CN110195130B - Method for detecting whether pig farm flies and living environment thereof carry African swine fever viruses - Google Patents
Method for detecting whether pig farm flies and living environment thereof carry African swine fever viruses Download PDFInfo
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Abstract
The invention discloses a method for detecting whether a pig farm fly and a living environment thereof carry African swine fever virus, which is characterized by comprising the following steps: step one, fly killing: killing flies in the whole pig farm in a living area, a garbage disposal area and a waste disposal area, and collecting all fly corpses in each area in the pig farm; step two, detection: mixing and mashing the fly corpses collected in the step one, detecting African swine fever pathogens in the fly corpses, and if the detection result is negative, indicating that the environment where the flies and the flies live in a pig farm do not carry African swine fever viruses, entering a step three to eliminate the existence of a false negative detection result; if the detection result is positive, the result indicates that the flies in the pig farm and the living environment of the flies carry African swine fever viruses; and step three, fruit fly putting test. The invention can accurately and quickly detect or prove whether the environment where the flies live carries the African swine fever virus or not in a pig farm so as to provide a basis for judging whether the pig farm is suitable for the reproduction or not.
Description
Technical Field
The invention belongs to the technical field of prevention, control and inspection of African swine fever, and particularly relates to a method for detecting whether a pig farm fly and a living environment thereof carry African swine fever viruses.
Background
African Swine Fever (ASF) is an acute, febrile, highly contagious disease of pigs caused by African swine fever virus infection, also called African swine fever or wart swine disease, and is clinically characterized by high fever, bleeding of skin and internal organs, and high mortality. African swine fever is endemic in Africa and, for a long time in the past, is not prevalent in the east. According to the literature, the epidemic situation of the African swine fever can be traced back to the beginning of the 20 th century. In 1921, african swine fever epidemic was first identified in kenia countries in eastern africa. Thereafter, the disease has spread epidemic in east, south, middle, west europe, america, and further east europe, with significant economic losses to these regions. In 2018, five communities in Shenbei street (New City) of Shenbei New zone Shenyang city in China find the first virus in China. By 12 months in 2018, 22 provinces in China occur that 87 pigs infect the African swine fever epidemic situation and 2 wild pigs infect the African swine fever epidemic situation, the current national African swine fever epidemic situation is still in a punctate emission state, large-area epidemic does not occur, and the overall occurrence of the epidemic situation is controllable.
The African swine fever is listed as a legal report animal epidemic disease by the world animal health Organization (OIE), and the disease is also a kind of animal epidemic disease which is mainly prevented in China. Since no vaccine effective for preventing african swine fever has been developed worldwide, rapid and accurate diagnosis and prevention are very important for preventing the spread and prevalence of the disease. At present, although the African swine fever does not outbreak on a large scale in China and only shows a tendency of spreading epidemic, the feeding amount of pig farms in China is huge, and a large number of pigs infected with the African swine fever virus in the pig farms are killed by the whole group of pigs, so that the direct economic loss caused by the African swine fever is still serious. Compared with the common swine fever virus, the African swine fever virus can survive for several weeks at room temperature, and no vaccine capable of effectively preventing the African swine fever exists at present, so that the risk of the African swine fever virus existing in a swine farm infected with the African swine fever virus or the environment around the swine farm is extremely high. In order to effectively and reasonably utilize idle pig farms, some pig farms can be introduced for reproduction again after being infected with African swine fever viruses and cleaned, disinfected and left unused for a period of time, but how to detect or ascertain whether the African swine fever viruses exist or are carried in the pig farms before reproduction is a big problem in the pig breeding industry.
