_Archives,,
Vi rology
Arch Virol (1991) 118:247-252
© Springer-Verlag 1991 Printed in Austria
Isolation and characterisation of a porpoise morbillivirus Brief R e p o r t S. J. McCullough 1, F. McNeilly l, G. M. Allan 1, S. Kennedy l, J. A. Smyth t, S. L. Cosby2, S. McQuaid 2, and B. K. R i m a 2 1Veterinary Research Laboratories, Belfast 2 Department of Biology, The Queen's University of Belfast, Belfast, Northern lreland
Accepted January 23, 1991 Summary. A morbillivirus was isolated from lung tissue of a porpoise which had lesions similar to those of phocine distemper. Porpoise and seal isolates differed in their reactions to monoclonal antibodies to canine distemper virus.
The type species of the genus Morbillivirus of the family' Paramyxoviridae is measles virus (MV) and the other members are canine distemper virus (CDV), rinderpest virus (RPV) and peste des petits ruminants virus (PPRV). An epizootic of morbillivirus infection killed thousands of harbour seals (Phoca vitulina) in European waters during 1988 [9]. The isolation of a morbillivirus from affected seals found on the coast of Northern Ireland and the lesions of distemper associated with the presence of morbillivirus antigen in these animals have been reported previously [4, 6]. We have suggested that the virus should be classified as a new member of the genus Morbillivirus, and have proposed the name phocine distemper virus (PDV) [1]. During the seat epizootic we also found evidence of morbillivirus infection in harbour porpoises (Phocoena phocoena) from the Irish Sea. Affected porpoises had lesions similar to those of PDV infected seals and we demonstrated morbillivirus antigen in many tissues using an immunoperoxidase technique [5]. We now report the first isolation of a morbiUivirus from the tissues of a porpoise and the results of comparison of this isolate with a PDV isolate by reaction with a panel of monoclonal antibodies (mAbs) against CDV. During the months of September to December 1988; six harbour porpoises which had been found stranded on the coast of Northern Ireland were submitted to our laboratories for necropsy. All six animals had distemper-like lesions [3a]. Samples of lung tissue were obtained for virus isolation from two carcases.
248
S.J. McCuUough et al.
Cryostat sections of lung tissue from both animals were stained by indirect immunofluorescence using serum from a human patient with subacute sclerosing panencephalitis (SSPE) as primary antibody, followed by fluorescein conjugated anti-human immunoglobulin. Immunofluorescent staining was observed in some of these cryostat sections, however, autolysis made it difficult to determine the specificity of the fluorescence seen when the sections were examined by incident UV light microscopy. Samples of lung tissue were ground in Eagle's minimum essential medium (MEM) containing 1000 IU penicillin, 1000 gg streptomycin and 100 IU nystatin
Fig. 1. a Negative contrast electron microscopy of lung homogenatefrom porpoise suspected of having a morbillivirus infection. Note viral nucleocapsid, x 200,000. b Indirect immunofluorescence staining of viral antigens in foetal bovine lung cells infected with porpoise morbillivirus and stained using SSPE antiserum, x 1,500.
