The methods used in the diagnosis of ND are detailed in the OIE Manual of Standards (OIE 2000a) and prescribed for European Union countries in Directive 92/66/EEC (CEC, 1992). Detailed descriptions of the tests which are outlined below can be obtained from these publications.
CLINICAL SIGNS AND LESIONS
For a definitive diagnosis of ND, both virus isolation and laboratory characterisation are necessary. Nevertheless, if the disease is known to be present in a given area, signs and lesions may be considered highly suggestive, especially for village chickens. Typical clinical signs are: a state of prostration and depression in the birds, with ruffled feathers; greenish white diarrhoea; and, in survivors, the head turned to one side, a condition known as torticollis is very often seen, as are paralysis of the legs, wings or other neurological signs. Other typical characteristics of the disease include: rapid spread; death within 2-3 days; a mortality rate of over 50 percent in naïve populations; and an incubation period of 3-6 days or, on rare occasions, 2-15 days (Beard and Hanson, 1984).
On necropsy, typical lesions are mucus in the trachea, and usually haemorrhages in the intestine, particularly in the proventriculus. It should be borne in mind that all the preceding signs and lesions can be caused by other diseases.
SEROLOGICAL DIAGNOSIS
In the absence of vaccination, the presence of specific antibodies against the ND virus indicates that the bird has been infected by the virus at some time, but not necessarily that it was suffering from the disease at the time of sampling. In practice, a high antibody titre is indicative of a recent infection. Two methods are used to measure antibody titres: the haemagglutination inhibition (HI) test, and the enzyme-linked immunosorbent assay (ELISA). For both, it is necessary to collect blood samples from the chickens. Catching village chickens for this purpose can present a problem. There are two approaches: where overnight housing is used, they can be retained in the morning; or children can be persuaded to catch them. Blood samples are taken from the wing veins - a detailed description of the method is given in Alders and Spradbrow (2001a). Cocks are usually harder to sample than hens. Blood can be drawn directly into a syringe, or collected into a tube after piercing the vein with a needle. In both cases, the sample is subsequently placed nearly horizontally to allow clotting and to permit separation of the serum sample, which should be straw coloured. The serum sample should be kept cool until it can be frozen in the laboratory.
The haemagglutination inhibition test
The HI test is based on the principle that the haemagglutinin on the viral envelope can bring about the agglutination of chicken red blood cells and that this can be inhibited by specific antibodies. V-bottomed microtitration plates are used. The serum samples are diluted in serial twofold dilutions in phosphate buffered saline and then a fixed quantity of viral antigen is added to each well. Usually 4 Haemagglutination Units are used, according to the method of Allan and Gough (1974). Following incubation, a suspension of red blood cells is added to each well and the plate is incubated again. In the absence of any antibody against the virus, haemagglutination occurs, appearing as a diffuse red colour at the bottom of the well. In the wells where the antibody against the virus is of a sufficient level, haemagglutination is inhibited and the red blood cells sediment and appear as a small pellet at the bottom of the well. The presence or absence of agglutination is accurately assessed by tilting the plates. Only those wells in which the RBCs stream at the same rate as the control wells (containing RBCs and PBS only) should be considered to show inhibition. The HI titre is the reciprocal of the highest dilution of serum which completely inhibits haemagglutination and is usually and most conveniently expressed as the logarithm to the base 2. Although the test is difficult to standardise between laboratories, the HI titre gives an indication of the immune status of the bird. A titre of log23 is indicative of protection and a titre of log26 or more suggests a recent infection by the virus. If no vaccination has taken place, diagnosis of the infection can be made on this basis, although it cannot be determined exactly when it took place. Sequential samples taken at different times can indicate whether the titre is rising - indicative of a recent infection - or declining.
When the HI titre is used as a measure of immunity (for example, when testing flock immunity following vaccination), it is recommended that an avirulent strain such as V4 or Ulster 2C be used as the viral antigen. The La Sota antigen has been found to be unsuitable for this purpose when vaccination is done by the same strain as it results in an overestimation of protective serum antibody titres (Maas, et al. 1998).
The ELISA works on the principle of recognition of anti-NDV antibodies, attached to a plate coated with viral antigen, by antibodies produced in another species against chicken antibodies. This anti-chicken antibody is conjugated to an enzyme that catalyses a reaction, causing a change of colour which can then be read quantitatively on a photo spectrometer designed to read microtitration plates. The Animal Production & Health Section of the Joint FAO/IAEA Division has produced an ELISA kit for antibodies against NDV which is designed to be easily transportable and give uniform results under widely varying ambient temperatures. HI and ELISA titres show a good degree of correlation and ELISA titres can be interpreted in a similar way to HI titres (Bell, et al., 1991).
VIRUS ISOLATION
The definitive diagnosis of ND is done through isolation and identification of the virus (Alexander, 1998). Tracheal and cloacal swabs are good sources of virus for isolation from living birds without having to kill them. A cotton-covered stick is inserted into the trachea or cloaca, and then put into a vial containing phosphate buffered saline plus penicillin and streptomycin. It is important to ensure that cloacal swabs are coated with faeces. These samples must be kept cool during transport to the laboratory where they should be stored at 4°C if they are to be processed within 48 hours or frozen at least at -20°C until the isolation attempt. Although cloacal swabs or faeces should always be sampled, virus can also be isolated from homogenised organs from dead birds, chosen to reflect the clinical signs. Nine-day-old embryonated fowls' eggs are injected with 0.1 mL of the suspension into the allantoic cavity and returned to incubation. The eggs are candled twice daily. As dead eggs occur, they are chilled, together with d all eggs after 5-7 days incubation, are chilled at 4°C at which point the allantoic fluid is then harvested and tested for its ability to haemagglutinate chicken red blood cells. Diagnosis is based on the inhibition of haemagglutination by specific anti-NDV serum. This proves infection of the bird by the virus, but does not indicate whether the virus is a pathogenic or avirulent strain.
PATHOGENICITY TESTS
The full diagnosis of ND requires an assessment of the virulence of the virus. As indicated above (Section 1.4), the current definition allows either molecular characterisation by nucleotide sequencing and deduction of the F0 cleavage site amino acid sequence or an in vivo estimation of virulence.
The recommended in vivo test is the intracerebral pathogenicity index (ICPI) test in day-old chicks (OIE 2000a). This involves the inoculation of virus derived from fresh infective allantoic fluid into the brain of ten day-old chicks from specific pathogen-free parents. Each bird is examined at 24-hour intervals for eight days and graded zero if normal, one if sick and two if dead. The index is the mean score per bird per observation over the 8-day period. The most virulent viruses give ICPI values approaching the maximum score of 2.0, while lentogenic viruses give values of, or close to, 0.0.
Where day-old chicks are not available, the mean death time (MDT) in eggs (i.e. the mean time in hours for the minimum lethal dose to kill all the inoculated embryos), can be used as a guide to virulence. The MDT has been used to classify ND virus strains into velogenic (taking under 60 hours to kill); mesogenic (taking 60 to 90 hours to kill); and lentogenic (taking more than 90 hours to kill).
