GB 16883-1997 Standards for determining natural plague foci and animal plague epidemics

This standard specifies the geographical, zoological, etiological and serological indicators for the determination of various types of natural plague foci and animal plague epidemics in my country. GB 16883-1997 Standard for the determination of natural plague foci and animal plague epidemics GB16883-1997 Standard download decompression password: www.bzxz.net

GB16883
Plague is a natural epidemic disease, forming a natural epidemic focus (referred to as the epidemic focus) in a certain geographical and ecological environment. By the end of 1995, it had been determined that there were plague epidemic focuses in 234 counties (cities, banners) in 17 provinces (regions) in my country. People are infected with plague due to direct contact with plague-infected animals or bites by infected fleas. Under certain conditions, it can cause the spread of plague among humans, which is serious. my country has classified it as a Class A infectious disease. Therefore, in order to timely and accurately determine the natural epidemic focus of plague and the epidemic situation of animal plague, and provide a basis for the correct and reasonable formulation of prevention and control measures, this standard is specially formulated
In the process of developing this standard, we strive to make full use of the field practice and theoretical research results of my country's investigation of plague epidemic focuses and research on plague animal diseases, and express them in relevant chapters. Appendix A and Appendix B of this standard are both appendices to the standard. This standard was proposed by the Ministry of Health of the People's Republic of China. The responsible drafting units of this standard are: Qinghai Provincial Institute of Endemic Disease Control and Prevention and Gansu Provincial Institute of Endemic Disease Control and Prevention; participating drafting units are: Xinjiang Uygur Autonomous Region Institute of Endemic Disease Control and Prevention, Yunnan Provincial Institute of Epidemiology Control and Prevention, Inner Mongolia Autonomous Region Institute of Epidemiology Control and Prevention. The main drafters of this standard are: Zhu Jinqin, Wang Wenshao, Zhang Hongxian, Huang Jianhua, Liu Jiyou, Wang Li, Li Zhilun. The Chinese Academy of Preventive Sciences, the technical unit entrusted by the Ministry of Health, is responsible for the interpretation of this standard. 500
1 Scope
National Standard of the People's Republic of China
Criteria for derterminating plaguenatural foci and plague epizootics
The criteria for derterminating plaguenatural foci and plague epizooticsGB16883---1997
This standard specifies the geographical, zoological, etiological and serological indicators for the determination of various types of plague natural foci and animal plague epizootics in my country.
The criteria for determining plague foci apply to areas with animal plague and positive results obtained through etiological tests, with counties (cities, banners) as units.
The criteria for determining animal plague epidemics apply to areas with counties (cities, banners) as units within the scope of the following ten types of natural plague foci in my country and positive results obtained through etiological and beach plague tests. 1) Natural plague foci of Himalayan marmots in the Qinghai-Tibet Plateau; 2) Natural plague foci of Mongolian marmots in the Hulunbuir Plateau; 3) Natural plague foci of long-tailed marmots in the Pamir Plateau; 4) Natural plague foci of gray marmots and long-tailed yellow squirrels in the Tianshan Mountains; 5) Natural plague foci of Daurian yellow squirrels in the Songliao Plain; 6) Natural plague foci of Alxa yellow squirrels in the Huangshi Plateau of Ganning; 7) Natural plague foci of long-clawed gerbils in the Inner Mongolia Plateau; 8) Natural plague foci of Brandt's voles in the Xilin Gol Plateau; 9) Natural plague foci of large woolly rats and Qi's field mice in the mountains of northwest Yunnan; 10) Natural plague foci of house mice in Yunnan and the southeast coast. 2 Referenced standards
The provisions contained in the following standards constitute the provisions of this standard by reference in this standard. When this standard is published, the versions shown are valid. All standards will be revised, and parties using this standard should explore the possibility of using the latest version of the following standards. GB15991-1995 Plague Diagnostic Standard
GB 15992—1995
Principles and Methods of Plague Control and Assessment
3 Definitions
This standard adopts the following definitions.
