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Serial inoculation of sheep with two bluetongue virus types
Research in Veterinary Science /986. 40. 386-392
Serial inoculation of sheep with two bluetongue virus types M. H. JEGGO, R. C. WARDLEY, Animal Virus Research Institute, Pirbright, J. BROWNLIE, Institute/or Research on Animal Diseases, Compton, Newbury, Berkshire, A. H. CORTEYN,
Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF
Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.
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BLUETONGUE virus (BTY) causes a disease of sheep, goats and cattle characterised by facial oedema and coronitis (Bowne 1971). There are at present some 22 known BTY serotypes and vaccination is the only satisfactory method of control once bluetongue has become endemic in an area. In South Africa this has given rise to a complex procedure involving the inoculation of several pentavalent vaccines (Howell 1979). However, the degree of protection afforded by these vaccines is limited. Although recent studies have indicated that heterotypic immunity can be induced by the serial inoculation of as few as two BTY types (Jeggo et al 1983a, 1984b) many aspects of this crossprotection still need elucidating. ·In these studies the outcome of inoculating, at various times afterwards, a second BTY type into animals previously inoculated with BTY, and the ability of BTY immune T cells and specific antibody generated by one BTY type to protect an identical twin from challenge with a different BTY type, were examined.
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FIG 1: Mean response following the inoculation of STV type 3 into three sheep. (a) Temperature response, (b) viraemia and (c) neutralising antibody
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FIG 2: Mean temperature responses of three sheep following the inoculation of BTY type 3 into animals previously inoculated with BTV type 4. (a) Seven days previously, (bl 14 days previously, (c) 30 days previously and (d) 70 days previously
Group I received only BTV type 3. The remaining animals all received BTV type 4 before the inoculation of type 3. In group 2 the BTV type 3 inoculation was given seven days after the inoculation of BTV type 4, while in groups 3, 4 and 5 the BTV type 3 was given 14, 30 and 70 days after the inoculation of BTV type 4, respectively. Monozygotic animals were kindly supplied by the Institute of Animal Physiology, Babraham. The donor twin was inoculated with !lTV type 3 and 14 days later the thoracic duct was cannulated, the lymph cells collected and purified as described previously (Jeggo et al 1984b). This T cell enriched population and 300 ml of serum from a sheep immune to BTV type 3 (Jeggo et a11984a) were simultaneously inoculated intravenously into the monozygotic recipient. This animal was challenged 12 hours later with BTV type 4. An animal receiving the immune serum alone and similarly challenged with !lTV type 4 was included as a control. All sheep were infected by the intradermal inoculation in the left ear of I ml containing approximately 1()6 median tissue infectious doses (TCIO 50) of BTV. Daily clinical examinations were performed and serum and heparinised blood (5 iu heparin ml I final
concentration) were collected for up to three-weeks after virus inoculation.
Viruses !lTV type 3 was obtained from the Veterinary Research Institute, Onderstepoort and had been passaged twice in embryonated eggs and three times in baby hamster kidney (BHK) cells. BTV type 4 was isolated from an outbreak of bluetongue in Cyprus in 1969 and had been passaged once in eggs and four times in BHK cells. Both BTV types were plaque purified three times in BHK cells and their type specificity verified in a microneutralisation test (Jeggo et al 1983a).
Virus isolation Virus isolation was carried out using heparinised blood andBHK monolayers as indicator cells (Jeggo et al 1983a). Viraemia levels were expressed as 10gIO TCID 50 ml : I of original packed cells, which constituted approximately half the total volume of the blood sample. In each experiment, virus isolates were retyped using the virus neutralisation test.
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FIG 3: Mean viraemia tit res from three sheep following the inoculation of BTV type 3 into animals previously inoculated with BTV type 4. (a) Seven days previously, (b) 14 days previously, (c) 30 days previously and (d) 70 days previously
Neutralising antibody detection Tests for neutralising antibodies to BTV types were carried out, using the microneutralisation system as described previously (Jeggo et al 1983a). Virus and positive and negative serum controls were included with each batch of sera tested. To determine which of the inoculated virus types might be present in the viraemic blood, box neutralisation tests were done on the blood samples collected on various days following inoculation of the second BTV type, as described previously (Jeggo et al 1984c).
