GB 16550-1996 Technical specification for Newcastle disease quarantine
Some standard content:
National Standard of the People's Republic of China
Technical code of quarantine for newcastle disease
Technical code of quarantine for newcastle disease1 Subject content and scope of application
GB16550-1996
This standard specifies the technical specifications for group quarantine, individual quarantine, laboratory testing and post-quarantine treatment of Newcastle disease. This standard applies to chickens and other poultry that can be infected with Newcastle disease.2 Referenced standards
GB16548--1996 Procedures for harmless treatment of livestock and poultry diseases and their products3 Group quarantine
Quarantine is implemented on poultry farms, independently raised poultry flocks, and poultry flocks sold or transported out. 3.1 Find out the background of the epidemic
By asking veterinary personnel or checking epidemic records, check whether the inspected poultry farms (groups, rooms) have had clinical cases of Newcastle disease in the past year, whether there is a history of contact with Newcastle disease cases, and whether Newcastle disease has occurred within a 2 km radius. 3.2 Check the immunization status
By checking the immunization certificate or querying other information, verify whether the inspected poultry flock has been injected with qualified vaccines as required. If the immunization is in accordance with the regulations and is within the prescribed protection period, the poultry is in good health and is considered to be immunized. If the immunization does not meet the prescribed requirements, it must be re-vaccinated with a qualified vaccine, isolated and raised for observation until the group antibody titer reaches 25 or above, and the whole group is healthy, and it is considered to be immunized. If suspected Newcastle disease poultry is found during the observation period, further inspection must be carried out in accordance with the requirements of Articles 3.3 and 3.4. 3.3 Group clinical examination
First, a static inspection is performed, allowing the poultry flock to be in a static state, and observing whether there are any abnormalities in nutritional status, mental state, respiration, feces, nasal and oral secretions. Then observe the movement of the poultry to see if there are any abnormal phenomena such as lameness, ataxia, and spasms. During the inspection, pay special attention to the following situations: a.
Depression or death without any symptoms; fever, loss of appetite, mental sluggishness, reduced or stopped egg production; increased oral and nasal secretions, fullness of the cyst; difficulty breathing, "gurgling" sounds in the throat (more obvious at night); diarrhea, green feces;
Head tilt, neck rotation, circling movements or ataxia. If a suspected case is found, further inspection shall be carried out according to the requirements of Article 3.4, and the entire group shall be isolated and raised at the same time. 3.4 Pathological anatomical examination
For suspected cases in clinical diagnosis, all or selected autopsies should be performed, and a preliminary judgment should be made based on the following lesions: a.
There is mucus in the mouth and throat, and bleeding in the pharyngeal mucosa; the glandular stomach papilla is swollen, and after squeezing, there is tofu-like necrotic fluid flowing out, and there are scattered bleeding spots on the papilla; b.
There are streaky or punctate bleeding in the subcutaneous layer of the muscular stomach, and sometimes irregular ulcers are seen, and there are bleeding spots and stripes at the junction of the glandular stomach and the muscular stomach; Approved by the State Bureau of Technical Supervision on October 3, 1996 and implemented on February 1, 1997
GB 16550--1996
There are large areas of scattered bleeding spots in the front part of the small intestine, or the intestinal mucosa has fibrous necrosis and pseudomembrane formation, red rough ulcers appear under the pseudomembrane, there is bleeding in the rectal folds, and the cecal tonsils (lymphoid follicles) are hemorrhagic and necrotic; the tracheal mucosa is congested and bleeding, and there is mucus in the trachea; there are pinpoint bleeding spots on the coronary fat, the outer membrane of the auricle and the apical fat. g.
4 Individual examination
4.1 Individual quarantine is required in situations where there are no group quarantine conditions. Individual quarantine Qualified poultry are generally used for food. If they are to be kept, they should be isolated for three weeks. Only after no abnormalities are found can they be mixed with other poultry. 4.1.1 Check whether they are from the epidemic area.
4.1.2 Check the immunization certificate.
4.1.3 Clinical examination and pathological anatomy examination
The examination contents shall refer to Articles 3.3 and 3.4. If poultry suspected of being infected with Newcastle disease is found, it shall be immediately disposed of harmlessly. 5 Laboratory test
5.1 Laboratory test shall be carried out in the following cases
Origin determination Quarantine during the quarantine period;
Monitoring immune status;
Qualification of suspected diseased chickens, or preliminary diagnosis of Newcastle disease during clinical and autopsy examinations, which requires further confirmation; d.