Isolation and characterisation of a porpoise morbillivirus
249
per ml. The resulting h o m o g e n a t e s were centrifuged at 3000 g for 30 min a n d the s u p e r n a t a n t fluids retained. The fluids were e x a m i n e d by negative c o n t r a s t electron m i c r o s c o p y which revealed the helical nucleocapsid characteristic o f the Paramyxoviridaein one o f the samples (Fig. l a). Cultures o f Vero cells, M a d i n D a r b y canine k i d n e y ( M D C K ) cells a n d a s e m i c o n t i n u o u s line o f bovine foetal lung (BFL) cells were inoculated with the supernatants o f porpoise lung tissue homogenates by adsorption onto cell m o n o layers for 1 h at 37 °C or by inoculation o f freshly trypsinised cells. I n o c u l a t e d cultures were harvested by trypsinisation at intervals o f 3-4 days a n d passaged with freshly trypsinised u n i n o c u l a t e d cells. A t each passage, coverslip cultures o f each cell line were p r e p a r e d a n d virus g r o w t h was m o n i t o r e d by indirect i m m u n o f l u o r e s c e n c e as described above. I m m u n o f l u o r e s c e n t staining o f m o r billivirus antigen was observed in cell cultures inoculated with the s u p e r n a t a n t fluid in which p a r a m y x o v i r u s particles h a d been detected by negative contrast electron m i c r o s o p y (Fig. lb). I n o c u l a t i o n by a d s o r p t i o n onto Vero or B F L m o n o l a y e r s resulted in earliest a n d m o s t rapid g r o w t h o f virus. Positive ceils a p p e a r e d in b o t h cell lines at second passage. By f o u r t h passage, 90% o f B F L cells and 10% o f Vero cells were positive by indirect i m m u n o f l u o r e s c e n c e . However, there was no evidence Table 1. Reactivity of anti-CDV monoclonal antibodies with seal and porpoise distemper viruses Anti-CDV mAbs
PDV a Vero adapted
PDV BFL adapted
Porpoise distemper virus
N1 (3.721) N2 (3.991) N3 (3.805) P1 (3.568) P2 (3.698) P3 (3.780) P4 (3.630) P5 (3.768) P6 (4.088) F1 (3.584) F2 (3.633) F3 (5.148) F4 (3.551) H1 (3.734) H2 (3.755) H3 (4.043) H4 (2.267)
+ + + + + + + + + + + +b
+ + + + + + + + + + + + +
+ + + + + + + + + + + _
_
_
_
b
PDV and porpoise distemper virus isolates adapted to BFL cells at the eighth passage. Scoring of immunofluorescent staining: + distinct; - none a Vero adapted PDV results reported previously [1] b H3 and H4 results previously appeared as: H3 negative, H4 positive due to printer's error
250
S.J. McCullough et al.
of virus growth in M D C K cells following adsorption onto established monolayers, though positive cells were detected at third passage in all cell lines when freshly trypsinised cells were inoculated. Infected BFL cells at fifth passage were lysed by freezing and thawing three times. The lysate was examined by negative contrast electron microscopy which revealed virus particles with typical paramyxovirus morphology. On the basis of morphology and reaction with SSPE antiserum, the isolated virus was characterised as a member of the genus Morbillivirus. Coverslip cultures of PDV- and porpoise distemper virus-infected BFL cells at the eighth passage level were examined by indirect immunofluorescence using a selected panel of mAbs to CDV (Convac strain, supplied by C. ~rvell, State Bacteriological Laboratory, Stockholm, Sweden). The reactions of porpoise morbillivirus with 11 of the panel of 17 mAbs were the same as those of PDV. However, it differed antigenically from PDV in epitopes on the nucleocapsid protein (N), the phosphoprotein (P), the fusion protein (F), and the haemagglutinin (H) (Table 1). These results, combined with reported findings on the reactivity of these mAbs with strains of CDV, RPV and MV [1, 13] indicate that the porpoise virus is more closely related to CDV and PDV than to any of the other morbitliviruses. Also, in CDV strains, there is a high degree of conservation at the particular epitopes examined. Isolates of MV and RPV were limited in their reactivity to the panel of mAbs, but there were strain differences. Thus, in order to assess the significance of the differences between PDV and the porpoise virus, it will be necessary to examine several isolates of each. PDV monoclonal antibodies could be used to determine differences more clearly. The results of the CDV mAb reactivity of the PDV isolate following adaption to Veto cells have been described previously [1] and are included in Table 1. The reaction to the H1 mAb differs from that reported here for BFL adapted virus. Antigenic changes occur as a result of in vitro passage and therefore it is important to only compare results where viruses are adapted to the same cell line after as few passages as possible and at the same passage level. Antigenic changes in lapinised RPV as a result of in vivo and in vitro passage have been described [-3] and, while it is possible that differences in CDV, PDV, and the porpoise virus are solely consequences of adaption to different hosts, previously reported nucleic acid hybridisation data [-8, 12] would suggest that this is highly unlikely in the case of the differences between PDV and CDV. It has recently been suggested that there is unlikely to have been an epidemiological link between outbreaks of distemper in Siberian fresh water seals (Phoca siberica) and in harbour seals in North Western Europe [-10]. This conclusion was based on differences in reactions to mAbs to CDV and later work on these two isolates indicated that the Siberian isolate was similar, if not identical, to CDV [14]. The results reported in the communication of the reactivity pattern of a panel of monoclonal antibodies to CDV with PDV and a porpoise morbillivirus are insufficient to propose that the porpoise morbil-
Isolation and characterisation of a porpoise morbillivirus
251
livirus originated from seals or vice versa. However, analysis of virus nucleic acid isolated directly from tissue samples of infected animals should elucidate the origins of these two marine m a m m a l morbilliviruses. The isolation of morbilliviruses from porpoises presents difficulties similar to those reported by other workers in the isolation of P D V [11, 2, 7]. It has been reported that P D V grows poorly in Vero cells and that primary dog and seal kidney cells are most sensitive [7]. However, if large numbers of isolates are to be made, it is essential that readily available cell cultures can be used. This paper provides evidence that porpoise morbillivirus can be isolated from porpoise tissues on continuous and semicontinuous cell lines, which is consistent with previous experience in the isolation o f P D V (S. McCullough, unpubl, obs.).
