F. These temperatures were noted between the 4th and 9th days after vaccination, depending on dosage and individual susceptibility, and they persisted for one to three days. More housed animals tended to show temperature reactions than those at grass. No other adverse signs or symptoms have been noted despite the fact that the majority of all vaccinations have been made during the local wet season when reactions to rinderpest vaccines are usually more severe. (ii) Antiho4J development following vaccination Following vaccination of White Fulani (Zebu) cattle with TCRV, antibodies to rinderpest, as determined by the SN test, first appear between
RINDERPEST IN TISSUE CULTURE, III
143
7 and 17 days. The higher the dose of virus the earlier can antibodies be detected within this period. Maximum levels are reached between two and four weeks and there is a slow decline up to 18 months when antibody is still present in fairly high concentration. Parallel estimations of antibody levels following vaccination with tissue culture virus (TCRV), caprinized (CRV), lapinized (LRV) and avianized (ARV) rinderpest viruses were made by complement fixation (CF) and SN tests on groups of cattle (Johnson & MacLeod, 1962). Results show that the use of TCRV results in the development of rinderpest antibodies to approximately the same degree as CRY. Antibody is formed at approximately the same rate following vaccination with field dosage of CRY, LRV and TCRV, but more slowly with ARV. Higher levels of antibody are produced by CRY and TCRV than with LRV or ARV. The decay rate in all four cases is approximately the same. (iii) Development of resistance to challenge Table I shows the reactions of White Fulani (Zebu) yearlings when challenged with 200 M1Dso CRY at 24-hourly mtervals following vaccination with low level doses of TCRV. At these levels of virus, antibody is not formed until ten to twelve days, but there is a resistance to challenge, without perceptible antibody present, at four days after vaccination. TABLE I DE VELOPMENT O P INTERPERENCE TO CHALLENGE
Animal No,
B8
Vaccine dosage
Time challenged after vaccination
Reaction after challenge
8
25
TCID ••
I
day
104'8° p
at
3
days
30
25
TCID ••
I
day
104'4°P
at
4
days
17
50
TCID ••
2
days
103'5° P
at
4
days
22
50
TCID 60
2
days
104,8 ° p
at
5
days
19
50
TCID••
3
days
103 '6° p
at
4
days
28
50
TCID ••
3
days
103'8°p
at
4
days
21
50
TCID ••
4 days
Nil
12
50
TCID ••
4 days
Nil
10
50
TCID••
5
days
Nil
26
25
TCID ••
5
days
102 '5°P /2
7
25
TCID ••
6
days
Nil
24
25
TCIDao
6
days
102 : 8°p / 2
29
25
TCID ••
7 days
Nil
31
25
TCID 60
7 days
Nil
days
days
144
BRITISH VETERINARY JOURNAL, 118, 4
Plowright & Ferris (1959b) demonstrated a similar interference phenomenon in East Mrica developing later than four days. Their method of challenge with setni-virulent rinderpest virus was superior to that used at Vom, where the attenuated CRV strain is used. CRV causes a typical marked hyperthermia in susceptible cattle. It will be obvious that a hyperthermia developing following challenge could conceivably be due to the original TCRV inoculation, as shown at six and seven days, although such reactions are comparatively rare and much milder. The absence of any temperature reactions at four and five days is, however, sufficient evidence that interference is present at this time. (iv) Duration of immuniry Seventy-two susceptible yearling cattle were vaccinated with 50 TCID 60 TCRV and run at grass with 12 controls. Antibody was demonstrable in undiluted sera (screening test) at two weeks after vaccination. At variable intervals following vaccination, three vaccinated animals with one control were bled for sera and challenged with 200 MIDso CRV. Up to 17 months all of 27 vaccinated cattle tested showed antibody on screening tests and all resisted challenge to 200MID 6o , the control animals at no time showing antibody and all responding typically to challenge. At 17 months, an outbreak of natural rinderpest on the station invalidated further work on this group. The remaining three controls died of rinderpest, whereas the remaining 45 TCRV vaccinated cattle continued healthy.
(v) Back passage of vaccine virus Attempts to demonstrate excretion of tissue culture vaccine virus from cattle vaccinated with 10,000 TCID 60 were made by housing 16 vaccinated cattle in close contact with six susceptible controls over a 28-day period. No evidence of excretion of TCRV could be found, as was shown by the complete susceptibility of the controls at the end of this period. Similarly, over a I7-month period, controls run at grass with TCRV-vaccinated cattle were still susceptible at the end of the period. To check for any increase in virulence should the strain be back-passaged in cattle, citrated blood samples were taken from a TCRV-vaccinated Zebu between the fourth and eighth days after vaccination. These were titrated for virus in vitro and samples showing the highest titre of virus were inoculated undiluted in 1 ml. samples into a second susceptible animal, the experiment being repeated through a further four passages. Over the five back passages, no apparent increase in virulence of the virus strain was demonstrated, no adverse response, other than mild hyperthermia, being noted at each back passage. (vi) Correlation of TCID 60 and M1D6o In order to have a firm basis for dosage estimation, the correlation between the in vitro and in vivo titre of virus was required. Plowright & Ferris (1959b) showed that in East Mrican cattle the relationship up to
RINDERPEST IN TISSUE CULTURE, III
the 45th virus passage was a very close one. Virus at the 67th, 68th and 70th passage levels, both wet and freeze dried, was titrated in tissue culture (Johnson, Ig62a) and the chilled dilutions were immediately inoculated in I ml. samples into groups of four rinderpest-susceptible Zebu cattle. Challenge with 200 M1D/iO CRY was carried out at two weeks after inoculation, presence of immunity being indicated by absence of a temperature response to challenge (Table II). Table II TABLE II CORRELATION OF TCID. o AND MID. o IN CATTLE
Vaccine passage level
MID.o/ml.
