A comparison of the pathogenicity of two strains of hog cholera virus
2. Virological studies S KAMOLSIRII?FUCHAIPORN*,CJ MORRISSY and HA WESTBURY Australian Animal Health Laboratory, CSIRO Division of Animal Health, PO Bag 24, Geelong, Victoria 3220 SUMMARY: Quantitative and qualitative differences were demonstratedin the amount of virus in a range of tissues from pigs infected with either the Weybridge or New South Wales (NSW) strains of hog cholera (HC) virus. The titre of the Weybridge strain in samples, as assessed by either virus titration in cell culture or by the density of specific fluorescing cells in tissue sections, was higher than that for the NSW strain. This correlated with the greater severity of the ciinico-pathologicalsyndrome induced by the Weybrldge strain. The implicationsof the differences in the virus content of tissues in the diagnosis of HC is discussed as is the use of monoclonal antibodies to differentiate HC and bovine virus diarrhoea viruses. Aust Vet J 69: 245 - 248
Introduction In a previous paper, the clinical signs and the gross and microscopic lesions were compared between groups of pigs experimentally infected with Weybridge and NSW strains of hog cholera (HC) virus, sometimes termed classical swine fever virus (Kamolsiriprichaipom et a2 1992). The Weybridge strain was found to be virulent by causing acute HC in all infected pigs, whereas the NSW strain caused only mild disease and was, therefore, considered to be of low virulence. The disease induced by the 2 strains can be easily differentiated by the seventy of the clinical signs, as well as by the nature and extent of the gross and microscopic lesions. Gross lesions, such as splenic infarcts, petechial haemorrhage in the kidney and on serosal and mucosal surfaces, swollen and necrotic tonsils, and congested and haemorrhagic lymph nodes, were found in pigs infected with the Weybridge strain, but not the NSW strain of the virus. Differences in the microscopic lesions were as marked as the differences in the gross lesions seen between the groups, with the brain, pancreas, tonsils, spleen and lymph nodes being, in particular, more severely affected in the pigs infected with the Weybridge strain. In this paper we describe the distribution and titre of virus and viral antigens, in tissues of pigs experimentally infected with either the Weybridge or NSW strains of the virus, as assessed by virus isolation in cell culture and by fluorescent antibody stainiig of frozen tissues samples, and the relationship between the presence of the virus and the development and extent of the histopathological lesions.
Present address: Foot and Mouth Disease Research Centre, Department of Livestock Development, PakChong, Nakhon Ratchasima, Thailand Correspondence to: Dr HA Westbury, Australian Animal Health Laboratory, CSIRO Division of Animal Health, PO Bag 24, Geelong, Victoria 3220
Australian VeterinaryJournal Vol. 69, No. 10, October 1992
Materials and Methods Experimental Design The HC virus strains used, the number and distribfition of pigs in experimental groups, the tissues collected and the time span of the study were as described by Kamolsiriprichaiporn et al (1992). Blood was also collected, the serum separated after clotting, and the clot, as well as the other tissues, used in virus isolation attempts.
Fluorescent Antibody Procedure Portions of fresh tissues were placed in cryomoulds with OCT compoundt covering the base of the mould, immersed in isopentane in liquid nitrogen for 8 sec and then transferred into a stainless-steel box containing dry ice for transport to the laboratory. Blocks were stored at -2OOC until sectioned in a cryostat. Tissue sections were mounted on poly-L-lysine coated slides and fixed in cold acetone for 5 min, allowed to drain and either processed further or stored at -20°C. Further processing involved washing with phosphate buffered saline (PBS), to remove the OCT compound, air drying and immunostaining. Tissues were stained with 50 p1 of a specific pig anti-pestivirus serum (Kamolsiriprichaipom, unpublished) diluted 1:5 in PBS, or a mouse monoclonal antibody (Mab) as ascitic fluid diluted 150 in PBS and incubated at 37OC for 30 min washed 3 times in PBS (5minper wash), and then stained with 50 p1 of biotinylated anti-pig IgG (Kamolsiriprichaipom, unpublished) diluted 1 5 in PBS, or an appropriate dilution of a fluorescein conjugated anti-mouse serum' at 37°C for 60 min in a moisture chamber. The slides were washed 3 times and 50 p1 of fluoresceinstreptavidin conjugate* diluted 150 was added to the series, using pig serum for the detection of virus and incubated as before. The slides were washed 3 times, mounted with 10%glycerin in PBS and examined for fluorescence.
*
Miles Australia Pty Ltd, Mulgrave, Victoria Silenus Laboratories, Hawthorn, Victoria Amersham International, Buckinghamshire,
UK 245
bovine serum albumen fraction v# and incubated at 37OC in a moist chamber for 30 min. Plates were washed 3 times in PBS containing0.05%Tween20(washingsolution)andtheincubated with 50 p1 of specific pig anti-HC virus serum diluted 1:300, or mouse monoclonal antibodies (Mab) diluted 1:lOO and 1:250 with 0.1M buffer, pH 8.0, containing 1% bovine serum albumin. Cells were washed 3 times and 50 pl of protein A peroxidase conjugate** diluted 1500, or an appropriate dilution of peroxidase conjugated anti-mouse serumtt in the above buffer was added for 60 min at 37OC. Freshly prepared substrate (Graham er af 1965) in 100 p1 amounts was added after washing and allowed to react for 15 to 20 min before it was removed by washing in tap water. Reading was done by eye, and microscopically, for specific cytoplasmic staining, with wells exhibiting this staining being recorded as positive, regardless of the number of cells in a well showing specific staining. Virus titres were calculated using the method of Spearman-Karber (Finney 1952).Stainedplateswere stored at 4OC ifa recordof the titration was required.