References 1. Cosby SL, McQuaid S, Duffy N, Lyons C, Rima BK, Allan GM, McCullough SJ, Kennedy S, Smyth JA, McNeilly F, Craig C, Orvell C (1988) Characterisation of a seal morbillivirus. Nature 336:115-116 2. Hofmeister R, Breuer E, Hentschke J, Molle G, Ludwig HJ (1988) Distemper-like disease in harbour seals: Virus isolation, further pathologic and serologic findings. J Vet Med B 35:765-769 3. Ishii H, Yoshikawa Y, Yamanouchi K (1986) Adaption of the lapinised rinderpest virus to in vitro growth and attenuation of its virulence in rabbits. J Gen Virol 67: 275-280 3a. Kennedy S, Smyth JA, Cush PF, McAliskey M, McCullough SJ, Rima BK (1991) Histopathologic and immunocytochemicalstudies of distemper in porpoises. Vet Pathol 28:1-7 4. Kennedy S, Smyth JA, Cush PF, Duignan P, Platten M, McCullough SJ, Allan GM (1989) Histopathotogic and immunocytochemical studies of distemper in seals. Vet Pathol 26:97-103 5. Kennedy S, Smyth JA, Cush PF, McCullough SJ, Allan GM, McQuaid S (1988) Viral distemper now found in porpoises. Nature 336:21 6. Kennedy S, Smyth JA, McCullough SJ, Allan GM, McI'~eilly F, McQuaid S (1988) Confirmation of cause of recent seal deaths. Nature 335:404 7. Liess B, Frey HR, Zagahwa A (1989) Morbillivirus in seals: isolation and some growth characteristics in cell cultures. Dtsch Tier/irztt Wochenschr 96:180-182 8. Mahy BWJ, Barrett T, Evans S, Anderson EC, Bostock CJ (1988) Characterisation of a seal morbillivirus. Nature 336:115 9. Osterhaus ADME (1989) A morbillivirus causing mass mortality in seals. Vaccine 7: 483 10. Osterhaus ADME, Broeders HWJ, Groen J, UytdeHaag FGC, Visser IKG, van de Bildt MWG, Orvell C, Kumarev VP, Zorin VL (1989) Different morbitliviruses in European and Siberian seals. Vet Rec 125:647-648 11. Osterhaus ADME, Groen J, De Vfies P, UytdeHaag FGCM, Klingeborn B, Zarnke R (1988) Canine distemper virus in seals. Nature 335:403404 12. Rima BK, Cosby SL, Duffy N, Lyons C, O'Loan D, Kennedy S, McCullough SJ, Smyth JA, McNeilly F (1990) Humoral immune responses in seals infected by phocine distemper virus. Res Vet Sci 49:114-116 13. Sheshberadaran H, Norrby E, McCullough KC, Carpenter WC, Orvell C (1986) The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies. J Gen Virol 67:1381-1392
252 S.J. McCullough et al.: Isolation and characterisation of a porpoise morbillivirus 14. Visser IKG, Kumarev VP, Orvell C, de Vries P, Broeders HWJ, van de Bildt MWG, Groen J, Teppema JS, Burger MC, UytdeHaag FGCM, Osterhaus ADME (1990) Comparison of two morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch Virol 111:149-164 Authors' address: S. J. McCullough, Veterinary Research Laboratories, Stormont, Belfast BT4 3SD, Northern Ireland. Received November 20, 1990
Vi rology
Arch Virol (1991) 118:247-252
© Springer-Verlag 1991 Printed in Austria
Isolation and characterisation of a porpoise morbillivirus Brief R e p o r t S. J. McCullough 1, F. McNeilly l, G. M. Allan 1, S. Kennedy l, J. A. Smyth t, S. L. Cosby2, S. McQuaid 2, and B. K. R i m a 2 1Veterinary Research Laboratories, Belfast 2 Department of Biology, The Queen's University of Belfast, Belfast, Northern lreland
Accepted January 23, 1991 Summary. A morbillivirus was isolated from lung tissue of a porpoise which had lesions similar to those of phocine distemper. Porpoise and seal isolates differed in their reactions to monoclonal antibodies to canine distemper virus.