69th wet virus 69th dry virus 69th dry virus stored for
12
months at
- 2 2°C
70th wet virus
shows that there is a close relationship between the TCID so in vitro and the cattle M1D/io in vivo. The relationship is shown, on average, as 2 TCID 50 to I M1Dso for Zebu cattle.
B. Use of the strain as a vaccine in hypersusceptible cattle Following the laboratory trials in Zebu cattle, small field trials in hypersusceptible non-humped cattle were done with the co-operation of the West, East and Northern Nigerian Governments. Five such pilot trials, each involving between 12 and 20 hypersusceptible animals, have been carried out using Ndama, Keteku, Muturu and cross-bred animals of these breeds. All trial experiments have been carried out during the wet season when reactions to LRV in hypersusceptible cattle are liable to be severe and vaccinations have previously had to be restricted to the dry season. The results of the first trial in Ndama cattle are shown in Table III. They are a fair representation of the results in the second Ndama trial and trials in Keteku, Muturu, Keteku X Muturu and Keteku X Zebu cross-bred animals. During these trials the normal temperature of control animals was taken as 102 ·5 of; all animals on test were weighed daily and challenge was carried out two weeks after vaccination with 200 MIDIiO CRY. The controls included were (a) two uninoculated animals; (b) two animals which received field dosage ofLRV; and (c) four animals which received 10,000 TCID 50 TCRV, two being rinderpest-immune. Table III shows that both animals given LRV developed typical wet season reactions of diarrhoea, lacrimation, anorexia and cessation of rumination. No animal inoculated with TCRV showed any reaction other than a mild hyperthermia. In hypersusceptible breeds this hyperthermia is more frequent than in Zebu cattle, 85 per cent of inoculated animals showing the reaction
14 6
BRITIS H V ETERINAR Y JOURNA L , 118, 4 TABLE III RESU LTS OF TCR V FIELD TR IAL IN NDAMA CATTL E
Animal No .
Vaccine and dosage
Peak temperature after vaccination
Weight change over 14 days
Challenge results at 14 days
849
T CRV; 50 T C ID. o
103·2°F a t 5 days
+ 10 lb.
Nil
862
TCRV; 50 T C ID 50
102·5°F at 7 days
+2 lb.
Nil
899
TCRV ; 50 TCID. o
103°F at 4 days
+ 6 lb.
Nil
9 15
T CRV ; 50 TCID ••
104°F at 5 days
Nil
Nil
93 0
TCRV ; 50 TCID ••
104·2°F at 6 days
+4 lb.
Nil
93 1
TCRV; 50 TCID ••
I04·4°F at 8 days
+ 4 lb.
Nil
937
TCRV; 50 TCID ••
I02 ·4°F at 3 days
+8 lb.
Nil
938
T CR V ; 50 TCID••
102 ·8°F at 7 days
+ 5 Ib .
Nil
946
T CRV; 50 TCID••
102·5°F at 3 days
Nil
Nil
934
TCRV; 10,000 T CID ••
I03·8°F at 4 days
+ 2 lb.
Nil
955
TCRV; 10,000 T CID ••
103·2°F at 5 days
Nil
Nil
CONTROLS 943
Nil
102·4°F / 1 day
+5 lb.
Died 6 days
956
Nil
102· 2°F / 2 days
+ 4 lb.
Died 5 days
888
LRV; Field dose
104°F / 6 days
-7 lb.
Nil
890
LRV ; Field dose
105°F / 5 days
-15 lb.
Nil
Immune
TCRV; 10,000 TCID ••
102 ·6°F /
Nil
Nil
Immune
TCRV ; 10,000 TCID ••
102·5°F / 3 days
+5 lb.
Nil
II
days
between the fourth and ninth days. The temperature rise averaged I ·5°F and persisted for two to four days. Following the successful pilot trial results, wider field trials were made. Six thousand hypersusceptible yearlings, including a few imported Friesian calves, have been vaccinated to date, all during the local wet season. The dosage rate was 50 doses per ampoule of freeze-dried virus and, as a check on the potency of the vaccine under field conditions, the last five animals vaccinated from each ampoule were bled for serum before and three weeks after vaccination. All but two serum samples showed antibody development at three weeks. The development of antibody in hypersusceptible cattle vaccinated with this field dosage was checked by removal of serum samples from 12 cattle at 24-hour intervals from the 7th to 21st day. The average time when the appearance of antibody was initially detected in each animal was ten days after vaccination. No experiments on duration of immunity have yet been completed in hypersusceptible cattle. Antibody has, however, been .shown in sera taken from
RINDERPEST IN TISSUE CULTURE, III
147
Muturu calves IS months after vaccination with TCRV. These calves, ranging in age from one day to five months, were vaccinated with TCRV when an outbreak of natural rinderpest occurred among their dams, none of which had ever been vaccinated with rinderpest vaccine. Two calf controls and two vaccinated calves, presumably infected before vaccination, died of rinderpest out of 40 calves vaccinated. No adverse reactions to vaccination were noted.
