Introduction
Newcastle disease (ND) is a viral disease of poultry caused by avian paramyxovirus serotype-1 (APMV-1) viruses belonging to the genus Avulavirus and family Paramyxoviridae. This disease is also known in certain regions as Ranikhet disease, Pseudo fowl pest, Pseudo poultry plague, Avian pest, Avian distemper, Avian pneumoencephalitis and Korean fowl plague. ND is prevalent worldwide and most rampant in Asian countries. ND affects over 250 species of birds (Alexander, Reference ALEXANDER1997) and is a reportable disease. It is economically important because of (i) restriction in trade (ii) loss due to a spectrum of clinical signs viz. drop in egg production, misshapen eggs, rough shelled eggs, paralysis of legs and wings, circling movements, respiratory distress, greenish white diarrhoea, and (iii) mass mortality in fully susceptible flocks. The disease is usually suspected in the field based on clinical signs. The virus can be detected by simple tests such as the hemagglutination (HA) test and the hemagglutination inhibition (HI) test. However, diagnosis of the disease is not always straight forward because of (i) wide use of live vaccines in the field (ii) lack of classical clinical signs in many cases (iii) involvement of concurrent infections viz., E. coli, Pasteurella multocida, Mycoplasma sp., infectious bursal disease virus, infectious bronchitis virus, chicken anaemia virus etc.
Definition for reporting an outbreak
The World Organisation for Animal Health, formerly the Office International des Epizootics, has defined ND as the infection of birds caused by Avian paramyxovirus serotype-1 (APMV-1) that meets one of the following criteria for virulence (i) the virus has an intracerebral pathogenicity index (ICPI) value of 0.7 or more in day old chicks (Gallus gallus) or (ii) the virus has multiple basic amino acids (at least three arginine or lysine residues between 113-116 at C-terminus of F2 protein and phenylalanine at residue 117 at the N- terminus of F1 protein (OIE, Reference OIE2008a).
Presumptive diagnosis is based on clinical signs and post mortem lesions but confirmatory diagnosis is based on laboratory tests. Conventional in vivo tests for the determination of virus virulence are used in many developing countries, but monoclonal antibody and molecular tests are preferred because of their rapidity and specificity.
Diagnosis of Newcastle's Disease
At field level, presumptive diagnosis of ND is usually based on clinical signs. The disease produces a variety of clinical signs involving respiratory, reproductive, nervous, and gastrointestinal systems. Beard and Hanson (Reference BEARD and HANSON1984) classified NDV strains into five pathotypes based on clinical signs observed in experimentally inoculated chickens. Viscerotropic velogenic (VV) NDV produces high mortality and hemorrhagic lesions mainly in the intestine (Doyle's form). Neurotropic velogenic (NV) NDV produces high mortality with respiratory and neurological signs (Beach's form). Mesogenic NDV produces low mortality with respiratory and nervous symptoms in young chickens (Baudette's form). Lentogenic NDV causes mild or in apparent infection in very young chicks (Hitchner's form) and asymptomatic enteric NDV causes inapparent intestinal infections. However, these classifications were shown to be arbitrary. For example, Hert's strain of NDV, which is classified as VVNDV, also produces severe respiratory signs (McFerran and McCracken, Reference MCFERRAN and MCCRACKEN1988).
When laboratory facilities are restricted veterinarians often diagnose the disease on clinical symptoms and the identification of post mortem lesions. But there is no pathognomonic lesion and several birds must be examined before arriving at a tentative diagnosis. Common lesions include haemorrhages on the tracheal mucosa, petechial haemorrhages in the mucosa of proventriculus, haemorrhages and ulcerations of the lymphoid tissues in the intestinal wall, and haemorrhages in the ovaries (McFerran and McCracken, Reference MCFERRAN and MCCRACKEN1988).
