CN112359144A - Method for monitoring and eliminating African swine fever virus in large-scale pig farm - Google Patents
Method for monitoring and eliminating African swine fever virus in large-scale pig farm Download PDFInfo
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
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- A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/02—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using physical phenomena
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- A—HUMAN NECESSITIES
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- A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
- A61L2/16—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor using chemical substances
- A61L2/18—Liquid substances or solutions comprising solids or dissolved gases
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- A—HUMAN NECESSITIES
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- A61L2202/00—Aspects relating to methods or apparatus for disinfecting or sterilising materials or objects
- A61L2202/20—Targets to be treated
- A61L2202/25—Rooms in buildings, passenger compartments
Abstract
The invention discloses a method for monitoring and eliminating African swine fever viruses in a large-scale pig farm, which comprises the steps of monitoring the environment outside the pig farm, monitoring the environment inside the pig farm, monitoring pigs, detecting samples, eliminating viruses and the like.
Description
Technical Field
The invention relates to the technical field of prevention and treatment of African swine fever, in particular to a method for monitoring and eliminating African swine fever viruses in a large-scale pig farm.
Background
African swine fever (ASF for short) is an acute, hemorrhagic and virulent infectious disease caused by the infection of domestic pigs and various wild pigs by African swine fever virus (ASFV for short). The world animal health organization classifies the animal epidemic disease as a legal report animal epidemic disease, and the disease is also a kind of animal epidemic situation which is mainly prevented in China. The onset process of African swine fever is short, the death rate of the most acute and acute infections reaches 100%, the clinical manifestations are fever, fast heartbeat, dyspnea, partial cough, serous or mucoid purulent secretion of eyes and nose, cyanosis of skin, and obvious bleeding of lymph nodes, kidney and gastrointestinal mucosa. The symptoms and lesions of African swine fever are similar to those of other hemorrhagic diseases of swine fever, and their subacute and chronic forms are practically indistinguishable at the production site and must be identified by laboratory methods.
ASFV enters pig body through mouth and upper respiratory tract system, and the virus spreads rapidly to the mandibular lymph node, spreading throughout the body through lymph and blood, when it is infected in nasopharynx or tonsil. Pigs, recovered pigs and recessively infected pigs are the main sources of infection. The virus is distributed in various tissues, body fluid, secretion, excrement and semen of the acute sick pigs, and is particularly detoxified from the nasopharynx. The live pig still keeps infectivity for several months and continuously expels toxin. Because recessive swine with virus and rehabilitation swine with virus for the whole life and specific antibodies in sick swine accumulate, the virus amount is not easy to detect.
Since 2007, African swine fever has occurred, spread, and prevailed in multiple countries around the world. China is a big country for raising pigs and consuming pork, the marketing amount, the stocking amount and the pork consumption amount of live pigs are all in the top of the world, the total amount of imported pigs and pork products is huge every year, and the trade is frequent in a plurality of countries. Therefore, the direct and indirect losses due to african swine fever would be immeasurable.
At present, no effective vaccine or medicine is available for preventing and controlling the African swine fever, a complete monitoring scheme is established for a critical control point of a pig farm, abnormal pigs and abnormal pig groups, a rapid pathogen detection technology is used, the virus infection condition of the pig farm is mastered in real time, and the method is an effective method for preventing and controlling the African swine fever by combining the fixed-point removal technology of the African swine fever virus.
Disclosure of Invention
The invention aims to provide a method for monitoring and eliminating African swine fever viruses in a large-scale pig farm, which can effectively prevent and control the African swine fever viruses in the breeding process of the pig farm by establishing a complete monitoring scheme for a key control point of the pig farm, abnormal pigs and abnormal pig groups, applying a rapid pathogen detection technology, mastering the virus infection condition of the pig farm in real time and combining the fixed-point elimination technology of the African swine fever viruses.
The invention is realized by the following technical scheme:
a method for monitoring and eliminating African swine fever virus in a large-scale pig farm comprises the following steps:
step one, monitoring the environment outside a pig farm: the method comprises the following steps of monitoring the entering personnel, the input articles, the vehicles coming and going and the surrounding environment respectively, and performing sample mixing and mixed inspection on samples in the same area, wherein the sample mixing and inspection is performed on not more than 5 samples, and the method specifically comprises the following steps:
(1) entering personnel: monitoring and downsampling by video, wiping samples such as mouth, nose, fingernails, hands, hair, clothes, trousers, shoes and the like by more than 5 cotton swabs, and putting the samples into a sampling tube;
(2) inputting articles: for each batch of solid raw materials, collecting 8-12 points, including 3-4 samples respectively at the head, tail and middle of the vehicle, collecting about 1kg of each point, and collecting 10 kg/batch in total; collecting more than 50ml of liquid by using a clean bottle for liquid raw materials, and storing the liquid at 2-8 ℃ for inspection; for the packaging box, collecting the surface of the packaging box by using gauze stained with physiological saline, and randomly collecting 70% of packaging box samples;
(3) the coming and going vehicles: the device comprises a cab, the inner side of a carriage, the roof of the carriage, the inner side of a door surface, different side connecting gaps, wheels, a mudguard and an oil tank, wherein a 'character' shaped sampling tool or a sterilized mop with gauze is used for all-dimensional mopping and sampling;
(4) ambient environment: sampling each floor of a workshop, and wiping, sampling and inspecting a raw material bin, a finished product bin, a premixing bin, a hardware bin, a bicycle shed, the ground where vehicles pass, a male and female bathing room and a discharge opening;
specifically, the monitoring frequency of the off-site environment of the pig in the first step is at least 1 time per week.