C. Viability of the virus Having demonstrated the safety and immunizing properties of the virus in susceptible and hypersusceptible cattle, it was necessary to determine the viability of the virus under varying conditions. All virus fluids used were HSH, adjusted to pH 7.2 with 2 M-tris buffer and both wet and freeze-dried virus was further buffered with 50 per cent Mist. desiccans LAH (Johnson, Ig62a). Titrations were made in twofold ranges as previously described. (i) Reconstituted freeze-dried fluids Ampoules of freeze-dried 50 per cent virus in buffer were diluted 50-fold in sterile chilled water and samples were set up chilled and unchilled in direct sunlight at 85°F and titrated every hour (Fig. I). (ii) Viability of the wet vaccine Samples of 50 per cent virus in buffer were set up at room temperature, 4°C, and -22 °C, and titrated at intervals (Fig. 2). (iii) Viability of the freeze-dried product Ampoules of freeze-dried 50 per cent virus in buffer were set up at room temperature, 4°c, and -22 °C and titrated at intervals (Fig. 3). The significance of these results is dealt with in the discussion of dosage factors below.
2·8 --0-0-
CHILLED
- - UNCHILL ED
2·6
2·0 L--_ _- ' -_ _-'--_
o
2
_
---'-_
3
_ _L-''---_..1.
4
5
TIME AFTER RECONSTITUTION IN HOURS
Fig.
I.
Viability of reconstituted freeze-dried virus.
BRITISH VETERINARY JOURNAL, 118, 4
•
••
~ ~
AT -22°C AT 4°C AT R.T
,....,
E
o
L/)
o
u
II
2
3
I
4 !I I I
~
I
!
I
o
246
10
12
TIME OF STORAGE IN
Fig.
2.
Viability of wet tissue culture rinderpest virus.
5
_3 E
---o
AT -22°C
l!)
~2 u
l-
--<>---0--.
AT 4 ° C
--tr--f:r-
AT RT
e>
o
...J
o
246 S TIME OF STORAGE IN MONTHS
m
12
Fig. 3. Viability of freeze-dried vaccine. DISCUSSION
For a number of years the control of rinderpest in Nigeria has depended on the use of ORV in humped cattle and LRV in non-humped cattle. Both vaccines have the disadvantage of causing reactions of varying severity, in particular in
RINDERPEST IN TISSUE CULTURE, III
149
calves and in the wet season (Johnson, Thorne & MacLeod, 196 I ). In trade cattle vaccinated with CRV such reactions are often associated with considerable weight loss. Severe reactions in cattle under one year old have, until recently, prevented the use of CRY in this age group. As a result, many of the outbreaks of rinderpest encountered in Nigeria are restricted to calves under .-' one year old. The use of the more highly attenuated LRV in Zebu trade cattle and calves is not practical, owing to the fragility of the reconstituted virus under local working conditions in the field. The need for a vaccine which causes minimal reactions, whilst fulfilling the requirements of potency, ease of production and high survival rate, when reconstituted, has been obvious for some years. Although AR V causes minimal reactions even in hypersusceptible cattle and is cheap to produce, it appears to have a relatively low antigenicity, resulting in low antibody levels and occasional breakdowns on challenge. TCRV, on initial results, appears to fulfil the requirements. It is economical to produce, safe in all ages and breeds of cattle tested, results in antibody levels comparable to those following use of LRV and can be used at concentrations where viability losses in the field are insufficient to affect the final immunization. It has the added advantage of very rapid reconstitution from the freeze-dried state (a decided advantage over other current rinderpest vaccines) and a freedom from bacterial contamination. The criticism which can be made ofTCRV is the possibility of contamination of rinderpest virus fluids with other latent viruses from kidney monolayers, but this danger is certainly no greater than the possibility of such virus contaminants from goat spleens (CRV) or rabbit tissues (LRV). The danger can be reduced by the use of embryo tissues for vaccine production and by adequate safety testing. The use of a cell line for virus production, particularly if from animals other than bovines, would be an obvious improvement and work is being carried out on this approach. TCRV is issued in packs of 50 doses per ampoule. Each ampoule contains 0·5 m!. offreeze-dried virus fluids buffered with 0·5 ml. of Mist. desiccans LAH. The contents of an ampoule are reconstituted and diluted to 100 m!. of chilled sterile water and each animal receives 2 m!. subcutaneously. The reconstituted vaccine should be used within one hour if not kept on ice, or within two hours if chilled. Some shade from direct sunlight is recommended. The 50-dose ampoule was decided upon on the following grounds: (I) The minimal TCID 50 of a batch is 10 3 • s/m!., which is approximately equivalent to 1,600 cattle MIDso (in Zebus). (2) A maximum laboratory storage time of six months at -22 °C would reduce this to a minimum of 800 cattle MIDso per ampoule. (3) Transport and field storage at 4°F for a maximum period of three months would cause a further fall to a minimum of 300 cattle MID60 per ampoule. (4) Field use of the reconstituted virus unchilled for one hour, or chilled for two hours, would reduce this to a minimum of 100 MIDso doses per ampoule. !)
