Rewu/"c" ill Veterinary Science /987. 41. /45-/49
Efficacy of a bivalent vaccine against Marek's disease A. K. PRUT~I, R. K. P. GUPTA, J. R. SADANA, Department of Veterinary Pathology . ' Haryana Agricultural University. Hissar-125004. India
A bivalent vaccine was prepared by combining inactivated Marek's disease virus and turkey herpesvirus. The efficacy of this vaccine, compared to turkey herpesvirus and inactivated Marek's disease virus separately, was studied in unsexed White Leghorn chicks which were vaccinated at one day old and then challenged at 21 days old with fowl blood infected with virulent Marek's disease virus. The ~ivalent vaccine appreciably delayed mortality result109 from Marek's disease and elicited the highest protective efficacy as judged on the basis of Marek's disease-specific mortality and percentage occurrence of lesions. The occurrence, extent and severity of gross lymphomas and microscopic Iymphoproliferative lesions in various organs of the bivalent vaccinated birds were less than in the other challenged groups. In addition, the level of viraemia remained consistently and significantl)' lower in the bivalent vaccinated birds.
THE turkey herpesvirus vaccine has been the vaccine m?st used to control Marek's disease (MD) in chickens. However, recently there have been increasing reports of this vaccine failing to protect even in correctly vaccinated flocks (Witter 1983). These vaccine failures have resulted in both economic losses caused by carcase condemnation in broilers with MD lymphomas and poor quality meat (Eidson 1982) and a high MD specific mortality (Ekperigin et al 1983). Prompted by this enigmatic situation in the control of MD, the present studies were undertaken to assess the e~ficacy of a bivalent vaccine against a virulent MD virus challenge. Materials and methods MD
The MD virus used was obtained from an outbreak of MD in chickens. The propagation of MD virus was carried out in living chicks as in the procedure of Gupta et al (1982). Blood from these chickens was used for the preparation of cell-free MD virus and for challenge.
Cell-free, freeze dried turkey herpesvirus vaccine obtained from a commercial source was used for preparing the bivalent vaccine. Inactivated MD virus
. For preparing inactivated MD virus, cell-free MD virus was extracted from feather follicles of known MD virus infected chickens employing sucrose phosphate ethylene diamine tetra-acetic acid buffer as in the method of Sharma (1980) and inactivated with 0'125 per cent glutaraldehyde (Powell 1975). The glutaraldehyde was allowed to react for 15 minutes and was inactivated by adding glycine to a final concentration of I per cent (Gormon and Scott 1976). The unreacted glutaraldehyde was removed by dialysing it against sucrose phosphate (sp) buffer for 24 hours at 4°C and changing the 51' buffer every two hours. The dialysing fluid from every change was tested for the presence of glutaraldehyde or glutaraldehyde glycine complex at 235 nm and 265 nrn, respectively, in an ultraviolet spectrophotometer (Gormon and Scott 1976) while the qualitative presence of glycine in the dialysing fluid was tested by the ninhydrin test (Oser 1965). The dialysate was filtered through a 0·45 lAm Millipore membrane. The filtrate was concentrated to II III of the original volume against polyethylene glycol 20,000 (Sigma). This concentrated preparation constituted the inactivated ~ID virus. Evaluation of inactivation
The inactivation of MD virus was evaluated in chicken embryos (Sharma 1980) and in day-old chicks (Murthy and Calnek 1979). None of the inoculated embryos revealed the development of MD pock lesions and none of the inoculated chicks exhibited either clinical signs or gross or microscopic lesions in any organ. There was no viraemia and no shedding of virus from feather follicles. These tests ensured that the inactivated MD virus was completely non-viable.
