- Email: [email protected]
The first isolation of Newcastle disease virus (NDV) from free-flying birds in Israel: Comparative studies on NDV strain isolated from migrating starlings (Sturnus vulgaris) wintering in Israel
Comp. Immun. Microbiol. infect. Dis. Vol. 10, No. I, pp. 65-70, 1987 Printed in Great Britain. All rights reserved
0147-9571/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
THE FIRST ISOLATION OF NEWCASTLE DISEASE VIRUS (NDV) FROM FREE-FLYING BIRDS IN ISRAEL: COMPARATIVE STUDIES ON N D V STRAIN ISOLATED FROM MIGRATING STARLINGS (STURNUS VULGARIS) WINTERING IN ISRAEL M. LIPKIND, B. RIVETZ* a n d E. SHIHMANTER Kimron Veterinary Institute, Beit Dagan, P.O. Box 12, 50250 Israel (Received 2 October 1986)
Almtraet--An NDV strain was isolated in Israel from a starling suffering from severe neurological affections. The strain appeared to be lentogenic to chickens, its hemagglutinin being thermostable and active toward horse erythrocytes. Comparative studies including LaSota and BI NDV reference strains showed that the isolate is not a vaccinal strain used for vaccination in the country. Key words: Newcastle disease virus, virulence, ecology
LE PREMIER ISOLEMENT EN ISRAEL DU VIRUS DE LA MALADIE DE NEWCASTLE (NDV) SUR DES OISEAUX S A U V A G E : E T U D E C O M P A R I ~ E A V E C LES S O U C H E S D E N D V ISOLt~ES S U R D E S E T O U R N E A U X ( S T U R N U S V U L G A R I S ) , MIGRATEUR HIVERNANT EN ISRAEL Rrsumr---Une souche du virus de la maladie de Newcastle a 6t6 isol6 d'un &ourneau souffrant de s6vdres troubles nerveux. La souche semble lentogdne pour les poultes. L'hemagglutination est thermostable et active envers les 6rythrocytes du cheval. On a dhmontr6 par des 6tudes comparhes avec la souche de rrfrrence La Sota et Bl NDV, que celle isolre fi partir de l'rtourneau n'&ait pas une des souches qu'on utilise pour la vaccination en Israel. Mots-clefs: Virus de la maladie de Newcastle, ecologie, virulance
INTRODUCTION A r e m a r k a b l y wide s p r e a d o f Newcastle disease virus ( N D V ) a m o n g the p o u l t r y all over the world [10, 1 l] a n d a sudden a n d practically s i m u l t a n e o u s r e a p p e a r a n c e o f the original type o f virulent N e w c a s t l e disease in S o u t h e a s t A s i a a n d in S o u t h A m e r i c a in 1962 a n d 2 years later in the M i d d l e East [10] with further r a p i d spread across continents [5, 8, 10] raised the question a b o u t the causes o f the spread. One i m p o r t a n t aspect o f this p r o b l e m is the s p r e a d o f N D V by feral avian hosts, m i g r a t o r y birds being o f m a i n interest. In fact, the virus was currently recovered from a n u m b e r o f v a r i o u s species o f free-flying birds since 1950 [9, 10] up to recent years [1,2, 15, 17, 18,21, 24]. Recently f o u n d i n v o l v e m e n t o f pigeon p o p u l a t i o n into so-called ' r a c i n g pigeon disease' caused by N D V - l i k e P M V - l virus [3] has further a g g r a v a t e d the situation. *Present address: Orgenics Ltd, P.O. Box 360, Yavne, 70650 Israel. 65