BRITISH VETERINARY JOURNAL, 118, 4
The above figures are based on minimal titre batches stored for the maximum periods of time and used, reconstituted, at the limit of the recommended period. Present batches average 10 4 • 7 TCID 5o /ml., and it is possible that some animals could receive up to 500 MID 5o , a level which has been shown to be innocuous. At present one pair of kidneys, yielding a minimum of ten Roux flask primary monolayers, gives 20 such monolayers at the first passage, of which ten are used for virus production, yielding an average total of 1,000 ml. of neat virus fluids (2,000 ml. with good monolayers). Diluted with buffer to 50 per cent, the yield is 2,000 ampoules or 100,000 doses. The remaining ten monolayers are again passaged and the process repeated up to at least the fifth passage level, giving a minimum yield per pair of kidneys of 500,000 freeze-dried doses over a six-week period. Up to ten cell passages have been used without obvious effect on the yield or vaccine virus properties. TCRV in Nigeria is at present used as a substitute for LRV for vaccination of young Zebu cattle and hypersusceptible breeds of all ages. Three quarters of a million doses have been used to date. The vaccine appears to be a valuable addition to the present campaign against rinderpest. SUMMARY
The use of a tissue culture attenuated strain of rinderpest vaccine in the main breeds of Nigerian cattle is described. Details are given of the reactions associated with the vaccine, the development of antibody and immunity of an interference phenomenon and back passage of the strain, the relationship between the TCID 50 titre and cattle MID 5o , and the viability of the strain under different conditions. The advantages of the vaccine are discussed and criteria for a dosage rate of freeze-dned virus are given. REFERENCES GREAVES, R. I. N. (1951).]. Hyg. (Lond.), 49, 220. JOHNSON, R. H. (1962a). Brit. vet.]., 118, 107. JOHNSON, R. H. (1962b). Brit. vet. ].,118, April, paper II. JOHNSON, R. H. & MAcLEOD, A. (1962). Unpublished data. jOHNtON, R. H., THORNE, A. L. C. & MACLEOD, A. j., (1961 ). Bull. PLOWRIGHT, W. & FERRIS, R. D. (1959a).]. compo Path., 6g, 152. PLOWRIGHT, W. & FERRIS, R. D. (1959b). compo Path., 69, 173.
J.
J.
(R eceived for publication, 8 December, 1961
epiz. D is. Afr·, 9, 233.
J . (1962),
ul, 141
RINDERPEST IN TISSUE CULTURE. III: USE OF THE ATTENUATED STRAIN AS A VACCINE FOR CATTLE By R. H.
JOHNSON
Federal Department of Veterinary Research, Vom, Nigeria
The two previous papers in this series have outlined the basic techniques and methods used in production and freeze drying of the rinderpest tissue culture virus and the use of qualitative and quantitative serum neutralization tests (Johnson, Ig62a, b). Plowright & Ferris (1959b) reported the use of the adapted and attenuated tissue culture rinderpest virus (TCRV) as a vaccine in East Mrican cattle, and the purpose of this report is to record the findings m West Mrican cattle vaccinated with t~e TORV strain. MATERIAL AND METHODS
The basic media and techniques have already been described (Johnson, 1962a, b). Virus for vaccine use is produced similarly to virus used for serum neutralization work. Bovine embryo kidney monolayers are used between the first and tenth passage levels, grown in a high sugar, high amino acid medium (HSH). The virus strain is that adapted and attenuated in tissue culture by Plowright & Ferris (I 959a, b) and is used between the 66th and 70th monolayer passages. Virus vaccine fluids are diluted 50 per cent in a modification of Mist. desiccans (Greaves, 1951; Johnson, Ig62b) and freeze dried in I ml. samples. SAFETY AND POTEN CY TESTING OF VACCINE BATCHES
Later in this paper it is shown that there is a very close relationship between the 50 per cent tissue culture infective dose (TCID so ) and the M1Dso for cattle. For this reason, cattle tests on vaccine batches concentrate on safety rather than potency. The concentration of virus in each batch of dried material is determined by titration in tissue culture (Johnson, 1962a). The following tests constitute the present checks on vaccine batches at Vom: A. In vitro tests Two I ml. ampoules of freeze-dried virus are titrated in tissue culture (Johnson, Ig62a) in 0'1 ml. samples over a twofold dilution range of 10 2 to 10 4 TCID 5o , being a corrected range per millilitre of 103 to 105 TCID so ' Undiluted virus is at the same time incubated with known positive and known negative rinderpest antisera in a serum neutrali7.ation (SN) test and the serumvirus mixtures are added to cell suspension.