Step two, monitoring the environment in the pig farm: the sampling tool in the shape of a Chinese character 'ji' or a disinfected mop with gauze clamped is used for carrying out all-dimensional wiping sampling, and no more than 5 samples in the same area are subjected to sample mixing and inspection;
specifically, the sampling points of the environment in the pig farm in the second step include: the pig farm living area comprises a pig farm living area public environment, a vacant stall, a dead pig platform, a pig loading platform, a pig transferring channel, a corridor, a fumigating room isolation stool, a transition room for replacing external clothes and shoes, and a bathing front area for replacing transition clothes and shoes.
Step three, monitoring pigs: checking whether abnormal pigs appear every day, if abnormal pigs appear in a pig farm, starting a whole-group sampling scheme, wherein the acquisition range comprises oral liquid, a nasal cavity, a water fountain/trough and the ground, and 100% of trough edges are subjected to inspection;
the performance of abnormal pigs in the third step comprises the following steps: fever, no food intake, abortion, cyanosis, bloody stool, blood coagulation failure, and mental retardation.
Specifically, in the third step, the full-group sampling scheme follows the following principle: the sampling personnel forbids cross-house/span sampling, and the next span sampling can be carried out only after bathing and changing clothes again; collecting normal pigs firstly and then collecting abnormal pigs; after sampling is finished, diluting and disinfecting public roads in the house by 10% sodium hypochlorite in a ratio of 1: 0.5; and (5) carrying out a video monitoring and sampling process in the whole shooting process.
Preferably, the specific method of the full-cluster sampling scheme comprises the following steps:
(1) preparation work before supervision sampling: preparing video shooting tools, sampling materials and materials for each pigsty to be ready for people to enter in advance;
preparing a video shooting tool in advance, wherein the video shooting tool comprises a memory card, a video shooting device, a mobile charger, a data line and the like; each pigsty is prepared with materials for personnel to enter, including disposable gloves, shoe covers, protective clothing, protective hats and the like; sufficient sampling supplies were prepared including a sampling rod, cotton string, cotton swab, saline, marker pen, EP tubing filled with 1ml saline, gauze, flame sterilization tools and other tools used in the sampling work.
(2) Positioning fence/obstetric table sampling:
each pig of the positioning fence/obstetric table uses a cotton swab to collect oral liquid of the pig firstly and then collect nasal liquid of the pig, one cotton swab collects the place where the oral cavity of the pig can contact, the fence frame, the water tank, the water fountain and drinking water are placed in an EP (EP) tube in which 1-2 ml of physiological saline is placed in advance; the pig samples sharing one water tank are put into one bag for packaging, and the name of the sample indicates the name of the pig farm, namely date, column number and ear number; when oral liquid is collected, putting the cotton swab into the oral cavity of the pig, taking out the pig after the pig bites the oral liquid for 3-4 seconds, or scraping the pig back and forth in the oral cavity of the pig for 2-3 times; when nasal cavity liquid is collected, a cotton swab is stretched into the nasal cavity of a pig for about 2-3 cm, and the nose of the pig is pressed downwards to collect the nasal liquid; the action is gentle during collection, so that the condition that the pigs are damaged or afraid of influencing the next sample collection due to violent insertion is avoided;
sampling to collect normal pigs firstly and then collect abnormal pigs; a new pair of long-arm gloves is replaced when the pigs in the collecting and positioning fence need to collect 1 pig, and shoe covers are replaced 1 time after one water tank is collected; the collection of pigs in the obstetric table requires only 1 pig to be collected and a new pair of long arm gloves is replaced.
(3) Large column sampling:
firstly, wetting gauze with physiological saline for later use before sampling; secondly, thoroughly sterilizing flame of the sampling hook of the 'shaped' sampling tool before sampling; thirdly, winding the disinfected sampling hook back and forth for three circles by using gauze, and then binding the gauze by using a sterilized nylon binding belt; when the gauze is bound and wound, the user does not need to touch the sampling hook by hands; the sampling hook is firstly wiped on the waterer back and forth for several times, and then wiped on the floor of the stall back and forth, so that the whole area of the stall is covered by 100 percent; taking a clean piece of wet gauze, and wiping 100% of the clean piece of wet gauze on a railing or a doorway (a place where a pig bites) of the stall for sampling; tying a cotton rope on a railing of a large fence in advance, and collecting saliva of the pigs;
(4) filling the collected and extruded liquid into a sampling pipe, and marking information such as a pig farm, a multi-span house, a column, an ear number and the like in detail; samples of two adjacent columns of the large column are uniformly collected and put into the same sampling tube.
