Diagnostic Methods and Technical Key Points for Newcastle Disease in Chickens

Since Newcastle disease (ND) was recognized in 1926, many countries have regarded ND as an endemic disease. Except for a few countries that produce poultry at a commercial level, all countries carry out preventive immunization.

One of the most characteristic properties of different strains of chicken Newcastle disease virus (NDV) is that their pathogenicity to chickens varies greatly. Based on the clinical symptoms caused in infected chickens, NDV strains can be divided into 5 groups or pathotypes, which are: the viscerotropic velogenic type, characterized by high pathogenicity, with intestinal hemorrhagic lesions commonly seen; the neurotropic velogenic type, characterized by high mortality, often with respiratory and nervous symptoms; the mesogenic type, characterized by respiratory symptoms, occasionally nervous symptoms, but low mortality; the lentogenic or respiratory type, characterized by mild symptoms or subclinical respiratory infection; the asymptomatic enteric type, characterized by subclinical enteric infection.

The division of pathotypes is difficult to clearly delineate, and even in infected specific-pathogen-free (SPF) chickens, overlapping of symptoms of various types can be seen. In addition, when there is mixed infection with other microorganisms or when the environment deteriorates, mild strains can also cause obvious clinical symptoms.

Because the clinical symptoms after infection in chickens vary greatly, and different hosts respond differently to infection, this makes diagnosis more complicated. Therefore, a single clinical symptom cannot serve as the basis for diagnosing ND. If symptoms and lesions related to the virulent pathotypes appear, the disease should be suspected.

●Pathogen identification:

1. Sample collection

Samples collected from dead poultry include oronasal swabs, as well as lung, air sac, intestine, spleen, brain, liver and heart tissues; these samples can be collected individually or mixed.

Samples collected from live poultry should include tracheal and cloacal swabs. In places where opportunities for sample collection are few, in addition to infected organs and tissues or those with related clinical symptoms, the main samples should be cloacal swabs (or feces) and tracheal swabs (or tracheal tissue).

2. Sample processing

Samples are placed in phosphate-buffered saline (pH 7.0 to 7.4) containing antibiotics; the antibiotics vary according to specific circumstances, but for tissue and tracheal swabs they should contain penicillin (2000 units/ml), streptomycin (2 mg/ml), kanamycin (50 μg/ml) and nystatin (1000 units/ml), while for feces and cloacal swabs the concentrations should be increased 5 times. After adding antibiotics, it is very important to adjust the pH to 7.0 to 7.4. Feces and homogenized tissues should be made into a 10 to 20% suspension with antibiotic solution. Samples should be processed as quickly as possible, first standing at room temperature for 1 to 2 hours. If conditions do not permit, samples can be stored at 4°C for several days.

3. Sample examination

After centrifugation of the feces or tissue suspension, the supernatant is collected and inoculated into the allantoic cavity of at least 5 9- to 11-day-old SPF chicken embryos, and incubated at 35°C to 37°C for 4 to 7 days. Allantoic fluid is collected from dead and dying embryos as well as from embryos surviving at the end of culture, chilled at 4°C, and then the HA activity of the allantoic fluid is tested. Negative samples should be passaged at least once more using another batch of eggs.

The presence of NDV can be confirmed with specific antisera, usually chicken antisera, which are prepared using one of the NDV strains.

In the test, the same serum is serially diluted twofold in PBS, 0.025 ml per well, then 0.025 ml of antigen containing 4 hemagglutinating units is added to each well; after 15 to 30 minutes, 0.025 ml of 1% chicken red blood cells is added, gently mixed, and the result is determined after 45 minutes. The highest dilution of serum that can completely inhibit 4 units of antigen is the hemagglutination inhibition (HI) titer. Tilting the plate for reading makes the agglutination test more accurate. Only when the red blood cells in those wells show the same "teardrop" flow as the red blood cells in the virus-free control wells is it considered hemagglutination inhibition.

Sera from some non-chicken avian species contain nonspecific agglutinins specific to chicken red blood cells; therefore, test sera can be absorbed with chicken red blood cells to remove the agglutinins, or red blood cells from the animal species under study can be used.