A. K. Pruthi, R. K. P. Gupta, J. R. Sadana
TABLE 1: Evaluation of herpesvirus of turkeys vaccine alone and in combination with inactivated MD virus for pock forming units in developing chicken embryos
Embryos inoculated 20
HVT HVTdiluent" HVT + IMDV SPbuffer
0-1 ml 0-1ml 0-2ml 0-2ml
Route Yolk Yolk Yolk Yolk
Embryos positive for pock lesions
Mean number of pock lesions ± SEM
14 Nil 12 Nil
925±114t Nil 945 ± 100t Nil
sac sac sac sac
Supplied by the manufacturer t Mean pock lesions do not differ significantly (P>0'051 SEM Standard error of the mean HVT Turkey herpesvirus IMDV Inactivated Marek's disease virus SP Sucrose phosphate
Bivalent vaccine The bivalent vaccine used in the present studies was constituted by combining equal volumes of turkey herpesvirus and inactivated MD virus. It was assayed for pock forming units in developing chicken embryos to determine the deleterious effect, if any, of inactivated MD virus on turkey herpesvirus. No significant difference between pock forming units of turkey herpesvirus and the bivalent vaccine (Table I) implied that there was no interaction between turkey herpesvirus and inactivated MD virus. The dose of live virus in the vaccines is shown in Table 2.
Protection test Three hundred and sixty day-old, unvaccinated White Leghorn chicks were divided randomly into six equal groups kept separately in different rooms. The details of treatment given to the different groups are indicated in Table 2. At 21 days old, the birds in groups 2 to 5 were challenged by inoculating each animal intraperitoneally with O·5 ml of infective whole blood (2500 pock forming units) from birds suffering from MD at 36 days old. The birds in all the groups were observed for 105 days for the development of clinical signs and mortality. All the surviving chickens were killed at the termination of the experiment (105th day). All the chickens which died during the experiment or were killed at its termination, were subjected to detailed post mortem examination. Tissues from apparently normal organs and those with gross lesions were collected in buffered 10 per cent formol saline for histopathological studies.
Viraemia in chickens The development of viraemia in chickens was tested at 14, 35, 56, 77 and 98 days of age. Pooled blood samples, collected in antibiotic-EDTA solution, from four randomly selected chickens of each group, were
inoculated into a set of 20, five-day-old chicken embryos. Each embryo received 0'2 ml of blood via the yolk sac route and later mean pock counts were calculated by the method of Biggs and Milne (1971).
Protective efficacy The protective efficacy of all three vaccines was calculated using the formulae of Witter (1982). For comparing the vaccines' protective efficacies, the interaction X2 test was applied and to test the relative statistical significance, data on viraemia were analysed by the analysis of variance test (Alder and Roessler 1964). Results
Clinical signs No clinical signs of the disease were observed among the birds in the turkey herpesvirus-vaccinatedunchallenged (group I), bivalent vaccinatedchallenged (group 3) and sp buffer-inoculated-unchallenged (group 6) birds. The earliest signs of MD were noticed at 31 days after challenge in group 5 birds, whereas in the inactivated MD virus-inoculatedchallenged (group 4) birds these appeared 42 days after challenge. In the turkey herpesvirus-vaccinatedchallenged group (group 2), a few birds were found to be weak, lame and emaciated 56 days after challenge. MD
MD specific mortality of 1'92 per cent was first observed in group 5 birds at seven weeks after challenge. Thereafter, the percentage of weekly mortality increased to 5' 88,8' 33,9'09, 12· 50, 11·40 at the eighth, ninth, 10th, II th and 12th week, respectively. In group 4 birds, the percentage mortality was I' 96 at eight weeks after challenge increasing to 2·0 per cent at nine weeks, 6·12 per cent at 10 weeks but decreasing slightly at the 12th week (4' 34 per cent). In
60 60 60 4 5 6
Pock forming units Pock forming units before inactivation t Challenge comprised injection of 0·5 ml infecred 12500pock forming units) whole blood C Challenged NC Not challenged HVT Herpesvirus of turkeys IMDV Inactivated Marek's disease virus Figures having superscripts in common do not differ significantly at 5 per cent level of probability as tested by analysis of variance test
7 (13'72) 21 (40·38) Nil 51
C C NC
50 51 49 NC C C
82·18a b 96'2 Nil 5 (9·8) 1 (2·04) 85·43a 94'94 b
1232 0·1 ml- 1 12320·1 ml- 1 924 + 308' 0·1 ml- 1 1232' 0·2 ml- 1 NiI0'2ml- 1 NiI0'2ml- 1
60 60 60
group 2, 3·92 per cent birds. died nine weeks after challenge and the mortality decreased to 2· 94 per cent at 10 weeks. But in group 3 birds, only 2· 04 per cent mortality was noticed II weeks after challenge. However, deaths from MD were not seen in group I or group 6 birds.