The mosquitoes and flies have the characteristics of rapid propagation, large quantity, miscellaneous species and wide distribution, and are the relaying media of a plurality of diseases in the live pig breeding, such as porcine reproductive and respiratory syndrome, African swine fever, porcine pseudorabies, porcine epidemic diarrhea, porcine transmissible gastroenteritis and the like. In the mosquito and fly control of the pig farm, no matter how thorough physical cleaning or biological security measures are, the existence of mosquitoes can not be avoided. Therefore, after the swine farm is infected with the African swine fever virus, the African swine fever virus can be spread and diffused by the mosquitoes and the flies, so that the African swine fever virus is left in the living environment of the mosquitoes and the flies, and although the swine farm can be cleaned, killed and disinfected to eliminate the virus carried by the mosquitoes and the flies as much as possible, for whether the African swine fever virus still exists or is completely killed in the living environment of the mosquitoes and the flies, the African swine fever virus cannot be detected or detected by means of the prior art, or how to obtain a detection sample of the African swine fever virus in the living environment of the mosquitoes and the flies is not disclosed or suggested in the prior art, so that a detection result can be accurately and quickly obtained.
Disclosure of Invention
Aiming at the problems, the invention provides a method for detecting whether the flies in the pig farm and the living environment thereof carry the African swine fever virus, which can accurately and quickly detect or prove whether the flies and the living environment thereof carry the African swine fever virus in the pig farm so as to provide a basis for whether the pig farm is suitable for reproduction.
The invention is realized by the following technical scheme.
The method for detecting whether the flies in the pig farm and the living environment thereof carry the African swine fever virus is characterized by comprising the following steps:
step one, fly killing
Killing flies in the whole pig farm in a living area, a garbage disposal area and a waste disposal area, and collecting all fly corpses in each area in the pig farm;
step two, detection
Mixing and mashing the fly corpses collected in the step one, detecting African swine fever pathogens in the fly corpses, and if the detection result is negative, indicating that the environment where the flies and the flies live in a pig farm do not carry African swine fever viruses, entering a step three to eliminate the existence of a false negative detection result; if the detection result is positive, the result indicates that the flies in the pig farm and the living environment of the flies carry African swine fever viruses;
step three, fruit fly throwing test
Under different seasons or sanitary conditions of culture environment, the number of flies in a pig farm is more or less, so that the number of fly corpses collected after killing flies in the first step is uncertain; under the condition that the number of collected fly bodies is small, even though African swine fever virus exists in the living environment of flies in a pig farm, the detection result is false negative due to the fact that the content of pathogen in a sample is lower than the detection limit, so that a fruit fly throwing test needs to be further adopted to eliminate the possibility of false negative result, and the specific method of the fruit fly throwing test is as follows:
the method comprises the following steps of respectively carrying out a drosophila throwing test on each region of a pig farm, wherein the drosophila throwing test is as follows: placing rotten food and a light source in a test area, sealing the test area, putting multiple batches of adult fruit flies in the test area, collecting the multiple batches of adult fruit flies after the adult fruit flies move in the test area for 1-3 days, mashing the collected adult fruit flies to be used as samples to be detected, and detecting African swine fever pathogens in the multiple batches of samples to be detected; if the detection results are negative, the result indicates that the environment where the flies live in the pig farm does not carry African swine fever viruses; if the detection is positive in some batches, the detection result in the step two is false negative, which can indicate that the region carries the African swine fever virus;
in addition, in the first step and the second step, because it is unclear which areas of the pig farm the flies have moved before being killed, after the flies are killed in the whole pig farm, the detection of the fly corpses cannot judge which areas of the piggery the viruses exist in or cover the areas, and in the second step, when the detection result is positive, the pig farm can be further disinfected in the whole field to thoroughly kill the African swine fever viruses; and whether the African swine fever virus is completely killed or not can still be detected by adopting a drosophila feeding test, and the drosophila feeding test is carried out by regions, so that the source region of the African swine fever virus can be accurately positioned and the positive region can be intensively disinfected.
As a preferred technical scheme, the adult drosophila is an artificially cultured adult drosophila which does not carry pathogens.
According to the preferable technical scheme, in the drosophila feeding test, 3-5 batches of adult drosophila are fed, and after the former batch of drosophila moves for 1-3 days and is collected, the later batch of drosophila can be fed.
As a preferred technical scheme, in the fruit fly throwing test, the number of fruit flies in each batch is not less than 300.
