Efficacy of a Killed gpX Deleted Pseudorabies Virus ... - Europe PMC

Efficacy of a Killed gpX Deleted Pseudorabies Virus Vaccine Donald R. Cook, Howard T. Hill and David R. Kinker

ABSTRACT Ten inactivated vaccines containing one of four adjuvants and varying concentrations of pseudorabies virus (PRV) antigens were compared in order to select a vaccine suitable for commercial production. A genetically engineered strain of PRV lacking the gene coding for glycoprotein X (gpX) was used in these vaccines. Vaccinated pigs were challenged intranasally with virulent PRV to determine the efficacy of vaccines. Vaccination of pigs with one dose of experimental vaccines adjuvanted with 50% Montanide ISA 50 or 20% Syntrogen induced a protective immunity at least equal to that induced by two commercially available killed PRV vaccines also evaluated. An experimental vaccine containing 20% Syntrogen was selected and further evaluated according to United States Department of Agriculture licensing requirements. None of the pigs vaccinated with this vaccine produced gpX antibodies detectable by the HerdChek®: AntiPRV-gpX assay. Therefore, this assay could differentiate PRV vaccine induced antibodies from antibodies induced by natural exposure when used in conjunction with this killed gpX deleted PRV vaccine. RESUME

Dans le but de mettre sur le marche un vaccin contre la pseudorage (PR), differentes concentrations du virus ont ete combinees 'a quatre differents adjuvants comparant ainsi dix difffrents vaccins chez le porc. La souche virale utilisee, deflciente du gene codant pour la glycoproteine X (gpX),

a ete obtenue par genie genetique. over MLPRV vaccines. A higher Suite a la vaccination, les animaux ont anamnestic response is induced in ete inoculis dans le groin avec une sensitized animals with KPRV vacsouche virulente du virus. La combi- cines (2,4,6,9). Also, inactivation naison d'une seule dose du vaccin avec prevents vaccine virus from replicat50% de Montanide ISA 50 ou 20% de ing in vaccinated animals. Replication Syntrogen a induit une protection of virus does occur in the pig after immunitaire au momns egale a deux MLPRV vaccination (5,8,10). There autres vaccins commerciaux conte- are potential problems that may result nant une souche turee du virus. Le from vaccine virus replication. Vacvaccin experimental contenant 20% de cine virus may be excreted (4), leading Syntrogen a par la suite ete evalue to a series of passages through pigs pour but d'approbation par le Minis- and possibly an increase in virus tere de l'agriculture des Etats-Unis. virulence by genetic mutation or Aucun des animaux vaccines avec ce recombination (2). Replication of vaccin n'a developpe d'anticorps some MLPRV in pigs causes mild contre la gpX lorsque verifle' a l'aide clinical signs (4), although it is possible du test Herd Check®. De plus, ce test that in young or pregnant animals permet de differencier si les anticorps virus replication may produce more produits font suite a la vaccination ou severe effects (2). For these reasons, a une infection avec une souche KPRV vaccine is considered by many sauvage du virus. (Traduit par Dr Pascal veterinarians as the vaccine of choice for boostering immunity, particularly Dubreuil). in pregnant sows (2). Vaccines that have an accompanyINTRODUCTION ing diagnostic test that can differentiate antibodies induced by vaccinaThe vaccination of pigs against tion from antibodies induced by field pseudorabies is commonly practiced virus infection are a major benefit to (1,2). Vaccination will not prevent PRV eradication programs (2). The infection with pseudorabies virus first vaccine available in the United (PRV); however, it will reduce the States with a differential diagnostic duration and severity of clinical signs test was PRV/ Marker® (SyntroVet of pseudorabies and the amount of Incorporated, Lenexa, Kansas). Pigs virus shed by infected pigs (3-6). vaccinated with this MLPRV vaccine Modified live pseudorabies virus can be distinguished from PRV (MLPRV) vaccines are generally infected pigs by using the HerdChek®: considered to provide greater protec- Anti-PRV-gpX assay (Anti-gpX tion than killed pseudorabies virus ELISA, IDEXX Incorporated, Port(KPRV) vaccines (4,6-8), even though land, Maine) (11). Specific modificathe antibody titers that are induced by tions have been made to the PRV/ one dose of KPRV vaccine are Marker vaccine virus, including the generally of similar magnitude and deletion of the gene coding for glycoduration as titers induced by one dose protein X (gpX) (information from of MLPRV vaccine (4). Killed PRV vaccine product insert, PRV/ vaccines do have some advantages Marker®, SyntroVet Incorporated,

Veterinary Diagnostic Laboratory, Iowa State University, Ames, Iowa 50011. Reprint requests to Dr. H.T. Hill. Submitted October 1, 1989.