A large column of samples must be collected, and a new pair of gloves is replaced (if the gloves need to be replaced when the gloves arrive at the wall of the stall). Sampling to collect normal pigs firstly and then collect abnormal pigs; after each sample is collected, disinfecting places such as walkways and the like which are possibly polluted in time.
Step four, sample detection: extracting nucleic acid from the samples collected in the first to third steps, and detecting whether the samples carry African swine fever viruses or not; the negative detection represents African swine fever virus, and the positive detection represents that the African swine fever virus is carried;
specifically, the sample detection method in the fourth step comprises the following steps: extracting nucleic acid by using a KingFisher Flex 96-hole nucleic acid extractor of Thermofisher, and performing fluorescent quantitative PCR amplification detection by using an ABI 7500FAST or Roche LightCycler96 fluorescent quantitative PCR instrument; or the nucleic acid is extracted manually, and amplification detection is carried out in a Tetracore T8 fluorescent quantitative PCR instrument.
For samples with large sample amount such as environment and abnormal pigs, nucleic acid extraction is carried out by a KingFisher Flex 96-hole nucleic acid extractor of Thermofish, and then fluorescent quantitative PCR amplification is carried out by an ABI 7500FAST or Roche LightCycler96 fluorescent quantitative PCR instrument, so that the detection result can be obtained within 3 hours.
Random scattered samples such as vehicles, raw materials and personnel can be amplified in a Tetracore T8 fluorescent quantitative PCR instrument by manually extracting nucleic acids, and detection results can be obtained within 3 hours. The 8 reaction channels of the Tetracore T8 fluorescent quantitative PCR instrument can be independently controlled, so that the requirement that a small amount of samples are detected at any time can be met.
The purpose that a large number of samples and random scattered samples in a pig farm can be rapidly detected by combining different instruments and reagents is achieved. The rapid diagnosis technology is applied to monitor production factors of the pig farm such as people, cars, things, pigs, environment and the like, the production activities of the pig farm are not influenced while the African swine fever is prevented and controlled, and the rapid diagnosis technology can be used for removing viruses at fixed points when epidemic situations occur in the pig farm, so that the loss of the pig farm is reduced.
Step five, virus removal: executing the principles of 'full-group detection, accurate killing, pairing rechecking and pathogen elimination';
(1) abnormal pigs are found and are sampled immediately, and the result must be detected on the same day; if positive pigs are isolated and eliminated in the same day, production is stopped in the same day, the pigs are detected in the whole group, and the detection result is obtained in the same day;
(2) dead pigs are bagged by 14-filament films, and the two ends of the dead pigs are tightly tied and sealed, so that the environment is not polluted; the positive pigs are detected to be subjected to off-site harmless treatment within 4 hours, the centralized air outlet of the off-site can not be realized, the isolation of the vertical position relative to the pigs is not realized, the overnight treatment is not allowed, the whole course of the road in the elimination process is closed, and the front and the back are thoroughly disinfected;
(3) spraying the pig manure and the ground in the empty stall after leaving the farm by using a 5-7% sodium hydroxide solution, bagging the pig manure with 12 threads after 2 hours, conveying the pig manure away, adding lime and deeply burying the pig manure; washing the ground with water (brush if necessary), drying the ground with a ground dryer, spraying 5% sodium hypochlorite, and keeping for more than 60 minutes; the sodium hypochlorite can rapidly degrade virus DNA in the environment, can reduce the detection cost and prevent dead virus false positive;
(4) and after the negative is detected for the first time, the environment is disinfected and purified comprehensively, the sampling interval is 3-5 days later, the African swine fever virus is not detected in the abnormal pig detection in the interval period, and the negative can be judged as negative after all negative is detected for 3 times of complete detection, which indicates the successful purification.
The sampling tool of the invention is shown in figure 1.
The invention has the beneficial effects that:
1. the invention can effectively prevent and control the African swine fever in the breeding process of the pig farm by formulating a perfect monitoring scheme for the key control points of the pig farm, abnormal pigs and abnormal pig groups, applying a rapid pathogen detection technology, mastering the virus infection condition of the pig farm in real time and combining the African swine fever virus fixed-point removal technology.
2. The invention adopts rapid pathogen detection technology, can carry out large-batch automatic extraction and detection, can also adopt artificial extraction of nucleic acid for random scattered samples, carries out amplification in a Tetracore T8 fluorescent quantitative PCR instrument, has 8 reaction channels which can be independently controlled, and can meet the requirement of a small amount of samples on detection at any time. The detection method is flexible and variable, the detection is rapid, and the detection result can be obtained within 3 hours.
3. The invention realizes the purpose that a large amount of samples and random scattered samples in a pig farm can be quickly detected by combining different instruments and reagents. The rapid diagnosis technology is applied to monitor production factors of the pig farm such as people, cars, things, pigs, environment and the like, the production activities of the pig farm are not influenced while the African swine fever is prevented and controlled, and the rapid diagnosis technology can be used for removing viruses at fixed points when epidemic situations occur in the pig farm, so that the loss of the pig farm is reduced.
