Early detection In order to detect ASF epidemic as early as possible, first of all, all employees in pig farms should be familiar with the clinical symptoms of ASF. Understand the situation of pig farm households, slaughter sites, pig trading places and harmless disposal sites within three kilometers of the surrounding area, pay close attention to the abnormal death of pigs in the surrounding area, and discover the hidden danger of the epidemic in time.
Always pay attention to the abnormal situation of pigs in all aspects of the farm, including the mental state, feeding situation, body temperature change, body surface change and performance of the sows in farrowing. Once suspicious symptoms are found, sample and send for examination (sampling priority: nasal swab > saliva > pubic swab > anal swab).
Mental status: promptly find pigs with abnormal eyes in the pen (pigs may not have detoxified yet at this time) and pigs with depressed spirit, and promptly sample and send them for inspection. Pig farms should actively mobilize frontline staff to pay attention to abnormal pigs, and give corresponding rewards to those who find abnormal pigs in advance and sample them in time with positive laboratory test.
Feeding situation: pay attention to the feeding situation of the group and individual pigs, and for pigs with slightly reduced feeding quantity (excluding feed factor), sample and send for inspection as soon as possible. Pigs with obvious decrease in feed intake will usually be accompanied by an increase in body temperature.
Body temperature change: normal nursery pigs and fattening pigs' body temperature is usually between 39℃-40℃; normal adult foundation pigs' body temperature is between 38℃-39℃ (among them, the body temperature of sows will rise to between 39℃-40℃ in the days before and after estrus and delivery). If pigs with body temperature above the normal range are found, they should be sampled and sent for inspection in time. In order to prevent cross contamination when measuring body temperature, the thermometer must be changed for one pig, and gloves and protective clothing must be worn; far-infrared thermometer can be used to measure temperature, and try not to enter the pen.
Body surface changes: Seeing red skin pigs indicates that the pigs are in a feverish state. In addition, some pigs have poor coagulation when they are given injections. For these abnormal pigs, take samples for testing in time.
Manifestation of trans-parturient sows: In addition to the above clinical manifestations, trans-parturient sows will also show abortion, which is different from the black or white fetuses common in other diseases, and will show homogeneous red fetus.
Rapid and accurate detection To achieve rapid and accurate detection, ASF sample collection is critical. When there is an outbreak (passive surveillance), sampling should be done for diseased pigs, dead pigs and the barn environment; while in routine surveillance, sampling should be done for ASFV susceptible pigs and the environment (e.g. pig-out platforms, farm gates and other risky places); if it is for a group in precise clearance, the number of samples can be increased appropriately. Attention should be paid to avoid cross-contamination during sampling and change disposable gloves after collecting each pig. Change disposable gloves in time after touching pigs, troughs and pens. When collecting blood, it must be done to replace 1 new needle for every 1 pig collected to avoid ASFV transmission. Relevant samples submitted to the diagnostic laboratory should have clear and long-lasting labels with good quality samples. Burn or autoclave all protective equipment after sampling.
(1) Sampling of suspected sick pig farms
Sampling preparation: prepare long cotton swabs (more than 15 cm), medical gauze, self-sealing bags, disposable syringes, protective clothing, disposable long-arm gloves, shoe covers, large plastic bags (or plastic sheeting for wrapping sick and dead pigs), markers, recording paper, pens, formaldehyde solution, Eppendorf tubes, zippered self-sealing bags, garbage bags, video recorders (cell phones), flashlights, mops, disinfectants, Flame gun, etc.
Sample types.
① suspected pigs mouth and nose swabs. Collect mouth and nose swabs from each pig in the same sampling tube in order to improve the accuracy of detection and reduce workload. Swabs are packed in Eppendorf tubes, and it is recommended to store the samples at 4°C and send them for testing immediately, and those without experimental conditions should be sent to a qualified laboratory within 24 hours.