Demonstration of NDV specific antigens present in specimens collected from diseased birds does not provide any information about the character of the virus, because the live vaccine virus may be detected instead of the suspected epizootic virus. Live vaccines are widely used to control the disease in many countries. However, demonstration of a virus specific antigen can be used for quick diagnosis in disease free countries or in unvaccinated flocks.
HA and HI tests, using specific antiserum (Alexander, Reference ALEXANDER1988), are widely used for detection of virus specific antigen in affected tissue suspensions or in the allantoic fluid of infected chicken embryos but the shelf-life of chicken red blood cells is short and poses a constraint in regular use. Chicken red blood cells fixed with glutaraldehyde or formaldehyde has been reported to increase shelf life. The fixed cells can be stored in the refrigerator and readily used in the HA and HI tests for at least eight weeks (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998a). The immunofluorescence test has been used for the detection of NDV specific antigen in tissue sections and impression smears (Maestrone and Coffin, Reference MAESTRONE and COFFIN1964; McNulty and Allan, Reference MCNULTY and ALLAN1986). Early infection before the onset of clinical signs can be diagnosed by detection of NDV specific antigen in blood smears by the immunofluorescence test (Roy et al., Reference ROY, VENUGOPALAN and MURALIMANOHAR1997a). Similarly, tests such as Dot-ELISA (Roy and Venugopalan, Reference ROY and VENUGOPALAN1999), agar gel immunodiffusion test and counter immunoelectrophoresis (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998b) have been used to demonstrate the presence of virus specific antigens.
Collection of samples
The collection of samples is another consideration. The samples should be collected from the affected organs of dead or live birds. Pooled samples of spleen, proventriculus, caecal tonsils, bursa and brain may be collected (Parede and Young, Reference PAREDE and YOUNG1990). In partially immunised birds, bursa is the preferred organ for virus isolation (Parede and Young, Reference PAREDE and YOUNG1990). Velogenic virus can invade the brain but some mesogenic strains may not (Singh and El-Zein, Reference SINGH and EL-ZEIN1973). The more virulent strains grow to high titres in kidneys (Alexander, Reference ALEXANDER1988). During a disease outbreak, intestinal contents were found to have the highest concentration of virus compared to other organs (Roy et al., Reference ROY, VENUGOPALAN and CHANDRAMOHAN1997b). In well-immunised birds, the virulent virus can grow in the intestinal tract (Alexander, Reference ALEXANDER1995) and avirulent viruses usually grow primarily in the alimentary tract (Alexander, Reference ALEXANDER1988). OIE (2009/2010) suggest collection of oro-nasal swabs, lung, kidney, intestine, spleen, brain, liver and heart from dead birds. Thus, spleen, proventriculus, caecal tonsils, bursa, brain, kidney and intestinal contents may be collected along with other affected organs from the dead birds. From live birds, cloacal swabs, tracheal swabs and tears are good samples for virus isolation (Alexander et al., Reference ALEXANDER, CAMPBELL, MANVELL, COLLINS, PARSONS and MCNULTY1992; Roy et al., Reference ROY, DHINAKAR RAJ, KOTEESWARAN, NACHIMUTHU and VENUGOPALAN1995; Roy et al., Reference ROY, PARIDA, KOTEESWARAN, VENUGOPALAN and MURALIMANOHAR2001; OIE, 2009/2010).
Virus isolation
Virus isolation is usually done in embryonated chicken eggs as described by OIE (Reference OIE2008a). Any inoculated embryos dying after 24 hours and live embryos beyond 96 hours post-inoculation should be chilled and the amino-allantoic fluid harvested. Virus isolation is confirmed by identifying the virus in amino-allantoic fluid (AAF) by HA tests and then HI tests with known NDV specific antiserum using 1% chicken red blood cells (CRBC) as an indicator system. Difficulties in virus isolation have been recorded. On rare occasions a newly isolated strain may show a minimum or questionable HA titre after a single passage through chicken embryos (Hanson, Reference HANSON1980). The likelihood of recovery increases with higher passage levels (Roy, Reference ROY1995). However, Roy et al. (Reference ROY, VENUGOPALAN and CHANDRAMOHAN1997b) reported cases in which the AAF was negative according to the HA test, but chorioallantoic membranes (CAM) from the same embryos were positive for NDV by the immunoperoxidase test (IPT). Virus was a detected in AAF by HA and HI tests during subsequent passages. Several cell lines have been employed for isolation of NDV. But the MDBK cell line has advantages as it selects the pathogenic isolates and eliminates lentogenic isolates within two or three serial passages (King, Reference KING1993; Roy et al., Reference ROY, VENUGOPALAN and KUMANAN1997c).
