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SN/T 2985-2020: (Technical Specifications for Equine Influenza Quarantine)
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SN/T 2985-2020	English	279	Add to Cart	3 days [Need to translate]	(Technical Specifications for Equine Influenza Quarantine)
SN/T 2985-2011	English	439	Add to Cart	3 days [Need to translate]	Quarantine protocol for equine influenza

SN/T 2985-2020: (Technical Specifications for Equine Influenza Quarantine)

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Quarantine protocol for equine influenza The People's Republic of China Entry-Exit Inspection and Quarantine Industry Standards Replace SN/T 2985-2011 Technical Specifications for Equine Influenza Quarantine 2020-12-30 release 2021-07-01 implementation Issued by the General Administration of Customs of the People's Republic of China

Foreword

This document was drafted in accordance with the rules given in GB/T 1.1-2020. This document replaces SN/T 2985-2011 "Technical Specification for Equine Influenza Quarantine". Compared with SN/T 2985-2011, the main technical differences of this document are as follows. --Refer to the "Handbook of Terrestrial Animal Diagnostic Tests and Vaccines" (2016 Edition) to optimize the test method; - Added one-way radiation hemolysis test; --- Added the H3N8 equine influenza virus nucleic acid detection fluorescent RT-PCR method. This document was proposed and managed by the General Administration of Customs of the People's Republic of China. Drafting organizations of this document. Beijing Customs of the People's Republic of China, Changchun Customs of the People's Republic of China, Tianjin of the People's Republic of China customs. The main drafters of this document. Shi Xiju, Bai Yaduo, Qi Wei, Wang Lin, Gao Zhiqiang, Meng Qingfeng, Zhang Lifeng, Zhang Wei, Wang Weili, Dong Zhizhen, Huai Shuo. The previous versions of the documents replaced by this document are as follows. - SN/T 2985-2011. Technical Specifications for Equine Influenza Quarantine

1 Scope

This document specifies the clinical diagnosis and laboratory diagnosis of equine influenza. This document is applicable to the quarantine of entry and exit equine influenza.

2 Normative references

The contents of the following documents constitute an indispensable clause of this document through normative references in the text. Among them, dated quotations Only the version corresponding to that date is applicable to this document; for undated references, the latest version (including all amendments) is applicable Used in this document. GB/T 19489 General requirements for laboratory biosafety SN/T 1687 Equine Influenza Hemagglutination Inhibition Test Operating Procedure 3 Terms, definitions and abbreviations There are no terms and definitions that need to be defined in this document. The following abbreviations apply to this document. EI. equine influenza (equine influenza) EIV. equine influenza virus HA. Haemagglutination (haemagglutination) HI. Haemagglutination inhibition test (haemagglutination inhibition) MDCK. canine kidney cells (madin-darby canine kidney) PBS. phosphate buffered saline RT-PCR. reverse-transcription polymerase chain reaction (reverse-transcription polymerase chain reaction) SRHT. single radial haemolysis test (single radial haemolysis test)

4 Clinical diagnosis

4.1 Clinical symptoms Equine influenza is composed of two influenza A subtypes with different antigenicity in the Orthomyxoviridae influenza A virus genus. (H7N7, called horse-1; H3N8, called horse-2) caused by an acute respiratory disease. When susceptible horses get sick, The clinical symptoms are fever, harsh dry cough, nasal flow and viscous purulent secretions; when a horse has a certain degree of immunity, the above symptoms The condition may not be fully manifested. 4.2 Pathological changes 4.2.1 Pathological anatomical changes The visual lesions are conjunctival flushing, edema, ectropion, brick red or light yellow, and corneal turbidity often occurs. Upper respiratory tract mucosal filling Blood, edema and exudation, epithelial cell shedding and focal erosion. Swollen lymph nodes in the head and neck. Lung congestion, edema, no expansion all. The liver generally has no obvious eye disease. 4.2.2 Histopathological changes It manifests as acute bronchitis and bronchiolitis, which are mainly characterized by the exudation of a large number of neutrophils in the lumen. Inflammation one Transient, usually only seen in the first 4 days after the onset of the disease, after which the inflammation subsides, and there are mild lymphocytes and plasma cell infiltration in the mucosa. Severe cases are acute bronchial interstitial pneumonia, which is characterized by general thickening of the alveolar mediastinum, and the proliferating cells are histiocytes and lymph cell. In the liver, diffuse or nodular lymphocytes and tissue cell infiltration can be found in the sinusoidal spaces. 4.3 Epidemiology Under natural conditions, only equine animals are susceptible, and there is no difference in age, sex and breed. A sick horse coughs and spews a virus-containing fly Mo, through the respiratory tract infection is the main mode of transmission of this disease. Experiments have shown that the disease can be spread through the air, or through contaminated feed. Oral transmission of food and drinking water. The virus exists in the semen of recovered horses for a long time, so sexual intercourse is also an important way of spreading the disease. The disease spreads extremely rapidly. The introduction of susceptible herds shows an explosive epidemic. After 1 week or a little longer, all susceptible horses are infected. disease. The disease can occur throughout the year, and it occurs more frequently in the spring and autumn in northern my country. In some areas, it mostly occurs in late winter and early spring, while another In some areas, it is popular in summer.

