J.
COMP.
PATH.
1977. VOL.
ATTEMPTS EXPERIMENTAL
87
591
TO IMMUNIZE INFECTION
MONKEYS AGAINST WITH HERPESVIRUS
SUIS
BY
and G. LLOYD
A. BASKERVILLE
Microbiological Research Establishment, Porton, Salisbury, Wiltshire, U.K.
INTRODUCTION In a recent communication we described the clinical, pathological and microbiological features of experimental infection of grivet and squirrel monkeys disease virus, ADV) (Baskerville and Lloyd, with Herjxsvims suis (Aujeszky’s 1977). The infection resulted in a fatal encephalomyelitis after an incubation period which varied from 7 to 13 days. The pathogenesis and pathology of the disease indicated that Aujeszky’s disease in subhuman primates might be a suitable experimental system for the study of human infection with (Her-esvirus simiae). Since there is considerable interest in “monkey B-virus” possible immunization of personnel occupationally exposed to infection with B-virus (Hull, 1973), a parallel experiment was carried out in which grivct monkeys were immunized with a living, avirulent strain of ADV and subsequently challenged with virulent virus.
MATERIALS
AND
METHODS
Five adult grivet monkeys (Cercopithecus aethiops) of either sex, which had no serum antibodies to H. simiae, were inoculated intranasaliy with a dose of IO6 TCD,, of the Bartha strain of ADV suspended in 1 ml of tissue-culture medium 199 containing 2 per cent foetal bovine serum. The virus was kindly supplied by Dr J. B. McFerran. The same dose was given again 2 weeks later and after a further 2 weeks the monkeys received a challenge dose intranasally of 1 ml of medium containing 1O6.5 TCD,, of the virulent NIA-2 strain of ADV (Baskerville, McCracken and McFerran, 1971). The Bartha and NIA-2 strains of virus were grown in Vero cells as described previously (McFerran and Dow, 1975; Baskerville and Lloyd, 1977). The monkeys in that part of the experiment already recorded served as unimmunized controls (Baskerville and Lloyd, 1977). The monkeys were anaesthetized with ketamine hydrochloride (“Vetalar”, Parke, Davis) given intramuscularly for all procedures such as infection and when samples were taken. Nasal swabs were taken at daily intervals after each vaccination and after challenge and body temperature was also recorded daily after chaIlenge. Blood samples were taken from the femoral vein at intervals for the determination of serum antibody levels. Virus isolation. Nasal swabs were expressed into 1 ml of transport medium containing bovine serum albumin and lightly centrifuged to remove debris. Necropsy was carried out immediately after death and the following specimens were taken for virus isolation: nasal mucosa, mandibular lymph nodes; olfactory bulbs and tracts, caudate nucleus, thalamus, cerebral cortex, medulla; cervical spinal cord. The tissues were homogenized and IO per cent suspensions were made in medium 199 containing 2 per cent foetal bovine serum, 0.1 per cent sodium bicarbonate, 1.4 per cent of Hepcs buffer and 60 mg penicillin and 100 mg streptomycin per ml. The tissue
592
A. BASKERVILLE
AND G. LLOYD
extracts and swab supernatants were inoculated on to monolayers of Vero cells in “Microtitre” plates. The cultures were examined for the cytopathic effects (CPE) of ADV daily for 14 days and any virus isolated was identified by neutralization with a specific anti-ADV anti-serum prepared in pigs. Serum antibody tests. For each sample serial two-fold dilutions were mixed with an equal volume of virus suspension of either the Bartha or the NIA-2 ADV strains containing 200 TCD,, and incubated at 37 “C for 1 h. The mixtures were inoculated on to Vero cells in “Microtitre” plates and the cultures were examined for CPE daily for 14 days. Histopatholopy. The following tissues were taken at necropsy and fixed in 10 per cent buffered neutral formalin: nasal mucosa, palatine tonsil, trzchea, lungs, heart, liver, kidneys, adrenal glands, brain, cervical cord. Tissues were processed by standard methods and embedded in paraffin wax, and 5-pm-thick sections were cut and stained with haematoxylin and eosin. RESULTS
Clinical Findings No abnormalities were detected after the first or second doses of vaccine virus. After challenge the incubation period before the onset of clinical signs in the group was similar to that in unimmunized monkeys and covered a period of 7 to 14 days from the onset of illness in the first and last affected monkeys. Body temperatures are shown in Table 1 and, in general, were little affected. A low temperature was usually associated with severe illness and TABLE 1 BODY
TEMPERATURE
(“c)
OF
IMMUNIZED
MONKEYS
AFTER
CHALLENGE
WITH
VIRULENT
MA-2
ADV
Days after challenge
6 ::
10
I1
I
2
3
-I
7
8
9
39.4
39.0
39.1
39.0
39.5
39.1 39.0
39.0 38.8
z2"
39.3 37,7* 37.2*
39.1"
38.8 38.8
38.7 38.3* 37.2"
40.0
39.2 38.4 38.7 39.6
38.6 38.8
39.2 38.3
38.8 38.4
38.7 38:8
39.3 36.6*
39.4 39.4*
36.6" 39.1
40.4
* Animal was clinically
I4
39.5*
affected.