66
M. LIPKIND et al.
Migratory starling (Sturnus vulgaris) wintering in Israel have some unique peculiarities [25, 26], in particular, a phenomenon of "daily and seasonal pulsation" [12] which makes this species an attractive object for studies on virus transmission and its re-passaging in a natural host reservoir which in this case is an over-saturated avian community reaching in the places of night spending 530 birds per cubic meter [25]. Therefore, this species was chosen for surveillance. The isolation of NDV from a starling was described in 1950 [4] but apart from diagnostics no property of the isolate has been reported. One isolation of NDV from starlings (among a large number of wild, semidomestic and exotic birds) in California was mentioned without any detail [17]. The paper presents results on characterization of a strain of NDV isolated during field surveillance of the migratory starlings. MATERIALS AND METHODS
Cloacal and tracheal swabs were taken and preserved as described earlier [13]. Propagation of swab materials in chick embryonated eggs, hemagglutinin (HA) titration, neuraminidase reaction and HA inhibition (HI) tests were performed as described in "Advanced Laboratory Techniques for Influenza Diagnosis" [16]. Mean death time of the minimal infectious dose ( M D T / M I D ) was carried out in SPF eggs as described in "Isolation and Identification of Avian Pathogens" [6]. Intracerebral pathogenicity index (ICPI) was determined using 1-day old (24-36h) chicks according to Lancaster and Alexander [11]. Primary chicken embryo cell (CEC) cultures were prepared from 10-day old chicken embryos and grown in Minimal Essential Medium (MEM) supplemented with 0.025 M Hepes buffer pH 7.4, 10% calf serum and 0.3% tryptose phosphate broth (TPB). CEC monolayers of 48 h were used for plaque assay. 0.2 ml of virus in dilution corresponding to 10 ~ to 104 EIDs0 were added, and after 60 min adsorption at 37°C an overlay including MEM, 0.85% Noble agar, 0.025 M Hepes buffer, pH 7.4, 5% calf serum and 0.15% TPB was added. A second overlay with 0.02% neutral red was added 3 days post infection. Plates were examined every day until 6 days post-infection. Rate of elution was determined according to the method described by Spalatin et al. [20]. Thermostability of HA was expressed as time period in minutes of heating at 56°C of virus preparations which was needed for inactivation of HA activity [6]. Virus-induced agglutination of equine red blood cells (RBC) was performed using 0.5% concentration of RBC in parallel to HA test with chicken RBC. RESULTS
Case description The field work was carried out in the kibbutz Neve Eitan, in Beit Shean Valley in November 1980. Masses of starlings spent nights in a neighbouring grove and during the daytime they spread over vicinage invading into farms. Since these birds caused much trouble, farmers spread ~-chloraloz for poisoning. In the framework of the study, 158 starlings were taken for swabbing. From this number, 122 were gathered dead on the ground throughout the village. The rest of the birds were captured stunned but alive and they fully revived in the laboratory. One bird, however, was found on the ground far away from the place of drug spreading. The bird's head was enormously swollen with protruding
N e w c a s t l e disease virus
67
Table 1. Hemagglutination inhibition cross reaction test using the isolate and two vaccinal NDV strains HI antibody titers to the following viruses Antisera
LaSota
B1
Isolate
LaSota B1 Isolate
640 320 320
640 320 640
480 320 640
*The figures show the reciprocal of the dilution inhibiting 8 H.A. units of virus.
bulging eyes. The bird displayed tremors, loss of orientation, evident blindness and inability to fly (only to flutter). No such symptoms were observed among the drugpoisoned but live birds. The bird died during transportation from the village to the laboratory. The cloacal and tracheal swabs were taken from all the birds, but only one HA agent was isolated from a tracheal swab taken from the above-described blind starling.
Identification The HA agent could be passaged in embryonated eggs after filtration through bacterial filter proving its viral nature. The HA agent was shown to have neuraminidase activity proving its belonging to myxoviruses. The HI test using the isolate and reference antiserum against NDV (LaSota strain) had shown evident inhibition of the isolate HA activity. Cross reaction HI test using anti-isolate serum (prepared in chickens) together with the reference NDV viruses (strains LaSota and B1) and corresponding antisera showed no significant difference between the homologous and heterologous HI titers (Table 1).
Properties of the isolate The M D T was determined using the highest dilution of inoculated virus (minimal infectious dose) which caused all the inoculated embryos to be HA-positive at 200 h post inoculation. M D T was found to be 142 h. ICPI of the isolate was found to fall into limits of 0.1~.2. The isolate showed no plaque-forming ability in CEC monolayer up to 6 days post inoculation. The rate of elution was very slow: hemagglutination pattern remained unchanged for more than 24 h and persisted after resuspension. The hemagglutinin of the isolate was remarkably thermostable: incubation of viruscontaining allantoic fluid at 56°C for 4 h did not change HA titer. The isolate was found to agglutinate horse RBC. The HA titer was the same as that with chicken RBC. All the above data are presented in Table 2. Table 2. Comparative characterization of the NDV/Starling/80 isolate
Viruses B1 LaSota Isolate
Pathogenicity indexes MDT (hours) ICPI* 120 103 142
0.14 0.19 0.15
Plaque-forming ability
Rate of elution
no no no
rapid slow slow
*Intracerebral pathogenicity index [maximum value = 2.0 (11)].
