ISOLATION AND CHARACTERISATION OF HERPES VIRUS FROM TUWEYS L. B. M.
PRASAD*
and P. B. SwbwBnow
Department of Veterinary Preventive Medicine, University of Queensland, Brisbane, Queensland, 4067 SUMMARY: Turkey h e r p e s v i r u s (HVT) was iso lat ed from t h e k i d n ey s of 6 of 8 t u r k ey p o u l t s from two flocks. T h e i s o l a t e s were identified by syncytiai-type of cy t o p at h o i o g y , inhibition of pla que formation by 5 - b r o m o d eo x y u r id in e, formation of Cowdry t y p e A intranuclear inclusions in cell cultures, a n d presence of h e r p e s - t y p e virions in negatively s t a i n e d p r e p a r a t i o n s a n d thin sections of in f ected cell c u l t u r e s . One of t h e s e isolates i n o cu l at ed into c h i c k e n s proved a p a t h o g e n i c over an o b ser v atio n p er io d of 10 w e e k s . Indirect immun~fiuorescenceand s e r u m neutralisation tests r ev eal ed ser o l o g i cal relations h i p b e t w e e n t h e s e isolates a n d t h e strain NSW 1 / 7 0 of HVT. Staining of HVT-infected cell culture by Marek's d i s e a s e h e r p e s v i r u s ( M D H V ) antiserum s h o w e d i n t r an u cl ear fluorescence but a t t e m p t s to p r e p a r e HVT precipitating an tig en or to d e m o n s t r a t e cross-precipitation betw e e n HVTand MDHV were unsuccessful.
Introduction
Following the initial description of turkey herpesvirus (HVT) in the U.S.A. (Kawarnura et al 1969; Witter et al 1970), the isolation of H V T has been reported from many countries, including Australia (Mustaffa-Babjee et al 1971; Sinkovic and Jones 1972). The quarantine regulations in this country stipulate local virus strains to be used for the preparation of vaccines, and the HVT strain NSW 1/70 is currently used as vaccine against Marek's disease (MD). However, there is a need for comparative studies o n the in vitro and in vivo characteristics of endemic H V T strains as information about the prevalence and properties of such strains is limited (Mustaffa-Babjee and Spradbrow 1973; Harman et al1973). This paper reports a study o n the isolation and characterisation of H V T from local turkey flocks. Materials and Methods Turkeys Primary isolation of HVT was attempted from 8 clinically healthy poults (White Holland or bronze-winged). Five poults, 6 to 8 months of age, were puchased from a private breeder in Queensland. The parent flock of these poults consisted of 42 birds that had not suffered from any serious clinical disease including neoplastic conditions. A further 3 poults were obtained from a commercial turkey flock in New South Wales when one day old. They were reared in isolation in the laboratory. Two were killed when 4Yz weeks of age and the third when 8 weeks old.
Cell Cultures Primary cell cultures of turkey kidney and of embryo fibroblasts from duck (DEF) and chick CEF) were prepared as described by Mustaffa-Babjee (1970) but using tricine-buffered medium 199 (T199) instead of Eagle's basal medium for cell culture.
Cloning of HVT Isolates Two of the HVT isolates (TAM-5, TAM-6) were purified through 3 cycles of cloning and propagation in DEF cultures. The use of cell cultures of chick origin was avoided to eliminate the risk of inadvertent contamination with vertically iransmittcd chicken viruses. Serial 10-fold dilutions of the primary isolates (infected turkey kidney cells) were inoculated into DEF in 55 mm plastic petri dishes which were overlayed with 0.So/u agarose. When the foci were clearly evident, usually on the 5th or 6th day of incubation. the agarose over an iboiatcd focus was removed with a capillary pipette and the well was filled w i t h irypsin-versene. The dispersed cells thus obtained were then inoculated into fresh 24-hour-old primary UEF in Roux flasks which were incubated at 38°C until the cytopathic effect (CPE) developed. This process was repeated 3 times. Later, the isolates were propagated to the 10th passage in DEF cultures. The preparations thus obtained did not show any evidence of contamination with viruses of chicken or duck origin by cultural and electron microscopic methods. Virus Assay l 'h e methods described by Calnek rt irl(1972) were followed. I,'ocus-forrning units (FFU) under agarose overlay and in some instances plaque-forming units (PFU) in liquid overlay in C EF cultures were counted at 5 or 6 days after inoculation. In a preliminary study, a comparison was made between the focus counts in cultures overlayed with agarose immediately and 18 hours after adsorption of cell-free VHT. The foci developing in the latter appeared slightly elongated but the focus counts in two cultures were not statistically different. Therefore, the latter procedure was not used in this study.