Results
Serial inoculation The outcome following inoculation of BTV type 3 alone into the sheep can be seen in Fig I, where the temperature, viraemia and antibody responses conform to patterns observed previously following inoculation of sheep with this virus (Jeggo et al 1983a). The outcome of the inoculation of BTV type 3 into sheep previously given BTV type 4 can be seen in
Figs 2, 3 and 4. In group 2 animals were inoculated with BTV type 3 seven days after the inoculation of BTV type 4, and although the sheep produced a typical temperature and viraemic response to BTV type 4 they did not develop further clinical signs after the inoculation of BTV type 3. However, examination of the blood on day 8 following this second virus inoculation, by box neutralisation, revealed O' 4 TClD 50 ml- I of BTV type 4 and O' 3 TCID 50 m',: I of BTV type 3. After five more days the viraemia consisted solely of BTV type 3. No virus was isolated from this group of animals beyond 35 days. Despite the isolation of BTV 3, there was a very much lower virus titre for a shorter duration than in control animals (Fig I b). In groups 3, 4 and 5 the previous inoculation of BTV type 4 at, respectively, 14,30 and 70 days before the inoculation of BTV type 3 had no effect on the pattern of response to BTV type 3 inoculation in terms of temperature or viraemia. Examination of the blood by box neutralisation, using various immune sera, revealed no alteration in the expected patterns of viraemias to the BTV 4 and subsequent BTV 3 inoculation. With a 14 day interval dual viraemias to the two serotypes were observed but at levels expected following the inoculation of each type alone. Thus, there
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was a gradual decline in the amount of BTY type 4 but a rapid increase in BTY type 3, peaking seven days after the inoculation of type 3. With 30 and 70 day intervals no BTY type 4 could be detected at the time of the inoculation of BTY type 3 and the pattern of the BTY 3 viraemia was similar in both groups to that of the control animals. The development of neutralising antibodies to BTY 4 in the various groups can be seen in Fig 4. The inoculation of the second serotype at various intervals after the primary inoculation had no effect on the production of antibodies to the first serotype. The development of antibodies to BTY 3 following the inoculation of this serotype can be seen in Fig Ic and Fig 4. In the control animals, antibodies to BTY type 3 were first detected on day 10 after infection at a titre of 1/100 and remained at this level. In groups 3, 4 and 5, which had an interval of 14 days or more between serotype inoculations, a similar pattern was obtained. However, with the inoculation of BTY type 3 seven days after the inoculation of BTY type 4, antibodies to BTY type 3 were not detected until 18 days after the inoculation of this second serotype and titres rose only slowly, although they eventually reached levels
comparable both to the other groups and to BTY type 4 specific antibodies. Sera from these animals were also examined for evidence of antibodies to the other 22 BTY serotypes 24 days after the inoculation of BTY type 3 (Fig 5). Previously this time interval had been shown to reveal the maximum heterologous antibody response after the inoculation of two BTY types (Jeggo et al 1983a). The animals inoculated with STY type 3 alone developed antibodies to only STY type 3 at tit res higher than 1/10, with a very low response to two other serotypes. With animals given two serotypes the heterologous nature of the antibody response increased both in spread and titres with this effect becoming more marked as the interval between the two inoculations widened.
