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Experimental studies in weaned pigs with three vaccines against Aujeszky's disease
Comp. lmmun. Microbiol. infect. Dis., Vol. 2, pp. 327 334. ,~ P e r g a m o n Press Ltd., 1979. Printed in G r e a t Britain
0147-9571/79/0901-0327 $02.00/0
E X P E R I M E N T A L S T U D I E S IN W E A N E D PIGS WITH T H R E E V A C C I N E S A G A I N S T A U J E S Z K Y ' S DISEASE J. B. McFERRAN, C. DOW a n d R. M. MCCRACKEN Veterinary Research Laboratories, Stormont, Belfast, BT4 3SD, Northern Ireland Abstract~Groups of pigs were vaccinated with two attenuated and one inactivated vaccine and then challenged 2 weeks later by intranasal inoculation of a virulent strain of Aujeszky's disease virus. Mortality rates varied from none in the group vaccinated with attenuated vaccine to 11°/oin the group given inactivated vaccine and 94~o in the controls. No vaccine prevented the development of clinical illness or excretion of virulent virus. However all vaccines reduced the number of days on which virus was excreted. Three weeks after challenge all 3 groups had recovered and there was no signifcant difference in mean weights. Key words." Aujeszky's disease, vaccination, pigs, attenuated live vaccine, inactivated vaccine E T U D E S E X P E R I M E N T A L E S C H E Z DES P O R C E L E T S SEVRES DE TROIS VACCINS C O N T R E LA M A L A D I E D ' A U J E S K Y Resam~-Des groupes de porcelets ont ere vaccinesavec deux vaccins attenues et un vaccin inactive puis soumis deux semaines plus tard ~il'inoculation intranasale d'une souche virulente du virus de la maladie d'Aujesky. Les taux de mortalite ont vari~ de zero dans le groupe vaccine avec un vaccin attenue ~i 11~ dans le groupe fi vaccin inactive et 94~odans le groupe de contr61e. Aucun vaccin n'a emp~che l'evolution clinique de la maladie ou l'excretion du virus virulent. Cependant tousles vaccins ont r~duit le nombre de jours pendant lesquels le virus a ~te excret~. Trois semaines apr~s l'epreuve, les trois groupes etaient gueris et aucune difference significativen'a ~te relevee dans les poids moyens. Mots-clefs: Maladie d'Aujesky, vaccination, porcelets, vaccin vivant attenu& vaccin inactive
INTRODUCTION Aujeszky's disease (AD) in N o r t h e r n I r e l a n d has traditionally caused sporadic disease in pigs, with occasional i n v o l v e m e n t o f cattle, dogs a n d cats. This p a t t e r n o f disease, which is associated with the virus strains o f the NIA-1 or N I A - 2 types, is n o r m a l l y confined to pigs u n d e r 4 weeks o f age a n d to p r e g n a n t sows, with i n a p p a r e n t infections in older animals. A n t i b o d y studies hav~ shown that whilst up to 3 0 ~ o f sows had a n t i b o d y [1] o n individual farms where sows a n d boars have a n t i b o d y , e x a m i n a t i o n o f the records frequently showed no evidence o f disease, indicating that economic loss due to this virus was m i n i m a l . However, in 1971 a m u c h m o r e virulent strain was isolated from 14-18-week-old pigs dying in a large fattening unit. Peak m o r t a l i t y was 2 - 4 weeks after the pigs arrived on the farm. This strain ( N I A - 3 ) is capable o f killing up to 100~ of 8-week-old pigs infected intranasally. It is n o t k n o w n if this strain entered from outside N o r t h e r n Ireland, which in view o f the veterinary regulations is unlikely, or whether it arose as a m u t a t i o n from the t r a d i t i o n a l strains. The second hypothesis is feasible, because the A D virus changes character c o m p a r a t i v e l y easily. T h u s a 26 pass level o f A D virus in pig kidney cells caused m a r k e d l y different lesions a n d virus d i s t r i b u t i o n in sheep t h a n did the 4 passage of the same isolate [2]. 327
328
J.B. McFERRAN,C. Dow and R. M. MCCRACKEN
Although at present this strain has only been recognised on one unit, in view of the intensification of pig husbandry, with 58 farms having more than 100 breeding sows, and 23 farms fattening 1000 or more purchased weaners [3] the possibility that similar strains could develop must be faced, and vaccination would be essential in units of this size. As there is no possibility of vaccinating the pigs before entry into these large units, it was decided to investigate the efficiency of 3 vaccines to protect pigs from early challenge with virulent virus. MATERIALS AND METHODS
Cell cultures Pig kidneys from 3 5-week-old pigs from the laboratory's pig herd were treated with 0.25~o trypsin solution. Growth was in Hanks' lactalbumin solution with I0~o ox serum and the cells were maintained in Earle's lactalbumin solution (LAE) with 2~o foetal calf serum.
Pigs A total of 71 pigs, 4-5 weeks of age from 9 litters were obtained from a minimal disease herd, which was free of Aujeszky's disease. They were divided, at random, into 3 groups of 18 and one group of 17. The pigs were bled and weighed at the intervals shown in Tables 1 and 3.
Vaccines An experimental batch of NIA-4 vaccine*, BUK-900 vaccine:~ and an inactivated vaccine§ were used. The 2 live vaccines were inoculated subcutaneously and the inactivated vaccine intramuscularly.