Major epidemics (such as avian influenza) are found during quarantine, and differential diagnosis is required; e
Laboratory diagnosis is required by the administrative department, breeding unit, seller or buyer, or both parties in dispute. 5.2 Laboratory test methods
Virus isolation and identification test, see Appendix A (Supplement). Hemagglutination inhibition test, used for immune monitoring, see Appendix Record B (Supplement). 6 Treatment after quarantine
6.1 After comprehensive judgment such as clinical examination, autopsy examination and laboratory diagnosis, the poultry flocks or poultry confirmed to be infected with Newcastle disease shall be treated in accordance with GB16548.
6.2 Poultry flocks (poultry) initially diagnosed as suspected of Newcastle disease shall be treated in one of the following ways: In addition to treating the suspected poultry in accordance with Article 6.1, the whole flock shall be isolated and raised, and emergency vaccination shall be carried out. If suspected poultry appears during the observation period after vaccination, it shall also be treated in accordance with Article 6.1. After 21 days of observation, poultry with clinical health and immune titer of more than 25 shall be treated as healthy poultry after body surface disinfection.
b. Poultry flocks (poultry) initially diagnosed as suspected of Newcastle disease shall be isolated and raised, and samples of suspected poultry shall be taken for laboratory diagnosis. If it is confirmed to be infected with Newcastle disease, the whole flock shall be treated in accordance with Article 6.1. 6.3 Within six months, poultry farms where Newcastle disease has occurred are not allowed to sell or transport poultry. 90
A7 Instruments and Equipment
Syringe: 1mL;
Injection Needle: No. 5-5;
Hemagglutination test plate: V-shaped, 96-well;
Micropipette: 50μl;
Constant Incubator;
Superclean bench or sterile room.
A2 Reagents
Sterile physiological saline;
Penicillin;
Streptomycin,
Standard positive serum.
A3 Sample collection and processing
GB 16550—1996
Appendix A
Virus isolation and identification test
(Supplement)
A3.1 Tracheal swabs and cloacal swabs (or feces) for live poultry. A3.2 For dead poultry, the brain is mainly used, and heart, liver, spleen, lung, kidney, air sac and other tissues can also be collected. A3.3 Grind the sample into a 1:5 emulsion with physiological saline; immerse the swab in 2-3mL of physiological saline, repeatedly suck and squeeze until no water drips out, and discard it. Add penicillin (to a final concentration of 1000 units/mL) and streptomycin (to a final concentration of 1mg/mL) to the solution. For cloacal swab (or feces) samples, the amount of penicillin and streptomycin is increased by 5 times. Then adjust the pH to 7.0~7.4, act at 37℃ for 1h, centrifuge at 1000r/min for 10min, take 0.1mL of the supernatant and inoculate 9-10 day old SPF chicken embryos through the allantoic cavity. A4 Collection and detection of culture
A4.1 The allantoic fluid cultured for 4-~7 days is collected aseptically and stored at -20℃. A4.2 The allantoic fluid is subjected to hemagglutination test according to Appendix B (Supplementary), and the hemagglutination inhibition test is performed with the standard positive serum to determine whether the Newcastle disease virus is multiplying.
A4.3 Determination of MDT (mean time of death of chicken embryo caused by the minimum lethal dose of virus): dilute the fresh allantoic fluid 10 times with physiological saline continuously, and inoculate 5 9-10 day old SPF chicken embryos at each dilution of 10-6~10-, 0.1mL per embryo, and incubate at 37℃. The remaining virus was stored at 4°C. After 8 hours, the second batch of chicken embryos were inoculated in the same way. The death time of the chicken embryos was observed and recorded for 7 consecutive days to determine the minimum lethal dose, that is, the maximum dilution factor that causes the death of the inoculated chicken embryos. Calculate MDT. A4.4 The MDT is used to determine the pathogenicity of the virus. Death after 40 to 70 hours is considered strong and death after more than 140 hours is considered weak. 91
B1 Instruments and Equipment
GB16550-1996bzxz.net
Appendix B
Newcastle Disease Microhemagglutination Inhibition Test (Supplement)
Microhemagglutination plate: V-shaped, 96-well;
Micro oscillator;
Plastic blood collection tube;
Pipette: 50μL.