Lyophilisation an d Storage Cell-associated HVT harvested on the 4th day of incubation was suspended in a freezing mixture consisting of 10% dimethyl sulphoxide and 10% calf serum in niedium TI99 and stored at either -70" or in liquid nitrogen. To prepare cell-free HVT. cells from an equal volume of infected culture suspension was suspended in the stabiliser SPGA (Bovarnick et a/ 1950) and sonicated. The extract was filtered (0.45 pm), lyophilised (Calnek e t a / 1970) and stored at 4OC.
De~erminarionof Viral Nucleic Acid Type
'Prcscnl address: Coniinoiiwcalth Suruin Laboialorics. Pnrkville. V i C t o i m . 3052.
The isolates were grown in C EF cultures in plastic dishes containing 5-bromodeoxyuridine ( B U D R ) in concentrations
582
Australian Veterinary Journal, Vol.
53, December, 1977
from 10 to 100 ,ug/ml in medium and the plaques were enumerated on the 5th day of incubation. The control consisted of infected cultures grown similarly in the absence of BUDR. Serology An indirect immunofluorescence test was employed using infected DEF cultures on coverslips or smears of buffy coat cells from viraemic chickens as the source of antigen. HVT antiserum used in the test was obtained from four 4-week-old chickens which had been vaccinated subcutaneously with 1,OOO FFU of cell-associated virus TAM-6) when one day of age. It had a neutralising antibody titre of 2 320 and was free of antibody to Marek's disease herpesvirus (MDHV) as determined by the agar-gel precipitin (AGP) test (Chubb and Churchill 1968). MDHV antiserum, as judged by the AGP test, was obtained from chickens in a flock with a high incidence of clinical MD. The staining procedures were the same as described by Ahmed et d(1970). The AGP antigens were prepared with the HVT isolates (TAM-5 and TAM-6) and MDHV (Mustaffa-Babjee 1970) by the method of Chubb and Churchill (1968). The serum neutralisation test was performed as described by Zygraich and Huygelen (1972), using 150 to 200 50% tissue culture infective doses of cell-free HVT (TAM-6). A 50% neutralisation endpoint was determined by the method of Reed and Muench (1938). Electron Microscopy Cell lysates from infected cultures showing characteristic herpesvirus CPE were negatively stained with 1.5% phosphotungstic acid (pH 6.6). For thin sectioning, the infected cells were removed with a rubber policeman, fixed in 5% buffered glutaraldehyde and embedded in Epon araldite. Thin sections were stained with uranyl acetate and lead citrate. Pathogenicity for Chickens Twenty four one-day-old chickens, free of maternal antibodies to HVT and MDHV, were inoculated subcutaneously with 1,280 FFU of cell-associated HVT (TAM-6). They were killed and autopsied in groups of 4 birds between 8 and 10 weeks after inoculation and cell cultures from the pooled samples of kidneys from each group were prepared for virus isolation. Histological samples were prepared from sciatic and brachial nerves, heart, spleen, liver, lung, bursa and gonads using conventional methods.
turkey kidney cultures but took a longer time to develop during the initial 2 to 3 passages. Later, all isolates produced plaques in 3 to 4 days in DEF. The affected cells varied in size and were spherical, flat, polygonal or fusiform in shape. Syncytia of varying sizes and angular or stellate outline developed and showed structureless homogeneous centres. A few cells remaining in the central clear areas or at the margin of plaques joined cells at the opposite edge by trailing strands of cytoplasm. Infected monolayers removed with collodion and stained with haematoxylin and eosin had multinucleated giant cells and Cowdry type A intranuclear inclusion bodies. In CEF cultures, the plaques were smaller in size and intranuclear inclusions were numerous but formation of a central clear area was not a constant feature. Plaque counts were reduced by 85% in the presence of 25 pg/ml of BUDR and plaques did not develop when the concentration of BUDR was increased to 50 pg/ml. The size of foci produced by isolates TAM-5 and TAM-6 was determined 5 days after inoculation by measuring 100 foci on each of 5 replicate CEF cultures. Their dimensions averaged 1.01 9 0.21 and 0.98 T 0.24 mm respectively and the difference between them was not statistically significant. A comparison of the titres of cell-associated and cell-free HVT recovered from an equal aliquot of culture suspension is shown in table 1. From 1.56 to 2.4% of viable cells were found to be infected with a mean approximating 2%. The infectiviy ratio of cell-associated to cell-free virus ranged between 1 : 1.67 and 1 : 2.40 with an average of 1 : 1.94.