pyrexia was transient and recorded on only one occasion in 2 of the 5 animals. On the 7th day monkey 33 had an epileptiform convulsion and on recovery was weak and into-ordinate, and showed the excessive salivation characteristic of this disease. After 4 h the monkey’s condition improved and it began to eat, though tremors of the arms and legs persisted. On day 8 monkeys 6 and 14 also had tremors of the limbs and neck. Monkey 33 suddenly became comatose and died on day 9 without regaining consciousness. Monkey 6 died at this stage after salivating for about 5 h. Monkey 14 started to salivate excessively, refused food and had muscular tremors of the whole body. It became comatose and died on day 10. At this time the remaining 2 animals, numbers 17 and 21, were normal. On the 11th day monkey 17 had several epileptiform convulsions,
593
Herpesvirus suis IN MONKEYS
each of a few minutes’ duration, and finally became comatose. It was killed by intravenous injection of pentobarbitone sodium while in this terminal state. On day 11 monkey 21 appeared normal but its temperature was 40.4 “C. However, its temperature fell and it remained normal until the 14th day when it began to salivate excessively. It became into-ordinate, had several convulsions, rapidly went into a state of coma, and died approximately 4 h after the onset of the salivation. Virus Isolation Nasal swabs. Vaccine virus could not be isolated from any of the animals at any stage after the first or second dose. MA-2 challenge virus was not recovered at any stage. Brain and other tissues. The detection and concentration of ADV in the various regions of the brain is presented in Table 2. TABLE 2 CONCENTRATION
14 (Day 10) 21 (Day 14)
OF NIA-2
--
ADV
(lOglo
TCDQO PER g) IN TISSUES
3.5
3.1
;:; 3.8 2.6
2.6 5.1 -,
OF MONKEYS
3.5 5.1
AT NECROPSY
2.6 3.5 5.1
2.6
no virus isolated.
Serum Antibody Kesponses No significant level of neutralizing antibody (less than 1 in 2) to the Bartha strain of virus was detected in the serum of any monkey after the first immunization or up to 14 days after the second. The antibody titres to the Bartha and NIA-2 strains of virus at various stages after challenge are shown in Table 3. 3
TABLE
SERUM
ANTIBODY IJ3VEI.S (RECIPROCAL OF TITRES) in IMMUNIZED CHALLENGE WITH NIA-2 ADV AND ON DAY OF DEATH
NEUTRALIZING
7
MONKEYS
DAYS
AFTER
Days Monh~y
17
3: 14 21
,,VIA-.?
t2 t2 t2
COMP.
PATH.
1977. VOL.
ATTEMPTS EXPERIMENTAL
87
591
TO IMMUNIZE INFECTION
MONKEYS AGAINST WITH HERPESVIRUS
SUIS
BY
and G. LLOYD
A. BASKERVILLE
Microbiological Research Establishment, Porton, Salisbury, Wiltshire, U.K.
INTRODUCTION In a recent communication we described the clinical, pathological and microbiological features of experimental infection of grivet and squirrel monkeys disease virus, ADV) (Baskerville and Lloyd, with Herjxsvims suis (Aujeszky’s 1977). The infection resulted in a fatal encephalomyelitis after an incubation period which varied from 7 to 13 days. The pathogenesis and pathology of the disease indicated that Aujeszky’s disease in subhuman primates might be a suitable experimental system for the study of human infection with (Her-esvirus simiae). Since there is considerable interest in “monkey B-virus” possible immunization of personnel occupationally exposed to infection with B-virus (Hull, 1973), a parallel experiment was carried out in which grivct monkeys were immunized with a living, avirulent strain of ADV and subsequently challenged with virulent virus.