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Can J Vet Res 1990; 54: 438-445

mercially available vaccines were compared in three separate trials. One experimental vaccine was selected and evaluated according to USDA licensing requirements (13). A summary of vaccine, adjuvant and challenge variables for each trial is presented in Table I. Experimental vaccines were supplied by SyntroVet Incorporated. The SyntroVet virus strain was propagated on Vero cells to obtain the viral antigens contained in the experimental vaccines. Infected cultures were harvested 48 to 72 h postinfection. The infectivity titer of pooled viral fluids was determined by plaque assay (14) prior to the inactivation of virus with binary ethyleneimine (15). After inactivation, viral fluids were mixed with one of four adjuvants. Three commercially available, oil based adjuvants were used, Emulsigen (Modern Veterinary Products Incorporated, Ralston, Nebraska) which is an oil-in-water emulsion, Amphigen Base (Hydronics Incorporated, Omaha, Nebraska) which is based on a light mineral oil and plant cell membrane amphipathic lipid combination, and Montanide ISA 50 (Seppic, Paris, France) which is an adjuvant based on a mannide oleate in mineral oil solution. The fourth adjuvant used was SyntrogenT (SyntroVet Incorporated, Lenexa, Kansas) which is a proprietary formulation. The final concentration of viral antigen in vaccine preparations was from the preinactivation estimated MATERIALS AND METHODS infectivity titer, and expressed as a I X VACCINES concentration (undiluted viral fluids) The efficacy of ten experimental or the respective fraction of the 1X vaccine preparations and two com- viral fluids.

Lenexa, Kansas). The deletion of this gene acts as a "negative marker." The Anti-gpX ELISA is specific for antibodies to gpX and ignores antibody titers in pigs vaccinated with PRV/ Marker. However, it detects gpX antibody in pigs infected with field strains of PRV (information from product insert, HerdChek®: Anti-PRV-gpX, IDEXX Incorporated, Portland, Maine). The development of a KPRV vaccine that does not induce gpX antibody in vaccinated pigs and that could be used in conjunction with the anti-gpX ELISA would be beneficial in eradicating PRV from infected herds. The purpose of this study was to evaluate inactivated vaccine preparations containing a gpX deleted strain of PRV, and to undertake United States Department of Agriculture (USDA) licensing trials with one of these vaccines. The virus strain was provided by SyntroVet Incorporated, and was especially developed for this study by deleting the gpX gene from the Iowa strain of PRV (12). The type of adjuvant and the concentration of viral antigen in vaccine preparations were varied to determine the effect these parameters had on the level of immunity induced in vaccinated pigs. The level of immunity was evaluated by measuring the humoral immune response to vaccination and by comparing the effect that intranasal challenge with PRV had on vaccinated and nonvaccinated pigs.

The two commercially available KPRV vaccines used, PR-Vac@Killed (Norden Laboratories, Lincoln, Nebraska) and Porci-Rab@ (Beecham Laboratories, Bristol, Tennessee) were purchased anonymously. All vaccines were administered as a 2 mL dose, intramuscularly (IM) in the neck. ANIMALS AND HOUSING

All pigs were obtained from pseudorabies free herds and housed in facilities secure for PRV exposure. The experiments followed the guidelines of the Guide to the Care and Use of Experimental Animals of the Canadian Council on Animal Care. CHALLENGE VIRUS

Pseudorabies virus strain VDL 4892 (1 1) was used to challenge vaccinated and nonvaccinated pigs. The virus was propagated and titrated on Madin Darby bovine kidney (MDBK) cells. One serial of challenge virus, which was stored at -70° C, was used throughout the study. TRIAL I