Serological Findings Results
HVT-infected cells treated with HVT antiserum had granular nuclear and diffuse cytoplasmic HVT was isolated from the kidneys of all five fluorescence. This pattern of fluorescence was Queensland turkeys (TAM-3 to TAM-7) and one noticed in cell cultures infected with all the NSW turkey, aged 8 weeks (TAM-8). HVT was isolates, and in the buffy coat cells from chickens not isolated from the other two NSW poults which were killed at 4% weeks of age. TABLE 1 Isolation o f H V T
Cellular and Cell-free Infectivity of Turkey Herpesvirus (TAM-6) in DEF Cell Cultures
Responses in Cell Cultures In primary turkey kidney cell cultures, plaques consisting of small round refractile cells were first noticed between 6 and 8 days after seeding. Two to 3 days later, the plaques grew in size, and the infected cells enlarged 3 to 5 times the normal size and became vacuolated. Later a clear central area devoid of cells remained in the centre of the. plaques. Between 8 and 10 days, several new plaques formed. Plaques were larger in DEF cultures than in Australian Veterinary J o u r n d , Vol. 53, December, 1977
FFU/ml Trial
Viable Cell Count
CA* Infectivity
1 2 3
23x10' 2.OX1Oe 1.6X10"
6.0X10' 33x10' 25x10'
CA
CF+ % Cells Ratio Infec- Infected CA-CF tivity Infectivity 2.5X 10' 2.OXlO' 1.5XlW
2.40 1.75 1.56
1:2.40 1:1.75 1:1.67
= Cell-associated.
+ CF = Cell-free
infectivity determined following lyophilisation
and reconstitution.
583
with HVT viraemia (Figure 1). Not infrequently, granular cytoplasmic and nuclear staining was also seen in cells from cultures with extensive CPE. When the cells were treated with MDHV antiserum, thick granular staining was confined to the nucleus only (Figure 2). Attempts to prepare AGP antigens with isolates TAM-5 and TAM-6 or to demonstrate cross-precipitation between the HVT and MDHV reactants were unsuccessful. The neutralising antibody titres of serums of turkeys from which the isolates were obtained ranged between 80 and 160. Furthermore, 16 serum samples collected from 4 chickens between 8 and 81 days after vaccination with 1,000 PFU of cell-free HVT (NSW 1/70) at one day of age (kindly supplied by Dr. B. Sinkovic, University of Sydney) showed mean neutralising antibody titres between 1 1 1 and 236 when titrated against TAM-6.
Pathogenicity The chickens inoculated with the TAM-6 isolate did not develop any clinical signs of disease, gross or histological lesions attributable to HVT or MDHV when examined between 8 and 10 weeks after inoculation. HVT was isolated in cell cultures from each of the pooled samples of kidneys from inoculated birds killed in groups during this period. Discussion
Figures 1 and 2. Fluorescence micropraphs of cells infected with HVT and stained by an indirect procedure: ( 1 ) A buffy coat cell (lymphocyte) from a viraemic chick 2 days after inoculation with TAM-6 and threated with HVT (TAM-6) antiserum showing diffuse cytoplasmic and grmular nuclear staining (X1.200); (2) A TAM-5 infected DEF cell treated with MDHV antiserum shows thick, clumpy granular fluorescence confined to nucleus (X600).
The characteristics of isolates reported here showed a close similarity to those of HVT (Witter et a1 1970; Purchase et a1 1971; Nazerian et a1 197 1) but enveloped virions or virus-related crystalline arrays were not detected. Failure of envelopment may be due to different virus strains used in these studies and may influence the degree of cell-association. In DEF cultures, about 2% of viable cells were infected when the CP E was not extensive, and a high proportion of the original cell-associated infectivity was recovered as cellfree virus after lyophilisation. It is difficult to make such comparisons between cell-associated and cell-free preparations since an infected cell will register only one plaque by in v i m assay although it may contain more than one infectious virus particle. Nevertheless, the results were similar to those reported by Calnek et al(l970). The relationship between the age of cultures and virus titres was not determined in the present study. It seems, as observed by Ash and Barnhart (1972), that harvesting of cells when cellular degeneration has not advanced may yield a high proportion of infected cells. Fluorescent reactions in HVT-infected cells treated with HVT and MDHV antiserum were similar to those described earlier (Witter er al 1970; Purchase et al 1971; Witter and Solomon 1971) and these reactions were able to distinguish between the antibodies to these viruses. It seemed likely that the granular cytoplasmic fluorescence was due to leakage of antigen through the nuclear membrane in cells with advanced cytopathology. Contrary to the findings of Witter et a1 (1970) but consistent with those in this study, failures to prepare HVT precipitating antigen (Melchior et a1 1973) or to demonstrate AGP reaction between HVT antibodies and MDHV antigen (Eidson and Anderson 1971) have been reported. However, neutralisation of antibody to NSW 1/70 by the isolate TAM-6 in this study indicated serological similarity between the HVT strains. Thus, these results showed serological relatedness between the different strains of HVT, and between them and MDHV. Apathogenicity of the isolate TAM-6 was
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Australian Veterinary Journal, Vol. 53, December, 1977
Electron Microscopic Observations Negatively stained preparations of infected cell lysates showed naked hexagonal nucleocapsids, 80 to 95 nm in diameter, with electron-dense nucleoids indicating penetration of the stain, and radially arranged hollow tubular capsomers. In thin sections, vacuolation of cytoplasm, margination of nuclear chromatin and intranuclear inclusions were seen more often in cells from CEF than DEF cultures. Non-enveloped hexagonal particles were seen either in nuclear vesicles, or in nucleoplasm and cytoplasm.