Characterisation of NDV isolates
Once the virus is isolated and identified as APMV-1, its virulence for chickens has to be assessed because of the lack of pathognomonic clinical signs even for infections with the most virulent viruses (Aldous and Alexander, Reference ALEXANDER2001), and the wide use of live vaccines in the field.
Widely accepted biological tests are:
(i) Mean death time (MDT) in embryonated hen's egg.
(ii) Intracerebral pathogenicity index (ICPI) in day-old chicks.
(iii) Intravenous pathogenicity index (IVPI) in six-week-old chickens.
With regard to MDT, death within 24 hours should not be considered. Velogenic viruses kill the embryos within 60 hours. Mesogenic viruses kill within 60-90 hours, and lentogenic viruses may or may not kill the embryo beyond 90 hours (OIE, Reference OIE2004a).
More virulent viruses will have ICPI values closer to 2.0 but lentogenic and asymptomatic enteric strains will give values close to zero. The ICPI test is the test accepted by OIE for assessment of the virulence of the virus. When an ICPI value is 0.7 or more, it is considered to be NDV of significance (OIE, 2009/2010).
Virulent NDVs will have IVPI values closer to 3.0, and low or intermediate virulent viruses will have values closer to zero (OIE, Reference OIE2004a).
However, such classification was not always found to be correct. Mesogenic viruses caused disease in six week old chickens during natural outbreaks (Roy and Venugopalan, Reference ROY and VENUGOPALAN1997). Viruses capable of causing severe disease may have IVPI values of zero. Because of this, ICPI values should be used for the assessment of virulence (OIE, Reference OIE2004a).
Monoclonal antibodies (MABS) in diagnosis
MABS provide a powerful tool to detect slight variations in antigenicity, such as single amino acid changes at the epitope to which the antibody is directed. MABS are very useful in diagnosis of disease, differentiation of virulent and vaccine viruses and epidemiological mapping, and are widely used for grouping the NDV isolates. However grouping with MABS was not always accurate. Two virulent NDVs were found to be closer to avirulent NDVs by MAB grouping (Alexander et al., Reference ALEXANDER, CAMPBELL, MANVELL, COLLINS, PARSONS and MCNULTY1992), and these viruses were genetically and antigenically close to viruses of low virulence from which it was assumed they were derived. One NDV isolate from pigeons, responsible for 23 outbreaks in Great Britain, was grouped as E (B1/Lasota viruses) with MABS (Russell, 1988), this was probably the result of vaccination, which was banned in Great Britain at the time. Six velogenic isolates were grouped under D (Komarov viruses), E (B1/Lasota viruses) and F (F-strain), and four mesogenic isolates were grouped under E (B1/Lasota viruses) with MABS (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998c). One isolate from epizootics in pigeon, which was found to be virulent for chickens but could not be grouped with the panel of MABS at the Central Veterinary Laboratory, UK (Roy et al., Reference ROY, VENUGOPALAN and MANVELL2000). Eight velogenic viruses isolated from gut contents of day-old chicks were grouped as lentogenic with MABS. This variation may be due to mutation of surface proteins (epitopes) of viral isolates by virtue of their long co-persistence in the presence of high levels of maternal antibodies in chicks (Roy and Venugopalan, Reference ROY and VENUGOPALAN2005). Possibly further research and development of more MABS will elucidate the present problem.
