1Centre for Animal Health Studies, Tamilnadu Veterinary and Animal Sciences. University, Madhavaram Milk Colony, Chenni-600 051, India; 2Avian Virology,.
Veterinary Research Communications, 24 (2000) 135^142 # 2000 Kluwer Academic Publishers. Printed in the Netherlands
Characterization of Newcastle Disease Viruses Isolated from Chickens and Ducks in Tamilnadu, India P. Roy1* A.T. Venugopalan1 and R. Manvell2 Centre for Animal Health Studies, Tamilnadu Veterinary and Animal Sciences University, Madhavaram Milk Colony, Chenni-600 051, India; 2Avian Virology, Veterinary Laboratory Agency, NewHaw, Addlestone, Surrey, UK *Correspondence: 917B South 17 Street (Hubbard-Apt), Birmingham, AL 35205, USA 1
Roy, P., Venugopalan, A.T. and Manvell, R., 2000. Characterization of Newcastle disease viruses isolated from chickens and ducks in Tamilnadu, India. Veterinary Research Communications, 24(2), 135^142 ABSTRACT During 1993, outbreaks of Newcastle disease occurred on many farms in Tamilnadu, India. Six Newcastle disease virus (NDV) isolates were obtained from the chickens on ¢ve di¡erent farms and from the birds on one duck farm during outbreaks of the disease. All the isolates were characterized as velogenic, based on the mean death time, intravenous pathogenicity index, intracerebral pathogenicity index (ICPI), stability of haemagglutinin at 568C, agglutination of equine erythrocytes, haemagglutination elution pattern and adsorption of haemagglutinin by chick brain cells. The isolate obtained from ducks resembled a group D strain, based on its ICPI and its reaction with a panel of monoclonal antibodies. The other ¢ve NDV isolates obtained from chickens were placed in groups B(1), C1(2) and D(2) on the basis of their binding patterns with the panel of monoclonal antibodies. In challenge experiments, it was found that LaSota vaccine provided 100% protection against each of these ¢eld isolates and against a local NDV strain obtained from the Institute of Veterinary Preventive Medicine, Tamilnadu, India, while unvaccinated chickens succumbed to challenge. The possible origin of epizootic viruses causing outbreaks in vaccinated £ocks is discussed. Keywords: chickens, ducks, Newcastle disease, protection, strain, vaccine, velogenic, virus Abbreviations: AAF, amnioallantoic £uid; EID50, dose infecting 50% of eggs; HA, haemagglutination; HI, haemagglutination inhibition; ICPI, intracerebral pathogenicity index; IVPI, intravenous pathogenicity index; IVPM, Institute of Preventive Medicine, Tamilnadu, India; mAb, monoclonal antibody; MDT, mean death time; ND, Newcastle disease; NDV, Newcastle disease virus
INTRODUCTION Newcastle disease (ND) was ¢rst reported in Tamilnadu, India, by Kylasamaier (1931) and since then the disease has been endemic. Several vaccines, mainly consisting of live lentogenic and live mesogenic viruses, are used to protect poultry against the disease and have reduced the number of outbreaks. Nevertheless, the disease continues to be reported (Roy and Balasubramaniam, 1992). Namakkal is the largest poultry-producing area in Tamilnadu and the second largest in India. About 18 million chickens are reared on around 3000 farms in an area of about 32 km radius in Namakkal. The farms are open, and in each village they are located in close proximity to each other. The common vaccination schedule against ND used in 135
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this locality was live F-strain given oculonasally at 7 days of age, live LaSota strain given in drinking water at 4 weeks of age and live R2B or RDVK (mesogenic strain) given subcutaneously at 8^12 weeks of age and again at 18^20 weeks of age. In 1993, ND outbreaks in Namakkal caused the death of about 10 million chickens on more than 2000 farms, with consequent large economic losses. Most of the outbreaks occurred during March in vaccinated £ocks. Outbreaks occurred in di¡erent age groups of chickens, the mortality ranging from 30% to 90%. The clinical signs shown by the a¡ected chickens included anorexia, listlessness, greenish-white diarrhoea, and paralysis of the legs and wings. On an unvaccinated commercial duck farm, the mortality was 10%. The a¡ected ducks showed signs such as anorexia and greenishwhite diarrhoea. At necropsy, the lesions in both chickens and ducks were mainly con¢ned to the intestinal tract. Button ulcers were seen in the intestine and haemorrhages in the proventriculus, resembling viscerotropic, velogenic ND. Intestinal homogenates showed a high haemagglutination (HA) titre compared to other organs and HA was inhibited by NDV speci¢c antiserum (Roy et al., 1997). This paper describes the antigenic and pathogenic characterization of six ND viruses isolated from six di¡erent farms during these outbreaks and the protection a¡orded against these viruses by a commercial vaccine. MATERIALS AND METHODS Virus isolation Viruses were isolated from chickens from ¢ve di¡erent commercial farms. Isolates nos. 642, 769, 791, 615 and 102 were from chickens aged 40 weeks, 33 weeks, 55 weeks, 5 weeks and 8 weeks, respectively. One isolate was obtained from 12-week-old ducks from a commercial duck farm and was designated no. 622. Virus isolation was done in 10-day-old embryonated hens' eggs as described by Alexander (1988). Antiserum Antiserum against NDV was prepared in speci¢c antibody-negative chickens (Allan et al., 1978). Ten 6-week-old chickens were injected with 0.5 ml formalinized amnioallantoic £uid (AAF) containing NDV (LaSota strain). Six weeks later, a dose of 109 EID50 of virulent NDV (IVPM) was injected into the birds. Sera were collected 2 weeks after the last injection, pooled and used as a positive control antiserum. Identi¢cation of virus AAF, harvested from embryos that died more than 24 h after inoculation, was tested for haemagglutination (HA) activity with 1% chicken erythrocytes. Identi¢cation was carried out by inhibiting the HA activity of AAF with NDV speci¢c antiserum in a haemagglutination inhibition (HI) test (Alexander, 1988).