5 Laboratory diagnosis

5.1 Pathogen isolation and identification 5.1.1 Laboratory biosafety requirements Isolation and identification of equine influenza virus pathogens should be carried out in a biosafety level 2 laboratory or a biosafety cabinet of the corresponding level. The conditions should meet the requirements of GB 19489. 5.1.2 Equipment, materials and reagents 5.1.2.1 Equipment High-speed bench-top refrigerated centrifuge, bench-top centrifuge, vortex shaker, refrigerator (4°C and -20°C), autoclave, microplate reader, Fluorescence PCR machine, PCR reaction tube or reaction plate, inverted microscope, micro adjustable pipette (10 μL,.200 μL, 1000 μL). 5.1.2.2 Materials and reagents 5.1.2.1 Sampling tools. cotton swabs, scissors, tweezers, syringes, 1.5 mL centrifuge tubes, mortars, sampling tools should be heated to 121℃±2℃, Autoclave for 15 minutes and dry, pH 7.2, 0.01 mol/L PBS, see Appendix A, antibiotics (penicillin and streptomycin) for the preparation method. 5.1.2.2 For chicken embryo inoculation. egg photoper, hole punch, alcohol lamp, 5% iodine tincture, 70% alcohol, 1.0 mL disposable sterile injection Ware, paraffin, 9~11 day old SPF chicken embryos. 5.1.2.3 Allantoic fluid activity determination. 96-well V-type micro-reaction plate, shaker, micropipette, Aldrich solution, chicken red blood cell suspension For the preparation method, see Appendix B. 5.1.2.4 For cell inoculation. canine kidney cells, newborn calf serum, cell culture flask, MEM culture medium. See Appendix C for the preparation method. 5.1.2.5 Virus antigen ELISA test. ELISA test kit. 5.1.2.6 For fluorescent RT-PCR. TriZol or other viral RNA extraction kits, reverse transcription magnetic beads (RT-PCR Ready-To- GoTM beads), Taq DNA polymerase, chloroform, isopropanol, absolute ethanol (analytical grade). 5.1.3 Sampling, sample delivery and processing 5.1.3.1 Take samples immediately after the appearance of clinical symptoms, including nasopharyngeal swabs or nasal and tracheal washes. 5.1.3.2 The swab is made of absorbent cotton or gauze wrapped around a metal wire. During sampling, the nasopharyngeal swab is sent into the nasopharynx through the anterior nasal passage And stay for about 1 minute, stick to the respiratory tract mucus, and immediately put it into the vial containing the transport culture medium. Nose and tracheal irrigation The objects are taken through nasal endoscopy. 5.1.3.3 The transport culture medium is PBS containing 40% glycerol, or phosphate broth containing 2% trypsin (plus 2% antibiotic) Solution (penicillin and streptomycin 10 000 IU/100 mL each) and amphotericin B (250 mg/mL mother liquor). 5.1.3.4 If the sample is inoculated within 1 to 2 days, it can be stored at 4°C; if it is to be stored for a long time, it should be stored at -70°C or more. Store at low temperature, and transport samples should also be refrigerated. 5.1.3.5 Squeeze the liquid from the nasopharyngeal swab, centrifuge at 1,500 g for 15 minutes, and take the supernatant for use. Remaining liquid Store at -70°C or lower. 5.1.4 Isolation of chicken embryo virus 5.1.4.1 Put the 9~11-day-old SPF chicken embryos on the egg finder for inspection, and use a pencil to draw the air chamber and embryo position, in the air chamber Make a mark on the edge 2 mm~8 mm away from the blood vessel, and mark the air chamber on the side of the chicken embryo chorioallantoic membrane with relatively few blood vessels. 5.1.4.2 Put the chicken embryo upright on the egg seat with the blunt end up. After disinfecting the eggshell gas chamber with 5% iodine tincture, use 70% alcohol to deiodize and disinfect, Use a sterile puncher to drill a small hole at the mark so as not to damage the shell membrane. 5.1.4.3 Use a 1.0 mL syringe to draw 0.2 mL of the processed sample, pierce the needle into the hole, and inject it into the allantoic cavity through the chorioallantoic membrane. Each sample was inoculated with 5 chicken embryos. 5.1.4.4 After inoculation, the holes are melted and sealed with paraffin, and then incubated in the incubator at 34°C~35°C for 3 days. Chickens that died within 24 hours after vaccination The embryo is discarded. 5.1.4.5 Transfer the chicken embryos that died 24 hours after inoculation and survived within 3 days to a 4℃ refrigerator for 4 hours or overnight to cause the chicken embryos to die. Collect allantoic fluid aseptically. 