similar to that reported for other strains of HVT (Witter e f a1 1970; Okazaki e t a f 1970). Few turkeys were examined in this study. However, in view of the fact that HVT was isolated from 6 of 8 poults examined in this study, it is reasonable to suggest that .infection with this virus is widespread in Australian turkey flocks. It is possible that the 5 isolates recovered from Queensland turkeys represent the same strain which had spread by direct contact or aerosol infection (Witter and Solomon 1971). Persistence of infection in turkeys in the presence of high levels of neutralising antibody, reported in this and other studies (Zygraich and Huygelen 1971), is analogous to MDHV infection in chickens. HVT spreads readily between turkeys (Witter and Solomon 1972). Isolation of HVT (TAM-8) from an 8-week old NSW poult but absence of infection in 41/2-week old birds lends support to the observations of Witter and Solomon (1971) who detected this virus in poults when 5 or 6 weeks of age under conditions of natural exposure. Whether this is related to the disappearance of maternal antibody is not known. As vertical transmission of HVT does not seem to occur (Paul e f al 1972), it seemed likely that the infection in NSW poults was acquired soon after hatching, probably at the NSW hatchery, since no turkey had been housed previously in the isolation premises in this laboratory.
References Ahrned, M., Jensen, K. E.,Slattery, S. M.,Leech, J. B. and Schidlovsky, G. ( 1 9 7 0 b A v i a n Dis. 14: 349. Ash, R. J. and Barnhart, R. E . (1972)-Avian Dis. 16: 285. Bovarnick, M. R., Miller, J. C. and Snyder, J. C. (1950) -J. Bact. 59: 509. Calnek, B. W., Hitchner, S. B. and Adldinger, H. G. (197O)-Appl. Microbiol. 20: 723. Calnek, B. W. Garrido, C., Okazaki, W. and Patrascu, I. V. (1972+Avian Dis. 16: 52. Chubb, R. C: and Churchill, A. E. ( 1 9 6 8 t V e t . Rec. 83: 4: Eidson, C. S. and Anderson, D. P. (1971)-Avian Dis. 15: 68. Harman, D., Jackson, C. A. W., Sinkovic, B. Webster, A. C., Jones, N. F. and Gilchrist, P . T. (1973)Aust. vet. 1. 49: 520. Kawamura. H., King, D. J. and Anderson, D. P . (1969) -Avian Dis. 14: 853. Melchior, F. W., Fredrickson, T. N. and Luginbuhl, R. E. (1973)-Appl. Microbiol. 26: 925. Mustaffa-Babjee, A. (1970)-Aust. vet. J . 46: 587. Mustaffa-Babjee, A., Ketterer, P. and Spradbrow, P. B. (1971)-Aust,. vet. J . 47: 125. Mustaffa-Babjee, A. and Spradbrow, P. B. (1973)Aust. vet. J . 49. 347. Nazerian, K., Lee, L. F., Witter, R. L. and Burrnester, B. R. (1971)-Virology 43: 442. Okazaki. W.. Purchase. H. G. and Burmester. B. R. ( 1 9 7 0 i A v i a n Dis. 14: 413. Paul, Piern; Larsen, C. T., Kamur, M.C.and Pomeroy, B. S . ( 1 9 7 2 h A v i a n Dis. 16: 27. Purchase, H. G., Burrnester, B. R. and Cunningham, C. H. (1971)-Am. J . vet. Res. 32: 1811. Reed, L. J. Muench, H. ( 1 9 3 8 b A m . J. Hyg. 27: 497
Sink&, B. and Jones, N. F. ( 1 9 7 2 j P r o c Aust. Poult. Sci. Conv.. Auckland, p. 2 (Abstr.). Witter, R. L., Nazerian, K., Purchase, H. G. and Burgoyne, G. H. ( 1 9 7 O b A m . J. Yet. Res. 31: 525. Witter, R. L. and Solomon, J. I. (1971)-Znfect. Zmmun. 4: 356. AcknowledgmentS Witter, R. L. and Solomon, J. J. (1972)-Avion Dis. 16: 34. We gratefully acknowledge the receipt of serums from Dr 8. Sinkovic, University of Sydney. The study was supported by a Zygraich, N. and Huygelen, C. ( 1 9 7 2 b A v i a n Dis. 16c 735. grant from the Poultry Research Advisory Committee of (Received for publication 7 October 1976) Australia.