Thermostability of hemagglutinin (rain) 5 0 240
Agglutination of horse RBC 0 + +
68
M. LIPKIND et al.
DISCUSSION The described case presents the first isolation of NDV from wild birds in Israel. According to the MDT, ICPI and plaque-forming ability tests, the isolate falls into the group of lentogenic NDV strains. This is not in conformity with the observed clinical picture which displayed an evidently severe form with prevailing neurological syndrome. Since the surveillance on starling population in Israel was carried out in the framework of the research on ecology of avian influenza viruses when the mass collection of birds was aimed for the isolation of the virus [12, 13], the carcasses of the birds were eradicated just after sampling and, thus, the present case has lacked pathological investigation. Therefore, it can be only suggested that the observed clinical picture was due to the NDV. However, the discrepancy between veiogenic character of certain NDV strains towards chickens and their avirulent character towards wild birds has been described [22], and, vice versa, embryos of the wild birds (waterfowl, in particular) were found to be more sensitive to the killing action of the lentogenic NDV strains of chicken origin as compared to chicken embryos [19]. This fact suggests that the whole classification of NDV strains by their virulence [11] which is based on the data on NDV-domestic fowl interaction does not seem to suit the NDV-wild bird interaction. The remarkable thermostability of the isolate HA supports the view that the virulence for chickens is not connected with the HA thermostability [7]. The latter, however, could serve as a useful marker for NDV strains and their origin in epizootiological investigations. Comparison of the isolate with two NDV reference strains, LaSota and BI, shows that, although all of them are lentogenic and do not differ antigenically (Table 1), they clearly differ according to the other markers used (Table 2). The isolate differs from B I by sensitivity to horse RBC, by the rate of elution from RBC, and by the HA thermostability, and it differs from LaSota only by the HA thermostability. That means that the isolate is not a vaccinal strain used in the country for poultry vaccination. Possible transmission of NDV from feral birds to poultry and vice versa which could mean preservation of the virus in natural avian reservoirs is widely discussed as the problem of high epizootiological importance. Although the whole view is rather speculative, it is seriously considered [11, 17], particularly in the Middle East region [1]. However, there is an opposite view which is based on found disparity between a rare practical opportunity for contact between waterfowl and domestic poultry and a rather high occurrence of anti-NDV antibodies among waterfowl including juvenile birds [19]. Besides, the isolates from waterfowl were of low virulence for chickens and had high HA thermostability, contrary to the NDV strains prevalent in poultry in U.S.A. [19]. In accordance with this, there are results of ecological studies performed in the U.S.S.R. [14]: a number of NDV strains were isolated from different avian species in Komandorskiye Islands with no poultry farm far around. This should be considered as a strong evidence for the existence of natural foci of NDV among wild avian reservoirs not connected with domesticated poultry. On the other side, however, the recently found "racing pigeon disease" caused by a NDV-like paramyxovirus-1 (PMV-1) has been shown to affect poultry [3]. These viruses have been isolated from pigeons in Israel, too [23], and it has been found that both "pigeon PMV-1" and "classic" NDV strains co-circulate in Israel avian reservoir (Lipkind et al., in preparation). Thus, the question about interconnections between NDV from natural reservoirs and poultry remains uncertain. Studies on isolation and characterization of NDV as well as
Newcastle disease virus
69
N D V - l i k e P M V - 1 v i r u s e s f r o m f l e e - f l y i n g ( e s p e c i a l l y m i g r a t i n g ) b i r d s in I s r a e l h a v e s o m e a d v a n t a g e s in e p i z o o t i o l o g i c a l a s p e c t o w i n g t o g e o g r a p h i c a l a n d e c o l o g i c a l p e c u l i a r i t i e s o f the country (location along the main fly-way of migrating birds and highly developed poultry industry). Acknowledgements--We are thankful to Mrs Tami Sabbag for typing the manuscript. The studies were supported by grants No. 1-42-79 and 1-194-80 from U.S. Israel Agricultural Research and Development Fund (BARD).