Molecular methods in diagnosis
Reverse transcriptase (RT) Polymerase Chain Reaction (PCR) is a very important and useful tool for detection and characterisation of NDV. Jestin et al. (Reference JESTIN, CHERBONNEL and ARNAULD1994) used nested PCR to amplify the F0 gene for identifying and characterising NDV isolates. They emphasised the sensitivity of nested PCR, but a disadvantage of nested PCR is the increased potential for contamination. The minimum time required was five days to obtain the nucleotide sequence. Kant et al. (Reference KANT, KOCH, VAN ROOZELAAR, BALK and TER HUURNE1997) used a hemi-nested PCR for detection and differentiation of NDV isolates. The advantages of the hemi-nested system are that the test could be done with tissue homogenates and the test could be completed within a day. However, the test could not detect some avirulent virus infections and could not detect virus isolated form waterfowl or the PMV-1 causing panzootics in pigeons.
Nucleotide sequence analysis of the F0 cleavage site is an accepted test for the identification of virulent viruses by OIE (2009/2010). Most pathogenic virulent viruses have at least three arginine or lysine (multiple basic amino acids) between residues 113-116 at the C-terminus of F2 protein and phenylalanine at residue 117 the N-terminus of the F1 protein (Collins et al., Reference COLLINS, BASHIRUDDIN and ALEXANDER1993; Oberdörfer and Werner, Reference OBERDÖRFER and WERNER1998); Lentogenic strains differed from the above (Collins et al., Reference COLLINS, BASHIRUDDIN and ALEXANDER1993; Toyoda et al., Reference TOYODA, SAKAGUCHI, HIROTA, GOTOH, KUMA, MIYATA and NAGAI1989; Alexander, Reference ALEXANDER2001). But In China, NDVs from three cases had the lentogenic motif 112-G-R-Q-G-R-L-117 in their F0 cleavage sites but ICPI (more than 1.75) and IVPI (more than 2.25) values indicated that they were velogenic (Tan, et al., 2008), and there may possibly have been a mixed populations of viruses. This is why the OIE definition does not allow confirmation of low virulence by cleavage site sequencing alone.
PCR- Restriction Enzyme (RE) analysis of various parts of NDV genome were attempted to differentiate the NDV strains. Wehmann et al. (Reference WEHMANN, HERCZEG, BALLAGI-PORDÁNY and LOMNICZI1997) differentiated nine B1 and thirteen La Sota viruses by amplifying matrix gene and RE analysis by MboI and HinfI. Nanthakumar et al. (Reference NANTHAKUMAR, KATARIA, TIWARI, BUTCHAIAH and KATARIA2000) amplified F gene and differentiated nine NDVs into different pathotypes using RE. The enzyme BglI made fragments of lentogenic strains only. HhaI made fragments of lentogenic and mesogenic strains only, whereas StyI made fragments for all the NDVs used in the experiments. In that study only nine isolates were used and two of them could not be typed. Therefore no conclusion can be arrived at based on this study with such a few isolates.
A real time reverse transcription PCR (RRT-PCR) assay system has been developed as a rapid diagnostic technique. This RRT–PCR assay showed promising specificity and sensitivity to various pathotypes but cannot replace virus isolation completely on an individual sample basis. However, RRT-PCR can be used for flock screening (Wise et al., Reference WISE, SUAREZ, SEAL, PEDERSEN, SENNE, KING, KAPCZYNSKI and SPACKMAN2004). Real time PCR has been used to detect and pathotype the NDV isolates (Farkas et al., 2009) and is used widely in many laboratories. Some NDV (15/15) isolates from pigeon and dove were classified as virulent based upon their fusion cleavage site motif (112-RRKKRF-117) and ICPI values (ranging from 0.98 to1.35) but they escaped detection by the fusion gene-based real-time PCR test (Kim et al., Reference KIM, AFONSO and SUAREZ2006). Based on genotype analysis, NDVs have been classified into nine class I viruses and ten class II viruses, and new classes are evolving which increases the possibility of diagnostic failures. RRT-PCR with matrix gene assay often fails to detect low virulence APMV-1 from waterfowl and RRT-PCR fusion gene assay often fails to detect certain virulent NDV genotypes (Miller et al., Reference MILLER, DECANINI and AFONSO2010). Sequence analysis and pathotyping of virulent NDVs in West and Central Africa revealed the circulation of a new genetic lineage, distinguishable from the other circulating viruses (Cattoli et al., Reference CATTOLI, FUSARO, MONNE, MOLIA, LEMENACH, MAREGEYA, NCHARE, BANGANA, MAINA, KOFFI, THIAM, BEZEID, SALVIATO, NISI, TERREGINO and CAPUA2010).