In the preferable technical scheme, in the fruit fly throwing test, after adult fruit flies move in a test area for 1 to 3 days, the number of the collected fruit flies is not less than 100.
In the fruit fly feeding test, the temperature in the test area is controlled to be maintained for 5-8 h at 22-28 ℃ and 1-2 h at 12-15 ℃ for circulation; the activity of the fruit flies is related to the environmental temperature, the activity of the fruit flies is strong within the range of 22-28 ℃, the fruit flies can fly more frequently and rapidly in a test area, so that the activity range of the fruit flies in the test area is expanded, the probability of pathogen contact in a pig farm environment is improved, but the fruit flies mainly forage for rotten food within the temperature range and rarely lie on walls, roofs, doors and windows; in the range of 12-15 ℃, the fruit flies are weak in moving ability, and most of the fruit flies are laid on walls, roofs, doors and windows and pig baths; the invention can make the fruit fly moving area better cover all positions in the pig farm environment, such as the putrid food placing position, the wall, the roof, the door and window and the pig trough, by controlling the temperature in the test area.
According to the preferable technical scheme, in the drosophila feeding test, the temperature in the test area is controlled to be maintained at 22-28 ℃ for 5-8 h, 12-15 ℃ for 1-2 h for circulation, and illumination is carried out at the same time when the temperature is maintained at 22-28 ℃ for 5-8 h.
As a preferred technical scheme, the detection of the African swine fever pathogen adopts fluorescence PCR (polymerase chain reaction).
The invention has the beneficial effects that:
1) in the prior art, whether healthy pigs infect the African swine fever virus again through flies or not is difficult to judge whether the healthy pigs are scientifically sampled to carry out pathogen detection from a pig farm environment, particularly a mosquito and fly living environment (such as a wall, a roof and a pig trough) or not during the re-production of the pig farm.
2) The method can quickly and accurately detect or ascertain whether the flies in the pig farm or the living environment of the flies carry the African swine fever virus, thereby providing the farmers with the basis of whether the pig farm can be reproduced, and reducing the possibility of the healthy pigs being reoccurred and the economic loss of the farmers caused by blind reproduction of the pig farm.
Detailed Description
The technical solutions in the present invention are described clearly and completely below, and it is obvious that the described embodiments are only some of the embodiments of the present invention, not all of them. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Example 1
Selecting a certain diseased pig farm published by the rural agricultural department as a site to be detected, and detecting whether the pig farm flies and the living environment thereof carry the African swine fever virus by adopting the method of the invention after unblocking the blocking for 2 months, wherein the method comprises the following specific steps:
step one, fly killing
Carrying out whole-plant fly killing on a living area, a garbage disposal area and a waste disposal area of a pig farm by using fenitrothion, collecting all fly corpses in each area of the pig farm, and collecting about 89 fly corpses in total;
step two, detection
Mixing and mashing the fly carcasses collected in the step one, sampling, and quantitatively detecting the African swine fever pathogen by adopting fluorescence PCR (polymerase chain reaction), wherein the detection result is shown in a table 1;
TABLE 1 detection results of fly cadaver African swine fever virus
| Group of | Results |
| Fly corpse | 36.48 |
| Negative control | NoCt |
| Positive control | 22.32 |
As can be seen from Table 1, the positive detection result of the collected fly corpse indicates that the swine fever virus is carried in the living environment of the flies and the African swine fever virus in the pig farm and needs to be disinfected and retested.