REFERENCES 1. Ahmed A. A. S., Sabban M, S., lbrahim A. M. M., Amin A., Khafagi A. R. and Sheble A. Some properties of Newcastle disease virus isolates recovered from migratory birds to Egypt, Zbl. Vet. Med. B27, 313 (1980). 2. Alexander D. J., Spackman D., Allan W. H. and Borland L. Isolation of Newcastle disease virus from a wild mallard duck (Arias platyrhynchos). Vet. Rec. 105, 328 (1979). 3. Alexander D. J., Russel P. H., Parsons G., Abu Elzein E. M. E., Ballough A., Cernik K., Engstrom B., Fevereiro M., Fleury H. J. A., Guittet M., Kaleta E. F., Kihm U., Kosters J., Lomniczi B., Meister J., Meulemans G., Nerome K., Petek M., Pokomunski S,. Polten B., Prip M,, Richter R., Saghy E., Samberg Y., Spanoghe L. and Tumova B. Antigenic and biological characterization of avian paramyxovirus type 1 isolates from p i g e o n - - a n international collaborative study. Avian Path. 14, 365 (1985). 4. Gillespie J. H., Kessel B. and Fabricant J. The isolation of Newcastle disease virus from a starling. Cornell Vet. 40, 93 (1950). 5. Hanson R. P. The reemergence of Newcastle disease. Adv. vet. Sci. Comp. Med. 18, 213 (1974). 6. Hanson R. P. Newcastle disease virus. In Isolation and Identification o f Avian Pathogens (Edited by Hitchner). Ithaca, N.Y. (1975). 7. Hanson R. P. and Spalatin J. Thermostability of the hemagglutinin of Newcastle disease virus as a strain marker in epizootiological studies. Avian Dis. 22, 660 (1978). 8. Hanson R. P., Spalatin J. and Jacobson G. S., The panzootic of viscerotropic Newcastle disease, an extract. Proe, 22nd Western Poultry Disease Conf. Davis, Calif. March 27-29 (1973). 9. Lancaster J. E. Newcastle Disease, A Review 1926 1964, Monograph No. 3, Canada Department of Agriculture, Ottawa, Ontario (1966). 10. Lancaster J. E. Newcastle d i s e a s ~ a review of the geographical incidence and epizootiology. Wld Poult. Sci. 33, 155 (1977). 11. Lancaster J. E. and Alexander D. J. Newcastle Disease Virus and Spread. Monograph No. 11, Canada Department of Agriculture, Ottawa, Ontario, (1975). 12. Lipkind M., Shihmanter E. and Shoham D. Further characterization of H7N7 avian influenza virus isolated from migrating starlings wintering in Israel. Zentbl. Vet. Med. B29, 566 (1982). 13. Lipkind M., Weisman Y., Shihmanter E. and Shoham D. The first isolation of animal influenza in Israel. Vet. Ree. 105, 510 (1979). 14. Lvov D. K., Syurin V. N., Nikiforov L. P., Portyanko N. V., Sazonov A. A., Andreev V. P., Chumakov V. M., Belousova R. V., Konstantinova L. A., Zabigailo N. M., Lvov N. D., Mikhtaryants E. A., Mymrin N. I. and Zhezmer V. Yu. Discovery of natural foci of Newcastle disease virus in the USSR. Vop. Virus. No. 3, 311 (1977). In Russian. 15. Onunkwo O. and Momoh M. A. Characterization of a Newcastle disease virus isolated from a parrot (Psittaeus erythracus) in Nigeria. J. Wildl. Dis. 17, 463 (1981). 16. Palmer D. F., Dowdle W. R., Coleman M. T. and Schild G. C. Advanced laboratory techniques for influenza diagnosis. Immunology Series No. 6, Procedural Guide, U.S. Dept of Health, Education and Welfare, Public Service, Center for Disease Control, Atlanta (1975). 17. Pearson G. U and McCann M. K. The role of indigenous wild, semi-domestic and exotic birds in the epizootiology of velogenic viscerotropic Newcastle disease in Southern California, 1972 1973, J. Am. vet. med. Ass. 167, 610 (1975). --~ 18. Shortridge K. F., Alexander D. J., Hu L. Y. and Kam S. L. IsolatiOn of Newcastle disease virus from Phasianide birds in Hong Kong. J. comp. Path. 88, 633 (1978). 19. Spalatin J. and Hanson R. P. Epizootiology of Newcastle disease in waterfowl, Avian Dis. 19, 573 (1975). 20. Spalatin J., Hanson R. P. and Beard P. D. The hemagglutination-elution pattern as a marker in characterizing Newcastle disease virus. Avian Dis. 14, 542 (1970). 21. Tumova B., Turek R., Kubinova I., Stumpa A. and Ciampor F. Incidence of paramyxoviruses in free-living birds in 1978 1982. Acta Virol. 28, 114 (1984). 22. Vickers M. L. and Hanson R. P. Experimental Newcastle disease virus infections in three species of wild birds. Avian Dis. 23, 70 (1979). C.I.M.ID. IO/I--E
70
M. LIPKIND et al.
23. Weisman Y., Aronovici A., Malkinson M., Shihmanter E. and Lipkind M. Isolation of paramyxoviruses from pigeons in Israel. Vet. Rec. llS, 23 (1984). 24. Yamane N., Idagiri T., Arikawa J., Morita M., Sukeno N. and Ishida N. Isolation of orthomyxoviruses from migrating and domestic ducks in Northern Japan in 1976-1977. Jap. J. reed. Sci. 31, 407 (1978). 25. Yom-Tov Y. Biology of starlings in Israel. Teva Va-Aretz (Nature and Country) 22, 98 (1980). In Hebrew. 26. Yom-Tov Y., Imber A. and Otterman J. The microclimate of winter roosts of the starling Sturnus vulgaris. Ibis 119, 366 (1977).