VirusIsolation Tissue samples and blood clots were ground in a mortar with sterilised sand to produce a 10% tissue suspension in tissue culture medium containing antibiotics. The suspension was centrifuged at loo0 g for 15 min, the supernatant harvested and used immediately for inoculation of cell cultures or stored at -8OOC. Four replicates of 100 pl of each serial dilution (10" to 10-6,of tissue supernatant were pipetted into 96-well, flat bottomed tissue culture platesn, together with 100 p1 of PK-15 cells at 200 OOO cells per ml of tissue culture medium. The plates were incubated at 37°C in a 5% C02 atmospherefor 4 days, then examined using the peroxidase-linked assay (see below) if cells had grown to near confluence.
Peroxidase-linked Assay (PLA) Tissue culture medium was removed, the cells rinsed with warm PBS, wells drained and 100 p1 of 5 1 0 % formaldehydesolution in PBS with 0.1% Nonidet P40 was added at room temperature for 15min, followed by rinsing and draining. Each well was then overlaid with 50 p1 of PBS containing 0.05% Tween 20 and 1%
Monoclonal Antibodies Ten Mab as asciticfluid were suppliedby Dr G Chappuis,Lyon, France. The Mab were universally or differentiallyreactive with bovine, ovine and porcine strains of pestivirus (G Chappuis, personal communication). They were diluted 1 5 0 in PBS and stored at -2OOC until used, when they were further diluted 1:2 and 1 5 in PBS.
Nunc, Post Box 280,Kamstrup-kd 4000,Roskilda, Denmark Calbiochem - Novabiochem Ply Ltd, Alexandria, New South Wales ** Sigma Chemical Company, St Louis, MO, USA tt Silenus Laboratories, Hawthorn, Victoria
'
TABLE 1 The mean infectivity titre of the Weybridge and NSW strains of hog cholera virus in various tissue samples compared with a qualitativeassessment of the degree of specific fluorescence in the same tissue Tissue
Virus strain Weybridge
NSW
VI'
FATt
VI
FAT
No of positive samples
Mean titreS
No of positive samples
Mean fluorescence
No of positive samples
Mean titre
No of positive samples
Mean flourescence
Spleen
8
4.41
8
3+
7
3.11
7
2+
Ileum
8
3.75
8
3+
4
4.00
5
2+
Mesenteric lymph node
6
3.50
8
3+
5
2.60
6
I+
Tonsil
5
3.25
7
3+
4
2.31
5
1+
Duodenum
4
3.44
8
3+
2
2.50
2
1+
Heart
7
3.29
3
2+
1
3.35
0
Pancreas
7
3.18
7
2+
-
0
Lung
6
3.67
8
2+
3.65
5
1+
Liver
3
2.58
6
2+
2.50
0
Kidney
8
3.39
7
1+
0 5 1 4
-
2.56
2
I+
Brain
4
3.25
7
1+
0
-
0
-
Bladder Ureter
4
3.00
4
1+
1.25
0
-
-
NT
-
8
1+
1 NT
-
0
Colon
5
2.85
NT
-
5
2.60
NT
Popliteal lymph node
7
3.36
NT
-
4
2.56
NT
8211 1 Z9
(73%)
891104 (87%)
43/98 (44%)
32/91 (35%)
NT Not tested Virus isolation Fluorescent antibody test Mean titre loglo TCIDso PER 1 ml
*
246
Australian VeterinaryJournal Vol. 69, No. 10, October 1992
Results
Isolation of Virusfrom B lood Clots Virus was detected in the blood of 2 of 8 pigs in the group infected with the Weybridge strain on day 3 after infection, and in all 8pigs on day 4.The titres rangedfrom 102.’ to 107.25 TCIDso perml. Viraemia, incontrast, wasnot detectedinpigsintheNSW group until day 6 (one pig), with all pigs in the group having a detectableviraemiabyday8. Therangeof titresinthesepigs was lo’.’ to lo4 TCDsO per ml. The mean virus titre in blood clots from pigs infected with the NSW strain was lO3.lZ TcD50 (12 samples collected from day 7 onwards) whereas the mean titre in the Weybridge group was 104.45TCIDso per ml (28 samples from day 4 onwards). The mean titre of virus in the Weybridge group was significantly higher than that in the NSW group (t 40 = 3.73, p < 0.001). The peak titre of the viraemia occurred 3 to 4 days after virus was first detected and in some it persisted until death or slaughter, though in others (2 pigs in the NSW and 1 in the other group) it later waned to undetectable levels. Virus was not isolated from control pigs.
TABLE 2 Qualitative assessment of the degree of fluorescenceof 5 monoclonalantibodies to pestivlrus when tested with 3 atralns of hog cholera and bovine virus diarrhoea virus ~~~
Mab’ no.
