This article was downloaded by: [Oregon State University] On: 22 January 2015, At: 09:53 Publisher: Taylor & Francis Informa Ltd Registered in England and Wales Registered Number: 1072954 Registered office: Mortimer House, 37-41 Mortimer Street, London W1T 3JH, UK
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Isolation of marek's disease virus from affected chickens a
G.W. Horner B.V.Sc. M.Sc. & M.P. James B.V.Sc.
a
a
Ruakura Animal Health Laboratory , Ministry of Agriculture and Fisheries , Private Bag, Hamilton Published online: 23 Feb 2011.
To cite this article: G.W. Horner B.V.Sc. M.Sc. & M.P. James B.V.Sc. (1975) Isolation of marek's disease virus from affected chickens, New Zealand Veterinary Journal, 23:6, 102-104, DOI: 10.1080/00480169.1975.34208 To link to this article: http://dx.doi.org/10.1080/00480169.1975.34208
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
102
NEW ZEALAND VETERINARY JOURNAL
VOL.
23
ISOLATION OF MAREK'S DISEASE VIRUS FROM AFFECTED CHICKENS G. W. HORNER and M. P. JAMES*
Downloaded by [Oregon State University] at 09:53 22 January 2015
INTRODUCTION
tures. The. kidney cells from each chicken were grown in 10 tubes containing coverslips. The cultures were observed daily and the cO'verslips removed at regular intervals and stained with haematoxylin and eosin (H & E). Before discarding the tubes, supernatant fluids were inoculated intO' 7-day-old embryonated eggs using the allantoic route and 4 blind passages made. Procedures followe:d to check for the presence of infectious bronchitis virus, chicken embryo lethal orphan (CELO) virus, and Newcastle disease virus have been describe:d (McCausland ·et al., 1972).
MAREK'S DISEASE is a common disease of . poultry in New Zealand, with annual flock losses ranging from 10 to" 30% (Anon., 1971). In a report on diseases of the domestic fowl in northern New Zealand, McCausland (1972) found that 12.9% of birds under 20 weeks of age submitted to the Ruakura Animal Health Laboratory were suffering from Marek's disease. Reports from England (Churchill and Biggs, 1967), United States (Witter et ai., 1969), and Australia (Mustaffa-Babjee, 1970; Smith and CO'akley, 1970) indicate that a cell-associated herpes virus is the RESULTS aetiological agent. Focal cytopathic effects were first obThis report records the primary isolation of the virus from two field cases of served in one culture on the 6th day, and in another on the 8th day after the monothe disease in New Zealand. layers be:r;ame confluent. One isolate was from .a bird with nerve MATERIALS AND METHODS lesions only, and the other from a bird with visceral tumours. Autogenous chicken kidney cell culThe cytopathogenic effects consisted of tures were prepared from 5 birds show- focal areas or clusters of spherical reing clinical and pathological signs of fractile ceUs (Fig. 1). Staining showed Marek's disease. In 3 of the birds (29 the presence of many small multiweeks old) gross. lesions were confined nucleated cells containing between 2 and to the sciatic nerves, portions of which 5 nuclei. Occasionally syncytia containing were enlarged and oedematous with loss up to 25 nuclei were present (Fig. 2) but of the cross striations and yellow dis- these were uncommon. Type A eosinocoloration. The other 2 birds (7 weeks philic intranuclear inclusions of various old) had lymphomatous lesions in the kidneys, spleen and gonads, characteristic of the acute form of Marek's disease (Calnek and Witter, 1972). The techniques used to prepare the cell cultures were based on those described by Churchill (1965). The medium used was a mixture of equal parts of medium 199 and HSLS medium (Johnson, 1962) supplemented with antibiotics, and 10% foetal calf serum for growth O'r 2% for maintenance of cul*G. W. Horner, B.V.Sc., M.Sc .• and M. P. James, B.V.Sc., Ruakura Animal Health Laboratory, Minis· try of A3riculture and Fisheries. Private Bag. Hamil· ton.
FIG. 1: Chicken kidney monolayer showing a focal area of CPE. The cluster of affected ce([s is staining very darkly. H & E X 65
1975
NEW ZEALAND VETERINARY JOURNAL
103
stages of development were present in many of the multinucleated and adjacent cells (Fig. 3). Between 2 and 5 foci were present on each coverslip and these were still visible up to 10 days after first being observed. Supernatant fluids from these cultures were negative for virus isolation in embryonated eggs.
