Systematic Veterinary Virology

NEWCASTLE DISEASE

Field diagnosis

• T entative diagnosis of ND made on the basis of history, clinical signs, and gross lesions

Virus isolation and identification

• Clinical materials: Samples from dead birds should consist of oro-nasal swabs, as well as samples collected from lung, kidneys, intestine (including contents), spleen, brain, liver and heart tissues. These may be collected separately or as a pool.
• Virus isolation: virus isolation is attempted by inoculating 9- to 11-day-old embryonating chicken eggs. Amnioallantoic fluid (AAF) is collected from all embryos dying after 24 hours postinoculation and tested for hemagglutination (HA) activity. If positive, the hemagglutination-inhibition (Hl) test is used with known NDV-positive serum to confirm the presence of NDV in the AAF. If NDV is found, it is characterized by inoculating 4- to 6-week-old chickens free of ND antibodies with the suspect AAF by swabbing the cloaca, instilling into the nares or conjuctival sac, or injecting into the thoracic air sac. If VVND virus is present, the inoculated chicks usually die in 3 to 7 days, revealing typical visceral lesions on postmortem examination. Neurotrophic viruses will cause severe neurologic and respiratory signs in inoculated chickens but no visceral lesions. If no bird dies in 10 days, the APMV-1 is not considered to be the velogenic, viscerotropic type but is either a lentogenic or mesogenic.
• Nucleic acid identification methods: The virus can also be identified from clinical materials and AAF by RT-PCR (Reverse transcriptase Polymerase chain reaction) using primers for F gene.
• Nucleic acid probes:
• ELISA techniques: Antigen capture and Antigen competitive ELISA techniques can also be used to identify APMV-1 from clinical materials and AAF.

Pathotyping

Newcastle disease virus are classified into Velogenic, mesogenic and lentogenic based on following three tests

• a. Intracerebral pathogenicity index (ICPI) in day old chickens. 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
• b. Intravenous pathogenicty index (IVPI) in 6 weeks old specific pathogen free (SPF) chickens. 0.1 ml of the diluted virus is injected intravenously into each of ten 6-week-old SPF chickens. Birds are examined at 24-hour intervals for 10 days and scored at each observation - 0 if normal, 1 if sick, 2 if paralysed or showing other nervous signs, and 3 if dead. The intravenous pathogenicity index (IVPI) is the mean score per bird per observation over the 10-day period. Lentogenic strains and some mesogenic strains will have IVPI values of 0, whereas the indices for virulent strains will approach 3.0.
• c. Mean death time (MDT) in embryonated eggs. The mean death time (MDT) in eggs means time in hours for the minimum lethal dose to kill all the inoculated embryos. 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).
• d. Molecular typing by studying the aminoacid between 112-119^th position in F gene.
• e. Panels of MAbs are also used to establish antigenic profiles of ND virus isolates based on whether or not they react with the viruses.

Serology

• A number of tests are performed to identify antibodies against NDV in chickens. The two common tests are haemagglutination inhibition (HI) test and ELISA. HI test is preformed in most of the laboratories. 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. A titre of log₂3 is indicative of protection and a titre of log₂6 or more suggests a recent infection by the virus. 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

Monoclonal antibody based HI

• Mouse monoclonal antibodies (MAbs) directed against strains of ND virus have been used in HI tests to allow rapid identification of ND virus without the possible cross-reactions with other APMV serotypes that may occur with polyclonal sera. MAbs have been produced that give reactions in HI tests that are specific for particular strains or variant ND virus isolates.