② Inguinal lymph nodes of sick and dead animals. Suddenly dead pigs carry a large amount of virus in their blood and organs, it is recommended to collect only inguinal lymph nodes, and then wrap the sick and dead pigs for harmless treatment.
③Whole blood. Use a vacuum blood collection tube containing EDTA anticoagulant to draw whole blood from jugular vein, anterior vena cava or ear vein. If the pig has died, blood can be taken from the heart immediately. Heparin should not be used as an anticoagulant as it can inhibit the PCR reaction and cause false negatives.
Micro blood samples can be collected and stored via dried blood spots (DBS) in remote areas or when cold chain transport is not possible. Dried blood spot (DBS) samples are collected by adding a few drops from the vein or skin of the animal by pricking the needle or sterile syringe needle into a specially designed absorbent filter paper. The blood sample should be completely saturated with filter paper and air dried for several hours. Store the sample in a low permeability plastic bag with desiccant to reduce humidity.
④ Serum. Collect blood samples from the jugular vein, anterior vena cava, auricular rim vein, or dissection procedure using a vacuum blood collection tube without anticoagulant.
⑤ Organ and tissue samples. Dissection sampling is not recommended because of the potential for ASFV spread. Dissection must be performed under biosecurity conditions. All pig organs and tissues can be used to detect ASFV, but the spleen, lymph nodes, liver, tonsils, heart, lungs, kidneys and bone marrow contain higher levels of toxicity.
(6) Vector samples such as soft ticks. Blunt-edged ticks can be used to detect ASFV. three collection techniques are available: manual collection, carbon dioxide trapping, and vacuum attraction capture. After collection, the ticks should be kept alive or stored directly in liquid nitrogen to avoid DNA degradation.
(vii) Environmental samples. Area sampling is performed on a block basis for risk levels. For example, all walls, floors, equipment and other surfaces in the pig farm are sampled for full coverage.
Production area: including all block unit walls, ground, fan, gutter, equipment, water line, material line, etc. in each block pig barn. Sampling should be fully covered including dead corners, bottom of troughs and other locations that are not easy to collect.
Outside the production area: Sampling of possible risk areas, including the pig out platform, farm gate, material tower, sewer, staff dormitory, storage room, bathroom, office, restaurant kitchen, vehicles, water source and all other areas that may be contaminated.
(2) Packing and transporting samples
Collected samples should be carefully packed, marked and delivered to the laboratory. Samples must be shipped with adequate amounts of cooling materials (e.g., ice packs, dry ice) to avoid spoilage. Samples should be packaged in a "triple packaging system" to ensure biosecurity during transport and to avoid contamination of samples. Sample transport must comply with the Ministry of Agriculture and Rural Affairs "highly pathogenic animal pathogenic microorganisms bacteria (viruses) transport packaging specifications" and other provisions.
(3) Waste disposal
After sampling, do a good job of disinfection and harmless treatment of carcasses, sites, items, personal protective equipment. Try to arrange different sampling personnel for different sites to avoid cross-contamination.
(4) Rapid and accurate detection
Real-time fluorescence quantitative PCR (qPCR) is a common method for rapid detection of ASFV in laboratory. Large pig breeding enterprises can build ASFV detection laboratories to do epidemic investigation and monitoring when pig farms around and their own pig farms are threatened, so as to provide technical support for epidemic disposal and precise removal. To carry out qPCR testing, special real-time fluorescent PCR instruments and corresponding detection reagents must be equipped. Laboratories can carry out qPCR assays for ASFV according to technical standards. Because qPCR detection technology is sensitive, extremely small amounts of contamination can cause false positives, the PCR laboratory for zoning management can avoid detection of contamination. The PCR laboratory should be divided into reagent preparation area, sample preparation area, product amplification area, etc. according to the conditions.
(5) Confirmation of the outbreak
The laboratory of China Animal Health and Epidemiology Center or provincial animal disease prevention and control center can confirm the diagnosis of ASF outbreak as required.