4. Evaluation of isolate virulence

The virulence of different NDV isolates varies greatly, and because live Newcastle disease vaccines are widely used, identifying the virus isolated only from diseased birds with clinical symptoms cannot constitute a diagnosis of ND; the virulence of the isolate should also be evaluated. The pathogenicity of NDV isolates cannot be determined in vitro, so virulence assessment is often determined based on one or more of the following tests.

a) Mean death time of chicken embryos (MDT)

Fresh infected allantoic fluid is serially diluted 10-fold in physiological saline to 6Log10 to 9Log10; each dilution is inoculated into 5 9- to 10-day-old SPF chicken embryos, 0.1 ml per embryo, and then placed at 37°C. The remaining virus dilutions are stored at 4°C; after 8 hours, each dilution is inoculated into another 5 chicken embryos, 0.1 ml per embryo, and cultured at 37°C. Observe twice daily for a total of 7 days. The time of death of each chicken embryo should be recorded at each observation.

Using the mean death time of chicken embryos, NDV strains can be divided into the velogenic type, with a death time less than or equal to 60 hours; the mesogenic type, with a death time between 61 and 90 hours; and the lentogenic type, with a death time greater than 90 hours.

b) Intracerebral pathogenicity index (ICPI)

Fresh infected allantoic fluid with a hemagglutination titer above 4Log2 is diluted 10-fold with sterile isotonic saline solution, without additives, and inoculated intracerebrally into 1-day-old SPF chicks, 10 in total, 0.05 ml per chick. Observe the chicks once every 24 hours for a total of 8 days. At each daily observation, the chickens should be scored: normal chickens are recorded as 0, sick chickens as 1, and dead chickens as 2; the intracerebral pathogenicity index is the average of all observation values for each chicken over 8 days. The intracerebral pathogenicity index of the most virulent viruses will approach the maximum value of 2.0, while that of lentogenic strains approaches 0.

c) Intravenous pathogenicity index (IVPI)

Fresh allantoic fluid with a hemagglutination titer above 4Log2 is diluted 1:10 with sterilized isotonic saline and inoculated intravenously into 6-week-old SPF chicks, 10 birds, 0.01 ml per bird. Observe once daily for a total of 10 days. At each observation, score: normal chickens as 0, sick chickens as 1, paralyzed chickens as 2, and dead chickens as 3; the intravenous pathogenicity index refers to the average of the records for each chicken at each observation over 10 days. The value for lentogenic strains and some mesogenic strains is 0, while virulent strains can reach 3.0. However, these tests may also have minor modifications. Instead of the above tests, undiluted allantoic fluid is used to swab the cloaca and conjunctiva of 6-week-old chickens, with the aim of distinguishing viscerotropic velogenic viruses from other velogenic viruses.

●Serological tests

ND virus can be used as an antigen in various serological tests, such as neutralization tests and enzyme-linked immunosorbent assay (ELISA). At present, the hemagglutination inhibition test is mostly used. Chicken serum rarely shows nonspecific reactions in this test, so the serum does not need pretreatment. Sera from avian species other than chickens sometimes agglutinate chicken red blood cells, so this property should first be determined before the test, and then the test serum should be absorbed with chicken red blood cells to remove nonspecific agglutination.

International ND reference serum can be obtained from the International Biological Standards Laboratory of the Central Veterinary Laboratory in Surrey, England. It is used to calibrate the laboratory's reference serum.

In diagnosis, the serological value is related to the immune status of the infected poultry. If serum diluted 1:8 or higher can inhibit 4 hemagglutinating units of antigen, it is judged as positive.

The hemagglutination inhibition test can be used to evaluate the immune status of a flock. When we are monitoring a vaccinated flock, it is possible to detect a suddenly rising antibody response, which is caused by wild virus infection, but attention should be paid to changes caused by other reasons; for example, when Newcastle disease virus-vaccinated turkeys are reinfected with PMV-3 virus, the titer will rise.