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National Standard of the People's Republic of China
Technical code of quarantine for swine fever
Technical code of quarantine for swine fever1 Subject content and scope of application
GB16551—1996
This standard specifies the technical specifications for group quarantine, individual quarantine, laboratory testing, comprehensive judgment and post-quarantine treatment of swine fever. This standard applies to the quarantine of swine fever.
2 Referenced standards
GB16548—1996 Procedures for harmless treatment of diseased carcasses of livestock and poultry and their products3 Group quarantine
3.1 After verification that qualified swine fever vaccines have been vaccinated in accordance with regulations, no suspected swine fever pigs have been found within one month after the injection, and the immunization is within the effective period, it can be considered that the immunization meets the requirements. If the vaccination is not done, or the vaccination does not meet the requirements, or if sick pigs suspected of swine fever are found after vaccination, supplementary vaccination must be carried out. If no abnormality occurs at the beginning of the effective period of vaccination, the vaccination can be considered qualified; if suspected swine fever pigs occur again after vaccination, clinical examination, autopsy and laboratory examination must be carried out. Only after the possibility of swine fever infection is ruled out can the vaccination be considered qualified. 3.2 Check the health status of the whole herd and see if there are any abnormal manifestations. If the body temperature of the inspected pigs in the herd is found to be above 40.5℃, fatigue, loss of appetite, mental depression, visible mucosal congestion, bleeding or abnormal secretions, hair, alternating constipation and diarrhea, or other suspected symptoms of swine fever, it should be treated as suspected swine fever, and the whole herd should be isolated for further diagnosis. In addition, when the piglets are weak, trembling or stunted, it can be suspected that the sows are carrying the swine fever virus, and laboratory confirmation should be carried out. 3.3 Suspected pigs detected in the group can be sampled for autopsy examination, and the following lesions are used as one of the bases for comprehensive diagnosis and qualitative analysis: the color of the renal cortex becomes lighter and there are punctate hemorrhages; a.
The lymph nodes are congested and swollen in appearance, and the periphery of the cut surface is bleeding, showing a red and white "marble-like" appearance; b.
The spleen is not enlarged, and a wedge-shaped infarction area is found on the edge; d. There are small spots of bleeding in the larynx and bladder;
Systemic hemorrhagic changes, mostly in small pieces or dots; e.
f. The ileocecal valve, ileum, and colon form "button-shaped swellings" (chronic swine fever); boar foreskin accumulates urine.
3.4 After quarantine, it is considered that the immunity is qualified and there is no abnormality in clinical examination; or suspected cases are found, and after comprehensive diagnosis such as autopsy examination and laboratory examination, it is proved that it is not swine fever, and it should be treated as a non-swine fever group. However, when raising them in a new place, they must be isolated for more than two weeks first, and only after observation that there is no abnormality can they be mixed with local pigs.
4 Individual quarantine
When pigs are taken as individual quarantine objects, they should be carefully quarantined individually. 4.1 The content of individual quarantine includes:
a. Check whether there is an epidemic situation in the place of origin or check the quarantine certificate of the place of origin; b. Check the epidemic prevention certificate;
Approved by the State Bureau of Technical Supervision on October 3, 1996
Implemented on February 1, 1997
Clinical examination.
GB16551-1996
4.2 If there is no doubt about the above three items, they can be treated as non-epidemic pigs of swine fever. 4.3 If there is no problem with item 4.1c, but there are problems with items 4.1a and b, they can be vaccinated again, and if there is no abnormality in isolation for 15 days, they should be treated as non-swine fever pigs. If there are suspicious symptoms after vaccination, they should continue to be isolated and observed. If the diagnosis cannot be confirmed, autopsy and laboratory tests should be carried out. 4.4 If there are no problems with 4.1a and b, and there are problems with 4.1c, the pigs should be isolated and observed. If the diagnosis cannot be confirmed, autopsy and laboratory tests should be performed. 5 Laboratory tests
5.1 Laboratory tests should be performed in any of the following situations: a.
Regular quarantine of the place of origin;
In the non-epidemic area, the whole herd is inspected or sampled for pigs imported from other places; suspected cases of swine fever or "atypical swine fever", sow reproductive disorders and congenital spasms in piglets must be diagnosed; c.