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Pathogenicity tests Fresh, bacteria-free AAF, obtained as above, was used for these tests. The mean death time (MDT) of 10-day-old embryonated hens' eggs, intracerebral pathogenicity index (ICPI) in day-old chicks and intravenous pathogenicity index (IVPI) in 6-week-old speci¢c antibody-negative chickens were assessed as described by Alexander (1988). Strain-di¡erentiating characteristics The thermostability of haemagglutinin at 568C (Hanson, 1972), HA of equine erythrocytes (Hanson, 1980), haemagglutination elution pattern (Hanson, 1980) and adsorption of haemagglutinin by chick brain cells (Hanson et al., 1967) were assessed as described by those authors. Monoclonal antibody (mAb) typing The isolates were grouped (by R.M.) with a panel of mAbs according to their reaction in the haemagglutination (HI) and immunoperoxidase (IPT) tests at the Central Veterinary Laboratory, Weybridge, UK as described by Russell and Alexander (1983). Vaccination and protection Sixty day-old chicks were divided into two equal groups (A and B) at 17 days of age. The chicks in group A received NDV vaccine (LaSota strain, IVPM, Tamilnadu) at 17 days of age by the oculonasal route, following the recommendations of the manufacturer. The birds in group B were kept as unvaccinated age-matched controls. At 38 days of age, subgroups of 5 chickens each from both groups were challenged intramuscularly with one of ¢ve of the di¡erent ¢eld isolates (excluding isolate 615) or with a standard local challenge strain of NDV (IVPM), as described by Nilakantan and colleagues (1969), each bird receiving about 105 minimal infective doses of the challenge virus. The chickens were observed for 21 days after challenge. Serum samples were collected at random from 10 chickens in each group at 38 days of age and from all the survivors 21 days after challenge, for measurement of NDV antibodies by the HI test (Alexander, 1988). RESULTS Virus isolation, identi¢cation and characterization Inoculated embryos died between 43 h and 50 h after inoculation, as shown in Table I. The harvested, fresh AAF showed HA activity with chicken erythrocytes and was
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inhibited by a known NDV speci¢c antiserum. The MDT, ICPI and IVPI values obtained in pathogenicity tests for each isolate are shown in Table I. The haemagglutinin in all the isolates was stable at 568C for at least 90 min. All the isolates eluted o¡ the HA slowly. All but one isolate (791) failed to agglutinate equine erythrocytes. The percentage of the haemagglutinin of each isolate absorbed by the chick brain cells was between 50 and 100. Details are presented in Table I. Monoclonal antibody typing All six isolates were con¢rmed as belonging to the PMV-1 serotype. High HI titres were obtained against PMV-1 polyclonal serum and mAb Ulster 85, which is speci¢c for most of the classical strains of NDV. Titres of 1:40 were obtained with mAb 7D4, which is speci¢c for F and LaSota strains of NDV, and also with mAb 617/161, which is speci¢c for the virus responsible for the pigeon panzootic. Con¢rmation of the HI results was obtained when the isolates were tested by the immunoprecipitation test. Isolates 622, 642 and 615 were placed in group D, isolate 796 was placed in group B and isolates 105 and 791 were placed in group C1 (Russell and Alexander, 1983). Vaccination and protection All the vaccinated birds withstood the challenge and remained healthy, but all the unvaccinated birds died within 4^5 days after challenge. The antibody levels against NDV before vaccination and the seroconversion that occurred before challenge and in the survivors are recorded in Table II. DISCUSSION In order to study the character of the epizootic virus that caused the devastating outbreaks in Namakkal in 1993, six isolates were obtained from six di¡erent farms on which the chickens or ducks showed typical signs of viscerotropic velogenic ND. Information about the antibody titres in the a¡ected £ocks before the outbreaks was not available. All six isolates were con¢rmed as NDV (PMV-1) based on the HA and HI tests. All the isolates had MDT values of less than 60 h, IVPI values of more than 2.0 and, except for the duck isolate (622), ICPI values of more than 1.5 (Table I), con¢rming them as velogenic NDVs (Hanson and Brandly, 1955; Alexander, 1988). In addition, their strain-di¡erentiating characters, including the stability of haemagglutinin at 568C (Hanson, 1972), failure to agglutinate equine erythrocytes (Hanson, 1980; Bell, 1986), slow elution from chicken erythrocytes (Sulochana et al., 1981) and avid adsorption to chick brain cells (Piraino and Hanson, 1960), con¢rmed that all six NDV isolates were velogenic. Isolate no. 791 agglutinated equine erythrocytes but was considered to be velogenic based on its pathogenicity and other strain-di¡erentiating tests, in agreement with the ¢ndings of Bell (1986).