BRITISH VETERINARY JOURNAL, lIB, 4
B. In vivo tests Two rinderpest-susceptible cattle checked by the SN test (Johnson, 1962b), are inoculated with the contents of one ampoule each of freeze-dried virus (50 doses). Two other susceptible bovines are inoculated with field dosage (1/50th of the contents of an ampoule). Morning temperatures and reactions ar{ recorded over a 14-day period. The four cattle, together with one control animal, are then challenged with 200 MID60 caprinized rinderpest virus (CRV) and temperatures taken for a further ten days. The criteria for passing a vaccine batch are: (a) An in vitro titre of not less than 103 . 6 TCID6o /ml. of the freeze-dried virus. The reason for this is associated with the dosage factor which is dealt with in the discussion. (b) No cytopathogenicity evident in the SN test containing positive serum. (c) No reactions other than mild hyperthermia in the four inoculated animals. (d) No reaction in the vaccinated cattle following challenge. RESULTS
A. Use of the strain as a vaccine in white Fulani (Zehu) cattle All initial work on TCRV was done on the humped White Fulani (Zebu) which is the only breed kept at these laboratories. Rinderpest-hypersusceptible breeds were later used in field trials. (i) Reactions foUowing vaccination Approximately 5,000 White Fulani (Zebu) cattle ranging in age from one day to three years have been vaccinated with doses of TCRV ranging from 1,000,000 to 10 TCID 60" Two hundred of these vaccinations were carried out at varying intervals using laboratory cattJe between 18 and 24 months old, which were kept under observation for periods up to 18 months after vaccination. The remaining 4,800 cattle were vaccinated in the field and the majority were calves varying from one day to nine months old. No adverse reactions have been reported from the field. In the 200 laboratory cattle there were no reactions other than a mild hyperthermia in occasional animals; 130 showed no observable rise in morning temperatures; 41 showed a rise of up to I ·5°F; i 9 a rise of between 1·5 and 3°F and loa rise of 3 to 4°F. These temperatures were noted between the 4th and 9th days after vaccination, depending on dosage and individual susceptibility, and they persisted for one to three days. More housed animals tended to show temperature reactions than those at grass. No other adverse signs or symptoms have been noted despite the fact that the majority of all vaccinations have been made during the local wet season when reactions to rinderpest vaccines are usually more severe. (ii) Antiho4J development following vaccination Following vaccination of White Fulani (Zebu) cattle with TCRV, antibodies to rinderpest, as determined by the SN test, first appear between
RINDERPEST IN TISSUE CULTURE, III
143
7 and 17 days. The higher the dose of virus the earlier can antibodies be detected within this period. Maximum levels are reached between two and four weeks and there is a slow decline up to 18 months when antibody is still present in fairly high concentration. Parallel estimations of antibody levels following vaccination with tissue culture virus (TCRV), caprinized (CRV), lapinized (LRV) and avianized (ARV) rinderpest viruses were made by complement fixation (CF) and SN tests on groups of cattle (Johnson & MacLeod, 1962). Results show that the use of TCRV results in the development of rinderpest antibodies to approximately the same degree as CRY. Antibody is formed at approximately the same rate following vaccination with field dosage of CRY, LRV and TCRV, but more slowly with ARV. Higher levels of antibody are produced by CRY and TCRV than with LRV or ARV. The decay rate in all four cases is approximately the same. (iii) Development of resistance to challenge Table I shows the reactions of White Fulani (Zebu) yearlings when challenged with 200 M1Dso CRY at 24-hourly mtervals following vaccination with low level doses of TCRV. At these levels of virus, antibody is not formed until ten to twelve days, but there is a resistance to challenge, without perceptible antibody present, at four days after vaccination. TABLE I DE VELOPMENT O P INTERPERENCE TO CHALLENGE
Animal No,
B8
Vaccine dosage
Time challenged after vaccination
Reaction after challenge
8
25
TCID ••
I
day
104'8° p
at
3
days
30
25
TCID ••
I
day
104'4°P
at
4
days
17
50
TCID ••
2
days
103'5° P
at
4
days
22
50
TCID 60
2
days
104,8 ° p
at
5
days
19
50
TCID••
3
days
103 '6° p
at
4
days
28
50
TCID ••
3
days
103'8°p
at
4
days
21
50
TCID ••
4 days
Nil
12
50
TCID ••
4 days
Nil
10
50
TCID••
5
days
Nil
26
25
TCID ••
5
days
102 '5°P /2
7
25
TCID ••
6
days
Nil
24
25
TCIDao
6
days
102 : 8°p / 2
29
25
TCID ••
7 days
Nil
31
25
TCID 60
7 days
Nil
days
days
144
BRITISH VETERINARY JOURNAL, 118, 4
Plowright & Ferris (1959b) demonstrated a similar interference phenomenon in East Mrica developing later than four days. Their method of challenge with setni-virulent rinderpest virus was superior to that used at Vom, where the attenuated CRV strain is used. CRV causes a typical marked hyperthermia in susceptible cattle. It will be obvious that a hyperthermia developing following challenge could conceivably be due to the original TCRV inoculation, as shown at six and seven days, although such reactions are comparatively rare and much milder. The absence of any temperature reactions at four and five days is, however, sufficient evidence that interference is present at this time. (iv) Duration of immuniry Seventy-two susceptible yearling cattle were vaccinated with 50 TCID 60 TCRV and run at grass with 12 controls. Antibody was demonstrable in undiluted sera (screening test) at two weeks after vaccination. At variable intervals following vaccination, three vaccinated animals with one control were bled for sera and challenged with 200 MIDso CRV. Up to 17 months all of 27 vaccinated cattle tested showed antibody on screening tests and all resisted challenge to 200MID 6o , the control animals at no time showing antibody and all responding typically to challenge. At 17 months, an outbreak of natural rinderpest on the station invalidated further work on this group. The remaining three controls died of rinderpest, whereas the remaining 45 TCRV vaccinated cattle continued healthy.