In addition, although the swine farm has been detected to have the African swine fever virus, it is difficult to judge which region or regions of the swine farm the virus exists in, so that the whole swine farm can be disinfected only and then rechecked; because the flies in the pig farm are killed in the whole pig farm in the first step, the flies in the pig farm are few or completely killed, the fly corpses cannot be collected during the re-inspection of the whole pig farm after the disinfection, the African swine fever virus in the fly living environment in the pig farm after the re-disinfection cannot be detected, and the fruit fly throwing test can be adopted to respectively perform the re-inspection on different areas of the pig farm, which is specifically as follows: placing rotten foods (such as rotten fruits) and arranging a light source in a test area, sealing the test area by adopting a gauze (mainly plugging doors, windows, wall openings and the like, and sealing a living area, a garbage treatment area and a waste treatment area to prevent adult drosophila clusters put among different areas from influencing the detection results of other test areas or carrying and transfecting viruses to other test areas), putting 3 batches of artificially cultured adult drosophila without pathogens into the test area, respectively collecting a plurality of batches of adult drosophila (using fly sticking paper or insecticides to kill the adult drosophila and then collecting adult drosophila corpses) after the adult drosophila moves for 2 days in the test area, mashing the adult drosophila corpses to be used as samples to be detected, detecting the African swine fever pathogens in the samples to be detected, wherein the detection results are shown in a table 2; in the adult fruit fly throwing process, after the former batch of fruit flies move for 2 days and are collected, the latter batch of fruit flies can be thrown, and 350 fruit flies are thrown in each batch.
TABLE 2 reinspection of the test results
As shown in Table 2, after the pig farm is disinfected, the African swine fever viruses are not detected in the living area, the waste treatment area and the garbage treatment area, and the virus killing effect in the pig farm is good. It should be noted that in other cases, only one area of the living area, the waste disposal area and the garbage disposal area may be detected to have the african swine fever virus, and then only the area needs to be disinfected in an important way, and then the drosophila throwing test is adopted to perform rechecking again until the detection is negative.
Example 2
Selecting a certain diseased pig farm published by the rural agricultural department as a detection site, and after unblocking for a period of time, detecting whether the pig farm flies and the living environment thereof carry the African swine fever virus by adopting the method of the invention to confirm the killing effect, the specific steps are as follows:
step one, fly killing
Carrying out whole-plant fly killing on a living area, a garbage disposal area and a waste disposal area of a pig farm by using fenitrothion, collecting all fly carcasses in each area of the pig farm, and collecting about 54 fly carcasses in total;
step two, detection
Mixing and mashing the fly carcasses collected in the step one, sampling, and detecting African swine fever pathogens by adopting fluorescence PCR (polymerase chain reaction), wherein the detection result is shown in a table 3;
TABLE 3 detection results of the fly cadaver African swine fever virus
| Group of | Results |
| Fly corpse | NoCt |
| Negative control | NoCt |
| Positive control | 21.06 |
As can be seen from Table 1, the collected fly corpse detection result is negative, which indicates that the environment in which flies and flies live in a pig farm do not carry African swine fever virus, and the third step is performed to eliminate the existence of false negative detection result;
step three, fruit fly throwing test
The method comprises the following steps of respectively carrying out a drosophila throwing test on each region of a pig farm, wherein the drosophila throwing test is as follows: placing rotten foods (such as rotten fruits) and arranging a light source in a test area, sealing the test area by adopting a gauze (mainly plugging doors, windows, wall openings and the like, and sealing a living area, a garbage treatment area and a waste treatment area to prevent adult drosophila clusters put among different areas from influencing the detection results of other test areas or carrying and transfecting viruses to other test areas), putting 4 batches of artificially cultured adult drosophila without pathogens into the test area, respectively collecting a plurality of batches of adult drosophila (using fly sticking paper or insecticides to kill the adult drosophila and then collecting adult drosophila corpses) after the adult drosophila moves for 3 days in the test area, mashing the adult drosophila corpses to be used as samples to be detected, detecting the African swine fever pathogens in the samples to be detected, wherein the detection results are shown in a table 4; in the adult fruit fly throwing process, after the former batch of fruit flies move for 3 days and are collected, the latter batch of fruit flies can be thrown, and 320 fruit flies are thrown in each batch;
as can be seen from Table 4, no African swine fever virus was detected in the pig farm living area, waste disposal area and waste disposal area (the negative detection result in step two is not false negative), and the killing effect of the pig farm was good. It should be noted that in other cases, only one area of the living area, the waste disposal area and the garbage disposal area may be detected to have the african swine fever virus (the detection result is positive, and the negative detection result in the second step is false negative), so that only the area needs to be disinfected in an important way, and then a drosophila throwing test is adopted for rechecking until the detection is negative.