HVT vaccine HVT vaccine HVT+IMDV vaccine IMDV vaccine SP buffer SP buffer
1 2 3.
Treatment (intramuscular) at day 1
Number of chickens treated
Challenge MD virust (day 21)
Number of chickens at risk
Nil 3 15·88) 1 12·04)
Percentage protective efficacy on gross lesions basis MD specific mortality through 105 days (%)
Number of birds with gross MD specific lesions 1%) Percentage vaccinal reduction in mortality (protective efficacy) TABLE 2: Protective efficacy of different vaccines against MD virus challenge on the basis of mortality and gross lesions
Marek's disease vaccine
No gross lesions were seen in birds of groups I and 6. In group 5 birds, the earliest gross lesions were seen in birds which died seven weeks after challenge and these were consistently observed up to 12 weeks after challenge. The affected visceral organs often revealed a diffuse enlargement with or without the development of nodular tumorous growths. In group 5 birds, gross lesions in thymus or bursa were characterised by atrophy of these organs whereas no such changes were observed in birds of other groups. Affected sciatic and brachial nerves and their plexuses appeared enlarged and oedematous with loss of striations. The overall frequency of gross lesions in birds of group 3 was less than in the other challenged groups (Table 2).
Histological lesions Microscopic lesions were observed in organs of birds from the groups in which gross lesions occurred. In general, the microscopic lesions were characterised by varying degrees of infiltration and proliferation of small to large lymphocytes, Iymphoblasts and reticular cells which were either focal or diffuse in distribution. In some cases, plasma cells were also occasionally observed. The normal architecture of the affected organs was obliterated in areas with extensive cellular proliferation. In addition, varying degrees of neuritic oedema, demyelination, swelling and fragmentation ofaxons and proliferation of Schwann cells was noticed in peripheral nerves. The extent and severity of microscopic lesions was least in the bivalent-vaccinated-challenged birds.
Viraemia The mean value of pock forming units per O' 2 ml of blood from birds in different groups at various intervals are given in Table 3. The level of viraemia in group 3 remained consistently low throughout, rangingbetween282'90and 10'31 pock"forming units per 0·2 ml at days 35 and 98, respectively. The highest number of pock forming units (1146 0·2 ml- I ) were detected at 35 days of age in group 5 birds and declined to 730' 37 at the 98th day of age.