Australian Veterinary Journal, Vol. 53, December. 1971
585
PRASAD*
and P. B. SwbwBnow
Department of Veterinary Preventive Medicine, University of Queensland, Brisbane, Queensland, 4067 SUMMARY: Turkey h e r p e s v i r u s (HVT) was iso lat ed from t h e k i d n ey s of 6 of 8 t u r k ey p o u l t s from two flocks. T h e i s o l a t e s were identified by syncytiai-type of cy t o p at h o i o g y , inhibition of pla que formation by 5 - b r o m o d eo x y u r id in e, formation of Cowdry t y p e A intranuclear inclusions in cell cultures, a n d presence of h e r p e s - t y p e virions in negatively s t a i n e d p r e p a r a t i o n s a n d thin sections of in f ected cell c u l t u r e s . One of t h e s e isolates i n o cu l at ed into c h i c k e n s proved a p a t h o g e n i c over an o b ser v atio n p er io d of 10 w e e k s . Indirect immun~fiuorescenceand s e r u m neutralisation tests r ev eal ed ser o l o g i cal relations h i p b e t w e e n t h e s e isolates a n d t h e strain NSW 1 / 7 0 of HVT. Staining of HVT-infected cell culture by Marek's d i s e a s e h e r p e s v i r u s ( M D H V ) antiserum s h o w e d i n t r an u cl ear fluorescence but a t t e m p t s to p r e p a r e HVT precipitating an tig en or to d e m o n s t r a t e cross-precipitation betw e e n HVTand MDHV were unsuccessful.
Introduction
Following the initial description of turkey herpesvirus (HVT) in the U.S.A. (Kawarnura et al 1969; Witter et al 1970), the isolation of H V T has been reported from many countries, including Australia (Mustaffa-Babjee et al 1971; Sinkovic and Jones 1972). The quarantine regulations in this country stipulate local virus strains to be used for the preparation of vaccines, and the HVT strain NSW 1/70 is currently used as vaccine against Marek's disease (MD). However, there is a need for comparative studies o n the in vitro and in vivo characteristics of endemic H V T strains as information about the prevalence and properties of such strains is limited (Mustaffa-Babjee and Spradbrow 1973; Harman et al1973). This paper reports a study o n the isolation and characterisation of H V T from local turkey flocks. Materials and Methods Turkeys Primary isolation of HVT was attempted from 8 clinically healthy poults (White Holland or bronze-winged). Five poults, 6 to 8 months of age, were puchased from a private breeder in Queensland. The parent flock of these poults consisted of 42 birds that had not suffered from any serious clinical disease including neoplastic conditions. A further 3 poults were obtained from a commercial turkey flock in New South Wales when one day old. They were reared in isolation in the laboratory. Two were killed when 4Yz weeks of age and the third when 8 weeks old.
Cell Cultures Primary cell cultures of turkey kidney and of embryo fibroblasts from duck (DEF) and chick CEF) were prepared as described by Mustaffa-Babjee (1970) but using tricine-buffered medium 199 (T199) instead of Eagle's basal medium for cell culture.
Cloning of HVT Isolates Two of the HVT isolates (TAM-5, TAM-6) were purified through 3 cycles of cloning and propagation in DEF cultures. The use of cell cultures of chick origin was avoided to eliminate the risk of inadvertent contamination with vertically iransmittcd chicken viruses. Serial 10-fold dilutions of the primary isolates (infected turkey kidney cells) were inoculated into DEF in 55 mm plastic petri dishes which were overlayed with 0.So/u agarose. When the foci were clearly evident, usually on the 5th or 6th day of incubation. the agarose over an iboiatcd focus was removed with a capillary pipette and the well was filled w i t h irypsin-versene. The dispersed cells thus obtained were then inoculated into fresh 24-hour-old primary UEF in Roux flasks which were incubated at 38°C until the cytopathic effect (CPE) developed. This process was repeated 3 times. Later, the isolates were propagated to the 10th passage in DEF cultures. The preparations thus obtained did not show any evidence of contamination with viruses of chicken or duck origin by cultural and electron microscopic methods. Virus Assay l 'h e methods described by Calnek rt irl(1972) were followed. I,'ocus-forrning units (FFU) under agarose overlay and in some instances plaque-forming units (PFU) in liquid overlay in C EF cultures were counted at 5 or 6 days after inoculation. In a preliminary study, a comparison was made between the focus counts in cultures overlayed with agarose immediately and 18 hours after adsorption of cell-free VHT. The foci developing in the latter appeared slightly elongated but the focus counts in two cultures were not statistically different. Therefore, the latter procedure was not used in this study.