Three vaccine preparations containing inactivated SyntroVet virus (KVSV) were used: vaccine 1 A-0. lX viral antigen/ 20% Emulsigen, vaccine 1 B0.1 X viral antigen/ 20% Emulsigen/ 67.5% Novalep®-5 (Coopers Animal Health Incorporated, Kansas City, Missouri), and vaccine 1C-0.iX viral antigen/ 10% Amphigen Base. In addition, two commercially available vaccines, PR-Vac@-Killed and PorciRab® were used. Twenty-nine pigs, four to five weeks of age, were randomly assigned to five groups of five pigs and one group of

TABLE I. Summary of variables for trials 1-4

Trial I

Vaccine KV-SV IA KV-SV IB KV-SV IC PR-Vac@-Killed

Porci-Rab®

KV-SV 2A KV-SV 2B KV-SV 2C KV-SV 2D KV-SV 3A 3 KV-SV 3B KV-SV 3C KV-SV 3D 4 KV-SV aPlaque-forming units 2

Vaccine dosage

0.lX (2 mL IM) 0.1X (2 mL IM) 0.1X (2 mL IM) (2 mL IM) (2 mL IM) IX (2 mL IM) IX (2 mL IM) 0.2X (2 mL IM) IX (2 mL IM) IX (2 mL IM) 0.5X (2 mL IM) 0.2X (2 mL IM) 0.5X (2 mL IM) 0.5X (2 mL IM)

Adjuvant 20% Emulsigen 20% Emulsigen/67.5% Novalep®-5 10% Amphigen Base 30% Emulsigen 10% Syntrogen 10% Syntrogen 50% Montanide ISA 50 50% Montanide ISA 50 50% Montanide ISA 50 50% Montanide ISA 50 50% Montanide ISA 50 20% Syntrogen

Challenge dosage 105.8 PFUa VDL 4892 105.8 PFU VDL 4892 105.8 PFU 105.8 PFU 1058 PFU 104.8 PFU 104.8 PFU 104.8 PFU 104.8 PFU 104.8 PFU 104.8 PFU 104.8 PFU 104.8 PFU 1045 PFU

VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892 VDL 4892

439

antigen/ 50% Montanide ISA 50, challenge and day 14 PC. Clinical vaccine 3B-0.5X viral antigen/ 50% observations were made and rectal Montanide ISA 50, vaccine 3C-0.2X temperatures taken daily for 14 days viral antigen/ 50% Montanide ISA 50, PC. Nasal swabs were collected on and vaccine 3D-0.5X viral antigen/ days 2, 4, 6, 8, 10, 12 and 14 PC. 20% Syntrogen. Twenty-five pigs, three to four LABORATORY PPOCEDURES weeks of age, were randomly assigned Nasal and tonsil swabs and tissue to five groups of five pigs. Four groups samples were assayed for virus. Nasal were vaccinated with one dose of and tonsil swabs were placed in tubes vaccine and the other group remained containing 0.5 mL of Earle's medium nonvaccinated controls. Twenty-one Island, New York). Grand (Gibco, intrawere PV challenged pigs days nasally with 104.8 PFU of PRV as a The tube was then vortexed, the swab 1 mL dose. Sera were collected on the removed and the tube centrituged at day of vaccination and days 7 and 14 2000 x g for 10 min. Tissue samples PV, and on the day of challenge (day were macerated in an equal volume of 21 PV) and days 7 and 14 PC. Pigs Earle's medium and centrifuged as were weighed on the day of vaccina- described for swabs. Supernatants tion, the day of challenge, and day 14 were assayed for PRV by inoculation PC. Pigs were observed for clinical of MDBK cells which were examined signs daily for 14 days PC and nasal for cytopathic effects (CPE) daily for swabs were collected on days 3, 6, 9 seven days. Negative cultures were and 12 PC. The vaccination site was _ and observed for seven TRIAL 2 subcultured palpated on days 7, 21 and 35 PV to Four KV-SV vaccine preparations detect any tissue reactions to the days. Pseudorabies isolates were identified by immunofluorescence were used: vaccine 2A-1X viral vaccines. (16). antigen/30% Emulsigen, vaccine 2B- TRIAL 4 (LICENSING TRIAL) Serum samples were assayed by the IX viral antigen/ 10% Syntrogen, Vaccine 3D-0.5X viral antigen/ 20% serum virus neutralization (SVN) test vaccine 2C-0.2X viral antigen/ 10% Syntrogen was used. as described by Hill et al (16), and the Syntrogen, and vaccine 2D-1X viral Twenty three-day-old pigs were anti-gpX ELISA (1 1). randomly selected from three litters antigen/ 50% Montanide ISA 50. Twenty-five pigs, three to four and vaccinated with a 2 mL dose of weeks of age were randomly assigned vaccine IM. Five pigs of the same age RESULTS remained nonvaccinated controls. At to five groups of five pigs. Four groups day 24 PV the pigs were weaned, and RESPONSE TO VACCINATION were vaccinated with one dose of at day 30 PV the pigs were challenged The group mean SVN antibody vaccine and the other group remained intranasally with 10 5PFU of PRV as nonvaccinated controls. Twenty-six a 1 mL dose. Sera were collected on titers of vaccinated pigs PV for trials 1, days PV pigs were challenged intra- the day of challenge and days 7 and 2 and 3 are shown in Figs. 1, 2 and 3 nasally with 104.8 PFU of PRV as a 14 PC. Pigs were weighed on the day of respectively. Comparing the SVN 1 mL dose. Sera were collected on the day of vaccination and days 7, 14 and 5 -_ PR-Vac -Killed 21 PV, and on the day of challenge O Porci-Rabe (day 26 PV) and days 7 and 14 PC. _ Vaccine 1A (M.AX viral antigen/20% Emulsigen) Pigs were weighed on the day of -C Vaccine 1B (O.1X viral antigen/20% Emulsigenl67.5% Novaleps 5) 4 vaccination, the day of challenge, and Vaccine 1C (M.AX viral antigen/10% Amphigen Base) day 14 PC. Pigs were observed for clinical signs daily for 14 days PC, and 3 nasal swabs were collected on days 3, SVN TITER (log 2) 6, 9 and 12 PC. Pigs that died during the trial were necropsied, and brain, tonsil, lung and spleen samples were collected for histological examination and virus isolation. Pigs vaccinated with vaccine 2D (KV-SV/ Montanide ISA 50) were necropsied, and the site of vaccination of each pig was examined grossly and histologically. 40 21 28 14 0 7

four pigs. Each of the groups containing five pigs were vaccinated with one dose of vaccine, and the group of four pigs remained nonvaccinated controls. Forty-two days postvaccination (PV) all pigs were challenged intranasally with 105.8 plaque-forming units (PFU) of PRV as a 1 mL dose. Sera were collected on the day of vaccination and then weekly until 14 days postchallenge (PC). Pigs were observed for clinical signs daily for 14 days PC, weighed on the day of challenge and day 14 PC, and nasal swabs were collected on days 7, 10 and 14 PC. Pigs that died during the trial were necropsied, and brain, tonsil, lung, and spleen samples were collected for histological examination and virus isolation.

-

-

-

1

0

TRIAL 3

Four KV-SV vaccine preparations were used: vaccine 3A-1X viral 440

DAYS POST-VACCINATION

Fig. 1. Serum virus neutralizing antibody mean ± SEM (n = 5).

response

in vaccinated pigs in trial 1. Each point is the

5 - -*_ Vaccine 2A (1X viral antigen/30% Emulsigen) -0- Vaccine 2B (1X viral antigen/10% Syntrogen) -_ Vaccine 2C (0.2X viral antigen/10% Syntrogen) 4 - -0- Vaccine 2D (1X viral antigen/50% Montanide ISA 50)

3

SVN TITER (log 2) 2

1

0

0

7

14

21

ci. 26

DAYS POST-VACCINATION

Fig. 2. Serum virus neutralizing antibody response in vaccinated pigs in trial 2. Each po int is the mean ± SEM (-o-, -D- n = 5; -0-, -*- n = 4).