0147-9571/87 $3.00+0.00 Copyright © 1987 Pergamon Journals Ltd
THE FIRST ISOLATION OF NEWCASTLE DISEASE VIRUS (NDV) FROM FREE-FLYING BIRDS IN ISRAEL: COMPARATIVE STUDIES ON N D V STRAIN ISOLATED FROM MIGRATING STARLINGS (STURNUS VULGARIS) WINTERING IN ISRAEL M. LIPKIND, B. RIVETZ* a n d E. SHIHMANTER Kimron Veterinary Institute, Beit Dagan, P.O. Box 12, 50250 Israel (Received 2 October 1986)
Almtraet--An NDV strain was isolated in Israel from a starling suffering from severe neurological affections. The strain appeared to be lentogenic to chickens, its hemagglutinin being thermostable and active toward horse erythrocytes. Comparative studies including LaSota and BI NDV reference strains showed that the isolate is not a vaccinal strain used for vaccination in the country. Key words: Newcastle disease virus, virulence, ecology
LE PREMIER ISOLEMENT EN ISRAEL DU VIRUS DE LA MALADIE DE NEWCASTLE (NDV) SUR DES OISEAUX S A U V A G E : E T U D E C O M P A R I ~ E A V E C LES S O U C H E S D E N D V ISOLt~ES S U R D E S E T O U R N E A U X ( S T U R N U S V U L G A R I S ) , MIGRATEUR HIVERNANT EN ISRAEL Rrsumr---Une souche du virus de la maladie de Newcastle a 6t6 isol6 d'un &ourneau souffrant de s6vdres troubles nerveux. La souche semble lentogdne pour les poultes. L'hemagglutination est thermostable et active envers les 6rythrocytes du cheval. On a dhmontr6 par des 6tudes comparhes avec la souche de rrfrrence La Sota et Bl NDV, que celle isolre fi partir de l'rtourneau n'&ait pas une des souches qu'on utilise pour la vaccination en Israel. Mots-clefs: Virus de la maladie de Newcastle, ecologie, virulance
INTRODUCTION A r e m a r k a b l y wide s p r e a d o f Newcastle disease virus ( N D V ) a m o n g the p o u l t r y all over the world [10, 1 l] a n d a sudden a n d practically s i m u l t a n e o u s r e a p p e a r a n c e o f the original type o f virulent N e w c a s t l e disease in S o u t h e a s t A s i a a n d in S o u t h A m e r i c a in 1962 a n d 2 years later in the M i d d l e East [10] with further r a p i d spread across continents [5, 8, 10] raised the question a b o u t the causes o f the spread. One i m p o r t a n t aspect o f this p r o b l e m is the s p r e a d o f N D V by feral avian hosts, m i g r a t o r y birds being o f m a i n interest. In fact, the virus was currently recovered from a n u m b e r o f v a r i o u s species o f free-flying birds since 1950 [9, 10] up to recent years [1,2, 15, 17, 18,21, 24]. Recently f o u n d i n v o l v e m e n t o f pigeon p o p u l a t i o n into so-called ' r a c i n g pigeon disease' caused by N D V - l i k e P M V - l virus [3] has further a g g r a v a t e d the situation. *Present address: Orgenics Ltd, P.O. Box 360, Yavne, 70650 Israel. 65