Control of Newcastle Disease
It is difficult to formulate a control strategy because of wide host range, various routes of transmission, and because the virus is quite stable at environmental temperatures. More than 250 species of birds are susceptible to NDV (Alexander, Reference ALEXANDER1997).Virulent virus has been isolated from ducks and chickens during a devastating disease outbreak in India, and grouped differently with monoclonal antibodies, but La Sota vaccine provided 100% protection (Roy et al., Reference ROY, VENUGOPALAN and MANVELL2000). Many psittacine birds harbour virulent NDV without showing clinical signs and shed the virus in droppings (Roy et al., Reference ROY, VENUGOPALAN, SELVARANGAM and RAMASAMY1998). Birds like the Amazon Parrot may act as carriers and shed the virus for longer than one year (Cubas, Reference CUBAS1993). Conures excreted virus up to 84 days post exposure and parrots excreted for up to 376 days (Erickson et al., Reference ERICKSON, NUN, MARE, GUSTAFSON, MILLER, PROTOR and CARBREY1978). The virus can survive for more than eight weeks in hot dry tropical areas at an ambient temperature of 40°C (Warner, Reference WARNER1989) and three months in temperature of 20-30°C (Lancaster, Reference LANCASTER1966). These birds carrying virulent NDV pose a potential threat to susceptible commercial chickens.
The control policy usually depends on several requirements. These include the following.
(i) Reporting of outbreaks: Outbreaks of NDV are to be reported to an international agency - e.g. OIE to undertake adequate measures.
(ii) Restriction of import and export of poultry and poultry products.
(iii) Veterinary administration in NDV-free countries prohibit importation or transit through their territory from NDV infected countries of domestic and wild birds, day-old birds, hatching eggs, semen of domestic or wild birds, fresh meat of domestic or wild birds, unprocessed meat products and products of animal origin used in animal feed or agriculture
(iv) Restriction in movement of personnel such as consultants may be restricted to not visiting NDV affected flocks and subsequently susceptible flocks (OIE - Terrestrial Animal Health Code, 2004b).
A country or a region may be considered free from ND when NDV infection has not been present for the past 12 months, based on surveillance. If an infection has occurred in a previously ND-free country or region, ND free status can be regained three months after an eradication policy (including disinfection of all affected establishments) is applied, providing that surveillance has been carried out during that three-month period (OIE - Terrestrial Animal Health Code, 2008b; 2009/2010)
At the farm level the control strategies include,
(i) Slaughtering or stamping out of affected flocks and cleaning infected premises.
(ii) Ring vaccination to control the spread of infection
(iii) Quarantine of premises for at least 35 days and seroconversion may be checked in SPF sentinel chickens after keeping them on the premises for 28 days.
(iv) Strict bio-security measures include proper disposal systems, restriction of visitors and movement of personnel, avoiding access of wild birds, aquatic birds and other animals inside the farm, strict sanitary measures, providing good quality feed and water and vaccination: The disease in most developing countries is controlled by vaccination (OIE - Terrestrial Animal Health Code, 2004b).