The type species of the genus Morbillivirus of the family' Paramyxoviridae is measles virus (MV) and the other members are canine distemper virus (CDV), rinderpest virus (RPV) and peste des petits ruminants virus (PPRV). An epizootic of morbillivirus infection killed thousands of harbour seals (Phoca vitulina) in European waters during 1988 [9]. The isolation of a morbillivirus from affected seals found on the coast of Northern Ireland and the lesions of distemper associated with the presence of morbillivirus antigen in these animals have been reported previously [4, 6]. We have suggested that the virus should be classified as a new member of the genus Morbillivirus, and have proposed the name phocine distemper virus (PDV) [1]. During the seat epizootic we also found evidence of morbillivirus infection in harbour porpoises (Phocoena phocoena) from the Irish Sea. Affected porpoises had lesions similar to those of PDV infected seals and we demonstrated morbillivirus antigen in many tissues using an immunoperoxidase technique [5]. We now report the first isolation of a morbiUivirus from the tissues of a porpoise and the results of comparison of this isolate with a PDV isolate by reaction with a panel of monoclonal antibodies (mAbs) against CDV. During the months of September to December 1988; six harbour porpoises which had been found stranded on the coast of Northern Ireland were submitted to our laboratories for necropsy. All six animals had distemper-like lesions [3a]. Samples of lung tissue were obtained for virus isolation from two carcases.
248
S.J. McCuUough et al.
Cryostat sections of lung tissue from both animals were stained by indirect immunofluorescence using serum from a human patient with subacute sclerosing panencephalitis (SSPE) as primary antibody, followed by fluorescein conjugated anti-human immunoglobulin. Immunofluorescent staining was observed in some of these cryostat sections, however, autolysis made it difficult to determine the specificity of the fluorescence seen when the sections were examined by incident UV light microscopy. Samples of lung tissue were ground in Eagle's minimum essential medium (MEM) containing 1000 IU penicillin, 1000 gg streptomycin and 100 IU nystatin
Fig. 1. a Negative contrast electron microscopy of lung homogenatefrom porpoise suspected of having a morbillivirus infection. Note viral nucleocapsid, x 200,000. b Indirect immunofluorescence staining of viral antigens in foetal bovine lung cells infected with porpoise morbillivirus and stained using SSPE antiserum, x 1,500.
Isolation and characterisation of a porpoise morbillivirus
249
per ml. The resulting h o m o g e n a t e s were centrifuged at 3000 g for 30 min a n d the s u p e r n a t a n t fluids retained. The fluids were e x a m i n e d by negative c o n t r a s t electron m i c r o s c o p y which revealed the helical nucleocapsid characteristic o f the Paramyxoviridaein one o f the samples (Fig. l a). Cultures o f Vero cells, M a d i n D a r b y canine k i d n e y ( M D C K ) cells a n d a s e m i c o n t i n u o u s line o f bovine foetal lung (BFL) cells were inoculated with the supernatants o f porpoise lung tissue homogenates by adsorption onto cell m o n o layers for 1 h at 37 °C or by inoculation o f freshly trypsinised cells. I n o c u l a t e d cultures were harvested by trypsinisation at intervals o f 3-4 days a n d passaged with freshly trypsinised u n i n o c u l a t e d cells. A t each passage, coverslip cultures o f each cell line were p r e p a r e d a n d virus g r o w t h was m o n i t o r e d by indirect i m m u n o f l u o r e s c e n c e as described above. I m m u n o f l u o r e s c e n t staining o f m o r billivirus antigen was observed in cell cultures inoculated with the s u p e r n a t a n t fluid in which p a r a m y x o v i r u s particles h a d been detected by negative contrast electron m i c r o s o p y (Fig. lb). I n o c u l a t i o n by a d s o r p t i o n onto Vero or B F L m o n o l a y e r s resulted in earliest a n d m o s t rapid g r o w t h o f virus. Positive ceils a p p e a r e d in b o t h cell lines at second passage. By f o u r t h passage, 90% o f B F L cells and 10% o f Vero cells were positive by indirect i m m u n o f l u o r e s c e n c e . However, there was no evidence Table 1. Reactivity of anti-CDV monoclonal antibodies with seal and porpoise distemper viruses Anti-CDV mAbs
PDV a Vero adapted
PDV BFL adapted
Porpoise distemper virus
N1 (3.721) N2 (3.991) N3 (3.805) P1 (3.568) P2 (3.698) P3 (3.780) P4 (3.630) P5 (3.768) P6 (4.088) F1 (3.584) F2 (3.633) F3 (5.148) F4 (3.551) H1 (3.734) H2 (3.755) H3 (4.043) H4 (2.267)