antibody titers of all pigs in tria and 3 at 21 days PV, pigs vacci with experimental vaccines vanted with Montanide ISA 50 SV/Montanide ISA 50) had s cantly (p < 0.05) higher SVN ody titers than other vaccinated with a range in titers from 1:4 to Pigs vaccinated with KV-SV Syntrogen, KV-SV/ 30% Emul or PR-Vac®-Killed had SVN ody titers significantly (p < higher than the remaining vacci The geometric mean SVN ant

titer of pigs in the licensintg trial vaccinated at three days of al.4 at 30 KV-SV/20% Syntrogen was 1: days PV, with a range in titers firom 1 2 to 1:8. No pigs vaccinated with vaccines had detectable gp) antiV bodies PV, whereas five of the 1ten pigs vaccinated with the comm(ercially available KPRV vaccines werre osi tive by anti-gpX ELISA. Pigs receiving vaccine 2D (KrV_SV/ Montanide ISA 50) were nec ropsied on day 14 PC. All pigs had lesrions in the muscle at the site of vaccine

KV-SV:

,

S Vaccine 3A (1X viral antigen/50% Montanide ISA 50) _ -0- Vaccine 3B (0.5X viral antigen/50% Montanide ISA 50)

5

4 -

_ Vaccine 3C (0.2X viral antigen/50% Montanide ISA 50) 0-U Vaccine 3D (0.5X viral antigen/20% Syntrogen)

3

-< SVN TITER

(log 2)

0

7

14

21

DAYS POST-VACCINATION

Fig. 3. Serum virus neutralizing antibody response in vaccinated pigs in trial 3. Each point is the = 5).

mean ± SEM (n

injection. Grossly an area of muscle 1.5 to 3 cm in diameter was pale and firm. When the lesion was cut numerous small globules of vaccine residue were evident. Histologically each lesion contained numerous residual oil globules, each encapsulated in connective tissue. In trial 3 intramuscular swelling at the site of injection was detected by palpation at day 7 PV in five pigs vaccinated with KV-SV/ Montanide ISA 50 and two pigs vaccinated with KV-SV/20% Syntrogen. At day 21 PV, reactions could still be palpated in four pigs that had received KV-SV/ Montanide ISA 50 vaccines, but by day 35 PV no reactions were detected by palpation. In trials 2 and 3 there was no evidence to suggest that vaccination had an effect on weight gain prior to challenge. RESPONSE TO CHALLENGE The group responses to challenge are summanzed in Tables II, III, IV and V. After challenge exposure, control pigs developed clinical signs typical of pseudorabies, with control pigs in trials 2 and 3 being more severely affected than control pigs in trials 1 and 4. In general, clinical signs of depression and reduced appetite were evident in control pigs three days PC. These signs worsened over the next three days, and all control pigs had mucous to purulent nasal discharge, laryngitis and dyspnea. Sixteen of 19 control pigs developed signs of central nervous system (CNS) dysfunction between days 5 and 8 PC. The severity of these signs varied from teeth grinding, muscle tremors and slight incoordination to head tilting, ataxia and convulsions. Three control pigs died. Histologically, there was evidence of moderate lymphocytic perivascular cuffing, focal gliosis and mild neuronal necrosis in the brains of all three pigs suggestive of viral encephalitis, and PRV was isolated from the brain, lung, tonsil and spleen of each pig. Recovery from clinical signs started at day 8 or 9 PC. However, not all pigs had recovered by day 14 PC, as three controls still showed signs of CNS dysfunction at thi1s time. 441

TABLE II. Summarized serological and clinical results used to evaluate the efficacy of killed pseudorabies virus vaccines in trial 1

lB IA Parameter 5 5 No. challenged Mean SVNa < 1:2 < 1:2 antibody |- 0 1-14 1:891 1:215 titer at PCb day Mean anti0.99 1.02 gpX ELISA |- 0 0.30 0.33 reactivity at PC day 1-14 20 0 Mortality % 40 40 CNS dysfunctc % 3/5 3/5 No. shedding |- 7 |-10 0/5 0/5 virus at PC day 1-14 0/4 0/5 4.6 -3.7 Weight gain (kg) postchallenge aSVN = serum virus neutralization bPC = postchallenge cCNS dysfunct = central nervous system dysfuncti

Clinical signs in vaccinates varied considerably. Generally, pigs vaccinated with KV-SV/ Montanide ISA 50, KV-SV/ 20% Syntrogen, and PRVac®-Killed showed only mild depression and mucous nasal discharge on days 4 and 5 PC. Pigs in other groups had a greater severity and duration of clinical signs, but were still less severely affected than controls. All pigs vaccinated with KV-SV/ Montanide ISA 50, KV-SV/ Syntrogen, KVSV/ Amphigen Base, and PR-Vac@Killed survived the challenge ex-