Virus
The virus used for challenge was the NIA-3 strain, used at the 5th cell culture passage, and diluted to give a titre of 1060 per 0.5 ml. The virus was administered by dropping 0.5 ml intranasally from a syringe. Virus isolation
Six pigs from each group were swabbed daily after challenge. Each swab was placed in 4 ml L A E + 1~ bovine serum albumin. Aliquots of olfactory bulbs, mesencephalon, * Supplied by Dr. N. Zygraich, R.I.T., Genval, Belgium. ~;Supptied by Dr. M. Krassalt, Mycofarm, De Bilt, The Netherlands: manufactured by Bioveta, Czechoslovakia. §"Geskyvac" supplied by Dr. J. F. Hepburn of Tasman Laboratories and manufactured by Laboratoire Roger Bellon, France.
Aujeszky's disease vaccines
329
medulla and tonsillar region of pharynx were taken from the pigs which died and made into 10~ suspensions using LAE as the diluent. Following low speed centrifugation the specimens were inoculated into 2 cultures of PK cells. If this was not possible, samples were stored at 4°C until cells were available. Herpesviruses were recognised by direct examination in the electron microscope, and a number o f isolates were shown to be Aujeszky's disease virus by neutralisation tests.
Serology The presence of antibody to Aujeszky's disease virus was detected by adding 200 tcids0 of virus to doubling dilutions of serum in microtitre plates. There were 2 wells for each serum dilution. Following 1-hr incubation at 37°C, Vero cells were added to each well. The plates were sealed with transparent tape and the test read at 2, 4 and 6 days. Sera known free of antibody and with low and high titres of antibody were included as controls. The titres are expressed as ~the final serum dilution which neutralised 100 tcidso (30-300 tcidso) virus in this test.
Histology Transverse sections o f anterior cerebrum, mid-cerebrum, posterior cerebrum, mesencephalon, cerebellum and medulla oblongata were taken from each pig and fixed in 10~o neutral buffered formalin. Tissues were processed by the paraffin technique and cut sections were stained with haematoxylin and eosin. RESULTS
Clinical Following vaccination. No illness was apparent during a 14-day observation period and all groups showed similar weight gains (Table 1). Following challenge. No signs o f illness were seen until the 3rd day when they developed a pyrexia, with all groups having a mean temperature at over 40.83 (Table 2) and individual temperatures as high as 41.78. All pigs were dull, constipated and food consumption was markedly reduced. On the 4th day the 3 vaccinated groups were still inactive, had a typical hoarse squeal and had poor appetites. The control group were depressed, tended not to squeal when handled and 2 had obvious clinical signs. Thus one pig was circling and the other was biting at boots and the food troughs, in between spells of pushing its head into the wall. All temperatures were at or above a mean of 40.83. By the 5th day one pig in the NIA-4 group had pruritis in the ear and shoulder. Pig 590 in the BUK group lay in sternal recumbency and was very depressed. A second pig in this group appeared to have difficulty in flexing its legs. It was incoordinated and collapsed periodically with all 4 legs extended. All the other pigs in these groups were lively and fighting over their food and the mean temperature had fallen. In contrast the pigs given the inactivated vaccine were still depressed, three pigs had nervous signs and one pig was
22.97 22.92 17.38 ---
Days after challenge 3 5 7 12 21 3.81 3.69 4.27 ---
2.16 2.99 3.51
S.D.
0.92 1.07 2.14 ---
0.52 0.73 0.85
S.E.
23.25 23.03 24.19 26.75 34.00
14.83 18.88 22.75
Mean
4.69 4.25 3.81 4.07 4.93
2.19 3.20 3.96
S.D.
NIA-4
1.01 1.00 0.90 0.96 1.16
0.52 0.75 0.93
S.E.
20.44 17.79 24.06 26.82 34.38
15.39 20.03 23.47
Mean
3.02 2.83 3.73 4.34 5.65
2.70 3.31 3.63
S.D.
BUK
0.71 0.67 0.90 1.05 1.37
0.64 0.78 0.86
S.E.
22.47 22.42 23.15 26.84 35.44
15.30 19.89 23.83
Mean
3.44 3.32 4.53 5.37 6.90
2.73 3.77 4.44
S.D.
Inactivated
40.88 40.94 40.72 --
3 4 5 6 7
40.88 40.83 40.05 39.00 39.28
40.94 40.83 39.83 39.11 39.11
BUK
Mean temperature-C NIA-4
40.83 41.00 41.00 40.56 39.61
Inactivated
0 0 5 8 13"
Control
*Further pigs died on day 8, 9 and 10, giving a final total of 16 dead of 17 inoculated.
Control
Days after challenge
0 0 0 0 0
NIA-4
0 0 0 0 1
BUK
0 0 1 1 2
Inactivated
Cumulative mortality
Table 2. Mean temperatures and mortality in groups of vaccinated and control pigs following intranasal challenge with a virulent (NIA-3) strain of Aujeszky's disease virus
*Weights are expressed in kg. S.D. = Standard deviation. S.E. = Standard error.
14.74 18.56 22.47
Mean*
Days after vaccination 0 7 14
Date
Control
0.81 0.81 1.13 1.34 1.73
0.64 0.89 1.05
S.E.