+ + + + + + + + + + + +b
+ + + + + + + + + + + + +
+ + + + + + + + + + + _
_
_
_
b
PDV and porpoise distemper virus isolates adapted to BFL cells at the eighth passage. Scoring of immunofluorescent staining: + distinct; - none a Vero adapted PDV results reported previously [1] b H3 and H4 results previously appeared as: H3 negative, H4 positive due to printer's error
250
S.J. McCullough et al.
of virus growth in M D C K cells following adsorption onto established monolayers, though positive cells were detected at third passage in all cell lines when freshly trypsinised cells were inoculated. Infected BFL cells at fifth passage were lysed by freezing and thawing three times. The lysate was examined by negative contrast electron microscopy which revealed virus particles with typical paramyxovirus morphology. On the basis of morphology and reaction with SSPE antiserum, the isolated virus was characterised as a member of the genus Morbillivirus. Coverslip cultures of PDV- and porpoise distemper virus-infected BFL cells at the eighth passage level were examined by indirect immunofluorescence using a selected panel of mAbs to CDV (Convac strain, supplied by C. ~rvell, State Bacteriological Laboratory, Stockholm, Sweden). The reactions of porpoise morbillivirus with 11 of the panel of 17 mAbs were the same as those of PDV. However, it differed antigenically from PDV in epitopes on the nucleocapsid protein (N), the phosphoprotein (P), the fusion protein (F), and the haemagglutinin (H) (Table 1). These results, combined with reported findings on the reactivity of these mAbs with strains of CDV, RPV and MV [1, 13] indicate that the porpoise virus is more closely related to CDV and PDV than to any of the other morbitliviruses. Also, in CDV strains, there is a high degree of conservation at the particular epitopes examined. Isolates of MV and RPV were limited in their reactivity to the panel of mAbs, but there were strain differences. Thus, in order to assess the significance of the differences between PDV and the porpoise virus, it will be necessary to examine several isolates of each. PDV monoclonal antibodies could be used to determine differences more clearly. The results of the CDV mAb reactivity of the PDV isolate following adaption to Veto cells have been described previously [1] and are included in Table 1. The reaction to the H1 mAb differs from that reported here for BFL adapted virus. Antigenic changes occur as a result of in vitro passage and therefore it is important to only compare results where viruses are adapted to the same cell line after as few passages as possible and at the same passage level. Antigenic changes in lapinised RPV as a result of in vivo and in vitro passage have been described [-3] and, while it is possible that differences in CDV, PDV, and the porpoise virus are solely consequences of adaption to different hosts, previously reported nucleic acid hybridisation data [-8, 12] would suggest that this is highly unlikely in the case of the differences between PDV and CDV. It has recently been suggested that there is unlikely to have been an epidemiological link between outbreaks of distemper in Siberian fresh water seals (Phoca siberica) and in harbour seals in North Western Europe [-10]. This conclusion was based on differences in reactions to mAbs to CDV and later work on these two isolates indicated that the Siberian isolate was similar, if not identical, to CDV [14]. The results reported in the communication of the reactivity pattern of a panel of monoclonal antibodies to CDV with PDV and a porpoise morbillivirus are insufficient to propose that the porpoise morbil-
Isolation and characterisation of a porpoise morbillivirus
251
livirus originated from seals or vice versa. However, analysis of virus nucleic acid isolated directly from tissue samples of infected animals should elucidate the origins of these two marine m a m m a l morbilliviruses. The isolation of morbilliviruses from porpoises presents difficulties similar to those reported by other workers in the isolation of P D V [11, 2, 7]. It has been reported that P D V grows poorly in Vero cells and that primary dog and seal kidney cells are most sensitive [7]. However, if large numbers of isolates are to be made, it is essential that readily available cell cultures can be used. This paper provides evidence that porpoise morbillivirus can be isolated from porpoise tissues on continuous and semicontinuous cell lines, which is consistent with previous experience in the isolation o f P D V (S. McCullough, unpubl, obs.).