TABLE I Characteristics of NDV isolates obtained from natural outbreaks
Farm/ isolate no. 642 796 791 615 105 622
Source
MDT (h)
ICPI
IVPI
Thermostability of HA at 568C (min)
Chicken Chicken Chicken Chicken Chicken Duck
48.0 50.2 48.0 48.0 43.2 50.0
1.6 1.8 2.0 1.8 1.6 1.4
2.50 2.63 2.82 2.10 2.90 2.09
120 120 120 120 120 90
Chick brain cell adsorption of HA (%)
HA of equine erythrocytes
HA elution pattern
96.9 87.5 98.5 50.0 100.0 88.0
^ ^ + ^ ^ ^
Slow Slow Slow Slow Slow Slow
Group based on reaction with mAbs D B C1 D C1 D
Remarks Velogenic Velogenic Velogenic Velogenic Velogenic Velogenic
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TABLE II Protection studies with LaSota vaccine against di¡erent ¢eld isolates
Challenge isolate nos.
Group A (vaccinated) ööööööööö GM-HI % titre Protected
Group B (unvaccinated) ööööööööö GM-HI % titre Protected
Group based on reaction with mAbs
Age of chickens (days)
^ D D C1 C1 B
38 59 59 59 59 59
4.2 +0.33 6.75+0.41 6.4 +0.21 7.2 +0.17 7.6 +0.21 7.4 +0.21
100 100 100 100 100 100
Nil ^ ^ ^ ^ ^
0 0 0 0 0 0
C1
59
7.6 +0.21
100
^
0
^ 642 622 105 791 796 Standard challenge NDV (IVPM)
GM-HI, geometric mean haemagglutination inhibition. Values expressed in log 2 base ^, no living chicks
Monoclonal antibodies are commonly used for studying antigenic variation among ND viruses and for epizootiological mapping. The NDV isolates were placed in mAb groups B, C1 and D (Table I), as de¢ned by Russell and Alexander (1983). C1 group isolates have been found to be prevalent in Tamilnadu (Kumanan et al., 1992). This paper reports the presence there of viruses of B and D groups. Thus, antigenically heterogeneous NDVs were found to be responsible for the outbreaks in Tamilnadu, but the source of these viruses remains unknown. They may have been prevalent for a long time in the ¢eld, restrained by extensive vaccination but growing in the intestine and perpetuating in the environment. It has been reported that, while vaccines protect birds from the more serious consequences of NDV infection, virulent epizootic virus may infect and replicate in the tissues and organs of these birds, which may then excrete the virus (Alexander, 1995). Vaccinated birds excreting virulent virus pose a threat of overt disease in unvaccinated birds (Alexander, 1995). Another possibility may be that the epizootic virulent viruses originated from the live vaccines that are widely used in the ¢eld, as group D viruses are closely related to the RDVK strain of vaccine. It has been suggested that the lentogenic and mesogenic live ND vaccines, which are used indiscriminately in the ¢eld, may be gaining virulence because of continuous passage (Roy and Venugopalan, 1998). Since LaSota vaccine is widely used in Namakkal, an experiment was designed to study the e¤cacy of the vaccine against each of the new isolates. Chickens vaccinated with the LaSota strain of NDV showed a log2 geometric mean HI titre of 4.2 at the time of challenge, but unvaccinated chickens did not then contain any antibody. All the
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vaccinated chickens withstood challenge against NDVs belonging to mAb groups B, C1 and D, but all the unvaccinated chickens succumbed to the challenge. This is in full agreement with Allan and colleagues (1978), who stated that there would be no mortality in chickens that were challenged when their HI titres ranged from 4 log2 to 6 log2, with a mean titre of 5.2 log2, but 100% mortality occurred on challenge when the HI titres were 2 log2 or less. Although antigenic heterogenicity was observed among the present ¢eld isolates, the LaSota vaccine provided 100% protection against each of the ¢eld isolates