Vaccine PR-Vac -Killed IC 5 5

Porci -Rab 5

< 1:2 < 1:2 1:304 1:45

< 1:2 1:256

1:2

1.00 0.44 0 40 4/5 0/5 0/5 0.1

0.79 0.13 0 0 0/5 0/5

0.73 0.11 20 20 3/5 1/4

0/5 10.8

0/4 2.4

1:2352

Control 4

1.00 0.33 0 50 4/4 2/4 2/4 -3.2

posure. Three pigs vaccinated with KV-SV/ Emulsigen and one vaccinated with Porci-Rab® died. Histologically, there was evidence of a viral encephalitis in all four pigs that died; however, PRV was only isolated from the tonsil of the pig vaccinated with Porci-Rab®. Signs of CNS dysfunction developed in pigs vaccinated with KV-SV/ Emulsigen (six of 15), KVSV/ Amphigen Base (two of five), KVSV/ Montanide ISA 50 (one of 19), KV- SV/ Syntrogen (one of 35), and Porci-Rab® (one of five).

TABLE III. Summarized serological and clinical results used to evaluate the efficacy of killed pseudorabies virus vaccines in trial 2 Parameter No. challenged Mean SVNa antibody titer at PC day Mean antigpX ELISA reactivity at PCb day

Mortality % CNS dysfunctc % No. shedding virus

2B 5

|- 0 1-14

< 1:2 1:16

< 1:2 1:56

1:2 1:14

I- 0 1-14

0.91 0.51 50 50 4/4 1/4 0/2 0/2 11.1 5.8

0.88 0.23 0 0 4/5 1/5 0/5 0/5 11.6 5.4

0.93 0.16 0 0 5/5

at

|- 3 |- 6 I- 9

PC day

1-12

Weight gain (kg) prechallenge Weight gain (kg) postchallenge aSVN = serum virus neutralization bPC = postchallenge cCNS dysfunct = central nervous system dysfunction

442

Vaccine 2C 5

2A 4

0/5 0/5

0/5 12.6 5.6

2D

Control 5

1:20 1:2048

< 1:2 1:8

4

0.85 0.44 0 0 3/4 0/4 0/4 0/4 13.2 9.0

0.96 0.49 60 100 5/5 2/2 2/2 1/2 15.0 -1.2

The majority of control pigs were still shedding PRV at day 12 PC. Vaccination did not prevent virus shedding, but it did decrease the duration of virus shedding compared to controls. In trials 1, 2 and 3, challenge exposure had severe effects on body weight gains of control pigs. Pigs vaccinated with PR-Vac®-Killed in trial 1 and with KV-SV/Montanide ISA 50 in trial 2 had significantly (p

Control

106 105 104

TEMP r 103 102

101

X-47 j//4

'

1 1 1 1

100 0

1

2

3

1 4

i

I

I

9 7 6 8 5 DAYS POST-CHALLENGE

I

i

10

11

12

I 13

14

Fig. 4. Mean body temperatures ± SEM after challenge of pigs in the licensing trial given vaccine KV-SV/20% Syntrogen (n = 20) and controls (n = 5).

virus shedding in vaccinated pigs, PRV vaccines can be effectively compared (4). If vaccines are tested at different times, meaningful comparisons can still be made if a standard response in control pigs is achieved. For PRV vaccines, a challenge dose of virus sufficient to cause CNS dysfunction or death in 80% of control pigs is desired. The severity of clinical signs following challenge exposure depends on the virulence of the viral strain, the age of the pigs, dose of virus and route of exposure (1). There have been no previous reports on the use of PRV strain VDL 4892 for challenge exposure of pigs. Therefore, the dose of challenge virus used initially was based on reports of the use of other PRV strains to challenge pigs intranasally (1,3,4,6). The dose of virus was changed between trials in response to the age of pigs being used. Virus shedding from vaccinated pigs PC was of much shorter duration than from control pigs. Virus shedding PC by pigs vaccinated with commercially available PRV vaccines has been reported to last up to eight days PC (10). The duration of shedding by pigs vaccinated with KV-SV/ Montanide ISA 50 or KV-SV/ Syntrogen compares favorably with this response. In trial 3, virus shedding could be detected in only one pig vaccinated with KV-SV/ Montanide ISA 50, and no vaccinates shed virus after day 3 PC. In the licensing trial no vaccinate shed virus after day 8 PC. 444