Suspected swine fever or virus carriers are found in the herd that was originally considered non-epidemic or in the herd introduced from the non-epidemic area, and they must be diagnosed; d.
If a suspected outbreak of swine fever is found, an accurate diagnosis must be made while taking emergency prevention and control measures, or the virus status of the pigs must be found out before lifting the blockade; e.
f. Differential diagnosis must be made if there is a suspicion of a major epidemic disease (such as African swine fever); g. The transaction parties jointly request or the agricultural and animal husbandry authorities require confirmation; h.
Those who need further confirmation after group quarantine or individual quarantine. 5.2 The test results of the following methods can be used as one of the bases for quarantine qualitative determination. 5.2.1 Rabbit cross-immunity test, see Appendix A (Supplement). 5.2.2 Immunoenzyme staining test, see Appendix B (Supplement). 5.2.3 Virus isolation and toxicity test, see Appendix C (Supplement). 5.2.4 Direct immunofluorescence antibody test, see Appendix D (Supplement). 6 Comprehensive judgment
The disease is not divided by age or season, the clinical symptoms are obvious, the lesions are typical in autopsy examination, and any of the tests in 5.2 is positive; or the clinical symptoms and disease situation are unknown, the lesions are typical in autopsy examination, and any of the tests listed in 5.2 is positive; the disease situation, clinical symptoms, and pathological changes are unknown, not obvious or atypical, but two of the tests listed in 5.2 are positive. These situations can be judged as pigs infected with swine fever virus.
7 Post-quarantine treatment
In group or individual quarantine, all groups or individuals that are comprehensively judged to be infected with swine fever shall be treated in accordance with GB16548. 95
GB165511996
Appendix A
Cross-immunity test for immune system
(Supplement)
Grind the lymph nodes and spleen of the sick pigs and dilute them with physiological saline at 1:10. Inject them intramuscularly into 3 healthy rabbits, 5mL/rabbit. Set up 3 control rabbits without disease injection. Intravenously inject 1:20 swine fever rabbit virus (lymph spleen virus) into all rabbits at intervals of 5 days, 1mL/rabbit. After 24 hours, measure the body temperature every 6 hours for 96 hours. If 2/3 of the control group show stereotyped fever or mild fever, the test is established. The test results of the test group are shown in Table A1.
Table A1 Determination of the results of the cross-immunity test in rabbits Body temperature reaction after inoculation of diseased materials
Body temperature reaction after inoculation of swine fever virus
Note: "ten" means that more than or equal to two-thirds of the animals have a reaction. Appendix B
Immunoenzyme staining test
(Supplement)
Result determination
Contains swine fever virus
Contains swine fever rabbit virus
Contains non-swine fever virus pyrogenic substances
B1 During the autopsy, tonsils, spleen, kidneys, and lymph nodes of the sick animals were collected for stamps or frozen sections. After the specimens were naturally dried, they were fixed in a mixture of 2% dialdehyde and formaldehyde in equal amounts for 10 minutes. After drying, they were placed in a refrigerator for inspection. Operation method:
Put the specimen into 0.01% hydrogen peroxide or 0.01% sodium nitrogen-supplemented Tris-HCl buffer for 30 minutes at room temperature. B1.1
Rinse with pH7.40.02mol/L phosphate buffered saline for 5 times, 3 minutes each time, and air dry. B1.2
Put the specimen in a wet box, add 1:10 enzyme-labeled antibody, cover the specimen surface, and place at 37C for 45 minutes. B1.3
Rinse with pH7.40.02mol/L phosphate buffered saline-1% Tween buffer for 5 times, 2~~3 minutes each time. B1.4
Put the specimen in DAB (4-dimethylaminoazobenzene) Tris-HCl solution, and place at 37C for 3 minutes. B1.5
B1.6 Rinse with pH 7.4 0.02 mol/L phosphate buffered saline for 3~~4 times, 2~3 minutes each time, then dehydrate with anhydrous alcohol and xylene, seal and inspect.