5.1.5 Determination of hemagglutination of allantoic fluid 5.1.5.1 Operation steps 5.1.5.1.1 Add PBS to wells 1-12 of the micro reaction plate, 0.025 mL per well. 5.1.5.1.2 Then draw 0.025 mL of chicken embryo allantoic fluid collected in step 5.1.4.5 (see SN/T for the treatment method of allantoic fluid virus antigen 1687-2005) Add to well 1 and mix well. 5.1.5.1.3 Draw 0.025 mL of liquid from the first hole and add it to the second hole. After mixing, draw 0.025 mL of the liquid and add it to the third hole. This ratio is diluted to the 12th well, and 0.025 mL is aspirated and discarded. 5.1.5.1.4 Add 0.025 mL PBS to each well. 5.1.5.1.5 Add another 0.025 mL of 1% chicken red blood cell suspension to each well. Before adding, the red blood cell suspension should be shaken thoroughly. 5.1.5.1.6 Gently shake and mix, and observe the results after standing at 20℃~25℃ for 40 minutes, or standing at 4℃ for 60 minutes Observe the results later. 5.1.5.2 Judgment of results When the red blood cells are agglutinated, the red blood cells are evenly distributed at the bottom of the reaction plate, and the reaction plate is tilted for a while and does not slide down; the red blood cells are not condensed When collecting, the reaction plate is tilted for a while, and the precipitated red blood cells can be seen sliding down to form a streamline. The highest dilution factor of agglutination appears The number is HA titer. The judgment is as follows. a) The sample has no hemagglutination activity. when the red blood cells have not agglutinated, the allantoic fluid of each sample needs to be collected together, and then passed through chicken embryo Blind transmission, when it is passed to the 5th generation without hemagglutination activity, it is judged to be negative for equine influenza virus; b) The sample has hemagglutination activity. All the samples with positive hemagglutination test are divided into small aliquots and stored for later use, and one part is tested for HA Titer. When the HA titer is greater than or equal to 1/16, it is judged to be positive for equine influenza virus; when the titer is low, continue to pass down. A total of 5 generations were passed blindly, and when the HA titer was still less than 1/16, it was judged to be negative for equine influenza virus. 5.1.6 Cell culture virus isolation 5.1.6.1 Operation steps 5.1.6.1.1 After the canine kidney cells (MDCK) grow into a monolayer in the cell culture flask, use a monolayer containing 2 μg/mL trypsin Wash tissue culture medium (without serum) at least 2 times. 5.1.6.1.2 Add 0.25 mL~0.5 mL of inoculation solution to each well, set three replicates, and incubate in a carbon dioxide incubator at 37°C for 1 h. 5.1.6.1.3 Then add 0.5 μg/mL~2 μg/mL trypsin culture medium without serum, and place it in a 37°C carbon dioxide incubator for culture. 5.1.6.1.4 Observe and record the cytopathic condition under the microscope every day. 5.1.6.2 Judgment of results After culturing for 48 h to 96 h, the cells swelled and became round, clumped, and finally disintegrated, which is a cytopathic condition. After cytopathic or culturing for 7 days, collect the supernatant and perform the HA test in accordance with 5.1.5.7.HA titer is greater than or equal to If it is less than 1/16, it is judged as positive; if there is no cytopathic disease and the HA titer is less than 1/16, continue subculture for at least 5 generations. 5.1.7 Virus antigen ELISA detection 5.1.7.1 Operation steps 5.1.7.1.1 Add 100 μL of the extract from the nasopharyngeal swab to each well of the microreaction plate coated with polyclonal rabbit anti-H3N8 subtype serum Inside, incubate at 22°C±2°C for 90 min, and wash the micro-reaction plate with PBS solution (PBST) containing 0.05% Tween-20°C for 3 times. 5.1.7.1.2 Add 100 μL of the horseradish peroxidase-monoclonal antibody (MAb) conjugate that has been diluted 1.50 with buffer diluent, Incubate at 37°C for 1 h, and wash the micro-reaction plate 6 times with PBST. 5.1.7.1.3 Add 100 μL of tetramethylbenzidine solution to each well of the micro reaction plate, incubate at 22℃±2℃ for 10 min, then add 100 μL 0.18 moL/L sulfuric acid terminates the reaction. Use a microplate reader to measure at a wavelength of 450 nm... ...