Hog cholera virus
Weybridge strain
Baker strain
Tobias strain
C24V strain
Non-Cytot strain
1
4+*
2+
3i
4+
3+
4+
2
0
0
0
4+
3+
4+
3
0
0
0
4+
4+
3+
4
0
0
0
4+
2+
3+
5
4+
3+
3+
0
0
0
’
*
NSW strain
~
Virus Bovine virus diarrhoea
Mab monoclonal antibody Non-cyto non-cytopathogenic Fluorescence intensity scored on a scale of 0 (absent) to 4+ (strongest)
Isolation of Virusfrom Tissue Samples Fourteen tissues were testedfor the presence of livevirus. Virus was isolated from all tissues, though not on all occasions, from pigs infected with the Weybridge strain but was never isolated from the brain and pancreas of pigs infected with the NSW strain, despite it being detected in all the other tissues, though again, not on all occasions. The number of times virus was isolated from each tissue sample in each group and the mean virus titre in these samples is shown in Table 1. The number of samples infected with the Weybridge strain (73%) was greater than that infected with the NSW strain (44%),though the mean titre of virus in the positive samples from both groups was similar. Spleen was the organ of choice for the isolation of HC virus. The number of tissues infected with virus and for each strain is shown in Table 1. The mean number of tissues infected with the NSW strain increased to a peak on day 9 after infection and subsequently declined, whereas the number of tissues infected with the other strain remained at 11or greater from the first day onwards. Virus was not isolated from control pigs.
Detection of ViralAntigen by Immunojluorescence Thirteen tissues were tested with the amounts of virus per sample being assessed on the basis of the density of fluorescent foci and scored from 0 to 4+,with increasing amounts of specific fluorescence. No specific fluorescence was seen in samples from control pigs. The number of times specific fluorescence was observed in each tissue sample and the mean score for the fluorescence for each of the virus strains is shown in Table 1. Specific fluorescence was found in 85% of the samples from pigs infected with the Weybridge strain, but in only 35% of samples from the NSW group. A mean score of 24-or more for fluorescent foci was determined for 9 tissues from the Weybridge group but in only one from the NSW pigs. If a tissue, in which more than 50% of the samples tested showed fluorescence, is considered to be a good choice for the direct demonstration of virus for diagnostic purposes, then the spleen, tonsil, mesenteric lymph node, ileum and lung were the best candidate tissues. The virus was detected by immunofluorescence in the pancreas, brain, ureter, bladder, liver and the heart of pigs infected with the Weybridge strain, but not in samples from pigs in the NSW group.
Comparison of VirusIsolation and Detection of Antigen by Direct Immunojluorescence There was good correlation between the 2 techniques in the number of virus-positive samples detected from pigs infected with the NSW strain of the virus. Forty percent and 38% of Australian VeterinaryJournal Vol. 69, No. 10,October 1992
84 paired samples were positive by isolation and immunofluorescence, respectively. By contrast 84%of 96 paired samples collected from pigs infected with the Weybridge strain were found to be positive by immunofluorescence compared with 73% by virus isolation, but these differences were not statistically significant.
Monoclonal Antibody Studies Five of the Mab were selected for use in immunofluorescent staining of cryostat sections after comparative testing with a small number of bovine virus diarrhoea (BVD)and HC virus strains grown in cell cultures. The score given in a range 0 to 4 for each Mab and virus strain is recorded in Table 2. The Mab were assessed, on their specific fluorescent reactivity with strains, to be cross-reactive (Mab l), BVD virus-reactive (Mab no. 2, 3 and 4) or HC virus-reactive (Mab 5). They performed according to this classification when used in direct immunostaining of cryostat sections, that is Mab 1 and 5 exhibited fluorescence with selected tissues from pigs infected with either the Weybridge or NSW strains, whereas Mab no. 2 , 3 and 4 showed no reaction.
Discussion Experimental infection of pigs demonstrated differences in the invasiveness of the Weybridge and NSW strains of HC virus. This difference was, perhaps, best exemplified by the isolation of the virus from the brain and pancreas of pigs infected with the Weybridge strain but not from animals with the NSW strain. However, it was also shown by the higher proportion of tissue samples from the Weybridge group in which virus was detccted. Thus, 73% and 44% of paired samples were positive for virus by virus isolation, and 87% and 35% of the same samples were positive by immunofluorescence in the Weybridge and NSW groups, respectively. Furthermore, the titre of virus in most tissues and in blood was significantly higher in the Weybridge pigs than in the NSW pigs. These qualitative and quantitative differences between the 2 strains were consistent with the severity of the clinico-pathological syndrome they induced in experimentally infected pigs (Kamolsiriprichaipom et a1 1992). The ability of a virus strain to quickly invade and multiply in a range of tissue seemed to be associated with the propensity to cause disease and provides a basis for grading the pathogencity of HC virus strains. However, there are indications that there are factors other than the strain of the virus that have some impact 241