Table 1. Mean weights and their standard deviations in groups of vaccinated and control pigs, subsequently challenged with a virulent (NIA-3) strain of Aujeszky's disease virus
('3
©
;>
z ('3 t~
Aujeszky'sdisease vaccines
331
Table 3. Titres of serum neutralising antibody found in pigs vaccinated and then challenged with a virulent (NIA-3) strain of Aujeszky'sdisease virus NIA-4 Days
Mean
After vaccination 0 7 14
<2* <2 3.4
After challenge 7 12 21
41.9 36.7 45.26
BUK Range --24
16-128 16-128 32 64
Mean
Range
<2 <2 2.9
--
55.7 66.7 52.2
Inactivated Mean Range
<2 8
<2 <2 5.7
-4-8
3~128 3~128 32-128
88.7 117.4 94.6
32-256 64-128 64-128
*Titres expressed as the reciprocal of the serum dilution which neutralised 100 tcids0 of virus. The control group died before antibodycould be detected, except pig No 534 whichsurvivedchallengeand had a titre of 1/32 at both the 12th and 21st day post-challenge bleeding. found dead. There were 5 dead pigs in the control group, and 6 pigs showed nervous signs. These included incoordination, short fits, circling, backing into corners, continual head movements and lateral recumbency. All the pigs vaccinated with BUK and NIA-4 strains were bright, no longer constipated and had normal mean temperatures by the 6th day except pig 590 in the BUK-vaccinated group. The group given inactivated vaccine were still depressed, some were incoordinated and only 2 were interested in food. Three more of the controls were dead and three of the remainder had severe nervous signs. One control pig (534), although it had a temperature of 40.72 stood out because it was alert and eating. Pig 590 in the BUK group finally died on the 7th day after challenge and a pig in the inactivated vaccine group died on the same day. All the other vaccinated pigs recovered quickly, whilst all the pigs in the control group, except pig 534, died. After challenge, all the groups showed the effect of the virus on weight gain, but 21 days after challenge all the groups had put on a mean weight gain of at least 10 kg, and there was no significant difference between the groups (Table 1). Serological response. All 3 groups of vaccinates responded with serum neutralising antibodies by the 14th day after vaccination (Table 3). Following challenge, there was a rapid response, especially in the group given inactivated vaccine.
Virus isolation Aujeszky's disease virus recoveries are shown in Table 4. Only 10 of the 30 samples taken from the N I A - 4 group contained virus, whilst 13 of 30 from the BUK-vaccinated group and 18 of 27 swabs from the group given inactivated vaccine had virus present. All the swabs collected from the control pigs up to 7 days after challenge had virus. Virus was recovered from the CNS of the dead pigs when examined, except from pigs 558 and 590. Pig 558 was given inactivated vaccine, and virus was recovered from the pharynx but not the CNS when it died on the 5th day. Pig 590 which died on the 7th day had been given B U K vaccine. N o virus was isolated from the CNS or the pharynx. However both of these pigs had severe CNS lesions.
J . B . MCFERRAN,C. D o w and R. M. MCCRACKEN
332
Table 4. Virus isolation from the nasal swabs of pigs challenged with NIA-3 strain of Aujeszky's disease virus Day following challenge Group
Pig number
3
4
5
6
7
Control
520 521 534 537 538 563
+ + + + + +
+ + + + + +
+ + + D + +
+ + +
D + +
+ D
D
517 560 562 567 572 603
. . + + +
-
+
-
-
BUK
533 539 556 582 586 607
+ + + + + .
Inactivated
515 523 541 543 558 601
NIA-4
+ + + +
+
+ + +
. . + -
+ + + + +
+ + + -
-
-
+ + + D +
+ +
-
+
+
. .
. .
.
. + + + + + +
. .
+
.
-
+ = virus detected. = no virus detected. D = pig dead. Virus was not detected in swabs taken from the vaccinated pigs after the 7th day post-challenge. -
Histological examination T h e b r a i n s o f 10 c o n t r o l p i g s d y i n g b e t w e e n 5 a n d 10 d a y s p o s t - i n f e c t i o n w e r e e x a m i n e d h i s t o l o g i c a l l y a n d l e s i o n s t y p i c a l o f A u j e s z k y ' s d i s e a s e w e r e s e e n in all [4]. I n t h e i n a c t i v a t e d v a c c i n e g r o u p 2 p i g s d i e d . L e s i o n s in t h e b r a i n o f p i g 558 w h i c h d i e d 5 d a y s p o s t - c h a l l e n g e w e r e s i m i l a r in d i s t r i b u t i o n t o t h o s e o f d e a d c o n t r o l p i g s b u t a pronounced and generalised glial and polymorph reaction was very evident throughout the b r a i n . I n t r a - n u c l e a r i n c l u s i o n b o d i e s w e r e r e a d i l y i d e n t i f i e d in n e u r o n s o f t h i s p i g a n d all the dead controls. Pig 604 which died 7 days post-challenge showed the presence of several m a l a c i c f o c i in t h e w h i t e m a t t e r o f t h e a n t e r i o r h a l v e s o f t h e c e r e b r a l h e m i s p h e r e s . T h e s e foci contained large numbers of macrophages which frequently were distended with cellular debris. Only one pig vaccinated with attenuated vaccine died. This pig showed l e s i o n s s i m i l a r in n a t u r e t o t h o s e in p i g 6 0 4 b u t w i t h a d i s t r i b u t i o n in t h e p o s t - c e r e b r u m mid-brain region.