Adoptive transfer experiments The inoculation of STY type 3 into the donor monozygotic animal resulted in a typical pyrexia and viraemic response (Fig 6). Thoracic duct lymphocytes (TDL) collected 12 days after virus inoculation from this animal and sera from an animal which had
M. H. Jeggo, R. C. Wardley, J. Brownlie, A. H. Corteyn
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recovered from a BTY type 3 infection were then inoculated into the recipient monozygotic animal before it was challenged with BTY type 4. The recipient failed to develop a pyrexia but a viraemia did occur which was of low titre and short duration (Fig 6). A control animal receiving immune sera to BTY type 3 and challenged with BTY type 4 produced a typical pyrexia and viraemia (results not shown). The modified response in the monozygotic sheep was
similar to that seen previously following the transfer OfTDLsalone without the simultaneous inoculation of immune sera (Jeggo et al 1984b). It thus appears that the immune sera did not add to the degree of protection against heterologous challenge. The recipient and control animal sera were also examined for the presence of antibodies other than those against BTY types 3 and 4; none could be detected. Examination of sera derived from a mono-
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zygotic animal used in a previous study (Jeggo et al 1984b) revealed antibodies to a large number of !lTV types. This animal was inoculated with BTV type 3 six weeks before the transfer of immune T cells to BTV 3 and challenge with BTV type 4. It failed to develop a pyrexia or viraemic response to the inoculation of BTV 4 but did produce a broad heterotypic antibody response. Discussion Initial studies with the,serial inoculation of BTV into sheep and cattle indicated that animals inoculated with one BTV type were fully susceptible to challenge 70 days later with a second BTV type (Jeggo et al 1983a, 1983b). These results indicate that for a short period after the administration of one BTV type animals are capable of reducing the effect of challenge with a second serotype, although this effect is short-lived. How is this protection likely to be mediated? Two
391
possible mechanisms have been identified; serum neutralising antibody and cytotoxic T lymphocytes (CTLs). Neutralising antibody does not appear until 10 to 12 days after infection and by definition sera produced by inoculation of one type will not protect against challenge with another type. However, antiBTV CTLs have been described both in mice (Jeggo and Wardley 1982a) and sheep (Jeggo et al 1984b) and these are capable of heterotypic lysis (Jeggo and Wardley 1982b). At this stage then during infection, it appears most likely that the protection is being afforded by CTLs and that the humoral component of the immune response has no part to play in this crossprotection. Previously it had been suggested that there was the possibility of some synergism between antibody and CTLs (Jeggo et al 1984b) because complete protection had been shown in an animal which had recovered from one BTV infection six weeks previously and was given 14 day TOLs against that type and then challenged with a second type. It was also argued that in that recipient, apart from anti-srv antibody, a range of other anti-srv memory cells would have been laid down. In the present experiments, antibody was passively transferred, rather than being induced by an active infection which would also have generated memory cells. The recipient was not totally protected but acted in the same way as animals given TOLs alone (Jeggo et al 1984b). This failure indicates that the postulated synergism between TOLs and antibody which gave complete protection did not occur and that this total protection was more likely due to the actively transferred TOLs and a non-humoral anti-urv memory component already present in the recipient. This study confirms previous findings that only two serotypes inoculated serially are needed to produce a heterotypic antibody response but it also demonstrates that it is necessary to inoculate both virus types as a prerequisite to the formation of this heterotypic response. Immune sera and early T cells to one type failed' to assist in the formation of a heterotypic humoral response in the monozygotic recipient. It would appear that this phenomenon relies on an immune memory response only initiated following the inoculation of virus and is not present until at least 14 days after virus inoculation. Further evidence for this is seen in the serially challenged animals where a significant heterologous antibody response did not occur until the two inoculations of virus were 30 days apart. How do these observations assist in the control of bluetongue virus infection? In areas in which a number of serotypes of the virus exist, the method of control has been to endeavour to induce as broad a heterotypic protection as possible, using multivalent vaccines containing the types present in that area. Thus in South Africa, where 17 serotypes of the virus