4. According to the invention, by a scientific sampling method and the principles of 'whole-group detection, accurate killing, pairing rechecking and pathogen removal', abnormal pigs are found to be sampled immediately, and the result is detected on the same day; if positive pigs are isolated and eliminated in the same day, production is stopped in the same day, the pigs are detected in the whole group, environmental protection and disinfection measures are taken until the pigs are all negative in the whole group for 3 times continuously, and virus purification is successful, so that the African swine fever is effectively prevented and controlled.
Drawings
1. FIG. 1 is a "dotted" sampling tool of the present invention.
Detailed Description
In order to make the technical solutions and advantages of the present invention clearer, the following describes the technical solutions of the present invention clearly and completely in combination with the embodiments of the present invention.
Example 1
A method for monitoring and eliminating African swine fever virus in a large-scale pig farm comprises the following steps:
step one, monitoring the environment outside a pig farm: the method comprises the following steps of monitoring the entering personnel, the input articles, the vehicles coming and going and the surrounding environment respectively, and performing sample mixing and mixed inspection on samples in the same area, wherein the sample mixing and inspection is performed on not more than 5 samples, and the method specifically comprises the following steps:
(1) entering personnel: monitoring and downsampling by video, wiping samples such as mouth, nose, fingernails, hands, hair, clothes, trousers, shoes and the like by more than 5 cotton swabs, and putting the samples into a sampling tube;
(2) inputting articles: for each batch of solid raw materials, 10 points are collected, wherein 3 samples are collected respectively at the head, the tail and the middle of the vehicle, 1kg of sample is collected at each point, and 10kg of sample is collected per batch; collecting 50ml of liquid by using a clean bottle for liquid raw materials, and storing the liquid at 2-4 ℃ for inspection; for the packaging box, collecting the surface of the packaging box by using gauze stained with physiological saline, and randomly collecting 70% of packaging box samples;
(3) the coming and going vehicles: the device comprises a cab, the inner side of a carriage, the roof of the carriage, the inner side of a door surface, different side connecting gaps, wheels, a mudguard and an oil tank, wherein a 'character' shaped sampling tool or a sterilized mop with gauze is used for all-dimensional mopping and sampling;
(4) ambient environment: sampling each floor of a workshop, and wiping, sampling and inspecting a raw material bin, a finished product bin, a premixing bin, a hardware bin, a bicycle shed, the ground where vehicles pass, a male and female bathing room and a discharge opening;
the detection scheme and the monitoring frequency of the off-site environment of the pig in the first step are shown in the following table 1:
TABLE 1 detection protocol for the outside environment of a pig farm
Step two, monitoring the environment in the pig farm: the sampling tool in the shape of a Chinese character 'ji' or a disinfected mop with gauze clamped is used for carrying out all-dimensional wiping sampling, and no more than 5 samples in the same area are subjected to sample mixing and inspection;
the sampling points of the environment in the pig farm comprise: the pig farm living area comprises a pig farm living area public environment, a vacant stall, a dead pig platform, a pig loading platform, a pig transferring channel, a corridor, a fumigating room isolation stool, a transition room for replacing external clothes and shoes, and a bathing front area for replacing transition clothes and shoes.
The detection scheme and the monitoring frequency of the environment in the pig farm in the second step are shown in the following table 2:
TABLE 2 protocol for the detection of the environment in a pig farm
Step three, monitoring pigs: checking whether abnormal pigs appear every day, if abnormal pigs appear in a pig farm, starting a whole-group sampling scheme, wherein the acquisition range comprises oral liquid, a nasal cavity, a water fountain/trough and the ground, and 100% of trough edges are subjected to inspection;
the performance of the abnormal pig comprises the following steps: fever, no food intake, abortion, cyanosis, bloody stool, blood coagulation failure, and mental retardation.
The step-wise full-group sampling scheme follows the following principles: the sampling personnel forbids cross-house/span sampling, and the next span sampling can be carried out only after bathing and changing clothes again; collecting normal pigs firstly and then collecting abnormal pigs; after sampling is finished, diluting and disinfecting public roads in the house by 10% sodium hypochlorite in a ratio of 1: 0.5; and (5) carrying out a video monitoring and sampling process in the whole shooting process.
The specific method of the full-group sampling scheme comprises the following steps:
(1) preparation work before supervision sampling: preparing video shooting tools, sampling materials and materials for each pigsty to be ready for people to enter in advance;
preparing a video shooting tool in advance, wherein the video shooting tool comprises a memory card, a video shooting device, a mobile charger, a data line and the like; each pigsty is prepared with materials for personnel to enter, including disposable gloves, shoe covers, protective clothing, protective hats and the like; sufficient sampling supplies were prepared including a sampling rod, cotton string, cotton swab, saline, marker pen, EP tubing filled with 1ml saline, gauze, flame sterilization tools and other tools used in the sampling work.