66
M. LIPKIND et al.
Migratory starling (Sturnus vulgaris) wintering in Israel have some unique peculiarities [25, 26], in particular, a phenomenon of "daily and seasonal pulsation" [12] which makes this species an attractive object for studies on virus transmission and its re-passaging in a natural host reservoir which in this case is an over-saturated avian community reaching in the places of night spending 530 birds per cubic meter [25]. Therefore, this species was chosen for surveillance. The isolation of NDV from a starling was described in 1950 [4] but apart from diagnostics no property of the isolate has been reported. One isolation of NDV from starlings (among a large number of wild, semidomestic and exotic birds) in California was mentioned without any detail [17]. The paper presents results on characterization of a strain of NDV isolated during field surveillance of the migratory starlings. MATERIALS AND METHODS
Cloacal and tracheal swabs were taken and preserved as described earlier [13]. Propagation of swab materials in chick embryonated eggs, hemagglutinin (HA) titration, neuraminidase reaction and HA inhibition (HI) tests were performed as described in "Advanced Laboratory Techniques for Influenza Diagnosis" [16]. Mean death time of the minimal infectious dose ( M D T / M I D ) was carried out in SPF eggs as described in "Isolation and Identification of Avian Pathogens" [6]. Intracerebral pathogenicity index (ICPI) was determined using 1-day old (24-36h) chicks according to Lancaster and Alexander [11]. Primary chicken embryo cell (CEC) cultures were prepared from 10-day old chicken embryos and grown in Minimal Essential Medium (MEM) supplemented with 0.025 M Hepes buffer pH 7.4, 10% calf serum and 0.3% tryptose phosphate broth (TPB). CEC monolayers of 48 h were used for plaque assay. 0.2 ml of virus in dilution corresponding to 10 ~ to 104 EIDs0 were added, and after 60 min adsorption at 37°C an overlay including MEM, 0.85% Noble agar, 0.025 M Hepes buffer, pH 7.4, 5% calf serum and 0.15% TPB was added. A second overlay with 0.02% neutral red was added 3 days post infection. Plates were examined every day until 6 days post-infection. Rate of elution was determined according to the method described by Spalatin et al. [20]. Thermostability of HA was expressed as time period in minutes of heating at 56°C of virus preparations which was needed for inactivation of HA activity [6]. Virus-induced agglutination of equine red blood cells (RBC) was performed using 0.5% concentration of RBC in parallel to HA test with chicken RBC. RESULTS
Case description The field work was carried out in the kibbutz Neve Eitan, in Beit Shean Valley in November 1980. Masses of starlings spent nights in a neighbouring grove and during the daytime they spread over vicinage invading into farms. Since these birds caused much trouble, farmers spread ~-chloraloz for poisoning. In the framework of the study, 158 starlings were taken for swabbing. From this number, 122 were gathered dead on the ground throughout the village. The rest of the birds were captured stunned but alive and they fully revived in the laboratory. One bird, however, was found on the ground far away from the place of drug spreading. The bird's head was enormously swollen with protruding
N e w c a s t l e disease virus
67
Table 1. Hemagglutination inhibition cross reaction test using the isolate and two vaccinal NDV strains HI antibody titers to the following viruses Antisera
LaSota
B1
Isolate
LaSota B1 Isolate
640 320 320
640 320 640
480 320 640
*The figures show the reciprocal of the dilution inhibiting 8 H.A. units of virus.
bulging eyes. The bird displayed tremors, loss of orientation, evident blindness and inability to fly (only to flutter). No such symptoms were observed among the drugpoisoned but live birds. The bird died during transportation from the village to the laboratory. The cloacal and tracheal swabs were taken from all the birds, but only one HA agent was isolated from a tracheal swab taken from the above-described blind starling.
Identification The HA agent could be passaged in embryonated eggs after filtration through bacterial filter proving its viral nature. The HA agent was shown to have neuraminidase activity proving its belonging to myxoviruses. The HI test using the isolate and reference antiserum against NDV (LaSota strain) had shown evident inhibition of the isolate HA activity. Cross reaction HI test using anti-isolate serum (prepared in chickens) together with the reference NDV viruses (strains LaSota and B1) and corresponding antisera showed no significant difference between the homologous and heterologous HI titers (Table 1).
Properties of the isolate The M D T was determined using the highest dilution of inoculated virus (minimal infectious dose) which caused all the inoculated embryos to be HA-positive at 200 h post inoculation. M D T was found to be 142 h. ICPI of the isolate was found to fall into limits of 0.1~.2. The isolate showed no plaque-forming ability in CEC monolayer up to 6 days post inoculation. The rate of elution was very slow: hemagglutination pattern remained unchanged for more than 24 h and persisted after resuspension. The hemagglutinin of the isolate was remarkably thermostable: incubation of viruscontaining allantoic fluid at 56°C for 4 h did not change HA titer. The isolate was found to agglutinate horse RBC. The HA titer was the same as that with chicken RBC. All the above data are presented in Table 2. Table 2. Comparative characterization of the NDV/Starling/80 isolate
Viruses B1 LaSota Isolate
Pathogenicity indexes MDT (hours) ICPI* 120 103 142
0.14 0.19 0.15
Plaque-forming ability
Rate of elution
no no no
rapid slow slow
*Intracerebral pathogenicity index [maximum value = 2.0 (11)].