on the outcome of infection of individual animals with HC virus (Mengeling and Cheville 1968; k i s s et ul 1976; Wood et d 1988). These authors observed acute to sub-acute HC, with classical lesions of haemorrhage and death, in pigs experimentally infected withvirulent strains of the virus, as well as minimal to no clinical disease and lesions in similar pigs inoculated with the same strain. Variation from acute to inapparent HC was also described in pigs inoculated with strains considered to be of low virulence. Similar variation was observed during the 196061 outbreak of HC in Australia (Golding 1962), though in most instances experimental infection resulted in mild to inapparenl disease (Keast et a1 1962). Some of these anomalies may be explicable if there was a greater understanding of the pathophysiology and immunopahtology of HC. The differences between strains demonstrated by us, and by Kamolsiriprichaipom et a1 (1992) could be used as the starting point for such a study. There have been a number of comparisons of virus isolation in cell culture and immunostaining of frozen tissue in the diagnosis of HC (Ressang and den Boer 1967; Torloneetd 1967; Meyling and Schjeming-Thiesen 1968; Kubin and Kolbl 1968; Carbrey et ul 1970). These studies described a good correlation between the results obtained, though there was a tendency, especiallywith field samples, for a lower proportion of samplesto be positive by immunostaining of tissue samples, probably because of tissue deterioration during transport to the laboratory. Where fresh samples were used for comparative testing, there were no statistically significant differences in the results obtained (Carbrey et al1970). Similar results were obtained in this study, with no significant difference in the results obtained in virus detectionby isolation or immunostabhg. A more important finding, from a diagnosticpoint of view, was the difference in the quantity of virus in different tissues of animals infected with the 2 strains. The Weybridge strain was detectedinmore tissues of more animals than was the NSW strain and, furthermore,usually at ahigher titre, either assessed qualitatively by the density of fluorescence or quantitatively by virus isolation and titration. This has implications for the selection of tissues to be used for diagnostic purposes. Biopsy of tonsillar tissue has been a recommended procedure in surveillance of HC (Anon 1981) but our results suggest that this procedure may be less applicable for disease caused by strains of low virulence. We found no microscopic lesions of HC in tonsils from pigs infected with the NSW strain (Kamolsiriprichaipom e t ul 1992) and markedly less specific fluorescence compared with that seen in tonsils from pigs infected with virulent virus. This also applied to other tissues often recommended for use in rapid diagnosis, such as lymph nodes, brain and pancreatic tissue. Examination of a number of animals and a range of tissues from pigs potentially infected with HC strains of low virulence is therefore very important, whereas it may be possible to use a restricted number of tissues, for example, tonsil and spleen in the diagnosis of‘ disease caused by strains of high virulence. A problem with immunostaining of tissues with polyclonal antisera to detect HC virus is that there can be cross-reactions
248
with other viruses of the Pestivirus genus that may infect pigs, for example, BVDvirus (Stewarterd 1971; Femeliusetul 1973; Cartrrey et ul1970; Leks et ul1976; M a n and Collery 1977). Panels of Mab have been used in the differentiation of viruses within this genus (Wensvmt et al1989) and they have also been usedfor this purpose in diagnostic tests (Terpstra and Wensvoort 1988). We used Mab that were universally or differentially reactive with bovine and porcine pestiviruses to probe tissue sections for HC virus and found one (Mab no. 5 ) to have some potential to differentiate HC virus from other members of the genus in a positive sense and others (Mab no. 2,3,4) to have some potential in anegative sense. However, testing with a wider range of pestivirus strains than was available to us is obviously necessary before these Mab could be used in strain indicative testing and epidemiological studies. Problems in using highly specific procedures such as Mab profiles or short gene probes in the characterisationof pestivirus strians have been alluded to by Littlejohns (1989) and we agree, though we think they are a useful addition to the range of tests used in the differentialdiagnosis of HC.
References Anon (1981) Hog Cholera and its Eradication, Animal and Plant Health Inspection Senice, USDA Publication No 91-55, US Depament of Agriculture,Washington DC C a r h y EA, StewartWC, KmseJI and SnyderML (1970) InDlbgnakand Epizootiology of Classical Swine Fever, EUR hbKcaation No 5486, Canmission of the European Communities, Brussels. Femelius AL, Amtower WL. Lambert G,MCClurkin AW and Matthews PJ (1973)CanadJCompMed3713 Finney DJ (1952)Siaristical Method in Biological Assay,Griffin, London Golding NK (1%2) Awr Vet J 3 8 123 GrahamRC,~dh~UandKamoskyMJ(1%S)JHirtochemcyrochem13:150 Kamolsiiprichaipm S , Hooper FT.Westbury HA and Momssy CJ (1992) Aust Vet J 69240 Keast JC, Littlejohns IR and Helwig DM (1%2)Ausr Vet J 38129 Kubin G and Kolbl 0 (1968)TierarztMschr 55578 h i s s B, Frey HR. Prager D. Hafez SM and Roeder P (1976) In Diagnmis and Epizootiology of Classical Swine Fever, EUR Publication No 5486, Canmission of the European Communities, Bmssels Lenihan D and Collery (1977) Proc CECIFAO Seminar on Hog CholeralClassical Swine Fever and African Swine Fever, Hannover 1976, edited by Leks B. EURPublicationNo 5904, Commissionofthe European Communities,Hannover Littlejohns IR (1989)Aut Ver J66:435 Mengeling WL and Cheville NF (1968) Proc 72nd Annu Meet US Anim Healrh Assoc, p 283 Meyling A and Schjeming-TheisenK (1968)Acra Vet Scad 9 5 0 Ressang AA and den Boer JL (1967)TijdschrDiergeneesk 92567 Stewart WC, Carbrey EA, Jenney EW, Brown CL and Kresse JI (1971)JAm Vet Med Assoc 159: 1556 TerpstraCand WensvoortG (1988)ResVetSci45137 TorfaR V.DiAntmioE,TitoliF.Gialle~LandPortllgalFL(I%’I)Vetffd 18634 Wensvoort G.Terpstra C, De Kluijver EP. Kragten C and Wamaar JC (1989) Vet Microbiol21:9 Wood L, Broclrman S, Harkness JW and Edwards S (1988) Vet Rec 122391
(Acceptedfor publication 16 July 1992)
Australian Veterinary Journal Vol. 69, No. 10, October 1992