(2) Positioning fence/obstetric table sampling:
each pig of the positioning fence/obstetric table uses a cotton swab to collect oral liquid of the pig firstly and then collect nasal liquid of the pig, one cotton swab collects the place which the oral cavity of the pig can contact, the fence frame, the water tank, the water fountain and the drinking water are arranged, and two cotton swabs are arranged in an EP pipe which is previously provided with 1ml of physiological saline; the pig samples sharing one water tank are put into one bag for packaging, and the name of the sample indicates the name of the pig farm, namely date, column number and ear number; when oral liquid is collected, the cotton swab is put into the oral cavity of the pig, and is taken out after the pig bites the oral liquid for 3 seconds, or is taken out after being scraped back and forth in the oral cavity of the pig for 2 times; when nasal cavity liquid is collected, a cotton swab is stretched into the nasal cavity of the pig by about 2 cm, and the nose of the pig is pressed downwards to collect the nasal liquid; the action is gentle during collection, so that the condition that the pigs are damaged or afraid of influencing the next sample collection due to violent insertion is avoided;
sampling to collect normal pigs firstly and then collect abnormal pigs; when the pigs in the positioning fence are collected, a new pair of long-arm gloves is replaced every time 1 pig is collected, and shoe covers are replaced 1 time after one water tank is collected; when only pigs in the obstetric table are collected, a new pair of long-arm gloves is replaced every time 1 pig is collected.
(3) Large column sampling:
firstly, wetting gauze with physiological saline for later use before sampling; secondly, thoroughly sterilizing flame of the sampling hook of the 'shaped' sampling tool before sampling; thirdly, winding the disinfected sampling hook back and forth for three circles by using gauze, and then binding the gauze by using a sterilized nylon binding belt; when the gauze is bound and wound, the user does not need to touch the sampling hook by hands; the sampling hook is firstly wiped on the waterer back and forth for several times, and then wiped on the floor of the stall back and forth, so that the whole area of the stall is covered by 100 percent; taking a clean piece of wet gauze, and wiping 100% of the clean piece of wet gauze on a railing or a doorway (a place where a pig bites) of the stall for sampling; tying a cotton rope on a railing of a large fence in advance, and collecting saliva of the pigs;
(4) filling the collected and extruded liquid into a sampling pipe, and marking information such as a pig farm, a multi-span house, a column, an ear number and the like in detail; samples of two adjacent columns of the large column are uniformly collected and put into the same sampling tube.
A large column of samples must be collected, and a new pair of gloves is replaced (if the gloves need to be replaced when the gloves arrive at the wall of the stall). Sampling to collect normal pigs firstly and then collect abnormal pigs; after each sample is collected, disinfecting places such as walkways and the like which are possibly polluted in time.
Step four, sample detection: extracting nucleic acid from the samples collected in the first to third steps, and detecting whether the samples carry African swine fever viruses or not; the negative detection represents African swine fever virus, and the positive detection represents that the African swine fever virus is carried;
for samples with large sample amount such as environment and abnormal pigs, nucleic acid extraction is carried out by a KingFisher Flex 96-hole nucleic acid extractor of Thermofish, then fluorescent quantitative PCR amplification is carried out by an ABI 7500FAST or Roche LightCycler96 fluorescent quantitative PCR instrument, and the detection result is obtained within 3 hours.
Randomly scattered samples of vehicles, raw materials, personnel and the like were amplified in a Tetracore T8 fluorescent quantitative PCR instrument by manually extracting nucleic acids, and the detection results were obtained within 3 hours.
Step five, virus removal: executing the principles of 'full-group detection, accurate killing, pairing rechecking and pathogen elimination';
(1) sampling abnormal pigs immediately, and detecting the result on the same day; if positive pigs are isolated and eliminated in the same day, production is stopped in the same day, the pigs are detected in the whole group, and the detection result is obtained in the same day;
(2) dead pigs are bagged by 14-filament films, and the two ends of the dead pigs are tightly tied and sealed, so that the environment is not polluted; the positive pigs are detected to be subjected to off-site harmless treatment within 4 hours, the centralized air outlet of the off-site can not be realized, the vertical positions of the opposite pigs are isolated, the whole course of the road in the elimination process is closed, and the front and the back are thoroughly disinfected;
(3) spraying pig manure and the ground by using a 5% sodium hydroxide solution in a vacant stall after leaving the farm, bagging the pig manure with 12 threads after 2 hours, transporting the pig manure away, and adding lime and deeply burying the pig manure; the ground is washed clean by water, the ground is dried by a ground dryer, and 5 percent sodium hypochlorite is sprayed for 60 minutes.
(4) And after negative full detection for the first time, the environment is comprehensively disinfected and purified, after 4 days of sampling interval each time, the African swine fever virus is not detected to be positive in abnormal pig detection in the interval period, and after 3 times of full-population detection, the result is judged to be negative, which indicates that the purification is successful.