Protective efficacy The
percentages of protective efficacy after
A. K. Pruthi, R. K. P. Gupta, J. R. Sadana
TABLE 3: Viraemia Imean pock forming units· 0·2 ml- 1 In chickens of different experimentally treated and MD challenged groups at various intervals Group number
Treatment (intramuscular) PFU Day 1
Challenge MD virus Viraemia: PFU' 0·2 ml- t (mean ± SEMI at different dayst of age Day 21 PFU 98 14 35 56
± 84·87 748·00 ±126·32 Nil Nil Nil
227·00 ± 38·25 538·70 ± 63·75 282·90 ± 79·97 391·20 ± 37·20 1146·00 ±198·67 Nil
15·00 ± 6·52 331·30 ± 37·54
± 16·74 492·28 ± 62·94 611·10 ±142·86 Nil
39'55 ± 12·76 15·90 ± 7·10 388·66 ± 94·64
44·46 ± 11·33 10·31 ± 5·30 225·20 ± 57'57 730·37 ±125'50 Nil
Average of 20 embryos inoculated with pooled blood from four randomly selected chickens t Data from 35 to 98 days were analysed after loglt transformations Figures having superscripts in common do not differ significantly at 5 per cent level of probability as tested by analysis of variance test PFU Pock forming units Pock forming units before inactivation C Challenged NC Not challenged SEM Standard error of the mean HVT Herpesvirus of turkeys IMDV Inactivated Marek's disease virus
different vaccinal preparations based on mortality and gross lesions are given in Table 2 and it is evident that the protective efficacy of bivalent vaccine was higher than either turkey herpesvirus or inactivated MD virus. Discussion The occurrence of excessive mortality or condemnation losses from MD in turkey herpesvirus vaccinated flocks caused by variant MD strains (Witter 1982, 1983) has necessitated developing more effective MD vaccines and bivalent or polyvalent vaccines combining turkey herpesvirus and non-pathogenic strains of MD virus have been used (Calnek et al1983, Witter et al 1984). In the present studies, a bivalent vaccine was prepared by mixing turkey herpesvirus with inactivated MD virus in order to avoid the use of viable non-oncogenic MD virus which has been reported to spread horizontally (Calnek 1982). It was observed in our study that no clinical signs of MD appeared in bivalent-vaccinated and challenged birds (group 3) compared to turkey herpesvirus-vaccinated and challenged birds (group 2). The protective efficacy of the bivalent vaccine on the basis of MD specific mortality and gross lesions was found to be significantly greater than that of turkey herpesvirus. A higher protection among chickens with bivalent or polyvalent vaccines has been observed previously (Witter 1982, Calnek et al 1983, Witter et al 1984). Although the bivalent vaccine in the present study protected chickens better than turkey herpesvirus, it was observed that inactivated MD virus alone was
significantly less efficacious than turkey herpesvirus. These observations contrast with the findings of Lesnik et al (1980) who observed a low reduction in mortality (45' 54 per cent) in turkey herpesvirusinoculated chickens compared to skin antigeninoculated chickens (69' 5 per cent). This difference may be because Lesnik et al (1980) used only 100 pock forming units of turkey herpesvirus for immunising chicks with maternal antibodies whereas 1232 pock forming units of turkey herpesvirus were employed in the present studies; Calnek (1980) recommended that 1000 or more pock forming units per dose should be employed to avoid interference by maternal antibodies. The mortality among turkey herpesvirus-vaccinated and challenged birds started at nine weeks after challenge whereas in bivalent-vaccinated and challenged birds it did not occur until II weeks after challenge, suggesting that the bivalent vaccine had greater efficacy. A comparison of MD virus viraemia following challenge in turkey herpesvirus- and bivalentvaccinated birds showed that the MD virus viraemia declined faster and was significantly lower in bivalentvaccinated birds. These findings support those of Schat et al (1982) of lowered MD virus isolation from spleen cells of bivalent-vaccinated chickens in comparison with turkey herpesvirus-vaccinated and challenged birds. The low level of MD virus viraemia further substantiates the additional protective efficacy of the bivalent vaccine compared to turkey herpesvirus alone. The viraemia in inactivated MD virus-inoculated and challenged birds was also con-