2. Virological studies S KAMOLSIRII?FUCHAIPORN*,CJ MORRISSY and HA WESTBURY Australian Animal Health Laboratory, CSIRO Division of Animal Health, PO Bag 24, Geelong, Victoria 3220 SUMMARY: Quantitative and qualitative differences were demonstratedin the amount of virus in a range of tissues from pigs infected with either the Weybridge or New South Wales (NSW) strains of hog cholera (HC) virus. The titre of the Weybridge strain in samples, as assessed by either virus titration in cell culture or by the density of specific fluorescing cells in tissue sections, was higher than that for the NSW strain. This correlated with the greater severity of the ciinico-pathologicalsyndrome induced by the Weybrldge strain. The implicationsof the differences in the virus content of tissues in the diagnosis of HC is discussed as is the use of monoclonal antibodies to differentiate HC and bovine virus diarrhoea viruses. Aust Vet J 69: 245 - 248
Introduction In a previous paper, the clinical signs and the gross and microscopic lesions were compared between groups of pigs experimentally infected with Weybridge and NSW strains of hog cholera (HC) virus, sometimes termed classical swine fever virus (Kamolsiriprichaipom et a2 1992). The Weybridge strain was found to be virulent by causing acute HC in all infected pigs, whereas the NSW strain caused only mild disease and was, therefore, considered to be of low virulence. The disease induced by the 2 strains can be easily differentiated by the seventy of the clinical signs, as well as by the nature and extent of the gross and microscopic lesions. Gross lesions, such as splenic infarcts, petechial haemorrhage in the kidney and on serosal and mucosal surfaces, swollen and necrotic tonsils, and congested and haemorrhagic lymph nodes, were found in pigs infected with the Weybridge strain, but not the NSW strain of the virus. Differences in the microscopic lesions were as marked as the differences in the gross lesions seen between the groups, with the brain, pancreas, tonsils, spleen and lymph nodes being, in particular, more severely affected in the pigs infected with the Weybridge strain. In this paper we describe the distribution and titre of virus and viral antigens, in tissues of pigs experimentally infected with either the Weybridge or NSW strains of the virus, as assessed by virus isolation in cell culture and by fluorescent antibody stainiig of frozen tissues samples, and the relationship between the presence of the virus and the development and extent of the histopathological lesions.
Present address: Foot and Mouth Disease Research Centre, Department of Livestock Development, PakChong, Nakhon Ratchasima, Thailand Correspondence to: Dr HA Westbury, Australian Animal Health Laboratory, CSIRO Division of Animal Health, PO Bag 24, Geelong, Victoria 3220
Australian VeterinaryJournal Vol. 69, No. 10, October 1992
Materials and Methods Experimental Design The HC virus strains used, the number and distribfition of pigs in experimental groups, the tissues collected and the time span of the study were as described by Kamolsiriprichaiporn et al (1992). Blood was also collected, the serum separated after clotting, and the clot, as well as the other tissues, used in virus isolation attempts.
Fluorescent Antibody Procedure Portions of fresh tissues were placed in cryomoulds with OCT compoundt covering the base of the mould, immersed in isopentane in liquid nitrogen for 8 sec and then transferred into a stainless-steel box containing dry ice for transport to the laboratory. Blocks were stored at -2OOC until sectioned in a cryostat. Tissue sections were mounted on poly-L-lysine coated slides and fixed in cold acetone for 5 min, allowed to drain and either processed further or stored at -20°C. Further processing involved washing with phosphate buffered saline (PBS), to remove the OCT compound, air drying and immunostaining. Tissues were stained with 50 p1 of a specific pig anti-pestivirus serum (Kamolsiriprichaipom, unpublished) diluted 1:5 in PBS, or a mouse monoclonal antibody (Mab) as ascitic fluid diluted 150 in PBS and incubated at 37OC for 30 min washed 3 times in PBS (5minper wash), and then stained with 50 p1 of biotinylated anti-pig IgG (Kamolsiriprichaipom, unpublished) diluted 1 5 in PBS, or an appropriate dilution of a fluorescein conjugated anti-mouse serum' at 37°C for 60 min in a moisture chamber. The slides were washed 3 times and 50 p1 of fluoresceinstreptavidin conjugate* diluted 150 was added to the series, using pig serum for the detection of virus and incubated as before. The slides were washed 3 times, mounted with 10%glycerin in PBS and examined for fluorescence.
*
Miles Australia Pty Ltd, Mulgrave, Victoria Silenus Laboratories, Hawthorn, Victoria Amersham International, Buckinghamshire,
UK 245
bovine serum albumen fraction v# and incubated at 37OC in a moist chamber for 30 min. Plates were washed 3 times in PBS containing0.05%Tween20(washingsolution)andtheincubated with 50 p1 of specific pig anti-HC virus serum diluted 1:300, or mouse monoclonal antibodies (Mab) diluted 1:lOO and 1:250 with 0.1M buffer, pH 8.0, containing 1% bovine serum albumin. Cells were washed 3 times and 50 pl of protein A peroxidase conjugate** diluted 1500, or an appropriate dilution of peroxidase conjugated anti-mouse serumtt in the above buffer was added for 60 min at 37OC. Freshly prepared substrate (Graham er af 1965) in 100 p1 amounts was added after washing and allowed to react for 15 to 20 min before it was removed by washing in tap water. Reading was done by eye, and microscopically, for specific cytoplasmic staining, with wells exhibiting this staining being recorded as positive, regardless of the number of cells in a well showing specific staining. Virus titres were calculated using the method of Spearman-Karber (Finney 1952).Stainedplateswere stored at 4OC ifa recordof the titration was required.