Lyophilisation an d Storage Cell-associated HVT harvested on the 4th day of incubation was suspended in a freezing mixture consisting of 10% dimethyl sulphoxide and 10% calf serum in niedium TI99 and stored at either -70" or in liquid nitrogen. To prepare cell-free HVT. cells from an equal volume of infected culture suspension was suspended in the stabiliser SPGA (Bovarnick et a/ 1950) and sonicated. The extract was filtered (0.45 pm), lyophilised (Calnek e t a / 1970) and stored at 4OC.
De~erminarionof Viral Nucleic Acid Type
'Prcscnl address: Coniinoiiwcalth Suruin Laboialorics. Pnrkville. V i C t o i m . 3052.
The isolates were grown in C EF cultures in plastic dishes containing 5-bromodeoxyuridine ( B U D R ) in concentrations
582
Australian Veterinary Journal, Vol.
53, December, 1977
from 10 to 100 ,ug/ml in medium and the plaques were enumerated on the 5th day of incubation. The control consisted of infected cultures grown similarly in the absence of BUDR. Serology An indirect immunofluorescence test was employed using infected DEF cultures on coverslips or smears of buffy coat cells from viraemic chickens as the source of antigen. HVT antiserum used in the test was obtained from four 4-week-old chickens which had been vaccinated subcutaneously with 1,OOO FFU of cell-associated virus TAM-6) when one day of age. It had a neutralising antibody titre of 2 320 and was free of antibody to Marek's disease herpesvirus (MDHV) as determined by the agar-gel precipitin (AGP) test (Chubb and Churchill 1968). MDHV antiserum, as judged by the AGP test, was obtained from chickens in a flock with a high incidence of clinical MD. The staining procedures were the same as described by Ahmed et d(1970). The AGP antigens were prepared with the HVT isolates (TAM-5 and TAM-6) and MDHV (Mustaffa-Babjee 1970) by the method of Chubb and Churchill (1968). The serum neutralisation test was performed as described by Zygraich and Huygelen (1972), using 150 to 200 50% tissue culture infective doses of cell-free HVT (TAM-6). A 50% neutralisation endpoint was determined by the method of Reed and Muench (1938). Electron Microscopy Cell lysates from infected cultures showing characteristic herpesvirus CPE were negatively stained with 1.5% phosphotungstic acid (pH 6.6). For thin sectioning, the infected cells were removed with a rubber policeman, fixed in 5% buffered glutaraldehyde and embedded in Epon araldite. Thin sections were stained with uranyl acetate and lead citrate. Pathogenicity for Chickens Twenty four one-day-old chickens, free of maternal antibodies to HVT and MDHV, were inoculated subcutaneously with 1,280 FFU of cell-associated HVT (TAM-6). They were killed and autopsied in groups of 4 birds between 8 and 10 weeks after inoculation and cell cultures from the pooled samples of kidneys from each group were prepared for virus isolation. Histological samples were prepared from sciatic and brachial nerves, heart, spleen, liver, lung, bursa and gonads using conventional methods.
turkey kidney cultures but took a longer time to develop during the initial 2 to 3 passages. Later, all isolates produced plaques in 3 to 4 days in DEF. The affected cells varied in size and were spherical, flat, polygonal or fusiform in shape. Syncytia of varying sizes and angular or stellate outline developed and showed structureless homogeneous centres. A few cells remaining in the central clear areas or at the margin of plaques joined cells at the opposite edge by trailing strands of cytoplasm. Infected monolayers removed with collodion and stained with haematoxylin and eosin had multinucleated giant cells and Cowdry type A intranuclear inclusion bodies. In CEF cultures, the plaques were smaller in size and intranuclear inclusions were numerous but formation of a central clear area was not a constant feature. Plaque counts were reduced by 85% in the presence of 25 pg/ml of BUDR and plaques did not develop when the concentration of BUDR was increased to 50 pg/ml. The size of foci produced by isolates TAM-5 and TAM-6 was determined 5 days after inoculation by measuring 100 foci on each of 5 replicate CEF cultures. Their dimensions averaged 1.01 9 0.21 and 0.98 T 0.24 mm respectively and the difference between them was not statistically significant. A comparison of the titres of cell-associated and cell-free HVT recovered from an equal aliquot of culture suspension is shown in table 1. From 1.56 to 2.4% of viable cells were found to be infected with a mean approximating 2%. The infectiviy ratio of cell-associated to cell-free virus ranged between 1 : 1.67 and 1 : 2.40 with an average of 1 : 1.94.