to
Aujeszky's disease vaccines
333
DISCUSSION Three vaccines were compared in this experiment. One attenuated vaccine investigated was produced using the BUK strain [5] and the other was prepared from an isolate made from a bovine in Northern Ireland. This virus is very attenuated, not even killing SPF rabbits [6]. The inactivated vaccine was a cell culture grown virus inactivated with glutaraldehyde in an oil in water emulsion [7]. All vaccines were highly efficient in their primary task o f protecting the pigs from death when subjected to a challenge which killed 94~o of the control group. The vaccines did not prevent the challenged pigs excreting virulent virus. Thus when swabs collected during the first 7 days after infection were tested, NIA-4 strain was most efficient; only 33~o of swabs had virus whilst 67~o of the swabs from pigs given inactivated vaccine contained virus. A comparison could not be undertaken with the control group because virus was not detected in swabs from the vaccinated pigs after 7 days, at which stage 13 of the control pigs were dead. However, using virulent strains, where unvaccinated pigs survived, virus excretion was virtually continuous for 10 days after infection and intermittently for another 7 days [8]. The pigs vaccinated with attenuated vaccine were much less ill, based not only on a subjective clinical assessment but also on the number of days they had an elevated temperature compared to the inactivated vaccinated group. Nevertheless 3 weeks after challenge there was no significant difference in the weights of the 3 groups. The serum neutralisation titres recorded 14 days after vaccination were low, but the titres produced by the attenuated vaccines were similar to those recorded for the Bartha vaccine [9]. The secondary response following challenge was considerably lower than previously recorded [9]. This may be a reflection o f the time between vaccination and challenge; 5 weeks in one experiment and 2 weeks in the second. It is probable that higher titres would have been recorded for the inactivated vaccine if a longer interval had been left before challenge [7]. However because of the type of husbandry practised, a vaccine would have to give adequate protection in 2 weeks. The choice of vaccine for control purposes is not easy. The inactivated vaccine produced a good immunity and, in theory at least, inactivated vaccines are safer. However inactivated vaccines can be dangerous, because the inactivation has not been effective, because a second unsuspected agent may be present which is more resistant to the inactivating agent than the vaccine virus, or because the vaccine may be contaminated with another agent following inactivation. Furthermore it is much more difficult to check inactivated vaccines for purity, because the adjuvent can preclude the use of cell cultures. To these hazards must also be added the increased cost of inactivated vaccine. This latter is unlikely to be important when vaccinating breeding animals, but could be a major factor in large fattening units. In theory the disadvantages of attenuated vaccines are that they are more liable to be contaminated, they may be excreted, they may cause disease and they may revert to virulence. The dangers of contamination can be minimised by growing the vaccine virus in a diploid cell line, which has been adequately tested, by using versene instead of trypsin to disperse the cells and by using serum obtained from a SPF source or sterilised by irradiation for cell culture growth. The BUK [5], the Bartha [9, 10] and the NIA-4 strains (McFerran and Dow, unpublished observations) are not excreted and therefore the risk o f reversion to virulence does not
334
J.B. MCFERRAN,C. Dow and R. M. MCCRACKEN
occur. F u r t h e r m o r e the N I A - 4 strain has very low virulence. The low serum neutralising titres stimulated by the a t t e n u a t e d vaccine m a y be a n advantage. Whilst any serum neutralising titre m u s t be regarded as indicative o f a potential carrier, a study o f a n u m b e r o f s e r a in a herd would in m a n y cases allow differentiation between a vaccinated herd a n d a herd infected with virulent virus. F o r good i m m u n i t y vaccinating doses o f 10 6 or greater for N I A - 4 are required ( M c F e r r a n a n d Dow, u n p u b l i s h e d observations) a n d J a m r i c h o v a a n d Skoda [5] also f o u n d a relationship between protection a n d vaccine virus titre. A n y c o m m e r c i a l a t t e n u a t e d vaccine should therefore have e n o u g h virus to stimulate a good i m m u n i t y . REFERENCES I. McFerran, J. B., Dow, C. and Hildebrand, W. R. P., Aujeszky's disease virus, Vet. Rec. 78, 700 (1966). 2. Dow, C. and McFerran, J. B., Experimental studies on Aujeszky's disease in sheep, Brit. vet. J. 122, 464-470 (1966). 3. Anonymous. Statistical Review of Northern Ireland Agriculture, 197:%1976. H.M.S.O., Belfast (1977). 4. Dow, C. and McFerran, J. B., The neuropathology of Aujeszky's disease in the pig, Res. vet. Sci. 3, 436-442 (1962). 5. Jamricbova, O. and Skoda, R., Multiplication and distribution of attenuated pseudorabies virus in the organism of vaccinated pigs, Acta Virologica 13, 42 51 (1969). 6. Dr. N. Zygraich, RIT, Genval, Belgium, personal communication (1978). 7. Delagneau, J. F., Toma, B., Vannier, P., Loquerie, R., Pruner, P. and Tillon, J. P., Immunisation contre la malades d'Aujeszky ~il'aide d'un nouveau vaccin huileux ~ virus inactive, Rec. Mbd. vbt. 151,567-575 (1975). 8. McFerran, J. B. and Dow, C., The excretion of Aujeszky's disease virus by experimentally infected pigs, Res. vet. Sci. 5, 405-410 (1964). 9. McFerran, J. B. and Dow, C., Studies on the immunisation of pigs with the Bartha strain of Aujeszky's disease virus, Res. vet. Sci. 19, 17-22 (1975). 10. Bartha, A. and Kojnok, J., Active immunisation against Aujeszky's disease, Proc. 17th Wld. vet. Cong., Hannover l, 531-533 (1963).