Vaccination had pronounced effects on the growth rate of pigs PC. In trials 1, 2 and 3, pigs receiving PR-Vac®Killed or KV-SV vaccines containing Montanide ISA 50 or 20% Syntrogen had comparable growth rates that were markedly higher than growth rates of control pigs. Vaccines containing 10% Syntrogen were slightly less effective. The development of body weight PC is considered an important parameter for evaluating vaccine efficacy because it is an

objective indicator of severity of illness and is directly related to economic losses (4). The failure to detect a difference in weight gain between vaccinates and controls in the licensing trial was probably a result of the lower challenge dose of PRV used in this trial. As the mortality, percent of pigs with CNS dysfunction, and weight gains of control pigs PC varied between trials, inter-trial comparisons of vaccine efficacy need to be done with care. Nevertheless, it seems reasonable to group the KV-SV vaccines into three categories according to their levels of efficacy: (i) the least effective were vaccines containing Emulsigen or Amphigen Base (vaccines l A, l B, l C and 2A); (ii) vaccines containing 10% Syntrogen (vaccines 2B and 2C) were of moderate efficacy, (iii) vaccines containing Montanide ISA 50 (vaccines 2D, 3A, 3B and 3C) or 20% Syntrogen (vaccine 3D) gave the best protection. The KVSV/ Montanlde ISA 50 and KV-SV/

20% Syntrogen vaccines conferred good protection against challenge with PRV strain VDL 4892. However, the depression and signs of CNS dysfunction seen in some pigs PC indicates that all vaccinates were not completely protected. The two commercially available vaccines-used in trial 2, PR-Vac®-Killed and PorciRab®, would be in categories (iii) and (i) respectively. The efficacy of vaccines 2D, 3A, 3B, 3C and 3D would make them suitable for use as killed vaccines against pseudorabies. However, factors such as cost, safety, stability and licensing difficulties need also to be considered (17), and for these reasons vaccine 3D (0.5X viral antigen/20% Syntrogen) was chosen for the licensing trial. Adjuvants added to veterinary vaccines must meet standards of purity, be nontoxic, not denature specific substances in the product through the dating period, and also not leave harmful residues in meat (18). Available evidence suggested that Syntrogen would meet these licensing requirements. In contrast, the likelihood of licensing vaccines containing Montanide ISA was uncertain. Montanide ISA 50 is an oil based adjuvant. The immunoenhancing effects of oil are related to its protection of antigens from host degradation, the transport of antigens through the lymphatic system where foci of antibody production can be established, and the progressive release of antigens from the site of injection (19). The manufacturer of Montanide ISA 50 reports that the product stimulates the immune response to many viral antigens, and that foot-and-mouth vaccines containing Montanide ISA have been used successfully in South America (20). In this study the vaccines containing Montanide induced the highest SVN antibody titers in vaccinated pigs. Pseudorabies virus vaccines that induce high antibody levels in the colostrum of sows, so that newborn piglets receive high maternal immunity, may be preferable. However, there are no reports of the use of Montanide ISA in USDA licensed vaccines, and the tissue reaction at the site of injection, although not uncommon with oil adjuvants, may be an obstacle to commercial acceptance.

The commercial availability of the KV-SV/20% Syntrogen vaccine would provide significant benefits to pseudorabies eradication programs. One dose of this killed vaccine confers good protection against pseudorabies and induces levels of circulating virus neutralizing antibody similar to other KPRV vaccines. However, KV-SV/ 20% Syntrogen has the advantage over these other killed vaccines in that the antibodies it induces can be differentiated from antibodies induced by field virus infection by the anti-gpX ELISA.

