Chicken Newcastle disease, also known as "pseudo-chicken plague" and "Asian chicken plague", its pathogen is paramyxovirus, and its main clinical features are neurological disorders, dyspnea, diarrhea, mucosal or serous hemorrhage. On-site quarantine includes epidemiological investigation, live quarantine, and carcass quarantine. The common test methods for the quarantine of Chicken Newcastle disease are virus isolation and identification, and blood coagulation inhibition test. The former is used for the diagnosis of Newcastle disease; the latter is used for immune antibody assays and immune surveillance for Newcastle disease.
1. Field quarantine
1. Epidemiology. Chickens are the most susceptible to the disease, and chickens of all breeds and ages are susceptible, and the susceptibility of young and middle chicks is higher than that of adult chickens. In recent years, there have been reports of diseases in quail, pigeons and ornamental birds in China. The main source of infection of the disease is sick chickens and poisonous birds during intermittent epidemics, which are transmitted by a large number of viruses excreted outward through the respiratory and digestive tracts. The disease can occur all year round, but it is more common in winter and spring. Newcastle disease may also occur in immunized flocks, with atypical symptoms, but the incidence and case fatality rates of chicks and adult chickens are not high.
2. Live quarantine. (1) The most acute type: sudden onset, rapid death without significant symptoms. (2) Acute and chronic type: sick chickens manifest as obvious cough, runny nose, difficulty breathing, breathing with their mouths open, and making a "clucking" sound. There is mucus in the mouth, and it is common to shake the head and swallow in an attempt to throw the mucus out. The sac has a liquid viscous substance that flows out of the mouth when lifted upside down. Stool is often yellow-green or yellowish-white, with occasional bloody stools. Sick chickens with a slightly longer course of illness have neurological symptoms such as paralyzed feet and wings, crooked head and neck, and unstable standing. (3) Atypical Newcastle disease: mainly occurs in immune flocks, due to low immune levels or immune disorders, young chickens and mature chickens have mild respiratory symptoms. Egg production in adult chickens declines, and sporadic deaths persist.
3. Quarantine of carcasses. The main lesions are swelling, bleeding, or necrosis of the lymphatic system, and bleeding spots and bleeding spots in the mucous membranes and/or serous membranes of the whole body. Typical lesions: (1) bleeding, ulceration or necrosis in and around the glandular gastric mucosal nipple, and bleeding spots or blood bands are common under the stratum corneum of the gastrointestinal tract; (2) enlargement, bleeding or necrosis of the cecal tonsils. The intestinal mucosa has bleeding spots of varying sizes as well as fibrous exudates. Some people with a longer course of illness form a false membrane, which becomes a "jujube nucleus-like" ulcer after the false membrane is detached. Tracheal mucosal hyperplasia with exudate. If the flock has been immunopathized, when Newcastle disease occurs, only catarrhal inflammation of the mucosa is seen, bleeding from the rectal mucosa and cecum tonsils, the lesions are relatively mild, and other lesions are atypical.
2. Laboratory inspection
1. Virus isolation and identification. (1) Sample collection and processing: take the brain or heart, liver, spleen, lung, kidney, air sac and other tissues of sick and dead birds, use normal saline to form a 1:5 emulsion, add penicillin (to the final concentration of 100 units / mL), streptomycin (so that the final concentration is 1000 μL / mL). Take a tracheal swab and a cloaca (or fecal swab) into 2 to 3 mL of normal saline containing cyanos and streptomycin (the amount of cyanol and streptomycin is increased by 5 times), and repeatedly squeeze until no water drops are discarded. Adjust the PH to 7.0~ 7.4, 37 °C for 1 hour, centrifuge at 1000 r/min for 10 min, take the supernatant at 4 °C and store for later use. (2) Virus isolation and identification: take 0.1 mL of the supernatant of the treated sample through the allicure cavity for 9 to 10 days old SPF chicken embryos, culture for 4 to 7 days, sterile collection of allantoic fluid; and standard positive serum for hemagglutination inhibition test to determine whether it is infected with Newcastle disease virus. (3) Determination of viral pathogenicity: dilute the allantoic fluid continuously 10 times with normal saline to 10-9, from 10-6 to 10-9, each dilution is inoculated with 5 SPF chicken embryos of 9 to 10 days old, 0.1 mL / embryo, and incubated at a temperature of 37 °C. After 8 h, the second batch of chicken embryos was inoculated with the same method. Observe the time of death of chicken embryos for 7 consecutive days and record. Basis for determination: The maximum dilution multiple causing the death of inoculated chicken embryos is the minimum lethal amount of the virus. (4) The result was determined: 40 to 70 hours of death is strong poison, and death of more than 140 hours is weak poison.
2. Blood coagulation inhibition test. (1) Equipment to be prepared: 96-well U-shaped micro plate, micro pipette, micro oscillator. pH 7.0 to 7.2 phosphate buffered saline, concentrated antigen or culture tested allantoic fluid (viral identification), positive and negative standard serum provided by the designated unit. Make 0.5% chicken red blood cell suspension: collect blood at the wing root of adult chicken, wash 3 to 4 times with 20 volumes of phosphate buffer, centrifuge at 2000 r/min for 3 to 4 min, aspirate the upper plasma, leukocytes, etc., and the last centrifugation is 5 min to make a 0.5% suspension. (2) Antigen hemocoagulation valence determination (hemagglutination test): Add 50 μL of phosphate buffer per well on the micropipette with a micropipette for a total of 4 rows. Add 1:5 dilution of antigen or urine (virus identification) to each row of well 1, 50 μL/well, and then dilute from left to right to well 11, and then pipette 50 μL from well 11 and discard. The last 1 well is without antigen and is intended to act as a blood cell control. Add 0.5% erythrocyte suspension 50 μL/well. The test liquid was placed in a micro-shaker, mixed for 1min, and then left for 30 min at room temperature of 18~20°C, and the blood coagulation image was carefully observed. The maximum dilution of the antigen in which full agglutination occurs is the hemagglutination titer of the antigen, i.e., the blood coagulation valence. Repeat in four rows each time, and the blood coagulation valence of the antigen is determined by the geometric mean. (3) Test operation: antigen dilution multiple = blood coagulation valence / 4. Add 50 μL of phosphate buffer to each cell of the microplate in well 1 of each row, 50 μL of antigen with a concentration of 8 units per well of row 2, and 50 μL of antigen with a concentration of 4 hemagglutination units in turn from well 3 to 12. Each row of 1st well was mixed with 50 μL of serum to be tested, and then 50 μL was added to the 2nd well; diluted to the 12th well in turn, and 50 μL was discarded after mixing. Placed at room temperature of 18 to 20 °C for 20 min. Add 0.5% erythrocyte suspension 50 μL/well dropwise, mix well and let stand for 30 min at room temperature and observe the results. (4) Judgment results: The results of red blood cell control and positive serum control should be non-agglutinative red blood cells; negative serum control and antigen control should have agglutination phenomenon.
3. Treatment of sick and dead chickens
If the internal organs of the sick and dead chicken are found to have lesions and the meat corpse has no lesions, its internal organs are used for industrial use or destruction, and the meat corpse can be used after high temperature treatment; in the case of systemic lesions, the meat corpse and internal organs are all used for industrial use or destruction, and the blood and feathers are disinfected and appear.
Author: Sun Zhenming Veterinarian