Serological Findings Results
HVT-infected cells treated with HVT antiserum had granular nuclear and diffuse cytoplasmic HVT was isolated from the kidneys of all five fluorescence. This pattern of fluorescence was Queensland turkeys (TAM-3 to TAM-7) and one noticed in cell cultures infected with all the NSW turkey, aged 8 weeks (TAM-8). HVT was isolates, and in the buffy coat cells from chickens not isolated from the other two NSW poults which were killed at 4% weeks of age. TABLE 1 Isolation o f H V T
Cellular and Cell-free Infectivity of Turkey Herpesvirus (TAM-6) in DEF Cell Cultures
Responses in Cell Cultures In primary turkey kidney cell cultures, plaques consisting of small round refractile cells were first noticed between 6 and 8 days after seeding. Two to 3 days later, the plaques grew in size, and the infected cells enlarged 3 to 5 times the normal size and became vacuolated. Later a clear central area devoid of cells remained in the centre of the. plaques. Between 8 and 10 days, several new plaques formed. Plaques were larger in DEF cultures than in Australian Veterinary J o u r n d , Vol. 53, December, 1977
FFU/ml Trial
Viable Cell Count
CA* Infectivity
1 2 3
23x10' 2.OX1Oe 1.6X10"
6.0X10' 33x10' 25x10'
CA
CF+ % Cells Ratio Infec- Infected CA-CF tivity Infectivity 2.5X 10' 2.OXlO' 1.5XlW
2.40 1.75 1.56
1:2.40 1:1.75 1:1.67
= Cell-associated.
+ CF = Cell-free
infectivity determined following lyophilisation
and reconstitution.
583
with HVT viraemia (Figure 1). Not infrequently, granular cytoplasmic and nuclear staining was also seen in cells from cultures with extensive CPE. When the cells were treated with MDHV antiserum, thick granular staining was confined to the nucleus only (Figure 2). Attempts to prepare AGP antigens with isolates TAM-5 and TAM-6 or to demonstrate cross-precipitation between the HVT and MDHV reactants were unsuccessful. The neutralising antibody titres of serums of turkeys from which the isolates were obtained ranged between 80 and 160. Furthermore, 16 serum samples collected from 4 chickens between 8 and 81 days after vaccination with 1,000 PFU of cell-free HVT (NSW 1/70) at one day of age (kindly supplied by Dr. B. Sinkovic, University of Sydney) showed mean neutralising antibody titres between 1 1 1 and 236 when titrated against TAM-6.
Pathogenicity The chickens inoculated with the TAM-6 isolate did not develop any clinical signs of disease, gross or histological lesions attributable to HVT or MDHV when examined between 8 and 10 weeks after inoculation. HVT was isolated in cell cultures from each of the pooled samples of kidneys from inoculated birds killed in groups during this period. Discussion
Figures 1 and 2. Fluorescence micropraphs of cells infected with HVT and stained by an indirect procedure: ( 1 ) A buffy coat cell (lymphocyte) from a viraemic chick 2 days after inoculation with TAM-6 and threated with HVT (TAM-6) antiserum showing diffuse cytoplasmic and grmular nuclear staining (X1.200); (2) A TAM-5 infected DEF cell treated with MDHV antiserum shows thick, clumpy granular fluorescence confined to nucleus (X600).
The characteristics of isolates reported here showed a close similarity to those of HVT (Witter et a1 1970; Purchase et a1 1971; Nazerian et a1 197 1) but enveloped virions or virus-related crystalline arrays were not detected. Failure of envelopment may be due to different virus strains used in these studies and may influence the degree of cell-association. In DEF cultures, about 2% of viable cells were infected when the CP E was not extensive, and a high proportion of the original cell-associated infectivity was recovered as cellfree virus after lyophilisation. It is difficult to make such comparisons between cell-associated and cell-free preparations since an infected cell will register only one plaque by in v i m assay although it may contain more than one infectious virus particle. Nevertheless, the results were similar to those reported by Calnek et al(l970). The relationship between the age of cultures and virus titres was not determined in the present study. It seems, as observed by Ash and Barnhart (1972), that harvesting of cells when cellular degeneration has not advanced may yield a high proportion of infected cells. Fluorescent reactions in HVT-infected cells treated with HVT and MDHV antiserum were similar to those described earlier (Witter er al 1970; Purchase et al 1971; Witter and Solomon 1971) and these reactions were able to distinguish between the antibodies to these viruses. It seemed likely that the granular cytoplasmic fluorescence was due to leakage of antigen through the nuclear membrane in cells with advanced cytopathology. Contrary to the findings of Witter et a1 (1970) but consistent with those in this study, failures to prepare HVT precipitating antigen (Melchior et a1 1973) or to demonstrate AGP reaction between HVT antibodies and MDHV antigen (Eidson and Anderson 1971) have been reported. However, neutralisation of antibody to NSW 1/70 by the isolate TAM-6 in this study indicated serological similarity between the HVT strains. Thus, these results showed serological relatedness between the different strains of HVT, and between them and MDHV. Apathogenicity of the isolate TAM-6 was
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Electron Microscopic Observations Negatively stained preparations of infected cell lysates showed naked hexagonal nucleocapsids, 80 to 95 nm in diameter, with electron-dense nucleoids indicating penetration of the stain, and radially arranged hollow tubular capsomers. In thin sections, vacuolation of cytoplasm, margination of nuclear chromatin and intranuclear inclusions were seen more often in cells from CEF than DEF cultures. Non-enveloped hexagonal particles were seen either in nuclear vesicles, or in nucleoplasm and cytoplasm.