TABLE 4 reinspection test results
Claims (2)
1. The method for detecting whether the flies in the pig farm and the living environment thereof carry the African swine fever virus is characterized by comprising the following steps:
step one, fly killing
Killing flies in the whole pig farm in a living area, a garbage disposal area and a waste disposal area, and collecting all fly corpses in each area in the pig farm;
step two, detection
Mixing and mashing the fly corpses collected in the step one, detecting African swine fever pathogens in the fly corpses, and if the detection result is negative, indicating that the environment where the flies and the flies live in a pig farm do not carry African swine fever viruses, entering a step three to eliminate the existence of a false negative detection result; if the detection result is positive, the result indicates that the flies in the pig farm and the living environment of the flies carry African swine fever viruses;
step three, fruit fly throwing test
The method comprises the following steps of respectively carrying out a drosophila throwing test on each region of a pig farm, wherein the drosophila throwing test is as follows: placing rotten food and a light source in a test area, sealing the test area, putting multiple batches of adult fruit flies in the test area, collecting the multiple batches of adult fruit flies after the adult fruit flies move in the test area for 1-3 days, mashing the collected adult fruit flies to be used as samples to be detected, and detecting African swine fever pathogens in the multiple batches of samples to be detected; if the detection results are negative, the result indicates that the environment where the flies live in the pig farm does not carry African swine fever viruses; if the detection is positive in some batches, the detection result in the second step is false negative, which indicates that the region carries the African swine fever virus; in the fruit fly feeding test, the temperature in a test area is controlled to be maintained at 22-28 ℃ for 5-8 h, 12-15 ℃ for 1-2 h for circulation, and illumination is carried out at the same time when the temperature is maintained at 22-28 ℃ for 5-8 h; the adult fruit fly is artificially cultured and does not carry pathogen; in the fruit fly feeding test, 3-5 batches of adult fruit flies are fed, the feeding quantity of each batch of fruit flies is not less than 300, and the feeding of the next batch of fruit flies can be started after the former batch of fruit flies move for 1-3 days and have collected not less than 100 fruit flies.
2. The method for detecting whether the flies in the pig farm and the living environment thereof carry the African swine fever virus according to claim 1, wherein the detection of the African swine fever pathogen adopts a fluorescence PCR method for detection.
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| CN105002297A (en) * | 2015-02-13 | 2015-10-28 | 河南科技学院 | Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN105002297A (en) * | 2015-02-13 | 2015-10-28 | 河南科技学院 | Multi-RT-PCR method for monitoring pollution of six viruses in pig farm environment through one system and application |
Non-Patent Citations (3)
| Title |
|---|
| Survival and localization of African swine fever virus in stable flies (Stomoxys calcitrans) after feeding on viremic blood using a membrane feeder;A. S. Olesen等;《Veterinary Microbiology》;20180619;第222卷;第25-29页 * |
| 家蝇体内猪繁殖与呼吸综合征病毒的初步分离与鉴定;路宪礼等;《中国畜牧兽医》;20120220;第39卷(第2期);第24-27页 * |
| 非洲猪瘟流行的背景下猪场生物安全管理;胡斌等;《广东畜牧兽医科技》;20190618;第44卷(第3期);第5-13页 * |
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Address after: No. 708, Changsha Avenue, Yuhua District, Changsha City, Hunan Province 410000 Patentee after: Hunan Animal Epidemic Prevention and Control Center Patentee after: Hunan Zhongjing Biotechnology Co.,Ltd. Address before: No. 708, Changsha Avenue, Yuhua District, Changsha City, Hunan Province 410000 Patentee before: Hunan Animal Epidemic Prevention and Control Center Patentee before: HUNAN NEW SOUTH BREEDING SERVICE Co.,Ltd. |