VirusIsolation Tissue samples and blood clots were ground in a mortar with sterilised sand to produce a 10% tissue suspension in tissue culture medium containing antibiotics. The suspension was centrifuged at loo0 g for 15 min, the supernatant harvested and used immediately for inoculation of cell cultures or stored at -8OOC. Four replicates of 100 pl of each serial dilution (10" to 10-6,of tissue supernatant were pipetted into 96-well, flat bottomed tissue culture platesn, together with 100 p1 of PK-15 cells at 200 OOO cells per ml of tissue culture medium. The plates were incubated at 37°C in a 5% C02 atmospherefor 4 days, then examined using the peroxidase-linked assay (see below) if cells had grown to near confluence.
Peroxidase-linked Assay (PLA) Tissue culture medium was removed, the cells rinsed with warm PBS, wells drained and 100 p1 of 5 1 0 % formaldehydesolution in PBS with 0.1% Nonidet P40 was added at room temperature for 15min, followed by rinsing and draining. Each well was then overlaid with 50 p1 of PBS containing 0.05% Tween 20 and 1%
Monoclonal Antibodies Ten Mab as asciticfluid were suppliedby Dr G Chappuis,Lyon, France. The Mab were universally or differentiallyreactive with bovine, ovine and porcine strains of pestivirus (G Chappuis, personal communication). They were diluted 1 5 0 in PBS and stored at -2OOC until used, when they were further diluted 1:2 and 1 5 in PBS.
Nunc, Post Box 280,Kamstrup-kd 4000,Roskilda, Denmark Calbiochem - Novabiochem Ply Ltd, Alexandria, New South Wales ** Sigma Chemical Company, St Louis, MO, USA tt Silenus Laboratories, Hawthorn, Victoria
'
TABLE 1 The mean infectivity titre of the Weybridge and NSW strains of hog cholera virus in various tissue samples compared with a qualitativeassessment of the degree of specific fluorescence in the same tissue Tissue
Virus strain Weybridge
NSW
VI'
FATt
VI
FAT
No of positive samples
Mean titreS
No of positive samples
Mean fluorescence
No of positive samples
Mean titre
No of positive samples
Mean flourescence
Spleen
8
4.41
8
3+
7
3.11
7
2+
Ileum
8
3.75
8
3+
4
4.00
5
2+
Mesenteric lymph node
6
3.50
8
3+
5
2.60
6
I+
Tonsil
5
3.25
7
3+
4
2.31
5
1+
Duodenum
4
3.44
8
3+
2
2.50
2
1+
Heart
7
3.29
3
2+
1
3.35
0
Pancreas
7
3.18
7
2+
-
0
Lung
6
3.67
8
2+
3.65
5
1+
Liver
3
2.58
6
2+
2.50
0
Kidney
8
3.39
7
1+
0 5 1 4
-
2.56
2
I+
Brain
4
3.25
7
1+
0
-
0
-
Bladder Ureter
4
3.00
4
1+
1.25
0
-
-
NT
-
8
1+
1 NT
-
0
Colon
5
2.85
NT
-
5
2.60
NT
Popliteal lymph node
7
3.36
NT
-
4
2.56
NT
8211 1 Z9
(73%)
891104 (87%)
43/98 (44%)
32/91 (35%)
NT Not tested Virus isolation Fluorescent antibody test Mean titre loglo TCIDso PER 1 ml
*
246
Australian VeterinaryJournal Vol. 69, No. 10, October 1992
Results
Isolation of Virusfrom B lood Clots Virus was detected in the blood of 2 of 8 pigs in the group infected with the Weybridge strain on day 3 after infection, and in all 8pigs on day 4.The titres rangedfrom 102.’ to 107.25 TCIDso perml. Viraemia, incontrast, wasnot detectedinpigsintheNSW group until day 6 (one pig), with all pigs in the group having a detectableviraemiabyday8. Therangeof titresinthesepigs was lo’.’ to lo4 TCDsO per ml. The mean virus titre in blood clots from pigs infected with the NSW strain was lO3.lZ TcD50 (12 samples collected from day 7 onwards) whereas the mean titre in the Weybridge group was 104.45TCIDso per ml (28 samples from day 4 onwards). The mean titre of virus in the Weybridge group was significantly higher than that in the NSW group (t 40 = 3.73, p < 0.001). The peak titre of the viraemia occurred 3 to 4 days after virus was first detected and in some it persisted until death or slaughter, though in others (2 pigs in the NSW and 1 in the other group) it later waned to undetectable levels. Virus was not isolated from control pigs.
TABLE 2 Qualitative assessment of the degree of fluorescenceof 5 monoclonalantibodies to pestivlrus when tested with 3 atralns of hog cholera and bovine virus diarrhoea virus ~~~
Mab’ no.