Downloaded by [Oregon State University] at 09:53 22 January 2015
DISCUSSION
FIG. 2: Chicken kidney monolayer showing a giant cell containing many nuclei. Most 0/ the nuclei contain intranuclear inclusions. H & E X 1000
The growth characteristics and cytopathogenic effe,cts of the agents described above are identical with those described by other workers for the Marek's disease virus (Churchill and Biggs, 1967; Witter et al., 1969; Mustaffa-Babjee, 1970; Smith and Coakley, 1970) and are typical of the group B cell-associated herpes viruses. The fact that the virus was not isolated from the cells from the other chickens may have been due to' the failure. of several of these cultures to form confluent monolayers. Multiple virus infections may cccur when anparently normal cells are cultured. However, in this study the use of both embryonated eggs and tissue cultures eliminated the, possibility that infectious bronchitis. chick embryo lethal orphan virus and Newcastle disease (McCausland et al., 1972) and reovirus (Mustaffa-Babjee and Spradbrow, 1973) were concurrently present in the cell cultures. Althou!!"h Marek's disease virus may cause pocks on the chorioallantoic membranes of chick embryos following yolk sac inocul~tion with cellular virus preparations (Calnek and Witter, 1972) this was not attempted in this study. SUMMARY
FIG. 3: Chicken kidney monolayer showing two nuclei which contain type A eosinophilic intranuclear inclusions. H & E X 1500
Primary isolation of Marek' s disease herpes virus was made from 2 out of 5 naturally infecterl chickens using autogenous .chicken kidney tissue cultures. The virus induced the formation of focal areas of cytopathoge.nic effect in the tissue cultures and staining showed the presence of multinucleated cells and intranuclear inclusions. 'fests for the presence, of infectious bronchitis virus, chick embryo lethal orphan virus, Newcastle disease virus and reovirus were negative.
104
NEW ZEALAND VETERINARY JOURNAL
ACKNOWLEDGEMENTS
We would like to thank Miss C. McLeod, B.Sc., for her technical assistance.
Downloaded by [Oregon State University] at 09:53 22 January 2015
REFERENCES Anon. (1971): Diseases of Domestic Animals in New Zealalld, 3rd ed. Editorial Services, Wellington. Calnek, B. W.; Witter, R. L. (1972): Marek's disease. In Diseases of Poultry (ed. M. S. Hofstad; B. W. Calnek; C. F. Helmboldt; W. M. Reid; H. W. Yoder), 6th ed. Iowa State Univ. Press, Ames. p.470. Churchill, A. E. (1965): The use of chicken kidney tissue culture in the study of the avian viruses of Newcastle disease, infectious laryngotracheitis and infectious bronchitis. Res. vet. Sci., 6: 162-9. Churchill, A. E.; Biggs, P. M. (1967): Agent of Marek's disease in tissue culture. Nature, Lond., 215: 528-30.
VOL.
23
Johnson, R. H. (1962): Rinderpest in tissue culture. 1. Methods of virus production. Br. vet. '., 118: 107-16. McCausland, I. P. (1972): Diseases of the domestic fowl in northern New Zealand. N.Z. vet. '., 20: 160-6. McCausland, I. P.; Carter, Margery E.; Hunter, R. (1972): Infectious bronchitis in New Zealand chickens. N.Z. vet. ,., 20: 120-4. Mustaffa-Babjee, A. (1970): Isolation of a cytopathogenic agent from acute Marek's disease. Aust. vet. '., 46: 345-6. Mustaffa-Babjee, A.; Spradbrow, P. B. (1973): Charaoterization of an avian reovirus isolated in Queensland. ,. compo Path., 83: 387-400. Smith, V. M.; Coakley, W. (1970): Isolation of infective agents from cases of Marek's in poultry. Aust. vet. !., 46: 184. Witter, R. L.; Solomon, J. J.; Burgoyne, G. H. (1969): Cell culture techniques for primary isolation of Marek's disease-associated herpesvirus. Avian Dis., 13: 101-18.
(Received for publication October 29, 1974)
New Zealand Veterinary Journal Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/tnzv20
Isolation of marek's disease virus from affected chickens a
G.W. Horner B.V.Sc. M.Sc. & M.P. James B.V.Sc.
a
a
Ruakura Animal Health Laboratory , Ministry of Agriculture and Fisheries , Private Bag, Hamilton Published online: 23 Feb 2011.