similar to that reported for other strains of HVT (Witter e f a1 1970; Okazaki e t a f 1970). Few turkeys were examined in this study. However, in view of the fact that HVT was isolated from 6 of 8 poults examined in this study, it is reasonable to suggest that .infection with this virus is widespread in Australian turkey flocks. It is possible that the 5 isolates recovered from Queensland turkeys represent the same strain which had spread by direct contact or aerosol infection (Witter and Solomon 1971). Persistence of infection in turkeys in the presence of high levels of neutralising antibody, reported in this and other studies (Zygraich and Huygelen 1971), is analogous to MDHV infection in chickens. HVT spreads readily between turkeys (Witter and Solomon 1972). Isolation of HVT (TAM-8) from an 8-week old NSW poult but absence of infection in 41/2-week old birds lends support to the observations of Witter and Solomon (1971) who detected this virus in poults when 5 or 6 weeks of age under conditions of natural exposure. Whether this is related to the disappearance of maternal antibody is not known. As vertical transmission of HVT does not seem to occur (Paul e f al 1972), it seemed likely that the infection in NSW poults was acquired soon after hatching, probably at the NSW hatchery, since no turkey had been housed previously in the isolation premises in this laboratory.
References Ahrned, M., Jensen, K. E.,Slattery, S. M.,Leech, J. B. and Schidlovsky, G. ( 1 9 7 0 b A v i a n Dis. 14: 349. Ash, R. J. and Barnhart, R. E . (1972)-Avian Dis. 16: 285. Bovarnick, M. R., Miller, J. C. and Snyder, J. C. (1950) -J. Bact. 59: 509. Calnek, B. W., Hitchner, S. B. and Adldinger, H. G. (197O)-Appl. Microbiol. 20: 723. Calnek, B. W. Garrido, C., Okazaki, W. and Patrascu, I. V. (1972+Avian Dis. 16: 52. Chubb, R. C: and Churchill, A. E. ( 1 9 6 8 t V e t . Rec. 83: 4: Eidson, C. S. and Anderson, D. P. (1971)-Avian Dis. 15: 68. Harman, D., Jackson, C. A. W., Sinkovic, B. Webster, A. C., Jones, N. F. and Gilchrist, P . T. (1973)Aust. vet. 1. 49: 520. Kawamura. H., King, D. J. and Anderson, D. P . (1969) -Avian Dis. 14: 853. Melchior, F. W., Fredrickson, T. N. and Luginbuhl, R. E. (1973)-Appl. Microbiol. 26: 925. Mustaffa-Babjee, A. (1970)-Aust. vet. J . 46: 587. Mustaffa-Babjee, A., Ketterer, P. and Spradbrow, P. B. (1971)-Aust,. vet. J . 47: 125. Mustaffa-Babjee, A. and Spradbrow, P. B. (1973)Aust. vet. J . 49. 347. Nazerian, K., Lee, L. F., Witter, R. L. and Burrnester, B. R. (1971)-Virology 43: 442. Okazaki. W.. Purchase. H. G. and Burmester. B. R. ( 1 9 7 0 i A v i a n Dis. 14: 413. Paul, Piern; Larsen, C. T., Kamur, M.C.and Pomeroy, B. S . ( 1 9 7 2 h A v i a n Dis. 16: 27. Purchase, H. G., Burrnester, B. R. and Cunningham, C. H. (1971)-Am. J . vet. Res. 32: 1811. Reed, L. J. Muench, H. ( 1 9 3 8 b A m . J. Hyg. 27: 497
Sink&, B. and Jones, N. F. ( 1 9 7 2 j P r o c Aust. Poult. Sci. Conv.. Auckland, p. 2 (Abstr.). Witter, R. L., Nazerian, K., Purchase, H. G. and Burgoyne, G. H. ( 1 9 7 O b A m . J. Yet. Res. 31: 525. Witter, R. L. and Solomon, J. I. (1971)-Znfect. Zmmun. 4: 356. AcknowledgmentS Witter, R. L. and Solomon, J. J. (1972)-Avion Dis. 16: 34. We gratefully acknowledge the receipt of serums from Dr 8. Sinkovic, University of Sydney. The study was supported by a Zygraich, N. and Huygelen, C. ( 1 9 7 2 b A v i a n Dis. 16c 735. grant from the Poultry Research Advisory Committee of (Received for publication 7 October 1976) Australia.
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