392
M. H. Jeggo, R. C. Wardley, J. Brownlie, A. H. Corteyn
have been identified, pentavalent vaccines are given three times in a two week period, exposing an animal to some 15 serological types. Despite this, each year outbreaks of bluetongue are common, indicating that this vaccine protocol is giving little protective immunity (Howell 1979). The findings in this study that a second serotype given within 14 days of the first fails to replicate fully adds to a previous study which also showed that not all types in a multivalent preparation are likely to grow (Jeggo et a11984c) and indicates further reasons why the BTV vaccine regime adopted in South Africa is unsuccessful. A simpler approach is the serial inoculation of two or three monovalent preparations separated by at least three to four weeks to give rise to a broad serotypic response. The distinct seasonality of BTV infection in many areas (Herniman et al 1983) and the fact that protection for two distinct groups of sheep need to be considered, long term protection for breeding animals and short term protection for meat animals, suggests that these immunological findings can further help BTV vaccine strategies. Hence, serial inoculation would appear to offer good control in breeding animals while the short term cross-reactive CTL response might be of use in meat animals. Studies presented here, however, indicate that this response is only effective for seven to 10 days. The reason for its rapid curtailment are not
clear although it may be due to the formation of either suppressor cells or anti-crt, anti-idiotypic antibody. From a commercial point of view, however, it will be necessary to understand and modify this response so that the few months protection needed in the field becomes available. References BOWNE, J. G. (1971) Advances in Veterinary Science and Comparative Medicine 15,1-16 HERNIMAN, K. A. J., BOORMAN, J. & TAYLOR, W. P. (1983) Journal of Hygiene 90, 177-193 HOWELL, P. G. (1979) Arbovirus Research in Australia, Proceedings of the 2nd Symposium, Vol I. Eds T. D. St George, E. L. French. Brisbane, Queensland Institute of Medical Research. pp 117-123 JEGGO, M. H. & WARDLEY, R. C. (1982a) Archives of Virology 71, 197-206 JEGGO, M. H. & WARDLEY, R. C. (1982b) Immunology 45, 629-634 JEGGO, M. H., GUMM, I. D. & TAYLOR, W. P. (1983a) Research in Veterinary Science 34,205-211 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1983b) Double-stranded RNA viruses. Vol I. Eds R. W. Compans, D. H. L. Bishop. Amsterdam, Elsevier. pp 353-359 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1984a) Research in Veterinary Science 36, 81-85 JEGGO, M. H., WARDLEY, R. C. & BROWNLIE. J. (1984b) Immunology 52, 403-410 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1984c)
Research in Veterinary Science 37, 368- 370
Accepted August I, 1985
Serial inoculation of sheep with two bluetongue virus types M. H. JEGGO, R. C. WARDLEY, Animal Virus Research Institute, Pirbright, J. BROWNLIE, Institute/or Research on Animal Diseases, Compton, Newbury, Berkshire, A. H. CORTEYN,
Animal Virus Research Institute, Pirbright, Woking, Surrey GU24 ONF
Groups of sheep inoculated with bluetongue virus type 4 were challenged at various intervals after inoculation (from seven to 70 days) with bluetongue virus type 3. Examination of the clinical and serological response showed that animals were protected from challenge with a second bluetongue virus for up to 14 days after the inoculation of the first virus type. An adoptive transfer experiment in monozygotic sheep involving both antibody and T lymphocytes was carried out. Only partial protection was observed against heterologous virus challenge, indicating that although the T cell response has a cross-protective component, antibody is not involved. These observations indicate that current vaccination procedures should be reappraised, particularly in terms of revaccination with multiple bluetongue virus type.
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BLUETONGUE virus (BTY) causes a disease of sheep, goats and cattle characterised by facial oedema and coronitis (Bowne 1971). There are at present some 22 known BTY serotypes and vaccination is the only satisfactory method of control once bluetongue has become endemic in an area. In South Africa this has given rise to a complex procedure involving the inoculation of several pentavalent vaccines (Howell 1979). However, the degree of protection afforded by these vaccines is limited. Although recent studies have indicated that heterotypic immunity can be induced by the serial inoculation of as few as two BTY types (Jeggo et al 1983a, 1984b) many aspects of this crossprotection still need elucidating. ·In these studies the outcome of inoculating, at various times afterwards, a second BTY type into animals previously inoculated with BTY, and the ability of BTY immune T cells and specific antibody generated by one BTY type to protect an identical twin from challenge with a different BTY type, were examined.