REFERENCES 1. GUSTAFSON DP. Pseudorabies. In: Leman AD, Straw B, Glock RD, Mengeling WL, Penny RHC, Scholl E, eds. Diseases of Swine. Ames: Iowa State University Press, 1986: 274-289. 2. MOLITOR T, THAWLEY D. Pseudorabies vaccines: Past, present and future. Compendium Food Animal 1987; 9: 409416. 3. ALVA-VALDES R, GLOCK RD, KLUGE JP, KEUNE CM. Effects of vaccination on lesion development in pseudorabies viruschallenged swine. Am J Vet Res 1983; 44: 588595. 4. DE LEEUW PW, VAN OIRSCHOT JT. Vaccines against Aujeszky's disease: Evaluation of their efficacy under standardized laboratory conditions. Vet Q 1985; 7: 191-197.

5. DONALDSON AI, WARDLEY RC, MARTIN S, HARKNESS JW. Influence of vaccination on Aujeszky's disease virus and disease transmission. Vet Rec 1984; 115: 121-124. 6. McFERRAN JB, DOW C, McCRACKEN RM. Experimental studies in weaned pigs with three vaccines against Aujeszky's disease. Comp Immunol Microbiol Infect Dis 1979; 2: 327-334. 7. FRESCURA T, CESSI D, VIVOLI P, DE SIMONE F. Immunoprophylaxis of Aujeszky's disease in Italy. In: Wittmann G, Hall SA, eds. Aujeszky's Disease. The Hague: Martinus Nijhoff Publishers, 1982: 139-142. 8. McFERRAN JB, DOW C. Studies on immunisation of pigs with the Bartha strain of Aujeszky's disease virus. Res Vet Sci 1975; 19: 17-22. 9. ZUFFA A, SALAJ J. Resistance of pigs immunized with live or inactivated Aujeszky's disease vaccines against experimental infection. Proc Int Pig Vet Soc Congr 1980: 113. 10. BASKERVILLE A, McFERRAN M, DOW C. Aujeszky's disease in pigs. Vet Bull 1973; 43: 465-479. 11. COOK D, HILL H, SNYDER M, McMAHON P, KINKER D. The detection of antibodies to the glycoprotein X antigen of pseudorabies virus. J Vet Diagnost Invest 1990; 2: 24-28. 12. PLATT KB, MARE CJ, HINZ PN. Differentiation of vaccine strains and field isolates of pseudorabies (Aujeszky's Disease) virus: Thermal sensitivity and rabbit virulence markers. Arch Virol 1979; 60: 13-23.

13. UNITED STATES DEPARTMENT OF AGRICULTURE. Code of federal regulations: Animals and animal products. Washington: Office of the Federal Register National Archives and Records Administration, 1989: 462. 14. DULBECCO R. Production of plaques in monolayer tissue cultures by single particles of an animal virus. Proc Natl Acad Sci 195; 38: 747-752. 15. BAHNEMANN HG. Inactivation of virus in serum with binary ethyleneimine. J Clin Microbiol 1976; 3: 209-210. 16. HILL HT, CRANDELL RA, KANITZ CL, McADARAGH JP, SEAWRIGHT GL, SOLORZANO RF, STEWARD WC. Recommended minimum standards for diagnostic tests employed in the diagnosis of pseudorabies (Aujeszky's disease). Proc 20th Annu Meet Am Assoc Vet Lab Diagnost 1978: 375-390. 17. REED DE. Future of carriers and adjuvants in veterinary biologics. In: Nervig RM, Gough PM, Kaeberle ML, Whetstone CA, eds. Advances in Carriers and Adjuvants for Veterinary Biologics. Ames: Iowa State University Press, 1986: 165-168. 18. ESPESETH DA. Licensing considerations for new adjuvants and carriers. In: Nervig RM, Gough PM, Kaeberle ML, Whetstone CA, eds. Advances in Carriers and Adjuvants for Veterinary Biologics. Ames: Iowa State University Press, 1986: 159-164. 19. McKERCHER PD. Oil adjuvants: Their use in veterinary biologics. In: Nervig RM, Gough PM, Kaeberle ML, Whetstone CA, eds. Advances in Carriers and Adjuvants for Veterinary Biologics. Ames: Iowa State University Press, 1986: 115-120. 20. SEPPIC. Montanide ISA 50: Incomplete Seppic adjuvant. Paris: Seppic, 1987.

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