References 1. Cosby SL, McQuaid S, Duffy N, Lyons C, Rima BK, Allan GM, McCullough SJ, Kennedy S, Smyth JA, McNeilly F, Craig C, Orvell C (1988) Characterisation of a seal morbillivirus. Nature 336:115-116 2. Hofmeister R, Breuer E, Hentschke J, Molle G, Ludwig HJ (1988) Distemper-like disease in harbour seals: Virus isolation, further pathologic and serologic findings. J Vet Med B 35:765-769 3. Ishii H, Yoshikawa Y, Yamanouchi K (1986) Adaption of the lapinised rinderpest virus to in vitro growth and attenuation of its virulence in rabbits. J Gen Virol 67: 275-280 3a. Kennedy S, Smyth JA, Cush PF, McAliskey M, McCullough SJ, Rima BK (1991) Histopathologic and immunocytochemicalstudies of distemper in porpoises. Vet Pathol 28:1-7 4. Kennedy S, Smyth JA, Cush PF, Duignan P, Platten M, McCullough SJ, Allan GM (1989) Histopathotogic and immunocytochemical studies of distemper in seals. Vet Pathol 26:97-103 5. Kennedy S, Smyth JA, Cush PF, McCullough SJ, Allan GM, McQuaid S (1988) Viral distemper now found in porpoises. Nature 336:21 6. Kennedy S, Smyth JA, McCullough SJ, Allan GM, McI'~eilly F, McQuaid S (1988) Confirmation of cause of recent seal deaths. Nature 335:404 7. Liess B, Frey HR, Zagahwa A (1989) Morbillivirus in seals: isolation and some growth characteristics in cell cultures. Dtsch Tier/irztt Wochenschr 96:180-182 8. Mahy BWJ, Barrett T, Evans S, Anderson EC, Bostock CJ (1988) Characterisation of a seal morbillivirus. Nature 336:115 9. Osterhaus ADME (1989) A morbillivirus causing mass mortality in seals. Vaccine 7: 483 10. Osterhaus ADME, Broeders HWJ, Groen J, UytdeHaag FGC, Visser IKG, van de Bildt MWG, Orvell C, Kumarev VP, Zorin VL (1989) Different morbitliviruses in European and Siberian seals. Vet Rec 125:647-648 11. Osterhaus ADME, Groen J, De Vfies P, UytdeHaag FGCM, Klingeborn B, Zarnke R (1988) Canine distemper virus in seals. Nature 335:403404 12. Rima BK, Cosby SL, Duffy N, Lyons C, O'Loan D, Kennedy S, McCullough SJ, Smyth JA, McNeilly F (1990) Humoral immune responses in seals infected by phocine distemper virus. Res Vet Sci 49:114-116 13. Sheshberadaran H, Norrby E, McCullough KC, Carpenter WC, Orvell C (1986) The antigenic relationship between measles, canine distemper and rinderpest viruses studied with monoclonal antibodies. J Gen Virol 67:1381-1392
252 S.J. McCullough et al.: Isolation and characterisation of a porpoise morbillivirus 14. Visser IKG, Kumarev VP, Orvell C, de Vries P, Broeders HWJ, van de Bildt MWG, Groen J, Teppema JS, Burger MC, UytdeHaag FGCM, Osterhaus ADME (1990) Comparison of two morbilliviruses isolated from seals during outbreaks of distemper in North West Europe and Siberia. Arch Virol 111:149-164 Authors' address: S. J. McCullough, Veterinary Research Laboratories, Stormont, Belfast BT4 3SD, Northern Ireland. Received November 20, 1990