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FIG 1: Mean response following the inoculation of STV type 3 into three sheep. (a) Temperature response, (b) viraemia and (c) neutralising antibody
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FIG 2: Mean temperature responses of three sheep following the inoculation of BTY type 3 into animals previously inoculated with BTV type 4. (a) Seven days previously, (bl 14 days previously, (c) 30 days previously and (d) 70 days previously
Group I received only BTV type 3. The remaining animals all received BTV type 4 before the inoculation of type 3. In group 2 the BTV type 3 inoculation was given seven days after the inoculation of BTV type 4, while in groups 3, 4 and 5 the BTV type 3 was given 14, 30 and 70 days after the inoculation of BTV type 4, respectively. Monozygotic animals were kindly supplied by the Institute of Animal Physiology, Babraham. The donor twin was inoculated with !lTV type 3 and 14 days later the thoracic duct was cannulated, the lymph cells collected and purified as described previously (Jeggo et al 1984b). This T cell enriched population and 300 ml of serum from a sheep immune to BTV type 3 (Jeggo et a11984a) were simultaneously inoculated intravenously into the monozygotic recipient. This animal was challenged 12 hours later with BTV type 4. An animal receiving the immune serum alone and similarly challenged with !lTV type 4 was included as a control. All sheep were infected by the intradermal inoculation in the left ear of I ml containing approximately 1()6 median tissue infectious doses (TCIO 50) of BTV. Daily clinical examinations were performed and serum and heparinised blood (5 iu heparin ml I final
concentration) were collected for up to three-weeks after virus inoculation.
Viruses !lTV type 3 was obtained from the Veterinary Research Institute, Onderstepoort and had been passaged twice in embryonated eggs and three times in baby hamster kidney (BHK) cells. BTV type 4 was isolated from an outbreak of bluetongue in Cyprus in 1969 and had been passaged once in eggs and four times in BHK cells. Both BTV types were plaque purified three times in BHK cells and their type specificity verified in a microneutralisation test (Jeggo et al 1983a).
Virus isolation Virus isolation was carried out using heparinised blood andBHK monolayers as indicator cells (Jeggo et al 1983a). Viraemia levels were expressed as 10gIO TCID 50 ml : I of original packed cells, which constituted approximately half the total volume of the blood sample. In each experiment, virus isolates were retyped using the virus neutralisation test.
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FIG 3: Mean viraemia tit res from three sheep following the inoculation of BTV type 3 into animals previously inoculated with BTV type 4. (a) Seven days previously, (b) 14 days previously, (c) 30 days previously and (d) 70 days previously
Neutralising antibody detection Tests for neutralising antibodies to BTV types were carried out, using the microneutralisation system as described previously (Jeggo et al 1983a). Virus and positive and negative serum controls were included with each batch of sera tested. To determine which of the inoculated virus types might be present in the viraemic blood, box neutralisation tests were done on the blood samples collected on various days following inoculation of the second BTV type, as described previously (Jeggo et al 1984c).
Results
Serial inoculation The outcome following inoculation of BTV type 3 alone into the sheep can be seen in Fig I, where the temperature, viraemia and antibody responses conform to patterns observed previously following inoculation of sheep with this virus (Jeggo et al 1983a). The outcome of the inoculation of BTV type 3 into sheep previously given BTV type 4 can be seen in
Figs 2, 3 and 4. In group 2 animals were inoculated with BTV type 3 seven days after the inoculation of BTV type 4, and although the sheep produced a typical temperature and viraemic response to BTV type 4 they did not develop further clinical signs after the inoculation of BTV type 3. However, examination of the blood on day 8 following this second virus inoculation, by box neutralisation, revealed O' 4 TClD 50 ml- I of BTV type 4 and O' 3 TCID 50 m',: I of BTV type 3. After five more days the viraemia consisted solely of BTV type 3. No virus was isolated from this group of animals beyond 35 days. Despite the isolation of BTV 3, there was a very much lower virus titre for a shorter duration than in control animals (Fig I b). In groups 3, 4 and 5 the previous inoculation of BTV type 4 at, respectively, 14,30 and 70 days before the inoculation of BTV type 3 had no effect on the pattern of response to BTV type 3 inoculation in terms of temperature or viraemia. Examination of the blood by box neutralisation, using various immune sera, revealed no alteration in the expected patterns of viraemias to the BTV 4 and subsequent BTV 3 inoculation. With a 14 day interval dual viraemias to the two serotypes were observed but at levels expected following the inoculation of each type alone. Thus, there