0147-9571/79/0901-0327 $02.00/0
E X P E R I M E N T A L S T U D I E S IN W E A N E D PIGS WITH T H R E E V A C C I N E S A G A I N S T A U J E S Z K Y ' S DISEASE J. B. McFERRAN, C. DOW a n d R. M. MCCRACKEN Veterinary Research Laboratories, Stormont, Belfast, BT4 3SD, Northern Ireland Abstract~Groups of pigs were vaccinated with two attenuated and one inactivated vaccine and then challenged 2 weeks later by intranasal inoculation of a virulent strain of Aujeszky's disease virus. Mortality rates varied from none in the group vaccinated with attenuated vaccine to 11°/oin the group given inactivated vaccine and 94~o in the controls. No vaccine prevented the development of clinical illness or excretion of virulent virus. However all vaccines reduced the number of days on which virus was excreted. Three weeks after challenge all 3 groups had recovered and there was no signifcant difference in mean weights. Key words." Aujeszky's disease, vaccination, pigs, attenuated live vaccine, inactivated vaccine E T U D E S E X P E R I M E N T A L E S C H E Z DES P O R C E L E T S SEVRES DE TROIS VACCINS C O N T R E LA M A L A D I E D ' A U J E S K Y Resam~-Des groupes de porcelets ont ere vaccinesavec deux vaccins attenues et un vaccin inactive puis soumis deux semaines plus tard ~il'inoculation intranasale d'une souche virulente du virus de la maladie d'Aujesky. Les taux de mortalite ont vari~ de zero dans le groupe vaccine avec un vaccin attenue ~i 11~ dans le groupe fi vaccin inactive et 94~odans le groupe de contr61e. Aucun vaccin n'a emp~che l'evolution clinique de la maladie ou l'excretion du virus virulent. Cependant tousles vaccins ont r~duit le nombre de jours pendant lesquels le virus a ~te excret~. Trois semaines apr~s l'epreuve, les trois groupes etaient gueris et aucune difference significativen'a ~te relevee dans les poids moyens. Mots-clefs: Maladie d'Aujesky, vaccination, porcelets, vaccin vivant attenu& vaccin inactive
INTRODUCTION Aujeszky's disease (AD) in N o r t h e r n I r e l a n d has traditionally caused sporadic disease in pigs, with occasional i n v o l v e m e n t o f cattle, dogs a n d cats. This p a t t e r n o f disease, which is associated with the virus strains o f the NIA-1 or N I A - 2 types, is n o r m a l l y confined to pigs u n d e r 4 weeks o f age a n d to p r e g n a n t sows, with i n a p p a r e n t infections in older animals. A n t i b o d y studies hav~ shown that whilst up to 3 0 ~ o f sows had a n t i b o d y [1] o n individual farms where sows a n d boars have a n t i b o d y , e x a m i n a t i o n o f the records frequently showed no evidence o f disease, indicating that economic loss due to this virus was m i n i m a l . However, in 1971 a m u c h m o r e virulent strain was isolated from 14-18-week-old pigs dying in a large fattening unit. Peak m o r t a l i t y was 2 - 4 weeks after the pigs arrived on the farm. This strain ( N I A - 3 ) is capable o f killing up to 100~ of 8-week-old pigs infected intranasally. It is n o t k n o w n if this strain entered from outside N o r t h e r n Ireland, which in view o f the veterinary regulations is unlikely, or whether it arose as a m u t a t i o n from the t r a d i t i o n a l strains. The second hypothesis is feasible, because the A D virus changes character c o m p a r a t i v e l y easily. T h u s a 26 pass level o f A D virus in pig kidney cells caused m a r k e d l y different lesions a n d virus d i s t r i b u t i o n in sheep t h a n did the 4 passage of the same isolate [2]. 327
328
J.B. McFERRAN,C. Dow and R. M. MCCRACKEN
Although at present this strain has only been recognised on one unit, in view of the intensification of pig husbandry, with 58 farms having more than 100 breeding sows, and 23 farms fattening 1000 or more purchased weaners [3] the possibility that similar strains could develop must be faced, and vaccination would be essential in units of this size. As there is no possibility of vaccinating the pigs before entry into these large units, it was decided to investigate the efficiency of 3 vaccines to protect pigs from early challenge with virulent virus. MATERIALS AND METHODS
Cell cultures Pig kidneys from 3 5-week-old pigs from the laboratory's pig herd were treated with 0.25~o trypsin solution. Growth was in Hanks' lactalbumin solution with I0~o ox serum and the cells were maintained in Earle's lactalbumin solution (LAE) with 2~o foetal calf serum.
Pigs A total of 71 pigs, 4-5 weeks of age from 9 litters were obtained from a minimal disease herd, which was free of Aujeszky's disease. They were divided, at random, into 3 groups of 18 and one group of 17. The pigs were bled and weighed at the intervals shown in Tables 1 and 3.
Vaccines An experimental batch of NIA-4 vaccine*, BUK-900 vaccine:~ and an inactivated vaccine§ were used. The 2 live vaccines were inoculated subcutaneously and the inactivated vaccine intramuscularly.