B1.7 Examination under a microscope, if the cytoplasm is stained dark brown, it is positive; yellow or colorless is negative. The cytoplasm of pig tissues inoculated with swine fever attenuated virus is slightly brown, which is obviously different from that infected with strong strains. Appendix C
Virus isolation and virus enhancement test
(Supplement)
This test can be used to separate viruses and amplify the number of viruses to improve the sensitivity of quarantine. C1 Cut 2g of tonsils or spleen into small pieces, add sterilized sand and grind them into a paste in a mortar. Use Hank's solution or MEM to make a 20% suspension, add penicillin (to a final concentration of 500 units/mL) and streptomycin (to a final concentration of 500ug/mL), place at room temperature for 1h, centrifuge at 3000r/min for 15min, and take the supernatant. C2 After the PK15 monolayer cells are digested and dispersed with trypsin, centrifuge at 800r/min for 10min, and prepare a 2X10° cell/mL suspension with MEM containing 5% fetal bovine serum without BVDV. C3 9 parts of cell suspension (C2) plus 1 part of disease material suspension (C1) are inoculated in a rotating bottle or a micro-cell culture plate. Set up several control bottles (wells) without disease material. 1, 2, and 3 days after inoculation, wash 2 bottles (wells) of culture and 1 bottle (well) of control with Hank's solution or MEM twice, each time for 5min, and fix with cold acetone for 10min.
C4 Perform immunoenzyme staining or immunofluorescence staining and examine under a microscope. Appendix D
Direct immunofluorescence antibody test
(Supplement)
D1 Sampling
During group quarantine, there shall be no less than three suspected pigs to be tested, of which at least two shall be early-stage diseased pigs. Oral tonsils shall be removed from the pigs after being killed or from living pigs. Tonsils and kidneys shall be collected from late-stage diseased pigs after being killed. In individual quarantine, suspected pigs shall be killed to collect tonsils and kidneys. The collected tissue samples must be fresh. D2 Submission for inspection
Sampling shall be sent for inspection as soon as possible after sampling. If it cannot be sent on the same day, it must be frozen and stored to avoid tissue corruption and autolysis. D3 Sectioning
Cut the sample tissue block into a 1cm×1cm surface, freeze it directly on the frozen section tray without any fixation (if the tissue block is too small, such as tonsils removed alive, it can be embedded with a special embedding agent or chemical paste for the frozen section machine), and then section it. The thickness of the section is required to be 5-7μm. Spread the slices on a clean glass slide with a thickness of 0.8 to 1 mm. D4 Fixation
Fix the slices in pure acetone for 15 minutes. Take out and immediately put them in 0.01 mol/L, pH7.2 phosphate buffered saline, and rinse gently 3 to 4 times. Take out, dry naturally, and then stain with fluorescent antibodies as soon as possible. D5 Fluorescent antibody staining
Drop the swine fever fluorescent antibody on the surface of the slices and place them in a humidified box at 37°C for 30 minutes. After taking out, rinse thoroughly in phosphate buffered saline, and then seal the coverslip (0.17 mm thick) with 0.5 mol/L, pH9.0~~9.5 carbonate buffered glycerol. After staining, microscopic examination should be carried out as soon as possible. If necessary, it can be stored at low temperature for inspection.
D6 Microscopic examination
Observe the stained slice specimens under a fluorescence microscope with blue-violet or ultraviolet excitation light. D7 judgment
In the field of view of fluorescence microscope, if the tonsil crypt epithelial cells or renal tubular epithelial cells show bright yellow-green fluorescence, it is judged as positive for swine fever virus infection.
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GB16551-1996
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 Animal Quarantine Standardization Technical Committee. This standard is drafted by the Animal Quarantine Institute of the Ministry of Agriculture. The main drafters of this standard are Yang Chengyu, Zheng Zhigang, Yang Huifen, and Shao Zhenhua. 98
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