and also against a local laboratory challenge strain. Vaccination against ND is practised in Namakkal, but there are no regulations on vaccination. The success of vaccination depends on several factors, including hygiene, the titre of the vaccine, mistakes in vaccination, etc. (Meulemans, 1988). Despite the supposedly extensive vaccination, in the absence of seromonitoring and with the presence in the ¢eld of immunosuppressive agents, such as infectious bursal disease, which is endemic in Namakkal, a situation appears to have arisen in which there was general breakdown of immunity, so that the prevalent, virulent NDVs were able to decimate the £ocks. Seromonitoring is essential to assess the e¤cacy of vaccines and vaccination, so such seromonitoring and strict biosecurity measures need to be mandatory to maintain the good health of £ocks. Continuous surveillance is also necessary to control ND, as diseased chickens and ducks are potential sources of infection to other poultry. REFERENCES Alexander, D.J., 1988. Newcastle disease diagnosis. In: J.D. Alexander (ed.), Newcastle Disease, (Kluwer Academic Publishers, Boston), 147^160 Alexander, D.J., 1995. Newcastle disease in countries of the European Union. Avian Pathology, 24, 3^10 Allan, W.H., Lancaster, J.E. and Toth, B., 1978. Newcastle Disease Vaccines, Their Production and Use, (FAO, Rome), 2, 63 Bell, J.G., 1986. Velogenic viscerotropic Newcastle disease virus strains in Morocco. Avian Diseases, 30, 231^233 Hanson, R.P., 1972. Newcastle disease virus. In: Hofstad M.S. et al., (eds), Diseases of Poultry, 6th edn, (Iowa State University Press, Ames, IA), 619^656 Hanson, R.P., 1980. Newcastle disease. In: S.B. Hitchner, C.H. Dobermuth, H.G. Purchase and J.E. Williams (eds), Isolation and Identi¢cation of Avian Pathogens, (AAAP, College Station, Texas, USA), 63^66 Hanson, R.P. and Brandly, C.A., 1955. Identi¢cation of vaccine strains of Newcastle disease virus. Science, 122, 156^157 Hanson, R.P., Spalation, J. and Dickinson, E.M., 1967. Criteria for determining the validity of a virus isolation. Avian Diseases, 11, 509^514 Kumanan, K., Elankumaran, S., Vijayarani, K., Palaniswami, K.S., Padmanaban, V.D., Manvell, R.J. and Alexander, D.J., 1992. Characterisation of Newcastle disease viruses isolated in India. Journal of Veterinary Medicine, B, 39, 383^387 Kylasamaier, K., 1931. A study on Madras fowl pest. Indian Veterinary Journal, 8, 340^342 Meulemans, G., 1988. Control by vaccination. In: D.J. Alexander (ed.), Newcastle Disease, (Kluwer Academic Publishers, Boston), 318^332 Nilakantan, R.P., Negi, S.K. and Sakkubai, P.R., 1969. Studies on potency testing of Newcastle (Ranikhet) disease vaccine. Indian Veterinary Journal, 46, 839^842 Piraino, F.P. and Hanson, R.P., 1960. An in vitro method for the identi¢cation of strains of Newcastle disease virus. American Journal of Veterinary Research, 21, 125^127
142 Roy, P. and Balasubramaniam, G.A., 1992. Outbreak of Newcastle disease in an organised farm. Indian Veterinary Journal, 69, 556^557 Roy, P. and Venugopalan, A.T., 1998. Virulence of Newcastle disease vaccine virus(es) in the ¢eld. Tropical Animal Health and Production, 30, 41^44 Roy, P., Venugopalan, A.T. and Chandramohan, A., 1997. Predominance of N.D. virus in the intestinal contents of chicks. Indian Veterinary Journal, 74, 550^558 Russell, P.H. and Alexander, D.J., 1983. Antigenic variation of Newcastle disease virus strains detected by monoclonal antibodies. Archives of Virology, 75, 243^353 Sulochana, S., Pillai, R.M., Nair, G.K., Sudharma, D. and Abulla, P.K., 1981. Epizootiology of Newcastle disease in Indian house crows. Veterinary Record, 109, 249^251 (Accepted: 9 August 1999)