Vaccination
ND is endemic in many countries and an eradication policy cannot be practiced because of wide and sporadic incidence of the disease. Several live lentogenic, live mesogenic, and inactivated vaccines are widely used to contain the disease. But the success of vaccination depends on several factors viz. quality and dose of vaccine, route of vaccination, age of birds at vaccination, interval between revaccination and intercurrent infection and presence of immunosuppressive agents. Although the use of live vaccines is being followed in many countries, the EU restricts the use of live vaccines in member states by legislation against viruses with an ICPI less than 0.4 [EEC directive (93/152/EEC)]. Use of live vaccine is an important concern. It is possible that live vaccine virus could gain virulence in the field because of continuous passage among chickens, as observed based on a study of antigenic relatedness (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998c). Disease outbreaks in Australia during 1998-2000 was due to NDVs closely related to the lentogenic strain known as Peat's Ridge strain and considered to be a precursor of virulent NDV (Dean Park strain) related based on amino acid sequence analysis of F protein which caused outbreaks in 1998-2000(Westbury, Reference WESTBURY2001). It is not advisable to use live vaccines with high ICPI values such as Komarov and Mukteswar. The OIE (2009/2010) recommend seed virus for live vaccine should have ICPI values of less than 0.4 i.e. viruses such as Ulster 2C, Hitchner B1 and V4.
Resistance to infection is usually associated with the development of antibodies against the hemagglutinin neuraminidase (HN) and fusion proteins (Meulemens et al., 1986). In endemic areas, it is advisable to use live lentogenic vaccines for primary vaccination through the oculonasal route to develop local immunity followed by booster vaccination with inactivated vaccine to develop systemic immunity. Antibodies appear in serum and in local secretions six to ten days after vaccination with live vaccine, and persist for various periods of time (Gough and Alexander, Reference GOUGH and ALEXANDER1973; Allen and Gough, 1976; Meulemens, 1988). The HI titre is a reliable indicator of immunity (Eidson and Kleven, Reference EIDSON and KLEVEN1980). In rare cases, protection against the disease can be observed even in the presence of low levels or absence of HI antibodies. This protection can be explained by cell-mediated immunity (CMI), which can be demonstrated as early as two days after vaccination (Ghumman and Bankowski, Reference GHUMMAN and BANKOWSKI1976; Timms and Alexander, Reference TIMMS and ALEXANDER1977). Among live lentogenic vaccines, La Sota strain has greater spreading potential. It is more advantageous to use B1 (milder strain than LaSota) for primary vaccination, and La Sota for subsequent vaccination (Meulemans, Reference MEULEMANS1988). Live mesogenic vaccines such as the Roakin, Komarov, H and Mukteswar strains are widely used in Africa, South East Asia and Middle East (Meulemans, Reference MEULEMANS1988).
Oil adjuvants have been used in live virus vaccines (Peleg et al., Reference PELEG, SAMINA and BRENNER1993; Roy and Venugopalan, Reference ROY and VENUGOPALAN1998d; Roy et al., Reference ROY, VENUGOPALAN and KOTEESWARAN1999). The advantages of adjuvanted live vaccines are that the live viruses escape from the oil environment, infect and initiate immune response. Alternatively, viruses trapped in the oil environment become killed and released at a constant rate to provide booster immunity (Peleg et al., Reference PELEG, SAMINA and BRENNER1993). Adjuvanted live vaccine, which is given by the parenteral route, is not preferred for primary vaccination because of poor local immunity (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998d). The advantages of live vaccine include no interference with the level of maternal antibodies, the live adjuvanted vaccines are processed by endogenous proteolytic pathways and antigens are presented in association with MHC class I molecule on target cells which stimulate CD8+ T cells. But for the killed viruses (trapped in an oil environment), uptake is slow and processed by exogenous pathways. The processed peptide is presented in association with class II molecules on the antigen presenting cells which stimulate CD4+ T cells to act as helper cells for B cells, thus causing greater antibody production (Roy and Venugopalan, Reference ROY and VENUGOPALAN1998d).