(v) Back passage of vaccine virus Attempts to demonstrate excretion of tissue culture vaccine virus from cattle vaccinated with 10,000 TCID 60 were made by housing 16 vaccinated cattle in close contact with six susceptible controls over a 28-day period. No evidence of excretion of TCRV could be found, as was shown by the complete susceptibility of the controls at the end of this period. Similarly, over a I7-month period, controls run at grass with TCRV-vaccinated cattle were still susceptible at the end of the period. To check for any increase in virulence should the strain be back-passaged in cattle, citrated blood samples were taken from a TCRV-vaccinated Zebu between the fourth and eighth days after vaccination. These were titrated for virus in vitro and samples showing the highest titre of virus were inoculated undiluted in 1 ml. samples into a second susceptible animal, the experiment being repeated through a further four passages. Over the five back passages, no apparent increase in virulence of the virus strain was demonstrated, no adverse response, other than mild hyperthermia, being noted at each back passage. (vi) Correlation of TCID 60 and M1D6o In order to have a firm basis for dosage estimation, the correlation between the in vitro and in vivo titre of virus was required. Plowright & Ferris (1959b) showed that in East Mrican cattle the relationship up to
RINDERPEST IN TISSUE CULTURE, III
the 45th virus passage was a very close one. Virus at the 67th, 68th and 70th passage levels, both wet and freeze dried, was titrated in tissue culture (Johnson, Ig62a) and the chilled dilutions were immediately inoculated in I ml. samples into groups of four rinderpest-susceptible Zebu cattle. Challenge with 200 M1D/iO CRY was carried out at two weeks after inoculation, presence of immunity being indicated by absence of a temperature response to challenge (Table II). Table II TABLE II CORRELATION OF TCID. o AND MID. o IN CATTLE
Vaccine passage level
MID.o/ml.
69th wet virus 69th dry virus 69th dry virus stored for
12
months at
- 2 2°C
70th wet virus
shows that there is a close relationship between the TCID so in vitro and the cattle M1D/io in vivo. The relationship is shown, on average, as 2 TCID 50 to I M1Dso for Zebu cattle.
B. Use of the strain as a vaccine in hypersusceptible cattle Following the laboratory trials in Zebu cattle, small field trials in hypersusceptible non-humped cattle were done with the co-operation of the West, East and Northern Nigerian Governments. Five such pilot trials, each involving between 12 and 20 hypersusceptible animals, have been carried out using Ndama, Keteku, Muturu and cross-bred animals of these breeds. All trial experiments have been carried out during the wet season when reactions to LRV in hypersusceptible cattle are liable to be severe and vaccinations have previously had to be restricted to the dry season. The results of the first trial in Ndama cattle are shown in Table III. They are a fair representation of the results in the second Ndama trial and trials in Keteku, Muturu, Keteku X Muturu and Keteku X Zebu cross-bred animals. During these trials the normal temperature of control animals was taken as 102 ·5 of; all animals on test were weighed daily and challenge was carried out two weeks after vaccination with 200 MIDIiO CRY. The controls included were (a) two uninoculated animals; (b) two animals which received field dosage ofLRV; and (c) four animals which received 10,000 TCID 50 TCRV, two being rinderpest-immune. Table III shows that both animals given LRV developed typical wet season reactions of diarrhoea, lacrimation, anorexia and cessation of rumination. No animal inoculated with TCRV showed any reaction other than a mild hyperthermia. In hypersusceptible breeds this hyperthermia is more frequent than in Zebu cattle, 85 per cent of inoculated animals showing the reaction
14 6
BRITIS H V ETERINAR Y JOURNA L , 118, 4 TABLE III RESU LTS OF TCR V FIELD TR IAL IN NDAMA CATTL E
Animal No .
Vaccine and dosage
Peak temperature after vaccination
Weight change over 14 days
Challenge results at 14 days
849
T CRV; 50 T C ID. o
103·2°F a t 5 days
+ 10 lb.
Nil
862
TCRV; 50 T C ID 50
102·5°F at 7 days
+2 lb.
