DIAGNOSIS

Clinical signs, gross and histopathological lesions lead to a presumptive diagnosis. Detection of viral antigens in tissues of the respiratory tract and central nervous system by indirect immunofluorescence is of supporting value.

Virus isolation is commonly done in embryonated chicken eggs by inoculation of the allantoic cavity of at least four eggs at day 9—11 of incubation. The inoculum consists of 10% w/v suspensions each derived from brain (cerebrum, cerebellum and medulla oblongata), nasal conchae, trachea, lung, spleen, liver, kidney and intestine. Tissue suspensions can be pooled. Inoculated eggs are further incubated at 37.8°C in a humidified atmosphere without turning. The chorioallantoic membranes, yolk sac and the embryo are checked for macroscopic lesions (haemorrhages, oedema). The obtained allantoic fluids (AF) are individually tested for haemagglutination (HA). The AF are tested in a semiquantitative manner for HA using phosphate- buffered saline as a diluent and a 1% suspension of washed chicken red blood cells. Most but not all AF agglutinate. If embryo death is noted at 24 hours post- inoculation or later but AF failed to aggluti­nate, a passage of AF derived from several embryos is required. The HA test is followed by HI tests using poly­clonal antisera (preferentially raised in chickens) against APMV1 and APMV2 to APMV9. The results of the HI tests allow the allocation of the new isolate to one of the nine APMVs.

As an alternative to the rather simple, rapid and inex­pensive HI test, virus neutralization tests in embryonated eggs or in permissive cell cultures (e.g. chicken embryo fibroblasts) may be used. If the results of the HI tests document the presence of an APMV1, direct electron microscopy of negatively contrasted AF without purification/concentration can be applied as a second independent line of evidence for confirmation of APMV.

Haemagglutinating, embryo-lethal isolates must be immediately submitted to the National Reference Labora­tories (NRL) of the EU member states for confirmation of the diagnosis and determination of virulence of the isolate.

Based on their virulence for chickens, the APMV1s are subdivided on the basis of three in vivo tests into five pathotypes (velogenic, neurotropic, mesogenic, lentogenic and avirulent-enteric). The commonly used three tests provide numerical index values for virulence and, there­fore, ease differentiation between pathotypes of deviating virulence. The intracerebral pathogenicity index (ICPI) test, which is the standard test to determine the patho­genicity, is performed in ten 1-day-old chicks and yields values between zero (avirulent) and 2.0 (highly virulent). The intravenous pathogenicity index (IVPI) test is per­formed in ten 6-week-old chickens and yields indices between zero (avirulent) and 3.0 (highly virulent). The third test is performed in embryonated chicken eggs and provides the time in hours that is required to cause embry­onic death (mean death time, MDT). Highly virulent viruses cause embryo death in less than 50 hours, whereas avirulent viruses do not cause regular embryo mortality within 6 days. These traditional in vivo tests have now been replaced or supplemented by molecular tests that focus on the F protein or proteins of other genes. These include the determination of multiple basic amino acids on the cleavage side of the fusion gene protein and other genes.

Most important are virulent APMV1 that cause high morbidity and mortality in domestic chickens and turkeys and are referred to as Newcastle disease, which is a notifi­able disease in most countries of the world.

The definition published in the Terrestrial Manual of the World Organisation for Animal Health (OIE)⁽¹¹⁾ for an outbreak of ND reflects the current understanding of the molecular basis of virulence:

Newcastle disease is defined as an infection of birds caused by a virus of avian paramyxovirus serotype 1 (APMVl) that meets one of the following criteria for virulence:

(a) The virus has an intracerebral pathogenicity index (ICPI) in day-old chicks (Gallus gallus) of 0.7 or greater.

(b) Multiple basic amino acids have been demonstrated in the virus ( either directly or by deduction) at the C-terminus of the F2-protein and phenylalanine at residue 117, which is the N-terminus of the F1 protein. The term “multiple basic amino acids” refers to at least three arginine or lysine residues between 113 and 116. Failure to demonstrate the characteristic pattern of amino acid residues as described above would require characterisation of the virus by an ICPI test. ”

The term ‘infection’ in this definition of ND means that the virus isolate must be derived from a bird that may or may not present signs of disease, including mortality, lesions or seroconversion. The term ‘birds’ includes all avian species and not only domestic poultry.

By contrast, the OIE definition of ND for the purpose of international trade as published in the Terrestrial Animal Health Codr (2009)(¹²) defines only an infection of poultry. Poultry is defined as ‘all domesticated poultry, including backyard poultry, used for the production of meat or eggs for consumption, for the production of other commercial products, for restocking supplies of game, or for breeding these categories of birds, as well as fighting cocks used for any other purpose.’ In annex III of the amended (in 2004) Council Directive 92/66EEC⁽¹³⁾ ‘Newcastle disease means an infection of poultry caused by any avian paramyxovirus 1 with an intracerebral pathogenicity index in day-old chicks greater than 0.7.’

ANTIBODY DETECTION

Virtually all species of birds produce antibodies that can be detected in serum but also in conjunctival fluid (tears), lavage from respiratory and digestive tracts and in yolk of laid eggs.