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*--*
was a gradual decline in the amount of BTY type 4 but a rapid increase in BTY type 3, peaking seven days after the inoculation of type 3. With 30 and 70 day intervals no BTY type 4 could be detected at the time of the inoculation of BTY type 3 and the pattern of the BTY 3 viraemia was similar in both groups to that of the control animals. The development of neutralising antibodies to BTY 4 in the various groups can be seen in Fig 4. The inoculation of the second serotype at various intervals after the primary inoculation had no effect on the production of antibodies to the first serotype. The development of antibodies to BTY 3 following the inoculation of this serotype can be seen in Fig Ic and Fig 4. In the control animals, antibodies to BTY type 3 were first detected on day 10 after infection at a titre of 1/100 and remained at this level. In groups 3, 4 and 5, which had an interval of 14 days or more between serotype inoculations, a similar pattern was obtained. However, with the inoculation of BTY type 3 seven days after the inoculation of BTY type 4, antibodies to BTY type 3 were not detected until 18 days after the inoculation of this second serotype and titres rose only slowly, although they eventually reached levels
comparable both to the other groups and to BTY type 4 specific antibodies. Sera from these animals were also examined for evidence of antibodies to the other 22 BTY serotypes 24 days after the inoculation of BTY type 3 (Fig 5). Previously this time interval had been shown to reveal the maximum heterologous antibody response after the inoculation of two BTY types (Jeggo et al 1983a). The animals inoculated with STY type 3 alone developed antibodies to only STY type 3 at tit res higher than 1/10, with a very low response to two other serotypes. With animals given two serotypes the heterologous nature of the antibody response increased both in spread and titres with this effect becoming more marked as the interval between the two inoculations widened.
Adoptive transfer experiments The inoculation of STY type 3 into the donor monozygotic animal resulted in a typical pyrexia and viraemic response (Fig 6). Thoracic duct lymphocytes (TDL) collected 12 days after virus inoculation from this animal and sera from an animal which had
M. H. Jeggo, R. C. Wardley, J. Brownlie, A. H. Corteyn
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(a) BlY 3 only 1/100
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FIG 5: Histograms depicting the mean level of serum neutralising antibodies to BTYtypes induced by the inoculation of BTV3 into groups of three sheep previously inoculated at various times with BTV4. All titres calculated from geometric means of reciprocalloglO VN50 values for each group
recovered from a BTY type 3 infection were then inoculated into the recipient monozygotic animal before it was challenged with BTY type 4. The recipient failed to develop a pyrexia but a viraemia did occur which was of low titre and short duration (Fig 6). A control animal receiving immune sera to BTY type 3 and challenged with BTY type 4 produced a typical pyrexia and viraemia (results not shown). The modified response in the monozygotic sheep was
similar to that seen previously following the transfer OfTDLsalone without the simultaneous inoculation of immune sera (Jeggo et al 1984b). It thus appears that the immune sera did not add to the degree of protection against heterologous challenge. The recipient and control animal sera were also examined for the presence of antibodies other than those against BTY types 3 and 4; none could be detected. Examination of sera derived from a mono-
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zygotic animal used in a previous study (Jeggo et al 1984b) revealed antibodies to a large number of !lTV types. This animal was inoculated with BTV type 3 six weeks before the transfer of immune T cells to BTV 3 and challenge with BTV type 4. It failed to develop a pyrexia or viraemic response to the inoculation of BTV 4 but did produce a broad heterotypic antibody response. Discussion Initial studies with the,serial inoculation of BTV into sheep and cattle indicated that animals inoculated with one BTV type were fully susceptible to challenge 70 days later with a second BTV type (Jeggo et al 1983a, 1983b). These results indicate that for a short period after the administration of one BTV type animals are capable of reducing the effect of challenge with a second serotype, although this effect is short-lived. How is this protection likely to be mediated? Two
391
