Diagnostic techniques of Newcastle disease in chickens

Since the recognition of Newcastle disease (ND) in 1926, many countries have regarded ND as an endemic epidemic. With the exception of a few countries that produce poultry at commercial grade, all countries are vaccinated.

One of the most characteristic properties of different strains of chicken Newcastle disease virus (NDV) is that its pathogenicity to chickens is very different. According to the clinical symptoms caused by infected chickens, NDV strains can be divided into five groups or disease types. they are: visceral virulence type, characterized by high pathogenicity, often seen intestinal bleeding; neurophilic virulence type, characterized by high disability rate, often with respiratory and neurological symptoms; moderate virulence type, characterized by respiratory symptoms, occasional neurological symptoms, but low disability rate. Mild or respiratory type characterized by mild or subclinical respiratory infection; asymptomatic intestinal type characterized by subclinical intestinal infection.

The classification of disease types is difficult to distinguish, and the overlap of symptoms can be seen even in infected pathogen-free (SPF) chickens. In addition, mild strains can also cause significant clinical symptoms when co-infected with other microorganisms or when the environment deteriorates.

As the clinical symptoms of chickens after infection are very different, different hosts have different responses to infection, which makes the diagnosis more complicated. Therefore, a single clinical symptom can not be used as the basis for the diagnosis of ND. If there are symptoms and pathological changes related to virulent disease, the disease should be suspected.

Pathogen identification of ●:

1. Sample collection

Samples collected from dead birds include oral and nasal swabs, as well as lungs, air bags, intestines, spleen, brain, liver and heart tissues, which can be collected individually or in combination.

Samples collected from live birds should include trachea and cloacal swabs. In places where there is little chance of sample collection, cloacal swabs (or feces) and tracheal swabs (or tracheal tissues) should be the main except for infected organs and tissues with related clinical symptoms.

two。 Sample treatment

The samples were placed in phosphate buffers containing antibiotics (pH7.0 to 7.4). Antibiotics varied according to specific conditions, but penicillin (2000 units / ml), streptomycin (2mg/ml), kanamycin (50 μ g / ml) and nystatin (1000 units / ml) should be contained in tissue and tracheal swabs, while the concentration of fecal and cloacal swabs should be increased by 5 times. It is important to adjust the pH value to 7.0 to 7.4 after adding antibiotics. Feces and chopped tissue are made into a 10 to 20% suspension with an antibiotic solution. The sample should be processed as quickly as possible, first standing at room temperature for 1 to 2 hours. If there are no conditions, the sample can be kept at 4 ℃ for a few days.

3. Sample inspection

After centrifugation, the supernatant of fecal or tissue suspension was taken, and at least five 9-to 11-day-old SPF embryos were inoculated into the allantoic cavity. The embryos were cultured at 35 ℃ ~ 37 ℃ for 4 to 7 days. The allantoic fluid of dead and dying chicken embryos and those who survived at the end of culture were collected and cooled at 4 ℃, then the HA activity of allantoic fluid was detected. Negative people pass on at least another batch of eggs.

The existence of NDV can be confirmed by specific antisera, usually chicken antisera, which are prepared from one of the NDV strains.

In the experiment, the same serum PBS was continuously doubly diluted, each well 0.025ml, and then 4 hemagglutination units of antigen 0.025ml were added to each well. After 15 to 30 minutes, 1% 0.025ml chicken red blood cells were added, gently mixed, and the results were determined for 45 minutes. The highest dilution of serum which can completely inhibit the four antigens is the titer of hemagglutination inhibition (HI). If the plate is tilted and interpreted, the agglutination test is more accurate. Hemagglutination inhibition is considered only if the red blood cells in those pores have the same "flow" as those without virus control holes.

Some non-chicken bird sera contain non-specific lectins specific to chicken red blood cells. therefore, chicken red blood cells can be used to absorb the test serum to exclude lectins or to use the red blood cells of the animal species studied.