Drinking water vaccination is useful for mass vaccination but does not always result in a high enough immune response in a significant portion of the flock. When vaccination is done through the oral route, infectivity of viruses reduce to 1000 fold as they pass through acidic pH (around 2.6) of the gizzard (Kohn, Reference KOHN1958). In practice, while drinking the birds dip their beak in the water and the vaccine virus passes through the nostrils, but the vaccines are often over diluted in drinking water and the birds do not receive the required dose. Aerosol vaccination is widely used in developing countries for large-scale vaccination, and can be used when maternal antibodies are present (Samberg et al., Reference SAMBERG, HORNSTEIN, CUPERSTEIN and GOTTFRIED1977). Aerosol vaccination is not used in Asia where poultry are reared in an open house system.
In ovo vaccination of chickens is an effective way to deliver vaccines and develop early post hatch protective immunity (Steel, 2008). Universal vaccination of commercial poultry delivered via the egg has been considered a major front in the war against NDV (Dilaveris et al., Reference DILAVERIS, CHEN, KAISER and RUSSELL2007).
A general approach to produce an NDV marker vaccine was to modify an immunogenic protein in such a way that it no longer induces antibodies to all of its epitopes. If a MAB against the particular epitope is available, it can be used to discriminate between vaccinated and infected animals in a suitable serological test. However, a major obstacle is that the biological function of the modified protein must be preserved. A vaccine that can be distinguished serologically from a field strain is called a marker vaccine or DIVA (Differentiation of Infected from Vaccinated Animals) vaccine. Marker vaccines were developed by deleting or replacing an immunodominant epitope on the nucleoprotein (NP) gene of NDV by a foreign epitope (Mebatsion et al., Reference MEBATSION, MARCK, KOOLEN, LEONIE, NIELS, MARIAN, ANGELA, PAUL and PIETER2002). The NP of NDV functions primarily to encapsidate the virus genome for the purpose of RNA transcription, replication, and packaging. This conserved multifunctional protein is also efficient in inducing NDV-specific antibody in chickens.
Several vaccines have been tried experimentally, but in many Asian countries where ND is endemic, live lentogenic, mesogenic and inactivated vaccines are widely used. Usually primary vaccination is done at five to seven days of age with B1 strain oculonasally, followed by La Sota in drinking water at four weeks age. The next vaccination is done with a mesogenic strain at 8-12 weeks of age subcutaneously. This vaccine is repeated just before lay or inactivated vaccine is used. There-after La Sota vaccine in drinking water is administered at 10-week intervals until 72 weeks of age.
Seromonitoring
In spite of vaccinations, birds often do not develop adequate immunity and the reasons are many fold. Hence, it is necessary to assess the level of immunity after vaccination to ensure that birds are protected adequately against the disease. The HI test is an OIE accepted test for the assessment of antibody levels against NDV. Allan et al. (Reference ALLAN, LANCASTER and TOTH1978) have described the relationship between the HI test and challenge. Birds with HI titre of 22 or less suffered 100% mortality on challenge, birds with HI titres of 22-25 had 10% mortality, 26-28 had serious egg production losses but those with titres of 29-211 remained normal on challenge.
Seromonitoring also helps in formulating a vaccination schedule. When chickens have high levels of maternal antibody, the primary vaccination should be delayed otherwise the vaccination may be a failure. The half-life of maternal antibody is 4.5 days (Allan et al., Reference ALLAN, LANCASTER and TOTH1978). In Taiwan, a co-operative program was organized between the poultry breeding and the livestock disease control centres (LDCC) and shipping chick boxes were labelled with the exact level of maternal antibodies against ND for the newly hatched chicks that was found helpful to decide vaccination schedule. The research indicated that if the chick's maternal antibody level was higher than 1:32 (log25), infection was prevented and vaccination was inappropriate. When the maternal antibody titre was lower than 1:32, it was appropriate to induce immunity by vaccination (Lee, Reference LEE1987).