possible mechanisms have been identified; serum neutralising antibody and cytotoxic T lymphocytes (CTLs). Neutralising antibody does not appear until 10 to 12 days after infection and by definition sera produced by inoculation of one type will not protect against challenge with another type. However, antiBTV CTLs have been described both in mice (Jeggo and Wardley 1982a) and sheep (Jeggo et al 1984b) and these are capable of heterotypic lysis (Jeggo and Wardley 1982b). At this stage then during infection, it appears most likely that the protection is being afforded by CTLs and that the humoral component of the immune response has no part to play in this crossprotection. Previously it had been suggested that there was the possibility of some synergism between antibody and CTLs (Jeggo et al 1984b) because complete protection had been shown in an animal which had recovered from one BTV infection six weeks previously and was given 14 day TOLs against that type and then challenged with a second type. It was also argued that in that recipient, apart from anti-srv antibody, a range of other anti-srv memory cells would have been laid down. In the present experiments, antibody was passively transferred, rather than being induced by an active infection which would also have generated memory cells. The recipient was not totally protected but acted in the same way as animals given TOLs alone (Jeggo et al 1984b). This failure indicates that the postulated synergism between TOLs and antibody which gave complete protection did not occur and that this total protection was more likely due to the actively transferred TOLs and a non-humoral anti-urv memory component already present in the recipient. This study confirms previous findings that only two serotypes inoculated serially are needed to produce a heterotypic antibody response but it also demonstrates that it is necessary to inoculate both virus types as a prerequisite to the formation of this heterotypic response. Immune sera and early T cells to one type failed' to assist in the formation of a heterotypic humoral response in the monozygotic recipient. It would appear that this phenomenon relies on an immune memory response only initiated following the inoculation of virus and is not present until at least 14 days after virus inoculation. Further evidence for this is seen in the serially challenged animals where a significant heterologous antibody response did not occur until the two inoculations of virus were 30 days apart. How do these observations assist in the control of bluetongue virus infection? In areas in which a number of serotypes of the virus exist, the method of control has been to endeavour to induce as broad a heterotypic protection as possible, using multivalent vaccines containing the types present in that area. Thus in South Africa, where 17 serotypes of the virus
392
M. H. Jeggo, R. C. Wardley, J. Brownlie, A. H. Corteyn
have been identified, pentavalent vaccines are given three times in a two week period, exposing an animal to some 15 serological types. Despite this, each year outbreaks of bluetongue are common, indicating that this vaccine protocol is giving little protective immunity (Howell 1979). The findings in this study that a second serotype given within 14 days of the first fails to replicate fully adds to a previous study which also showed that not all types in a multivalent preparation are likely to grow (Jeggo et a11984c) and indicates further reasons why the BTV vaccine regime adopted in South Africa is unsuccessful. A simpler approach is the serial inoculation of two or three monovalent preparations separated by at least three to four weeks to give rise to a broad serotypic response. The distinct seasonality of BTV infection in many areas (Herniman et al 1983) and the fact that protection for two distinct groups of sheep need to be considered, long term protection for breeding animals and short term protection for meat animals, suggests that these immunological findings can further help BTV vaccine strategies. Hence, serial inoculation would appear to offer good control in breeding animals while the short term cross-reactive CTL response might be of use in meat animals. Studies presented here, however, indicate that this response is only effective for seven to 10 days. The reason for its rapid curtailment are not
clear although it may be due to the formation of either suppressor cells or anti-crt, anti-idiotypic antibody. From a commercial point of view, however, it will be necessary to understand and modify this response so that the few months protection needed in the field becomes available. References BOWNE, J. G. (1971) Advances in Veterinary Science and Comparative Medicine 15,1-16 HERNIMAN, K. A. J., BOORMAN, J. & TAYLOR, W. P. (1983) Journal of Hygiene 90, 177-193 HOWELL, P. G. (1979) Arbovirus Research in Australia, Proceedings of the 2nd Symposium, Vol I. Eds T. D. St George, E. L. French. Brisbane, Queensland Institute of Medical Research. pp 117-123 JEGGO, M. H. & WARDLEY, R. C. (1982a) Archives of Virology 71, 197-206 JEGGO, M. H. & WARDLEY, R. C. (1982b) Immunology 45, 629-634 JEGGO, M. H., GUMM, I. D. & TAYLOR, W. P. (1983a) Research in Veterinary Science 34,205-211 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1983b) Double-stranded RNA viruses. Vol I. Eds R. W. Compans, D. H. L. Bishop. Amsterdam, Elsevier. pp 353-359 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1984a) Research in Veterinary Science 36, 81-85 JEGGO, M. H., WARDLEY, R. C. & BROWNLIE. J. (1984b) Immunology 52, 403-410 JEGGO, M. H., WARDLEY, R. C. & TAYLOR, W. P. (1984c)
Research in Veterinary Science 37, 368- 370
Accepted August I, 1985