Virus
The virus used for challenge was the NIA-3 strain, used at the 5th cell culture passage, and diluted to give a titre of 1060 per 0.5 ml. The virus was administered by dropping 0.5 ml intranasally from a syringe. Virus isolation
Six pigs from each group were swabbed daily after challenge. Each swab was placed in 4 ml L A E + 1~ bovine serum albumin. Aliquots of olfactory bulbs, mesencephalon, * Supplied by Dr. N. Zygraich, R.I.T., Genval, Belgium. ~;Supptied by Dr. M. Krassalt, Mycofarm, De Bilt, The Netherlands: manufactured by Bioveta, Czechoslovakia. §"Geskyvac" supplied by Dr. J. F. Hepburn of Tasman Laboratories and manufactured by Laboratoire Roger Bellon, France.
Aujeszky's disease vaccines
329
medulla and tonsillar region of pharynx were taken from the pigs which died and made into 10~ suspensions using LAE as the diluent. Following low speed centrifugation the specimens were inoculated into 2 cultures of PK cells. If this was not possible, samples were stored at 4°C until cells were available. Herpesviruses were recognised by direct examination in the electron microscope, and a number o f isolates were shown to be Aujeszky's disease virus by neutralisation tests.
Serology The presence of antibody to Aujeszky's disease virus was detected by adding 200 tcids0 of virus to doubling dilutions of serum in microtitre plates. There were 2 wells for each serum dilution. Following 1-hr incubation at 37°C, Vero cells were added to each well. The plates were sealed with transparent tape and the test read at 2, 4 and 6 days. Sera known free of antibody and with low and high titres of antibody were included as controls. The titres are expressed as ~the final serum dilution which neutralised 100 tcidso (30-300 tcidso) virus in this test.
Histology Transverse sections o f anterior cerebrum, mid-cerebrum, posterior cerebrum, mesencephalon, cerebellum and medulla oblongata were taken from each pig and fixed in 10~o neutral buffered formalin. Tissues were processed by the paraffin technique and cut sections were stained with haematoxylin and eosin. RESULTS
Clinical Following vaccination. No illness was apparent during a 14-day observation period and all groups showed similar weight gains (Table 1). Following challenge. No signs o f illness were seen until the 3rd day when they developed a pyrexia, with all groups having a mean temperature at over 40.83 (Table 2) and individual temperatures as high as 41.78. All pigs were dull, constipated and food consumption was markedly reduced. On the 4th day the 3 vaccinated groups were still inactive, had a typical hoarse squeal and had poor appetites. The control group were depressed, tended not to squeal when handled and 2 had obvious clinical signs. Thus one pig was circling and the other was biting at boots and the food troughs, in between spells of pushing its head into the wall. All temperatures were at or above a mean of 40.83. By the 5th day one pig in the NIA-4 group had pruritis in the ear and shoulder. Pig 590 in the BUK group lay in sternal recumbency and was very depressed. A second pig in this group appeared to have difficulty in flexing its legs. It was incoordinated and collapsed periodically with all 4 legs extended. All the other pigs in these groups were lively and fighting over their food and the mean temperature had fallen. In contrast the pigs given the inactivated vaccine were still depressed, three pigs had nervous signs and one pig was
22.97 22.92 17.38 ---
Days after challenge 3 5 7 12 21 3.81 3.69 4.27 ---
2.16 2.99 3.51
S.D.
0.92 1.07 2.14 ---
0.52 0.73 0.85
S.E.
23.25 23.03 24.19 26.75 34.00
14.83 18.88 22.75
Mean
4.69 4.25 3.81 4.07 4.93
2.19 3.20 3.96
S.D.
NIA-4
1.01 1.00 0.90 0.96 1.16
0.52 0.75 0.93
S.E.
20.44 17.79 24.06 26.82 34.38
15.39 20.03 23.47
Mean
3.02 2.83 3.73 4.34 5.65
2.70 3.31 3.63
S.D.
BUK
0.71 0.67 0.90 1.05 1.37
0.64 0.78 0.86
S.E.
22.47 22.42 23.15 26.84 35.44
15.30 19.89 23.83
Mean
3.44 3.32 4.53 5.37 6.90
2.73 3.77 4.44
S.D.
Inactivated
40.88 40.94 40.72 --
3 4 5 6 7
40.88 40.83 40.05 39.00 39.28
40.94 40.83 39.83 39.11 39.11
BUK
Mean temperature-C NIA-4
40.83 41.00 41.00 40.56 39.61
Inactivated
0 0 5 8 13"
Control
*Further pigs died on day 8, 9 and 10, giving a final total of 16 dead of 17 inoculated.
Control
Days after challenge
0 0 0 0 0
NIA-4
0 0 0 0 1
BUK
0 0 1 1 2
Inactivated
Cumulative mortality
Table 2. Mean temperatures and mortality in groups of vaccinated and control pigs following intranasal challenge with a virulent (NIA-3) strain of Aujeszky's disease virus
*Weights are expressed in kg. S.D. = Standard deviation. S.E. = Standard error.
14.74 18.56 22.47
Mean*
Days after vaccination 0 7 14
Date
Control
0.81 0.81 1.13 1.34 1.73
0.64 0.89 1.05
S.E.