4. Virulence evaluation of isolates

The virulence of different NDV isolates is very different, and because of the wide application of live Newcastle disease vaccine, only the virus isolated from diseased birds with clinical symptoms can not constitute the diagnosis of ND, and the virulence of the isolates should be evaluated. The pathogenicity of NDV isolates cannot be carried out in vitro, so the evaluation of pathogenicity is often based on one or more of the following tests.

A) the average time of death of chicken embryos (MDT)

The fresh infected allantoic fluid was continuously diluted to 6Log10~9Log10 by 10 times of normal saline. Five 9-to 10-day-old SPF chicken embryos were inoculated at each dilution, and each chicken embryo was inoculated with 0.1ml, then placed at 37 ℃. The remaining virus dilution was stored in 4 ℃. After 8 hours, another 5 chicken embryos were inoculated with each dilution. Each chicken embryo was inoculated with 0.1ml and cultured at 37 ℃. The rats were observed twice a day for 7 days. The time of death of each chicken embryo was recorded in each observation.

According to the average death time of chicken embryo, NDV strain can be divided into strong virulence type, moderate virulence type, moderate virulence type and mild type.

B) Intracranial pathogenicity index (ICPI)

The fresh infected allantoic fluid with hemagglutination titer above 4Log2 was diluted 10 times with isotonic aseptic salt solution without additive and was inoculated into the brain of 1-day-old SPF chicks. A total of 10 chickens were inoculated with 0.05ml. The chickens were observed every 24 hours for 8 days. Chickens should be scored every day. Normal chickens should be marked as 0, sick chickens as 1, and dead chickens as 2. The intracerebral pathogenicity index is the average of all observed values in each chicken within 8 days. The intracerebral pathogenicity index of the most virulent virus will be close to the maximum value of 2.0, while the value of mild strain will be close to 0.

C) intravenous pathogenicity index (IVPI)

The fresh allantoic fluid whose hemagglutination titer was higher than 4Log2 was diluted with sterilized isotonic saline (1 ∶ 10). Six-week-old SPF chicks were inoculated intravenously with 10 chicks, each of which was inoculated with 0.01ml. They were observed once a day for 10 days. The score of each observation should be recorded as 0 for normal chickens, 1 for sick chickens, 2 for paralyzed chickens and 3 for dead chickens. The intravenous pathogenicity index refers to the average number of each chicken observed within 10 days. The value of mild strain and some moderately virulent strain is 0, while the value of virulent strain can reach 3.0. But there can also be minor changes to these tests. The cloaca and conjunctiva of 6-week-old chickens were wiped with undiluted allantoic fluid instead of the above test in order to distinguish visceral virulent viruses from other virulent viruses.

● serological test

ND virus can be used as an antigen in various serological tests, such as neutralization test and enzyme-linked immunosorbent assay (ELISA). At present, hemagglutination inhibition test is mostly used. Chicken serum rarely has a non-specific reaction in this test, so the serum should not be pretreated. The sera of other poultry breeds except chicken sometimes have agglutination effect on chicken red blood cells, so this characteristic should be determined before the experiment, and then chicken red blood cells should be used to adsorb the test serum to eliminate non-specific agglutination.

The international ND reference serum is available from the International Biological Standards Laboratory of the Central Veterinary Laboratory in Suriname, UK. Use it to calibrate the laboratory reference serum.

The serological value in diagnosis is related to the immune status of infected poultry. If the serum diluted at 1 ∶ 8 or higher than this dilution can inhibit 4 hemagglutination units, it is judged to be positive.

Hemagglutination inhibition test can be used to evaluate the immune status of chicken flocks. When we are monitoring immunized chickens, it is possible to detect a sudden increase in antibody response, which is caused by wild virus infection, but we should pay attention to changes caused by other causes, such as when turkeys immunized with Newcastle disease virus are reinfected with PMV-3 virus, the titer will increase.