Example 2
A method for monitoring and eliminating African swine fever virus in a large-scale pig farm comprises the following steps:
step one, monitoring the environment outside a pig farm: the method comprises the following steps of monitoring the entering personnel, the input articles, the vehicles coming and going and the surrounding environment respectively, and performing sample mixing and mixed inspection on samples in the same area, wherein the sample mixing and inspection is performed on not more than 5 samples, and the method specifically comprises the following steps:
(1) entering personnel: monitoring and downsampling by video, wiping samples such as mouth, nose, fingernails, hands, hair, clothes, trousers, shoes and the like by more than 5 cotton swabs, and putting the samples into a sampling tube;
(2) inputting articles: for each batch of solid raw materials, 10 points are collected, including 4 samples are collected at the head, the tail and the middle of the vehicle respectively, 1kg of sample is collected at each point, and 10kg of sample is collected per batch; collecting 60ml of liquid by using a clean bottle for liquid raw materials, and storing at 4-8 ℃ for inspection; for the packaging box, collecting the surface of the packaging box by using gauze stained with physiological saline, and randomly collecting 70% of packaging box samples;
(3) the coming and going vehicles: the device comprises a cab, the inner side of a carriage, the roof of the carriage, the inner side of a door surface, different side connecting gaps, wheels, a mudguard and an oil tank, wherein a 'character' shaped sampling tool or a sterilized mop with gauze is used for all-dimensional mopping and sampling;
(4) ambient environment: sampling each floor of a workshop, and wiping, sampling and inspecting a raw material bin, a finished product bin, a premixing bin, a hardware bin, a bicycle shed, the ground where vehicles pass, a male and female bathing room and a discharge opening;
step two, monitoring the environment in the pig farm: the sampling tool in the shape of a Chinese character 'ji' or a disinfected mop with gauze clamped is used for carrying out all-dimensional wiping sampling, and no more than 5 samples in the same area are subjected to sample mixing and inspection;
the sampling points of the environment in the pig farm comprise: the pig farm living area comprises a pig farm living area public environment, a vacant stall, a dead pig platform, a pig loading platform, a pig transferring channel, a corridor, a fumigating room isolation stool, a transition room for replacing external clothes and shoes, and a bathing front area for replacing transition clothes and shoes.
Step three, monitoring pigs: checking whether abnormal pigs appear every day, starting a whole-group sampling scheme when the abnormal pigs appear in a pig farm, wherein the acquisition range comprises oral liquid, a nasal cavity, a water fountain/trough and the ground, and 100% of trough edges are subjected to inspection;
the performance of the abnormal pig comprises the following steps: fever, no food intake, abortion, cyanosis, bloody stool, blood coagulation failure, and mental retardation.
The step-wise full-group sampling scheme follows the following principles: the sampling personnel forbids cross-house/span sampling, and the next span sampling can be carried out only after bathing and changing clothes again; collecting normal pigs firstly and then collecting abnormal pigs; after sampling is finished, diluting and disinfecting public roads in the house by 10% sodium hypochlorite in a ratio of 1: 0.5; and (5) carrying out a video monitoring and sampling process in the whole shooting process.
The specific method of the full-group sampling scheme comprises the following steps:
(1) preparation work before supervision sampling: preparing video shooting tools, sampling materials and materials for each pigsty to be ready for people to enter in advance;
preparing a video shooting tool in advance, wherein the video shooting tool comprises a memory card, a video shooting device, a mobile charger, a data line and the like; each pigsty is prepared with materials for personnel to enter, including disposable gloves, shoe covers, protective clothing, protective hats and the like; sufficient sampling supplies were prepared including a sampling rod, cotton string, cotton swab, saline, marker pen, EP tubing filled with 1ml saline, gauze, flame sterilization tools and other tools used in the sampling work.
(2) Positioning fence/obstetric table sampling:
each pig of the positioning fence/obstetric table uses a cotton swab to collect oral liquid of the pig firstly and then collect nasal liquid of the pig, one cotton swab collects the place which the oral cavity of the pig can contact, the fence frame, the water tank, the water fountain and the drinking water are arranged, and two cotton swabs are arranged in an EP pipe which is previously provided with 2ml of physiological saline; the pig samples sharing one water tank are put into one bag for packaging, and the name of the sample indicates the name of the pig farm, namely date, column number and ear number; when oral liquid is collected, the cotton swab is put into the oral cavity of the pig and taken out after the pig bites for 4 seconds, or the pig is scraped back and forth in the oral cavity of the pig for 2 times and taken out; when nasal cavity liquid is collected, a cotton swab is stretched into the nasal cavity of the pig by about 3 cm, and the nasal cavity of the pig is pressed downwards to collect the nasal liquid; the action is gentle during collection, so that the condition that the pigs are damaged or afraid of influencing the next sample collection due to violent insertion is avoided;
sampling to collect normal pigs firstly and then collect abnormal pigs; when the pigs in the positioning fence are collected, a new pair of long-arm gloves is replaced every time 1 pig is collected, and shoe covers are replaced 1 time after one water tank is collected; when only pigs in the obstetric table are collected, a new pair of long-arm gloves is replaced every time 1 pig is collected.
(3) Large column sampling:
firstly, wetting gauze with physiological saline for later use before sampling; secondly, thoroughly sterilizing flame of the sampling hook of the 'shaped' sampling tool before sampling; thirdly, winding the disinfected sampling hook back and forth for three circles by using gauze, and then binding the gauze by using a sterilized nylon binding belt; when the gauze is bound and wound, the user does not need to touch the sampling hook by hands; the sampling hook is firstly wiped on the waterer back and forth for several times, and then wiped on the floor of the stall back and forth, so that the whole area of the stall is covered by 100 percent; taking a clean piece of wet gauze, and wiping 100% of the clean piece of wet gauze on a railing or a doorway (a place where a pig bites) of the stall for sampling; tying a cotton rope on a railing of a large fence in advance, and collecting saliva of the pigs;
(4) filling the collected and extruded liquid into a sampling pipe, and marking information such as a pig farm, a multi-span house, a column, an ear number and the like in detail; samples of two adjacent columns of the large column are uniformly collected and put into the same sampling tube.