Nil
899
TCRV ; 50 TCID. o
103°F at 4 days
+ 6 lb.
Nil
9 15
T CRV ; 50 TCID ••
104°F at 5 days
Nil
Nil
93 0
TCRV ; 50 TCID ••
104·2°F at 6 days
+4 lb.
Nil
93 1
TCRV; 50 TCID ••
I04·4°F at 8 days
+ 4 lb.
Nil
937
TCRV; 50 TCID ••
I02 ·4°F at 3 days
+8 lb.
Nil
938
T CR V ; 50 TCID••
102 ·8°F at 7 days
+ 5 Ib .
Nil
946
T CRV; 50 TCID••
102·5°F at 3 days
Nil
Nil
934
TCRV; 10,000 T CID ••
I03·8°F at 4 days
+ 2 lb.
Nil
955
TCRV; 10,000 T CID ••
103·2°F at 5 days
Nil
Nil
CONTROLS 943
Nil
102·4°F / 1 day
+5 lb.
Died 6 days
956
Nil
102· 2°F / 2 days
+ 4 lb.
Died 5 days
888
LRV; Field dose
104°F / 6 days
-7 lb.
Nil
890
LRV ; Field dose
105°F / 5 days
-15 lb.
Nil
Immune
TCRV; 10,000 TCID ••
102 ·6°F /
Nil
Nil
Immune
TCRV ; 10,000 TCID ••
102·5°F / 3 days
+5 lb.
Nil
II
days
between the fourth and ninth days. The temperature rise averaged I ·5°F and persisted for two to four days. Following the successful pilot trial results, wider field trials were made. Six thousand hypersusceptible yearlings, including a few imported Friesian calves, have been vaccinated to date, all during the local wet season. The dosage rate was 50 doses per ampoule of freeze-dried virus and, as a check on the potency of the vaccine under field conditions, the last five animals vaccinated from each ampoule were bled for serum before and three weeks after vaccination. All but two serum samples showed antibody development at three weeks. The development of antibody in hypersusceptible cattle vaccinated with this field dosage was checked by removal of serum samples from 12 cattle at 24-hour intervals from the 7th to 21st day. The average time when the appearance of antibody was initially detected in each animal was ten days after vaccination. No experiments on duration of immunity have yet been completed in hypersusceptible cattle. Antibody has, however, been .shown in sera taken from
RINDERPEST IN TISSUE CULTURE, III
147
Muturu calves IS months after vaccination with TCRV. These calves, ranging in age from one day to five months, were vaccinated with TCRV when an outbreak of natural rinderpest occurred among their dams, none of which had ever been vaccinated with rinderpest vaccine. Two calf controls and two vaccinated calves, presumably infected before vaccination, died of rinderpest out of 40 calves vaccinated. No adverse reactions to vaccination were noted.
C. Viability of the virus Having demonstrated the safety and immunizing properties of the virus in susceptible and hypersusceptible cattle, it was necessary to determine the viability of the virus under varying conditions. All virus fluids used were HSH, adjusted to pH 7.2 with 2 M-tris buffer and both wet and freeze-dried virus was further buffered with 50 per cent Mist. desiccans LAH (Johnson, Ig62a). Titrations were made in twofold ranges as previously described. (i) Reconstituted freeze-dried fluids Ampoules of freeze-dried 50 per cent virus in buffer were diluted 50-fold in sterile chilled water and samples were set up chilled and unchilled in direct sunlight at 85°F and titrated every hour (Fig. I). (ii) Viability of the wet vaccine Samples of 50 per cent virus in buffer were set up at room temperature, 4°C, and -22 °C, and titrated at intervals (Fig. 2). (iii) Viability of the freeze-dried product Ampoules of freeze-dried 50 per cent virus in buffer were set up at room temperature, 4°c, and -22 °C and titrated at intervals (Fig. 3). The significance of these results is dealt with in the discussion of dosage factors below.
2·8 --0-0-
CHILLED
- - UNCHILL ED
2·6
2·0 L--_ _- ' -_ _-'--_
o
2
_
---'-_
3
_ _L-''---_..1.
4
5
TIME AFTER RECONSTITUTION IN HOURS
Fig.
I.
Viability of reconstituted freeze-dried virus.
BRITISH VETERINARY JOURNAL, 118, 4
•
••
~ ~
AT -22°C AT 4°C AT R.T
,....,
E
o
L/)
o
u
II
2
3
I
4 !I I I
~
I
!
I
o
246
10
12
TIME OF STORAGE IN
Fig.
2.
Viability of wet tissue culture rinderpest virus.
5
_3 E
---o
AT -22°C
l!)
~2 u
l-
--<>---0--.