Hog cholera virus
Weybridge strain
Baker strain
Tobias strain
C24V strain
Non-Cytot strain
1
4+*
2+
3i
4+
3+
4+
2
0
0
0
4+
3+
4+
3
0
0
0
4+
4+
3+
4
0
0
0
4+
2+
3+
5
4+
3+
3+
0
0
0
’
*
NSW strain
~
Virus Bovine virus diarrhoea
Mab monoclonal antibody Non-cyto non-cytopathogenic Fluorescence intensity scored on a scale of 0 (absent) to 4+ (strongest)
Isolation of Virusfrom Tissue Samples Fourteen tissues were testedfor the presence of livevirus. Virus was isolated from all tissues, though not on all occasions, from pigs infected with the Weybridge strain but was never isolated from the brain and pancreas of pigs infected with the NSW strain, despite it being detected in all the other tissues, though again, not on all occasions. The number of times virus was isolated from each tissue sample in each group and the mean virus titre in these samples is shown in Table 1. The number of samples infected with the Weybridge strain (73%) was greater than that infected with the NSW strain (44%),though the mean titre of virus in the positive samples from both groups was similar. Spleen was the organ of choice for the isolation of HC virus. The number of tissues infected with virus and for each strain is shown in Table 1. The mean number of tissues infected with the NSW strain increased to a peak on day 9 after infection and subsequently declined, whereas the number of tissues infected with the other strain remained at 11or greater from the first day onwards. Virus was not isolated from control pigs.
Detection of ViralAntigen by Immunojluorescence Thirteen tissues were tested with the amounts of virus per sample being assessed on the basis of the density of fluorescent foci and scored from 0 to 4+,with increasing amounts of specific fluorescence. No specific fluorescence was seen in samples from control pigs. The number of times specific fluorescence was observed in each tissue sample and the mean score for the fluorescence for each of the virus strains is shown in Table 1. Specific fluorescence was found in 85% of the samples from pigs infected with the Weybridge strain, but in only 35% of samples from the NSW group. A mean score of 24-or more for fluorescent foci was determined for 9 tissues from the Weybridge group but in only one from the NSW pigs. If a tissue, in which more than 50% of the samples tested showed fluorescence, is considered to be a good choice for the direct demonstration of virus for diagnostic purposes, then the spleen, tonsil, mesenteric lymph node, ileum and lung were the best candidate tissues. The virus was detected by immunofluorescence in the pancreas, brain, ureter, bladder, liver and the heart of pigs infected with the Weybridge strain, but not in samples from pigs in the NSW group.
Comparison of VirusIsolation and Detection of Antigen by Direct Immunojluorescence There was good correlation between the 2 techniques in the number of virus-positive samples detected from pigs infected with the NSW strain of the virus. Forty percent and 38% of Australian VeterinaryJournal Vol. 69, No. 10,October 1992
84 paired samples were positive by isolation and immunofluorescence, respectively. By contrast 84%of 96 paired samples collected from pigs infected with the Weybridge strain were found to be positive by immunofluorescence compared with 73% by virus isolation, but these differences were not statistically significant.
Monoclonal Antibody Studies Five of the Mab were selected for use in immunofluorescent staining of cryostat sections after comparative testing with a small number of bovine virus diarrhoea (BVD)and HC virus strains grown in cell cultures. The score given in a range 0 to 4 for each Mab and virus strain is recorded in Table 2. The Mab were assessed, on their specific fluorescent reactivity with strains, to be cross-reactive (Mab l), BVD virus-reactive (Mab no. 2, 3 and 4) or HC virus-reactive (Mab 5). They performed according to this classification when used in direct immunostaining of cryostat sections, that is Mab 1 and 5 exhibited fluorescence with selected tissues from pigs infected with either the Weybridge or NSW strains, whereas Mab no. 2 , 3 and 4 showed no reaction.
Discussion Experimental infection of pigs demonstrated differences in the invasiveness of the Weybridge and NSW strains of HC virus. This difference was, perhaps, best exemplified by the isolation of the virus from the brain and pancreas of pigs infected with the Weybridge strain but not from animals with the NSW strain. However, it was also shown by the higher proportion of tissue samples from the Weybridge group in which virus was detccted. Thus, 73% and 44% of paired samples were positive for virus by virus isolation, and 87% and 35% of the same samples were positive by immunofluorescence in the Weybridge and NSW groups, respectively. Furthermore, the titre of virus in most tissues and in blood was significantly higher in the Weybridge pigs than in the NSW pigs. These qualitative and quantitative differences between the 2 strains were consistent with the severity of the clinico-pathological syndrome they induced in experimentally infected pigs (Kamolsiriprichaipom et a1 1992). The ability of a virus strain to quickly invade and multiply in a range of tissue seemed to be associated with the propensity to cause disease and provides a basis for grading the pathogencity of HC virus strains. However, there are indications that there are factors other than the strain of the virus that have some impact 241