Marek's disease vaccine
sistently and significantly lower than the SP bufferinoculated and challenged birds as has been observed by Lesnik and Ross (1975). A comparison of the protective efficacy of the three vaccines indicates that the major protection against MD in the bivalent vaccine was probably afforded by herpesvirus of turkeys, and that the inactivated MD virus component only enhanced protection since inactivated MD virus alone was found to be poorly protective. It is suggested that the additional protection of the bivalent vaccine compared to herpesvirus of turkeys vaccine may be caused by amelioration of the immunodepressive phase which virulent MD virus induces since viraemia following challenge was lower in bivalent-vaccinated birds. Another reason for the increased protection afforded by the bivalent vaccine in the present study may be that the turkey herpesvirus and inactivated MD virus combination induced different serotype specific immune responses, particularly when the inactivated MD virus was prepared after extraction of MD virus from feather follicle epithelium of known MD virusinfected chickens. It has been well established that feather follicle epithelium of MD virus-infected birds is a rich natural source of MD virus-associated antigens and the only site where the majority of viral particles are complete and enveloped (Calnek et al 1970). It is also well known that virus envelope proteins are mainly immunogenic (Mohanty and Dutta 1981). Acknowledgements
We are grateful to the Indian Council of Agricultural Research, New Delhi, for the financial assistance provided to the first author in the form of a senior research fellowship. References ALDER. H. L. & ROESSLER. E. B. (1964) Introduction 10
Probability and Statistics. 3rd edn, San Francisco and London. W. H. Freeman and Company. pp 194-196,247-254 BIGGS, P. M. & MILNE, B. S. (\971) American Journal of Veterinarv Research 32. 1795-1800 CALNEK, ·B. W. (\980) Marek's Disease Virus and Lymphoma. Oncogenic Herpesviruses, Vol I. Ed F. Rapp. Florida, CRC Press. pp 103-143 CALNEK, B. W. (1982) Marek's disease vaccines. 171h International Congress of Herpesvirus of Man and Animals: Standardisation of Immunological Procedures. Lyon, France. 1981. Developments in Biological Standardisation. Basel, S. Karger 52. pp 401-405 CAINEI'. B. W .. ADIDINGER, H. K. & KAHN, D. E. (1970) A "ian Diseases 14. 219-233 CALNEK, B. W., SCHAT, K. A., PECKHAM. M. C. & FABRICANT. J. (1983) Avian Diseases 27. 844-849 EIDSON. C. S. (\982) Marek's Disease Tumours in Chickens Vaccinated with Turkey Herpesvirus Vaccine. 171h 100ernationat Congress on Herpesvirus of Man and Animals: Standardisarion of Immunological Procedures. Lyon. France. 1981. Developments in Biological Standardisation, S. Karger. Basel 52. pp 437 -446 EKPERIGIN. H. E., FADLY. A.M .• LEE. L. F .• L1U, X. & McCAPES, R. H. (\983) A "ian Diseases 27.503-512 (iORMON. S. P. & SCOTT, E. M. (1976) Microbios Letters 1. 197-204 GUPTA. S. 1'.• KHAROLE. M. U. & KALRA, D. S. (1982) Avian Diseases 26. 7- 13 LESNIK. F. & ROSS, L. J. N. (\975) lnternatlonal Journal of Cancer 16.153-163 LESNIK, F.• CHUDY. D., VRTIAK. O. J.• KONRAD. V., DANIHEL. 1'.1 •• RAGAC, P. & POLACEK. M. (1980) Comparative Immunology Microbiology and Infectious Diseases 2. 491-500 MOHANTY. S. B. & DUTTA. S. K. (1981) Structure of Viruses. Veterinary Virology. Philadelphia. Lea and Febiger. pp 6-8 MURTHY. K. K. & CALNEK. B. W. (\979) lnfection and Immunity 26.547-553 OSER. B. L. (Ed) (1965) Hawk's Physiological Chemistry, 141h edn. Bombay. Tala McGraw Hill. p 181 POWELL. P. C. (1975) Nature, London 257.684-685 SCHAT, K. A.. CALNEK. B. W. & FABRICANT, J. (\982) A"ian Palhology II. 593-605 SHARMA, J. M. (1980) lsolarion and ldentificarion of Avian Pathogens, Eds S. B. Hitchner, C. A. Dornerrnuth, H. G. Purchase & J. E. Williams. New York. American Association of Avian Pathologists. pp 91-95 WITTER, R. L. (1982) A vian Pathology 11,49-62 WITTER. R. L. (\983) A vian Diseases 27. 113-132 WITTER, R. L.. SHARMA. J. M. & LEE. 1.. F. (1984) Al'lim Diseases 28. 44-60
Accepted January /3, /986