Table 1. Mean weights and their standard deviations in groups of vaccinated and control pigs, subsequently challenged with a virulent (NIA-3) strain of Aujeszky's disease virus
('3
©
;>
z ('3 t~
Aujeszky'sdisease vaccines
331
Table 3. Titres of serum neutralising antibody found in pigs vaccinated and then challenged with a virulent (NIA-3) strain of Aujeszky'sdisease virus NIA-4 Days
Mean
After vaccination 0 7 14
<2* <2 3.4
After challenge 7 12 21
41.9 36.7 45.26
BUK Range --24
16-128 16-128 32 64
Mean
Range
<2 <2 2.9
--
55.7 66.7 52.2
Inactivated Mean Range
<2 8
<2 <2 5.7
-4-8
3~128 3~128 32-128
88.7 117.4 94.6
32-256 64-128 64-128
*Titres expressed as the reciprocal of the serum dilution which neutralised 100 tcids0 of virus. The control group died before antibodycould be detected, except pig No 534 whichsurvivedchallengeand had a titre of 1/32 at both the 12th and 21st day post-challenge bleeding. found dead. There were 5 dead pigs in the control group, and 6 pigs showed nervous signs. These included incoordination, short fits, circling, backing into corners, continual head movements and lateral recumbency. All the pigs vaccinated with BUK and NIA-4 strains were bright, no longer constipated and had normal mean temperatures by the 6th day except pig 590 in the BUK-vaccinated group. The group given inactivated vaccine were still depressed, some were incoordinated and only 2 were interested in food. Three more of the controls were dead and three of the remainder had severe nervous signs. One control pig (534), although it had a temperature of 40.72 stood out because it was alert and eating. Pig 590 in the BUK group finally died on the 7th day after challenge and a pig in the inactivated vaccine group died on the same day. All the other vaccinated pigs recovered quickly, whilst all the pigs in the control group, except pig 534, died. After challenge, all the groups showed the effect of the virus on weight gain, but 21 days after challenge all the groups had put on a mean weight gain of at least 10 kg, and there was no significant difference between the groups (Table 1). Serological response. All 3 groups of vaccinates responded with serum neutralising antibodies by the 14th day after vaccination (Table 3). Following challenge, there was a rapid response, especially in the group given inactivated vaccine.
Virus isolation Aujeszky's disease virus recoveries are shown in Table 4. Only 10 of the 30 samples taken from the N I A - 4 group contained virus, whilst 13 of 30 from the BUK-vaccinated group and 18 of 27 swabs from the group given inactivated vaccine had virus present. All the swabs collected from the control pigs up to 7 days after challenge had virus. Virus was recovered from the CNS of the dead pigs when examined, except from pigs 558 and 590. Pig 558 was given inactivated vaccine, and virus was recovered from the pharynx but not the CNS when it died on the 5th day. Pig 590 which died on the 7th day had been given B U K vaccine. N o virus was isolated from the CNS or the pharynx. However both of these pigs had severe CNS lesions.
J . B . MCFERRAN,C. D o w and R. M. MCCRACKEN
332
Table 4. Virus isolation from the nasal swabs of pigs challenged with NIA-3 strain of Aujeszky's disease virus Day following challenge Group
Pig number
3
4
5
6
7
Control
520 521 534 537 538 563
+ + + + + +
+ + + + + +
+ + + D + +
+ + +
D + +
+ D
D
517 560 562 567 572 603
. . + + +
-
+
-
-
BUK
533 539 556 582 586 607
+ + + + + .
Inactivated
515 523 541 543 558 601
NIA-4
+ + + +
+
+ + +
. . + -
+ + + + +
+ + + -
-
-
+ + + D +
+ +
-
+
+
. .
. .
.
. + + + + + +
. .
+
.
-
+ = virus detected. = no virus detected. D = pig dead. Virus was not detected in swabs taken from the vaccinated pigs after the 7th day post-challenge. -
Histological examination T h e b r a i n s o f 10 c o n t r o l p i g s d y i n g b e t w e e n 5 a n d 10 d a y s p o s t - i n f e c t i o n w e r e e x a m i n e d h i s t o l o g i c a l l y a n d l e s i o n s t y p i c a l o f A u j e s z k y ' s d i s e a s e w e r e s e e n in all [4]. I n t h e i n a c t i v a t e d v a c c i n e g r o u p 2 p i g s d i e d . L e s i o n s in t h e b r a i n o f p i g 558 w h i c h d i e d 5 d a y s p o s t - c h a l l e n g e w e r e s i m i l a r in d i s t r i b u t i o n t o t h o s e o f d e a d c o n t r o l p i g s b u t a pronounced and generalised glial and polymorph reaction was very evident throughout the b r a i n . I n t r a - n u c l e a r i n c l u s i o n b o d i e s w e r e r e a d i l y i d e n t i f i e d in n e u r o n s o f t h i s p i g a n d all the dead controls. Pig 604 which died 7 days post-challenge showed the presence of several m a l a c i c f o c i in t h e w h i t e m a t t e r o f t h e a n t e r i o r h a l v e s o f t h e c e r e b r a l h e m i s p h e r e s . T h e s e foci contained large numbers of macrophages which frequently were distended with cellular debris. Only one pig vaccinated with attenuated vaccine died. This pig showed l e s i o n s s i m i l a r in n a t u r e t o t h o s e in p i g 6 0 4 b u t w i t h a d i s t r i b u t i o n in t h e p o s t - c e r e b r u m mid-brain region.