The HI test is an easy and widely accepted test, however problems can arise collection of serum from birds because of non co-operation by the farmers. Storage and transportation from the field to the laboratory pose additional problems. A filter paper sampling technique (Brugh and Beard, Reference BRUGH and BEARD1980; Roy et al., Reference ROY, NACHIMUTHU and VENUGOPALAN1992) has proven to be a solution to this problem. Blood is adsorbed on to Whatman no.1 filter paper, air dried and dispatched by ordinary mail, without preservative, to a nearby laboratory for screening antibodies. Brij-35 (Polyoxyethylene 23 lauryl ether) solution was used to eluate antibody from the sample area of the filter paper and was shown to be equivalent to 1: 16 dilution of whole serum (Roy et al., Reference ROY, NACHIMUTHU and VENUGOPALAN1992). The eluate was used as alternative to serum in the HI test. About 35,000 blood samples were collected on filter paper from a poultry-producing region in India where ND is endemic and layer birds were vaccinated regularly. The samples were screened for antibody levels against NDV by the HI test. There was no drop in egg production in flocks when the HI titre was 27 or more (Roy et al., Reference ROY, CHANDRAN, RAJA and MANICKAM2005).
The passive hemagglutination (PHA) test yielded similar results to the HI test for assessments of antibodies against NDV. PHA has several advantages in that tagged chicken red blood cells (CRBC) can be stored for more than 8 weeks, variation in preparation of 4 HA or 8 HA unit and 1% CRBC can be avoided and results can be obtained within 40 minutes (Roy and Venugopalan, Reference ROY and VENUGOPALAN2000).
For layer flocks, antibody levels in egg yolks can be assessed by the HI test. Antibodies from egg yolk can be extracted using chloroform (Piela et al., Reference PIELA, GULKA, YATES and CHANG1984) or Brij-35 solution (Roy et al., Reference ROY, ANANDAN, RAVIKUMAR, KOTEESWARAN and VENUGOPALAN1997d) and used as an alternative to serum in the HI test. Once egg yolk is extracted it can be adsorbed onto Whatman no 1 filter paper and after air drying can be screened for antibody levels after 7 days (Roy et al., Reference ROY, ANANDAN, RAVIKUMAR, KOTEESWARAN and VENUGOPALAN1997d) This is helpful for screening samples from remote places.
Conclusions
Newcastle disease continues to be a problem in many developing countries. Conventional methods of diagnosis are slow but affordable in field level laboratories. HA and HI tests are widely used but regular collection and preparation of chicken red blood cells is cumbersome and can be replaced with fixed chicken red blood cells where facilities are not available. The correct specimens should be collected from live or dead birds for effective isolation and identification of virus. Molecular tests are quick, sensitive and do not require use of live animals or birds or embryos but are costly for small field level laboratories. They have failed to determine the virulence of the virus correctly in some reported cases, thus virus isolation remains the gold standard. Monoclonal antibodies are useful for quick epidemiological mapping but on some occasions this was found to be not effective. This paper discussed issues including the correct specimens to be collected, different sero-diagnostic tests and their modifications for effective diagnosis of the disease at small field level laboratories in developing countries and control by vaccination and seromonitoring. Future work is needed for an accurate multiplex PCR for quick diagnosis and pathotyping of field isolates. Possibly tests such as lateral flow with different monoclonal antibodies will be more useful for field level diagnosis of NDV. Control requires strict biosecurity, thermostable effective vaccines for wild and feral birds, a vaccine for aquatic birds and DIVA vaccine for commercial chickens. Seromonitoring is highly essential and filter paper sampling followed by HI test is promising.
Acknowledgements
The author is thankful to Dr D.J. Alexander, Avian Virology, AHVLA Weybridge, Addlestone, Surrey KT15 3NB, UK for reading and offering valuable comments in this paper.