Thermostability of hemagglutinin (rain) 5 0 240
Agglutination of horse RBC 0 + +
68
M. LIPKIND et al.
DISCUSSION The described case presents the first isolation of NDV from wild birds in Israel. According to the MDT, ICPI and plaque-forming ability tests, the isolate falls into the group of lentogenic NDV strains. This is not in conformity with the observed clinical picture which displayed an evidently severe form with prevailing neurological syndrome. Since the surveillance on starling population in Israel was carried out in the framework of the research on ecology of avian influenza viruses when the mass collection of birds was aimed for the isolation of the virus [12, 13], the carcasses of the birds were eradicated just after sampling and, thus, the present case has lacked pathological investigation. Therefore, it can be only suggested that the observed clinical picture was due to the NDV. However, the discrepancy between veiogenic character of certain NDV strains towards chickens and their avirulent character towards wild birds has been described [22], and, vice versa, embryos of the wild birds (waterfowl, in particular) were found to be more sensitive to the killing action of the lentogenic NDV strains of chicken origin as compared to chicken embryos [19]. This fact suggests that the whole classification of NDV strains by their virulence [11] which is based on the data on NDV-domestic fowl interaction does not seem to suit the NDV-wild bird interaction. The remarkable thermostability of the isolate HA supports the view that the virulence for chickens is not connected with the HA thermostability [7]. The latter, however, could serve as a useful marker for NDV strains and their origin in epizootiological investigations. Comparison of the isolate with two NDV reference strains, LaSota and BI, shows that, although all of them are lentogenic and do not differ antigenically (Table 1), they clearly differ according to the other markers used (Table 2). The isolate differs from B I by sensitivity to horse RBC, by the rate of elution from RBC, and by the HA thermostability, and it differs from LaSota only by the HA thermostability. That means that the isolate is not a vaccinal strain used in the country for poultry vaccination. Possible transmission of NDV from feral birds to poultry and vice versa which could mean preservation of the virus in natural avian reservoirs is widely discussed as the problem of high epizootiological importance. Although the whole view is rather speculative, it is seriously considered [11, 17], particularly in the Middle East region [1]. However, there is an opposite view which is based on found disparity between a rare practical opportunity for contact between waterfowl and domestic poultry and a rather high occurrence of anti-NDV antibodies among waterfowl including juvenile birds [19]. Besides, the isolates from waterfowl were of low virulence for chickens and had high HA thermostability, contrary to the NDV strains prevalent in poultry in U.S.A. [19]. In accordance with this, there are results of ecological studies performed in the U.S.S.R. [14]: a number of NDV strains were isolated from different avian species in Komandorskiye Islands with no poultry farm far around. This should be considered as a strong evidence for the existence of natural foci of NDV among wild avian reservoirs not connected with domesticated poultry. On the other side, however, the recently found "racing pigeon disease" caused by a NDV-like paramyxovirus-1 (PMV-1) has been shown to affect poultry [3]. These viruses have been isolated from pigeons in Israel, too [23], and it has been found that both "pigeon PMV-1" and "classic" NDV strains co-circulate in Israel avian reservoir (Lipkind et al., in preparation). Thus, the question about interconnections between NDV from natural reservoirs and poultry remains uncertain. Studies on isolation and characterization of NDV as well as
Newcastle disease virus
69
N D V - l i k e P M V - 1 v i r u s e s f r o m f l e e - f l y i n g ( e s p e c i a l l y m i g r a t i n g ) b i r d s in I s r a e l h a v e s o m e a d v a n t a g e s in e p i z o o t i o l o g i c a l a s p e c t o w i n g t o g e o g r a p h i c a l a n d e c o l o g i c a l p e c u l i a r i t i e s o f the country (location along the main fly-way of migrating birds and highly developed poultry industry). Acknowledgements--We are thankful to Mrs Tami Sabbag for typing the manuscript. The studies were supported by grants No. 1-42-79 and 1-194-80 from U.S. Israel Agricultural Research and Development Fund (BARD).