to
Aujeszky's disease vaccines
333
DISCUSSION Three vaccines were compared in this experiment. One attenuated vaccine investigated was produced using the BUK strain [5] and the other was prepared from an isolate made from a bovine in Northern Ireland. This virus is very attenuated, not even killing SPF rabbits [6]. The inactivated vaccine was a cell culture grown virus inactivated with glutaraldehyde in an oil in water emulsion [7]. All vaccines were highly efficient in their primary task o f protecting the pigs from death when subjected to a challenge which killed 94~o of the control group. The vaccines did not prevent the challenged pigs excreting virulent virus. Thus when swabs collected during the first 7 days after infection were tested, NIA-4 strain was most efficient; only 33~o of swabs had virus whilst 67~o of the swabs from pigs given inactivated vaccine contained virus. A comparison could not be undertaken with the control group because virus was not detected in swabs from the vaccinated pigs after 7 days, at which stage 13 of the control pigs were dead. However, using virulent strains, where unvaccinated pigs survived, virus excretion was virtually continuous for 10 days after infection and intermittently for another 7 days [8]. The pigs vaccinated with attenuated vaccine were much less ill, based not only on a subjective clinical assessment but also on the number of days they had an elevated temperature compared to the inactivated vaccinated group. Nevertheless 3 weeks after challenge there was no significant difference in the weights of the 3 groups. The serum neutralisation titres recorded 14 days after vaccination were low, but the titres produced by the attenuated vaccines were similar to those recorded for the Bartha vaccine [9]. The secondary response following challenge was considerably lower than previously recorded [9]. This may be a reflection o f the time between vaccination and challenge; 5 weeks in one experiment and 2 weeks in the second. It is probable that higher titres would have been recorded for the inactivated vaccine if a longer interval had been left before challenge [7]. However because of the type of husbandry practised, a vaccine would have to give adequate protection in 2 weeks. The choice of vaccine for control purposes is not easy. The inactivated vaccine produced a good immunity and, in theory at least, inactivated vaccines are safer. However inactivated vaccines can be dangerous, because the inactivation has not been effective, because a second unsuspected agent may be present which is more resistant to the inactivating agent than the vaccine virus, or because the vaccine may be contaminated with another agent following inactivation. Furthermore it is much more difficult to check inactivated vaccines for purity, because the adjuvent can preclude the use of cell cultures. To these hazards must also be added the increased cost of inactivated vaccine. This latter is unlikely to be important when vaccinating breeding animals, but could be a major factor in large fattening units. In theory the disadvantages of attenuated vaccines are that they are more liable to be contaminated, they may be excreted, they may cause disease and they may revert to virulence. The dangers of contamination can be minimised by growing the vaccine virus in a diploid cell line, which has been adequately tested, by using versene instead of trypsin to disperse the cells and by using serum obtained from a SPF source or sterilised by irradiation for cell culture growth. The BUK [5], the Bartha [9, 10] and the NIA-4 strains (McFerran and Dow, unpublished observations) are not excreted and therefore the risk o f reversion to virulence does not
334
J.B. MCFERRAN,C. Dow and R. M. MCCRACKEN
occur. F u r t h e r m o r e the N I A - 4 strain has very low virulence. The low serum neutralising titres stimulated by the a t t e n u a t e d vaccine m a y be a n advantage. Whilst any serum neutralising titre m u s t be regarded as indicative o f a potential carrier, a study o f a n u m b e r o f s e r a in a herd would in m a n y cases allow differentiation between a vaccinated herd a n d a herd infected with virulent virus. F o r good i m m u n i t y vaccinating doses o f 10 6 or greater for N I A - 4 are required ( M c F e r r a n a n d Dow, u n p u b l i s h e d observations) a n d J a m r i c h o v a a n d Skoda [5] also f o u n d a relationship between protection a n d vaccine virus titre. A n y c o m m e r c i a l a t t e n u a t e d vaccine should therefore have e n o u g h virus to stimulate a good i m m u n i t y . REFERENCES I. McFerran, J. B., Dow, C. and Hildebrand, W. R. P., Aujeszky's disease virus, Vet. Rec. 78, 700 (1966). 2. Dow, C. and McFerran, J. B., Experimental studies on Aujeszky's disease in sheep, Brit. vet. J. 122, 464-470 (1966). 3. Anonymous. Statistical Review of Northern Ireland Agriculture, 197:%1976. H.M.S.O., Belfast (1977). 4. Dow, C. and McFerran, J. B., The neuropathology of Aujeszky's disease in the pig, Res. vet. Sci. 3, 436-442 (1962). 5. Jamricbova, O. and Skoda, R., Multiplication and distribution of attenuated pseudorabies virus in the organism of vaccinated pigs, Acta Virologica 13, 42 51 (1969). 6. Dr. N. Zygraich, RIT, Genval, Belgium, personal communication (1978). 7. Delagneau, J. F., Toma, B., Vannier, P., Loquerie, R., Pruner, P. and Tillon, J. P., Immunisation contre la malades d'Aujeszky ~il'aide d'un nouveau vaccin huileux ~ virus inactive, Rec. Mbd. vbt. 151,567-575 (1975). 8. McFerran, J. B. and Dow, C., The excretion of Aujeszky's disease virus by experimentally infected pigs, Res. vet. Sci. 5, 405-410 (1964). 9. McFerran, J. B. and Dow, C., Studies on the immunisation of pigs with the Bartha strain of Aujeszky's disease virus, Res. vet. Sci. 19, 17-22 (1975). 10. Bartha, A. and Kojnok, J., Active immunisation against Aujeszky's disease, Proc. 17th Wld. vet. Cong., Hannover l, 531-533 (1963).