AT 4 ° C
--tr--f:r-
AT RT
e>
o
...J
o
246 S TIME OF STORAGE IN MONTHS
m
12
Fig. 3. Viability of freeze-dried vaccine. DISCUSSION
For a number of years the control of rinderpest in Nigeria has depended on the use of ORV in humped cattle and LRV in non-humped cattle. Both vaccines have the disadvantage of causing reactions of varying severity, in particular in
RINDERPEST IN TISSUE CULTURE, III
149
calves and in the wet season (Johnson, Thorne & MacLeod, 196 I ). In trade cattle vaccinated with CRV such reactions are often associated with considerable weight loss. Severe reactions in cattle under one year old have, until recently, prevented the use of CRY in this age group. As a result, many of the outbreaks of rinderpest encountered in Nigeria are restricted to calves under .-' one year old. The use of the more highly attenuated LRV in Zebu trade cattle and calves is not practical, owing to the fragility of the reconstituted virus under local working conditions in the field. The need for a vaccine which causes minimal reactions, whilst fulfilling the requirements of potency, ease of production and high survival rate, when reconstituted, has been obvious for some years. Although AR V causes minimal reactions even in hypersusceptible cattle and is cheap to produce, it appears to have a relatively low antigenicity, resulting in low antibody levels and occasional breakdowns on challenge. TCRV, on initial results, appears to fulfil the requirements. It is economical to produce, safe in all ages and breeds of cattle tested, results in antibody levels comparable to those following use of LRV and can be used at concentrations where viability losses in the field are insufficient to affect the final immunization. It has the added advantage of very rapid reconstitution from the freeze-dried state (a decided advantage over other current rinderpest vaccines) and a freedom from bacterial contamination. The criticism which can be made ofTCRV is the possibility of contamination of rinderpest virus fluids with other latent viruses from kidney monolayers, but this danger is certainly no greater than the possibility of such virus contaminants from goat spleens (CRV) or rabbit tissues (LRV). The danger can be reduced by the use of embryo tissues for vaccine production and by adequate safety testing. The use of a cell line for virus production, particularly if from animals other than bovines, would be an obvious improvement and work is being carried out on this approach. TCRV is issued in packs of 50 doses per ampoule. Each ampoule contains 0·5 m!. offreeze-dried virus fluids buffered with 0·5 ml. of Mist. desiccans LAH. The contents of an ampoule are reconstituted and diluted to 100 m!. of chilled sterile water and each animal receives 2 m!. subcutaneously. The reconstituted vaccine should be used within one hour if not kept on ice, or within two hours if chilled. Some shade from direct sunlight is recommended. The 50-dose ampoule was decided upon on the following grounds: (I) The minimal TCID 50 of a batch is 10 3 • s/m!., which is approximately equivalent to 1,600 cattle MIDso (in Zebus). (2) A maximum laboratory storage time of six months at -22 °C would reduce this to a minimum of 800 cattle MIDso per ampoule. (3) Transport and field storage at 4°F for a maximum period of three months would cause a further fall to a minimum of 300 cattle MID60 per ampoule. (4) Field use of the reconstituted virus unchilled for one hour, or chilled for two hours, would reduce this to a minimum of 100 MIDso doses per ampoule. !)
BRITISH VETERINARY JOURNAL, 118, 4
The above figures are based on minimal titre batches stored for the maximum periods of time and used, reconstituted, at the limit of the recommended period. Present batches average 10 4 • 7 TCID 5o /ml., and it is possible that some animals could receive up to 500 MID 5o , a level which has been shown to be innocuous. At present one pair of kidneys, yielding a minimum of ten Roux flask primary monolayers, gives 20 such monolayers at the first passage, of which ten are used for virus production, yielding an average total of 1,000 ml. of neat virus fluids (2,000 ml. with good monolayers). Diluted with buffer to 50 per cent, the yield is 2,000 ampoules or 100,000 doses. The remaining ten monolayers are again passaged and the process repeated up to at least the fifth passage level, giving a minimum yield per pair of kidneys of 500,000 freeze-dried doses over a six-week period. Up to ten cell passages have been used without obvious effect on the yield or vaccine virus properties. TCRV in Nigeria is at present used as a substitute for LRV for vaccination of young Zebu cattle and hypersusceptible breeds of all ages. Three quarters of a million doses have been used to date. The vaccine appears to be a valuable addition to the present campaign against rinderpest. SUMMARY
The use of a tissue culture attenuated strain of rinderpest vaccine in the main breeds of Nigerian cattle is described. Details are given of the reactions associated with the vaccine, the development of antibody and immunity of an interference phenomenon and back passage of the strain, the relationship between the TCID 50 titre and cattle MID 5o , and the viability of the strain under different conditions. The advantages of the vaccine are discussed and criteria for a dosage rate of freeze-dned virus are given. REFERENCES GREAVES, R. I. N. (1951).]. Hyg. (Lond.), 49, 220. JOHNSON, R. H. (1962a). Brit. vet.]., 118, 107. JOHNSON, R. H. (1962b). Brit. vet. ].,118, April, paper II. JOHNSON, R. H. & MAcLEOD, A. (1962). Unpublished data. jOHNtON, R. H., THORNE, A. L. C. & MACLEOD, A. j., (1961 ). Bull. PLOWRIGHT, W. & FERRIS, R. D. (1959a).]. compo Path., 6g, 152. PLOWRIGHT, W. & FERRIS, R. D. (1959b). compo Path., 69, 173.
J.
J.
(R eceived for publication, 8 December, 1961
epiz. D is. Afr·, 9, 233.