To cite this article: G.W. Horner B.V.Sc. M.Sc. & M.P. James B.V.Sc. (1975) Isolation of marek's disease virus from affected chickens, New Zealand Veterinary Journal, 23:6, 102-104, DOI: 10.1080/00480169.1975.34208 To link to this article: http://dx.doi.org/10.1080/00480169.1975.34208
PLEASE SCROLL DOWN FOR ARTICLE Taylor & Francis makes every effort to ensure the accuracy of all the information (the “Content”) contained in the publications on our platform. However, Taylor & Francis, our agents, and our licensors make no representations or warranties whatsoever as to the accuracy, completeness, or suitability for any purpose of the Content. Any opinions and views expressed in this publication are the opinions and views of the authors, and are not the views of or endorsed by Taylor & Francis. The accuracy of the Content should not be relied upon and should be independently verified with primary sources of information. Taylor and Francis shall not be liable for any losses, actions, claims, proceedings, demands, costs, expenses, damages, and other liabilities whatsoever or howsoever caused arising directly or indirectly in connection with, in relation to or arising out of the use of the Content. This article may be used for research, teaching, and private study purposes. Any substantial or systematic reproduction, redistribution, reselling, loan, sub-licensing, systematic supply, or distribution in any form to anyone is expressly forbidden. Terms & Conditions of access and use can be found at http://www.tandfonline.com/page/terms-and-conditions
102
NEW ZEALAND VETERINARY JOURNAL
VOL.
23
ISOLATION OF MAREK'S DISEASE VIRUS FROM AFFECTED CHICKENS G. W. HORNER and M. P. JAMES*
Downloaded by [Oregon State University] at 09:53 22 January 2015
INTRODUCTION
tures. The. kidney cells from each chicken were grown in 10 tubes containing coverslips. The cultures were observed daily and the cO'verslips removed at regular intervals and stained with haematoxylin and eosin (H & E). Before discarding the tubes, supernatant fluids were inoculated intO' 7-day-old embryonated eggs using the allantoic route and 4 blind passages made. Procedures followe:d to check for the presence of infectious bronchitis virus, chicken embryo lethal orphan (CELO) virus, and Newcastle disease virus have been describe:d (McCausland ·et al., 1972).
MAREK'S DISEASE is a common disease of . poultry in New Zealand, with annual flock losses ranging from 10 to" 30% (Anon., 1971). In a report on diseases of the domestic fowl in northern New Zealand, McCausland (1972) found that 12.9% of birds under 20 weeks of age submitted to the Ruakura Animal Health Laboratory were suffering from Marek's disease. Reports from England (Churchill and Biggs, 1967), United States (Witter et ai., 1969), and Australia (Mustaffa-Babjee, 1970; Smith and CO'akley, 1970) indicate that a cell-associated herpes virus is the RESULTS aetiological agent. Focal cytopathic effects were first obThis report records the primary isolation of the virus from two field cases of served in one culture on the 6th day, and in another on the 8th day after the monothe disease in New Zealand. layers be:r;ame confluent. One isolate was from .a bird with nerve MATERIALS AND METHODS lesions only, and the other from a bird with visceral tumours. Autogenous chicken kidney cell culThe cytopathogenic effects consisted of tures were prepared from 5 birds show- focal areas or clusters of spherical reing clinical and pathological signs of fractile ceUs (Fig. 1). Staining showed Marek's disease. In 3 of the birds (29 the presence of many small multiweeks old) gross. lesions were confined nucleated cells containing between 2 and to the sciatic nerves, portions of which 5 nuclei. Occasionally syncytia containing were enlarged and oedematous with loss up to 25 nuclei were present (Fig. 2) but of the cross striations and yellow dis- these were uncommon. Type A eosinocoloration. The other 2 birds (7 weeks philic intranuclear inclusions of various old) had lymphomatous lesions in the kidneys, spleen and gonads, characteristic of the acute form of Marek's disease (Calnek and Witter, 1972). The techniques used to prepare the cell cultures were based on those described by Churchill (1965). The medium used was a mixture of equal parts of medium 199 and HSLS medium (Johnson, 1962) supplemented with antibiotics, and 10% foetal calf serum for growth O'r 2% for maintenance of cul*G. W. Horner, B.V.Sc., M.Sc .• and M. P. James, B.V.Sc., Ruakura Animal Health Laboratory, Minis· try of A3riculture and Fisheries. Private Bag. Hamil· ton.