REFERENCES 1. Ahmed A. A. S., Sabban M, S., lbrahim A. M. M., Amin A., Khafagi A. R. and Sheble A. Some properties of Newcastle disease virus isolates recovered from migratory birds to Egypt, Zbl. Vet. Med. B27, 313 (1980). 2. Alexander D. J., Spackman D., Allan W. H. and Borland L. Isolation of Newcastle disease virus from a wild mallard duck (Arias platyrhynchos). Vet. Rec. 105, 328 (1979). 3. Alexander D. J., Russel P. H., Parsons G., Abu Elzein E. M. E., Ballough A., Cernik K., Engstrom B., Fevereiro M., Fleury H. J. A., Guittet M., Kaleta E. F., Kihm U., Kosters J., Lomniczi B., Meister J., Meulemans G., Nerome K., Petek M., Pokomunski S,. Polten B., Prip M,, Richter R., Saghy E., Samberg Y., Spanoghe L. and Tumova B. Antigenic and biological characterization of avian paramyxovirus type 1 isolates from p i g e o n - - a n international collaborative study. Avian Path. 14, 365 (1985). 4. Gillespie J. H., Kessel B. and Fabricant J. The isolation of Newcastle disease virus from a starling. Cornell Vet. 40, 93 (1950). 5. Hanson R. P. The reemergence of Newcastle disease. Adv. vet. Sci. Comp. Med. 18, 213 (1974). 6. Hanson R. P. Newcastle disease virus. In Isolation and Identification o f Avian Pathogens (Edited by Hitchner). Ithaca, N.Y. (1975). 7. Hanson R. P. and Spalatin J. Thermostability of the hemagglutinin of Newcastle disease virus as a strain marker in epizootiological studies. Avian Dis. 22, 660 (1978). 8. Hanson R. P., Spalatin J. and Jacobson G. S., The panzootic of viscerotropic Newcastle disease, an extract. Proe, 22nd Western Poultry Disease Conf. Davis, Calif. March 27-29 (1973). 9. Lancaster J. E. Newcastle Disease, A Review 1926 1964, Monograph No. 3, Canada Department of Agriculture, Ottawa, Ontario (1966). 10. Lancaster J. E. Newcastle d i s e a s ~ a review of the geographical incidence and epizootiology. Wld Poult. Sci. 33, 155 (1977). 11. Lancaster J. E. and Alexander D. J. Newcastle Disease Virus and Spread. Monograph No. 11, Canada Department of Agriculture, Ottawa, Ontario, (1975). 12. Lipkind M., Shihmanter E. and Shoham D. Further characterization of H7N7 avian influenza virus isolated from migrating starlings wintering in Israel. Zentbl. Vet. Med. B29, 566 (1982). 13. Lipkind M., Weisman Y., Shihmanter E. and Shoham D. The first isolation of animal influenza in Israel. Vet. Ree. 105, 510 (1979). 14. Lvov D. K., Syurin V. N., Nikiforov L. P., Portyanko N. V., Sazonov A. A., Andreev V. P., Chumakov V. M., Belousova R. V., Konstantinova L. A., Zabigailo N. M., Lvov N. D., Mikhtaryants E. A., Mymrin N. I. and Zhezmer V. Yu. Discovery of natural foci of Newcastle disease virus in the USSR. Vop. Virus. No. 3, 311 (1977). In Russian. 15. Onunkwo O. and Momoh M. A. Characterization of a Newcastle disease virus isolated from a parrot (Psittaeus erythracus) in Nigeria. J. Wildl. Dis. 17, 463 (1981). 16. Palmer D. F., Dowdle W. R., Coleman M. T. and Schild G. C. Advanced laboratory techniques for influenza diagnosis. Immunology Series No. 6, Procedural Guide, U.S. Dept of Health, Education and Welfare, Public Service, Center for Disease Control, Atlanta (1975). 17. Pearson G. U and McCann M. K. The role of indigenous wild, semi-domestic and exotic birds in the epizootiology of velogenic viscerotropic Newcastle disease in Southern California, 1972 1973, J. Am. vet. med. Ass. 167, 610 (1975). --~ 18. Shortridge K. F., Alexander D. J., Hu L. Y. and Kam S. L. IsolatiOn of Newcastle disease virus from Phasianide birds in Hong Kong. J. comp. Path. 88, 633 (1978). 19. Spalatin J. and Hanson R. P. Epizootiology of Newcastle disease in waterfowl, Avian Dis. 19, 573 (1975). 20. Spalatin J., Hanson R. P. and Beard P. D. The hemagglutination-elution pattern as a marker in characterizing Newcastle disease virus. Avian Dis. 14, 542 (1970). 21. Tumova B., Turek R., Kubinova I., Stumpa A. and Ciampor F. Incidence of paramyxoviruses in free-living birds in 1978 1982. Acta Virol. 28, 114 (1984). 22. Vickers M. L. and Hanson R. P. Experimental Newcastle disease virus infections in three species of wild birds. Avian Dis. 23, 70 (1979). C.I.M.ID. IO/I--E
70
M. LIPKIND et al.
23. Weisman Y., Aronovici A., Malkinson M., Shihmanter E. and Lipkind M. Isolation of paramyxoviruses from pigeons in Israel. Vet. Rec. llS, 23 (1984). 24. Yamane N., Idagiri T., Arikawa J., Morita M., Sukeno N. and Ishida N. Isolation of orthomyxoviruses from migrating and domestic ducks in Northern Japan in 1976-1977. Jap. J. reed. Sci. 31, 407 (1978). 25. Yom-Tov Y. Biology of starlings in Israel. Teva Va-Aretz (Nature and Country) 22, 98 (1980). In Hebrew. 26. Yom-Tov Y., Imber A. and Otterman J. The microclimate of winter roosts of the starling Sturnus vulgaris. Ibis 119, 366 (1977).