B2 reagent
B2.1 diluent
pH7.0~7.2 phosphate buffered saline
sodium chloride (GB126677)
potassium dihydrogen phosphate (GB1274-77)
sodium hydroxide (GB629-77)
distilled water
autoclave, store at 4'℃, dilute 20 times when usedB2.2 concentrated antigen
B2.30.5% red blood cell suspension
1000ml
Collect adult chicken blood, wash with 20 times phosphate buffered saline for 3 to 4 times, centrifuge at 2000r/min for 3 to 4 minutes each time, and the last time for 5 minutes, and make a 0.5% suspension with phosphate buffered saline. B2.4 Standard positive serum
B3 Test serum
Randomly collect 20 to 30 blood samples from each group of chickens and separate the serum. Blood collection method: First puncture the sub-wing vein with a three-edged needle, and then drain the blood with a plastic tube to a length of 6 to 8 cm. Melt one end of the tube and seal it. After the serum is coagulated and precipitated, centrifuge it at 1000r/min for 5 minutes, cut the plastic tube, and pour the serum into a small hole on a plastic plate. If it needs to be stored for a long time, cut off one end of the clot after centrifugation, seal it with melted paraffin, and store it at 0℃. B4 Operation method
B4.1 Microhemagglutination test
B4.1.1 Add 50μL of diluent (B2.1) to each well of the microhemagglutination plate (B1.1), and drop four rows in total. B4.1.2 Pipette 1:5 diluted antigen and drip it into the first column of wells, 50μL per well, then dilute it in multiple proportions from left to right to the 11th column of wells, then pipette 50μL from each of the 11th column of wells and discard it. No antigen is added to the last column as a control. B4.1.3 Add 50μL of 0.5% red blood cell suspension (B2.3) to each well. B4.1.4 Place on a micro-oscillator (B1.2) and oscillate for 1 minute, or hold the blood coagulation plate in a circle to mix. B4.1.5 Place at room temperature (18~20℃) for 30~~40 minutes, and determine the result based on the blood coagulation image. The maximum dilution of the antigen that shows complete agglutination is the blood coagulation titer of the antigen. Repeat four rows each time, and express the result as the geometric mean. B4.1.6 Calculate the concentration of the antigen containing 4 blood coagulation units. Calculate according to formula (B1): 92
B4.2 Microhemagglutination inhibition test
GB16550—1996
Hemagglutination titer
Antigen should be diluted by multiples of one
B4.2.1 First take 50μuL of diluent (B2.1) and add it to the first well of the microhemagglutination plate (B1.1), then take 4 hemagglutination units of antigen and add them to wells 3 to 12 in sequence, 50μL per well, and add 8 hemagglutination units of antigen to well 2 in sequence. B4.2.2 Pipette 50μL of the test serum (B3) into well 1 (serum control), squeeze and mix, then pipette 50μL into well 2, dilute it to well 12 in multiples, and finally discard 50μL.
B4.2.3 Place it at room temperature (18-20℃) for 20 minutes. B4.2.4 Add 50μL of 0.5% red blood cell suspension (B2.3) to each well, shake and mix, let stand at room temperature for 30~~40min, and determine the result.
B4.2.5 A standard positive serum (B2.4) with a known titer should be set as a control for each measurement. B5 Result determination
B5.1 When the control shows a correct result, the maximum dilution that completely inhibits red blood cell agglutination is the hemagglutination inhibition titer of the serum.
B5.2 If more than 10% of the chickens have a high hemagglutination inhibition titer of more than 11 (log2), it means that the chickens have been infected with Newcastle disease virus. B5.3 If the immune level of the chickens is monitored, the protection rate of the chickens with a hemagglutination inhibition titer of 4 (log2) is about 50%; the protection rate of those above 4 (log2) is 90%~100%; the protection rate of non-immune chickens below 4 (log2) is about 9%, and the protection rate of immunized chickens is about 43%. The hemagglutination inhibition titer of the chicken group is expressed as the geometric mean of the hemagglutination inhibition titer of the sampled samples. If the average level is above 4 (1log2), it means that the chicken group is immune.
Additional Notes:
This standard is proposed by the Ministry of Agriculture of the People's Republic of China. This standard is under the jurisdiction of the National Technical Committee for Animal Quarantine Standardization. This standard is drafted by the Animal Quarantine Institute of the Ministry of Agriculture. The main drafters of this standard are Ma Hongchao, Yang Chengyu, Zheng Zhigang, Yang Huifen, and Wang Zelin. 93
Tip: This standard content only shows part of the intercepted content of the complete standard. If you need the complete standard, please go to the top to download the complete standard document for free.