MATERIALS
AND
METHODS
Five adult grivet monkeys (Cercopithecus aethiops) of either sex, which had no serum antibodies to H. simiae, were inoculated intranasaliy with a dose of IO6 TCD,, of the Bartha strain of ADV suspended in 1 ml of tissue-culture medium 199 containing 2 per cent foetal bovine serum. The virus was kindly supplied by Dr J. B. McFerran. The same dose was given again 2 weeks later and after a further 2 weeks the monkeys received a challenge dose intranasally of 1 ml of medium containing 1O6.5 TCD,, of the virulent NIA-2 strain of ADV (Baskerville, McCracken and McFerran, 1971). The Bartha and NIA-2 strains of virus were grown in Vero cells as described previously (McFerran and Dow, 1975; Baskerville and Lloyd, 1977). The monkeys in that part of the experiment already recorded served as unimmunized controls (Baskerville and Lloyd, 1977). The monkeys were anaesthetized with ketamine hydrochloride (“Vetalar”, Parke, Davis) given intramuscularly for all procedures such as infection and when samples were taken. Nasal swabs were taken at daily intervals after each vaccination and after challenge and body temperature was also recorded daily after chaIlenge. Blood samples were taken from the femoral vein at intervals for the determination of serum antibody levels. Virus isolation. Nasal swabs were expressed into 1 ml of transport medium containing bovine serum albumin and lightly centrifuged to remove debris. Necropsy was carried out immediately after death and the following specimens were taken for virus isolation: nasal mucosa, mandibular lymph nodes; olfactory bulbs and tracts, caudate nucleus, thalamus, cerebral cortex, medulla; cervical spinal cord. The tissues were homogenized and IO per cent suspensions were made in medium 199 containing 2 per cent foetal bovine serum, 0.1 per cent sodium bicarbonate, 1.4 per cent of Hepcs buffer and 60 mg penicillin and 100 mg streptomycin per ml. The tissue
592
A. BASKERVILLE
AND G. LLOYD
extracts and swab supernatants were inoculated on to monolayers of Vero cells in “Microtitre” plates. The cultures were examined for the cytopathic effects (CPE) of ADV daily for 14 days and any virus isolated was identified by neutralization with a specific anti-ADV anti-serum prepared in pigs. Serum antibody tests. For each sample serial two-fold dilutions were mixed with an equal volume of virus suspension of either the Bartha or the NIA-2 ADV strains containing 200 TCD,, and incubated at 37 “C for 1 h. The mixtures were inoculated on to Vero cells in “Microtitre” plates and the cultures were examined for CPE daily for 14 days. Histopatholopy. The following tissues were taken at necropsy and fixed in 10 per cent buffered neutral formalin: nasal mucosa, palatine tonsil, trzchea, lungs, heart, liver, kidneys, adrenal glands, brain, cervical cord. Tissues were processed by standard methods and embedded in paraffin wax, and 5-pm-thick sections were cut and stained with haematoxylin and eosin. RESULTS
Clinical Findings No abnormalities were detected after the first or second doses of vaccine virus. After challenge the incubation period before the onset of clinical signs in the group was similar to that in unimmunized monkeys and covered a period of 7 to 14 days from the onset of illness in the first and last affected monkeys. Body temperatures are shown in Table 1 and, in general, were little affected. A low temperature was usually associated with severe illness and TABLE 1 BODY
TEMPERATURE
(“c)
OF
IMMUNIZED
MONKEYS
AFTER
CHALLENGE
WITH
VIRULENT
MA-2
ADV
Days after challenge
6 ::
10
I1
I
2
3
-I
7
8
9
39.4
39.0
39.1
39.0
39.5
39.1 39.0
39.0 38.8
z2"
39.3 37,7* 37.2*
39.1"
38.8 38.8
38.7 38.3* 37.2"
40.0
39.2 38.4 38.7 39.6
38.6 38.8
39.2 38.3
38.8 38.4
38.7 38:8
39.3 36.6*
39.4 39.4*
36.6" 39.1
40.4
* Animal was clinically
I4
39.5*
affected.
pyrexia was transient and recorded on only one occasion in 2 of the 5 animals. On the 7th day monkey 33 had an epileptiform convulsion and on recovery was weak and into-ordinate, and showed the excessive salivation characteristic of this disease. After 4 h the monkey’s condition improved and it began to eat, though tremors of the arms and legs persisted. On day 8 monkeys 6 and 14 also had tremors of the limbs and neck. Monkey 33 suddenly became comatose and died on day 9 without regaining consciousness. Monkey 6 died at this stage after salivating for about 5 h. Monkey 14 started to salivate excessively, refused food and had muscular tremors of the whole body. It became comatose and died on day 10. At this time the remaining 2 animals, numbers 17 and 21, were normal. On the 11th day monkey 17 had several epileptiform convulsions,
593
Herpesvirus suis IN MONKEYS
each of a few minutes’ duration, and finally became comatose. It was killed by intravenous injection of pentobarbitone sodium while in this terminal state. On day 11 monkey 21 appeared normal but its temperature was 40.4 “C. However, its temperature fell and it remained normal until the 14th day when it began to salivate excessively. It became into-ordinate, had several convulsions, rapidly went into a state of coma, and died approximately 4 h after the onset of the salivation. Virus Isolation Nasal swabs. Vaccine virus could not be isolated from any of the animals at any stage after the first or second dose. MA-2 challenge virus was not recovered at any stage. Brain and other tissues. The detection and concentration of ADV in the various regions of the brain is presented in Table 2. TABLE 2 CONCENTRATION
14 (Day 10) 21 (Day 14)
OF NIA-2
--
ADV
(lOglo
TCDQO PER g) IN TISSUES
3.5
3.1
;:; 3.8 2.6
2.6 5.1 -,
OF MONKEYS
3.5 5.1
AT NECROPSY
2.6 3.5 5.1
2.6
no virus isolated.
Serum Antibody Kesponses No significant level of neutralizing antibody (less than 1 in 2) to the Bartha strain of virus was detected in the serum of any monkey after the first immunization or up to 14 days after the second. The antibody titres to the Bartha and NIA-2 strains of virus at various stages after challenge are shown in Table 3. 3
TABLE
SERUM
ANTIBODY IJ3VEI.S (RECIPROCAL OF TITRES) in IMMUNIZED CHALLENGE WITH NIA-2 ADV AND ON DAY OF DEATH
NEUTRALIZING
7
MONKEYS
DAYS
AFTER
Days Monh~y
17
3: 14 21
,,VIA-.?
t2 t2 t2