A large column of samples must be collected, and a new pair of gloves is replaced (if the gloves need to be replaced when the gloves arrive at the wall of the stall). Sampling to collect normal pigs firstly and then collect abnormal pigs; after each sample is collected, disinfecting places such as walkways and the like which are possibly polluted in time.
Step four, sample detection: extracting nucleic acid from the samples collected in the first to third steps, and detecting whether the samples carry African swine fever viruses or not; the negative detection represents African swine fever virus, and the positive detection represents that the African swine fever virus is carried;
the sample detection method comprises the following steps:
for samples with large sample amount such as environment and abnormal pigs, nucleic acid extraction is carried out by a KingFisher Flex 96-hole nucleic acid extractor of Thermofish, then fluorescent quantitative PCR amplification is carried out by an ABI 7500FAST or Roche LightCycler96 fluorescent quantitative PCR instrument, and the detection result is obtained within 3 hours.
Randomly scattered samples of vehicles, raw materials, personnel and the like were amplified in a Tetracore T8 fluorescent quantitative PCR instrument by manually extracting nucleic acids, and the detection results were obtained within 3 hours.
Step five, virus removal: executing the principles of 'full-group detection, accurate killing, pairing rechecking and pathogen elimination';
(1) sampling abnormal pigs immediately, and detecting the result on the same day; detecting positive pigs, isolating and eliminating the positive pigs in the same day, stopping production in the same day, detecting the positive pigs in the whole group, and obtaining detection results in the same day;
(2) dead pigs are bagged by 14-filament films, and the two ends of the dead pigs are tightly tied and sealed, so that the environment is not polluted; the positive pigs are detected to be subjected to off-site harmless treatment within 4 hours, the centralized air outlet of the off-site can not be realized, the vertical positions of the opposite pigs are isolated, the whole course of the road in the elimination process is closed, and the front and the back are thoroughly disinfected;
(3) spraying pig manure and the ground by using a 5% sodium hydroxide solution in a vacant stall after leaving the farm, bagging the pig manure with 12 threads after 2 hours, transporting the pig manure away, and adding lime and deeply burying the pig manure; the ground is washed clean by water, the ground is dried by a ground dryer, and 5 percent sodium hypochlorite is sprayed for 80 minutes.
(4) And after negative full detection for the first time, the environment is comprehensively disinfected and purified, after sampling for each time, the interval is 5 days, the African swine fever virus is not detected to be positive when abnormal pigs are detected in the interval, and all negative full group detection is continuously carried out for 3 times, and the result is judged to be negative, which indicates that the purification is successful.
Claims (7)
1. A method for monitoring and eliminating African swine fever virus in a large-scale pig farm is characterized by comprising the following steps: the method comprises the following steps:
step one, monitoring the environment outside a pig farm: the method comprises the following steps of monitoring the entering personnel, the input articles, the vehicles coming and going and the surrounding environment respectively, and performing sample mixing and mixed inspection on samples in the same area, wherein the sample mixing and inspection is performed on not more than 5 samples, and the method specifically comprises the following steps:
(1) entering personnel: monitoring and downsampling by video, wiping samples such as mouth, nose, fingernails, hands, hair, clothes, trousers, shoes and the like by more than 5 cotton swabs, and putting the samples into a sampling tube;
(2) inputting articles: for each batch of solid raw materials, collecting 8-12 points, including 3-4 samples respectively collected at the head, the tail and the middle of the vehicle; collecting liquid by using a clean bottle for liquid raw materials, and storing the liquid at 2-8 ℃ for inspection; for the packaging box, collecting the surface of the packaging box by using gauze stained with physiological saline, and randomly collecting 70% of packaging box samples;
(3) the coming and going vehicles: the device comprises a cab, the inner side of a carriage, the roof of the carriage, the inner side of a door surface, different side connecting gaps, wheels, a mudguard and an oil tank, wherein a 'character' shaped sampling tool or a sterilized mop with gauze is used for all-dimensional mopping and sampling;
(4) ambient environment: sampling each floor of a workshop, and wiping, sampling and inspecting a raw material bin, a finished product bin, a premixing bin, a hardware bin, a bicycle shed, the ground where vehicles pass, a male and female bathing room and a discharge opening;
step two, monitoring the environment in the pig farm: the sampling tool in the shape of a Chinese character 'ji' or a disinfected mop with gauze clamped is used for carrying out all-dimensional wiping sampling, and no more than 5 samples in the same area are subjected to sample mixing and inspection;
step three, monitoring pigs: checking whether abnormal pigs appear every day, if abnormal pigs appear in a pig farm, starting a whole-group sampling scheme, wherein the acquisition range comprises oral liquid, a nasal cavity, a water fountain/trough and the ground, and 100% of trough edges are subjected to inspection;
step four, sample detection: extracting nucleic acid from the samples collected in the first to third steps, and detecting whether the samples carry African swine fever viruses or not; the negative detection represents African swine fever virus, and the positive detection represents that the African swine fever virus is carried;
step five, virus removal: executing the principles of 'full-group detection, accurate killing, pairing rechecking and pathogen elimination';
(1) abnormal pigs are found and are sampled immediately, and the result must be detected on the same day; if positive pigs are isolated and eliminated in the same day, production is stopped in the same day, the pigs are detected in the whole group, and the detection result is obtained in the same day;
(2) dead pigs are bagged by 14-filament films, and the two ends of the dead pigs are tightly tied and sealed, so that the environment pollution is avoided; the positive pigs are detected to be subjected to off-site harmless treatment within 4 hours, the centralized air outlet of the off-site can not be realized, the isolation of the vertical position relative to the pigs is not realized, the overnight treatment is not allowed, the whole course of the road in the elimination process is closed, and the front and the back are thoroughly disinfected;
(3) spraying pig manure and the ground by using a 5% sodium hydroxide solution in a vacant stall after leaving the farm, bagging the pig manure with 12 threads after 2 hours, transporting the pig manure away, and adding lime and deeply burying the pig manure; washing the ground with water, drying the ground, spraying 5-7% of sodium hypochlorite, and keeping for more than 60 minutes;
(4) and after the negative is detected for the first time, the environment is disinfected and purified comprehensively, the sampling interval is 3-5 days later, the African swine fever virus is not detected in the abnormal pig detection in the interval period, and the negative can be judged as negative after all negative is detected for 3 times of complete detection, which indicates the successful purification.
2. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: in the first step, the monitoring frequency of the off-site environment of the pig is at least 1 time per week.
3. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: in the second step, the sampling points of the environment in the pig farm comprise: the pig farm living area comprises a pig farm living area public environment, a vacant stall, a dead pig platform, a pig loading platform, a pig transferring channel, a corridor, a fumigating room isolation stool, a transition room for replacing external clothes and shoes, and a bathing front area for replacing transition clothes and shoes.
4. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: the performance of abnormal pigs in the third step comprises the following steps: fever, no food intake, abortion, cyanosis, bloody stool, blood coagulation failure, and mental retardation.
5. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: in the third step, the full-group sampling scheme follows the following principle: the sampling personnel forbids cross-house/span sampling, and the next span sampling can be carried out only after bathing and changing clothes again; collecting normal pigs firstly and then collecting abnormal pigs; after sampling is finished, diluting and disinfecting public roads in the house by 10% sodium hypochlorite in a ratio of 1: 0.5; and (5) carrying out a video monitoring and sampling process in the whole shooting process.
6. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: in the third step, the specific method of the full-group sampling scheme comprises the following steps:
(1) preparation work before supervision sampling: preparing video shooting tools, sampling materials and materials for each pigsty to be ready for people to enter in advance;
(2) positioning fence/obstetric table sampling:
each pig of the positioning fence/obstetric table uses a cotton swab to collect oral liquid of the pig firstly and then collect nasal liquid of the pig, one cotton swab collects the place which the oral cavity of the pig can contact, the fence frame, the water tank, the water fountain and the drinking water are arranged, and the two cotton swabs are arranged in an EP pipe which is previously filled with physiological saline; the pig samples sharing one water tank are put into one bag for packaging, and the name of the sample indicates the name of the pig farm, namely date, column number and ear number; when oral liquid is collected, putting the cotton swab into the oral cavity of the pig, taking out the pig after the pig bites the oral liquid for 3-4 seconds, or scraping the pig back and forth in the oral cavity of the pig for 2-3 times; when nasal cavity liquid is collected, a cotton swab is stretched into the nasal cavity of a pig for about 2-3 cm, and the nose of the pig is pressed downwards to collect the nasal liquid;
(3) large column sampling:
firstly, wetting gauze with physiological saline for later use before sampling; secondly, thoroughly sterilizing flame of the sampling hook of the 'shaped' sampling tool before sampling; thirdly, winding the disinfected sampling hook back and forth for three circles by using gauze, and then binding the gauze by using a sterilized nylon binding belt; when the gauze is bound and wound, the user does not need to touch the sampling hook by hands; the sampling hook is firstly wiped on the waterer back and forth for several times, and then wiped on the floor of the stall back and forth, so that the whole area of the stall is covered by 100 percent; taking a clean wet gauze, and wiping 100% of the railings or doorways of the stall for sampling; tying a cotton rope on a railing of a large fence in advance, and collecting saliva of the pigs;
(4) filling the collected and extruded liquid into a sampling pipe, and marking information such as a pig farm, a multi-span house, a column, an ear number and the like in detail; samples of two adjacent columns of the large column are uniformly collected and put into the same sampling tube.
7. The method for scale-up pig farm African swine fever virus monitoring and clearance according to claim 1, wherein: the sample detection method in the fourth step comprises the following steps: extracting nucleic acid by using a KingFisher Flex 96-hole nucleic acid extractor of Thermofisher, and performing fluorescent quantitative PCR amplification detection by using an ABI 7500FAST or Roche LightCycler96 fluorescent quantitative PCR instrument; or the nucleic acid is extracted manually, and amplification detection is carried out in a Tetracore T8 fluorescent quantitative PCR instrument.
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