FIG. 1: Chicken kidney monolayer showing a focal area of CPE. The cluster of affected ce([s is staining very darkly. H & E X 65
1975
NEW ZEALAND VETERINARY JOURNAL
103
stages of development were present in many of the multinucleated and adjacent cells (Fig. 3). Between 2 and 5 foci were present on each coverslip and these were still visible up to 10 days after first being observed. Supernatant fluids from these cultures were negative for virus isolation in embryonated eggs.
Downloaded by [Oregon State University] at 09:53 22 January 2015
DISCUSSION
FIG. 2: Chicken kidney monolayer showing a giant cell containing many nuclei. Most 0/ the nuclei contain intranuclear inclusions. H & E X 1000
The growth characteristics and cytopathogenic effe,cts of the agents described above are identical with those described by other workers for the Marek's disease virus (Churchill and Biggs, 1967; Witter et al., 1969; Mustaffa-Babjee, 1970; Smith and Coakley, 1970) and are typical of the group B cell-associated herpes viruses. The fact that the virus was not isolated from the cells from the other chickens may have been due to' the failure. of several of these cultures to form confluent monolayers. Multiple virus infections may cccur when anparently normal cells are cultured. However, in this study the use of both embryonated eggs and tissue cultures eliminated the, possibility that infectious bronchitis. chick embryo lethal orphan virus and Newcastle disease (McCausland et al., 1972) and reovirus (Mustaffa-Babjee and Spradbrow, 1973) were concurrently present in the cell cultures. Althou!!"h Marek's disease virus may cause pocks on the chorioallantoic membranes of chick embryos following yolk sac inocul~tion with cellular virus preparations (Calnek and Witter, 1972) this was not attempted in this study. SUMMARY
FIG. 3: Chicken kidney monolayer showing two nuclei which contain type A eosinophilic intranuclear inclusions. H & E X 1500
Primary isolation of Marek' s disease herpes virus was made from 2 out of 5 naturally infecterl chickens using autogenous .chicken kidney tissue cultures. The virus induced the formation of focal areas of cytopathoge.nic effect in the tissue cultures and staining showed the presence of multinucleated cells and intranuclear inclusions. 'fests for the presence, of infectious bronchitis virus, chick embryo lethal orphan virus, Newcastle disease virus and reovirus were negative.
104
NEW ZEALAND VETERINARY JOURNAL
ACKNOWLEDGEMENTS
We would like to thank Miss C. McLeod, B.Sc., for her technical assistance.
Downloaded by [Oregon State University] at 09:53 22 January 2015
REFERENCES Anon. (1971): Diseases of Domestic Animals in New Zealalld, 3rd ed. Editorial Services, Wellington. Calnek, B. W.; Witter, R. L. (1972): Marek's disease. In Diseases of Poultry (ed. M. S. Hofstad; B. W. Calnek; C. F. Helmboldt; W. M. Reid; H. W. Yoder), 6th ed. Iowa State Univ. Press, Ames. p.470. Churchill, A. E. (1965): The use of chicken kidney tissue culture in the study of the avian viruses of Newcastle disease, infectious laryngotracheitis and infectious bronchitis. Res. vet. Sci., 6: 162-9. Churchill, A. E.; Biggs, P. M. (1967): Agent of Marek's disease in tissue culture. Nature, Lond., 215: 528-30.
VOL.
23
Johnson, R. H. (1962): Rinderpest in tissue culture. 1. Methods of virus production. Br. vet. '., 118: 107-16. McCausland, I. P. (1972): Diseases of the domestic fowl in northern New Zealand. N.Z. vet. '., 20: 160-6. McCausland, I. P.; Carter, Margery E.; Hunter, R. (1972): Infectious bronchitis in New Zealand chickens. N.Z. vet. ,., 20: 120-4. Mustaffa-Babjee, A. (1970): Isolation of a cytopathogenic agent from acute Marek's disease. Aust. vet. '., 46: 345-6. Mustaffa-Babjee, A.; Spradbrow, P. B. (1973): Charaoterization of an avian reovirus isolated in Queensland. ,. compo Path., 83: 387-400. Smith, V. M.; Coakley, W. (1970): Isolation of infective agents from cases of Marek's in poultry. Aust. vet. !., 46: 184. Witter, R. L.; Solomon, J. J.; Burgoyne, G. H. (1969): Cell culture techniques for primary isolation of Marek's disease-associated herpesvirus. Avian Dis., 13: 101-18.
(Received for publication October 29, 1974)