on the outcome of infection of individual animals with HC virus (Mengeling and Cheville 1968; k i s s et ul 1976; Wood et d 1988). These authors observed acute to sub-acute HC, with classical lesions of haemorrhage and death, in pigs experimentally infected withvirulent strains of the virus, as well as minimal to no clinical disease and lesions in similar pigs inoculated with the same strain. Variation from acute to inapparent HC was also described in pigs inoculated with strains considered to be of low virulence. Similar variation was observed during the 196061 outbreak of HC in Australia (Golding 1962), though in most instances experimental infection resulted in mild to inapparenl disease (Keast et a1 1962). Some of these anomalies may be explicable if there was a greater understanding of the pathophysiology and immunopahtology of HC. The differences between strains demonstrated by us, and by Kamolsiriprichaipom et a1 (1992) could be used as the starting point for such a study. There have been a number of comparisons of virus isolation in cell culture and immunostaining of frozen tissue in the diagnosis of HC (Ressang and den Boer 1967; Torloneetd 1967; Meyling and Schjeming-Thiesen 1968; Kubin and Kolbl 1968; Carbrey et ul 1970). These studies described a good correlation between the results obtained, though there was a tendency, especiallywith field samples, for a lower proportion of samplesto be positive by immunostaining of tissue samples, probably because of tissue deterioration during transport to the laboratory. Where fresh samples were used for comparative testing, there were no statistically significant differences in the results obtained (Carbrey et al1970). Similar results were obtained in this study, with no significant difference in the results obtained in virus detectionby isolation or immunostabhg. A more important finding, from a diagnosticpoint of view, was the difference in the quantity of virus in different tissues of animals infected with the 2 strains. The Weybridge strain was detectedinmore tissues of more animals than was the NSW strain and, furthermore,usually at ahigher titre, either assessed qualitatively by the density of fluorescence or quantitatively by virus isolation and titration. This has implications for the selection of tissues to be used for diagnostic purposes. Biopsy of tonsillar tissue has been a recommended procedure in surveillance of HC (Anon 1981) but our results suggest that this procedure may be less applicable for disease caused by strains of low virulence. We found no microscopic lesions of HC in tonsils from pigs infected with the NSW strain (Kamolsiriprichaipom e t ul 1992) and markedly less specific fluorescence compared with that seen in tonsils from pigs infected with virulent virus. This also applied to other tissues often recommended for use in rapid diagnosis, such as lymph nodes, brain and pancreatic tissue. Examination of a number of animals and a range of tissues from pigs potentially infected with HC strains of low virulence is therefore very important, whereas it may be possible to use a restricted number of tissues, for example, tonsil and spleen in the diagnosis of‘ disease caused by strains of high virulence. A problem with immunostaining of tissues with polyclonal antisera to detect HC virus is that there can be cross-reactions
248
with other viruses of the Pestivirus genus that may infect pigs, for example, BVDvirus (Stewarterd 1971; Femeliusetul 1973; Cartrrey et ul1970; Leks et ul1976; M a n and Collery 1977). Panels of Mab have been used in the differentiation of viruses within this genus (Wensvmt et al1989) and they have also been usedfor this purpose in diagnostic tests (Terpstra and Wensvoort 1988). We used Mab that were universally or differentially reactive with bovine and porcine pestiviruses to probe tissue sections for HC virus and found one (Mab no. 5 ) to have some potential to differentiate HC virus from other members of the genus in a positive sense and others (Mab no. 2,3,4) to have some potential in anegative sense. However, testing with a wider range of pestivirus strains than was available to us is obviously necessary before these Mab could be used in strain indicative testing and epidemiological studies. Problems in using highly specific procedures such as Mab profiles or short gene probes in the characterisationof pestivirus strians have been alluded to by Littlejohns (1989) and we agree, though we think they are a useful addition to the range of tests used in the differentialdiagnosis of HC.
References Anon (1981) Hog Cholera and its Eradication, Animal and Plant Health Inspection Senice, USDA Publication No 91-55, US Depament of Agriculture,Washington DC C a r h y EA, StewartWC, KmseJI and SnyderML (1970) InDlbgnakand Epizootiology of Classical Swine Fever, EUR hbKcaation No 5486, Canmission of the European Communities, Brussels. Femelius AL, Amtower WL. Lambert G,MCClurkin AW and Matthews PJ (1973)CanadJCompMed3713 Finney DJ (1952)Siaristical Method in Biological Assay,Griffin, London Golding NK (1%2) Awr Vet J 3 8 123 GrahamRC,~dh~UandKamoskyMJ(1%S)JHirtochemcyrochem13:150 Kamolsiiprichaipm S , Hooper FT.Westbury HA and Momssy CJ (1992) Aust Vet J 69240 Keast JC, Littlejohns IR and Helwig DM (1%2)Ausr Vet J 38129 Kubin G and Kolbl 0 (1968)TierarztMschr 55578 h i s s B, Frey HR. Prager D. Hafez SM and Roeder P (1976) In Diagnmis and Epizootiology of Classical Swine Fever, EUR Publication No 5486, Canmission of the European Communities, Bmssels Lenihan D and Collery (1977) Proc CECIFAO Seminar on Hog CholeralClassical Swine Fever and African Swine Fever, Hannover 1976, edited by Leks B. EURPublicationNo 5904, Commissionofthe European Communities,Hannover Littlejohns IR (1989)Aut Ver J66:435 Mengeling WL and Cheville NF (1968) Proc 72nd Annu Meet US Anim Healrh Assoc, p 283 Meyling A and Schjeming-TheisenK (1968)Acra Vet Scad 9 5 0 Ressang AA and den Boer JL (1967)TijdschrDiergeneesk 92567 Stewart WC, Carbrey EA, Jenney EW, Brown CL and Kresse JI (1971)JAm Vet Med Assoc 159: 1556 TerpstraCand WensvoortG (1988)ResVetSci45137 TorfaR V.DiAntmioE,TitoliF.Gialle~LandPortllgalFL(I%’I)Vetffd 18634 Wensvoort G.Terpstra C, De Kluijver EP. Kragten C and Wamaar JC (1989) Vet Microbiol21:9 Wood L, Broclrman S, Harkness JW and Edwards S (1988) Vet Rec 122391
(Acceptedfor publication 16 July 1992)
Australian Veterinary Journal Vol. 69, No. 10, October 1992