3.1 Plague Natural Foci Plague is a typical natural foci disease. Areas where animal plague exists and spread are plague natural foci. Plague natural foci are solid unity formed by hosts, vectors and pathogens through long-term survival competition, adaptation and natural selection under corresponding geographical landscape conditions and in the long history of biological evolution. Such areas with specific ecosystems in which plague bacteria circulate are called plague natural foci approved by the State Administration of Technical Supervision on June 16, 1997 and implemented on January 1, 1998
3.2 Main reservoir
CB16883-1997
Host animals that play a decisive role in the long-term preservation of plague bacteria. The common characteristics of main hosts are that they are susceptible and sensitive to plague bacteria, which means they can be infected with plague or die of septicemia; they are often the dominant species in a certain focus of plague, with high density; they have a wide distribution area and are distributed continuously; they have a medium suitable for the spread of plague, that is, fleas that can form bacterial plugs. 3.3 Main vector
Fleas that have a high transmission ability and play a major role in causing and maintaining the plague epidemic in the main host and maintaining the natural focus of plague. 4 Criteria for determining natural plague foci
4.1 There are corresponding natural geographical landscapes suitable for the existence of various plague foci. 4.2 In the landscape, there are major host animals distributed in continuous patches, whose population coverage usually accounts for more than 20% of the landscape, and whose density (see GB15992 for details) is high and constant
4.3 The flea index of the major host's major vector should be ≥1. 4.4 Plague bacteria are detected in the major host or its major external parasites in the landscape (see GB15991 for details). 5 Standards for determining the epidemic of animal plague in the epidemic source area 5.1 Standards for the current epidemic of animal plague
5.1.1 Plague bacteria are detected in the major host and its major vector or other animals in the epidemic source area. 5.1.2 Although plague bacteria have not been detected, plague F antibodies are detected by indirect hemagglutination in the serum of the main hosts or shepherd dogs during continuous testing in the epidemic source area, and the positive rate or the hemagglutination titer of one or more of them reaches one of the following standards, indicating that an animal plague epidemic has occurred or is occurring in the detection area that year. 5.1.2.1 In the epidemic source area of ​​Himalayan marmot and Mongolian marmot, in the habitat of marmots, the detection area is more than 10,000 hectares, the number of inspected marmots is not less than 10% of the predicted number of marmots, the indirect hemagglutination positivity rate is more than 5%, and the highest hemagglutination titer is more than 1:160; the number of inspected shepherd dogs is not less than 50, the positive rate is more than 5%, and the highest hemagglutination titer is more than 1:320. 5.1.2.2 In the foci of the gray morning otter, long-tailed ground squirrel and long-tailed marmot, in their respective habitats, the total inspection area is more than 10,000 ha. The number of marmots to be inspected shall not be less than 10% of the predicted number of otters, and the number of long-tailed ground squirrels shall not be less than 5% of the predicted number of rats. The indirect hemagglutination positive rate shall reach 1% for marmots and more than 10% for long-tailed ground squirrels. The highest hemagglutination titer shall reach 1:40 for morning otter and more than 1:160 for long-tailed ground squirrels. No less than 50 shepherd dogs shall be inspected, with a positive rate of 5% and a highest hemagglutination titer of more than 1:160. 5.1.2.3 In the foci of the Daurian ground squirrel, in the habitats of ground squirrels, the total inspection area is more than 2,500 ha. The number of Daurian ground squirrels to be inspected shall not be less than 10% of the predicted number of rats. The indirect hemagglutination positive rate shall reach more than 3% and the highest hemagglutination titer shall reach more than 1:160. 5.1.2.4 In the Alashan yellow squirrel epidemic source, in the yellow squirrel habitat, the total inspection area is more than 1000ha, and the Alashan yellow squirrel is inspected for no less than 20% of the predicted rat population, with an indirect hemagglutination positive rate of more than 3% and a hemagglutination maximum titer of more than 1:160. 5.1.2.5 In the long-clawed gerbil epidemic source, in the gerbil habitat, the total inspection area is more than 1000ha, and the long-clawed gerbil is inspected for no less than 10% of the predicted rat population, with an indirect hemagglutination positive rate of more than 2% and a hemagglutination maximum titer of more than 1:160. 5.1.2.6 In the Brandt's vole epidemic source, in the vole habitat, the total inspection area is more than 1000ha, and the Brandt's vole is inspected for no less than 5% of the predicted rat population, with an indirect hemagglutination positive rate of more than 2% and a hemagglutination maximum titer of more than 1:160. 5.1.2.7 In the plague source area of ​​the Great Downy Rat and the Apodemus chevnii, in their habitats, the total inspection area is more than 1000ha, and no less than 300 Great Downy Rat and Apodemus chevnii are inspected, with the indirect hemagglutination positive rate reaching more than 3%, and the highest hemagglutination titer reaching more than 1:320. The canine serum positive rate is more than 10%, and the highest hemagglutination titer is more than 1:160. 5.1.2.8 In the plague source area of ​​the domestic rat, in the habitat of the yellow-breasted rat or other main hosts, no less than 500 yellow-breasted rats or other main hosts are inspected, with the indirect hemagglutination positive rate reaching more than 1%, and the highest hemagglutination titer reaching more than 1:160. 5.2 Criteria for determining the prevalence of animal plague in the plague source area: Any animal that does not meet the requirements of 5.1.1~5.1.2, but in the case of continuous testing in each epidemic source, the area where the serum of the main host or dog is positive by indirect hemagglutination (see GB15991 for details). 502
A1 Collection of animal materials
GB16883-1997
Appendix A
(Standard Appendix)
The collection method of animal and insect materials to be inspected should select representative areas within the scope of the investigation to capture the required animals and collect self-sufficient animals in the entire range for inspection at the same time. A1.1 Collection method: All the animals to be inspected will be registered with their classification numbers, and each animal will be placed in a small cloth bag. After the live animals are killed, the external parasites will be cleaned, and the animals will be classified and identified, and then dissected according to the following method. A1.2 Self-destructed, infected and eroded animals: After dissection according to the conventional method, observe whether there are lesions in the glands, liver, spleen, lungs, heart, etc., and take the corresponding materials for bacteriological examination. The instruments used must be disinfected after each use. A1.3 Captured animals: In principle, only liver, spleen or diseased tissues are taken for examination. A1.4 Decayed animals: Bone marrow and brain tissues are often taken for examination. A2 Collection of insect materials
Insect materials include: fleas, ticks, mites, lice, etc., with fleas as the focus. Collection of fleas:
A2.1 Collection of animal fleas: Collected fleas and other insects are placed in a vial containing 0.5×10-5 gentian violet and 2% saline, and the host, collection location, and collection date are marked.
A2.2 Collection of tunnel fleas: Collect with an egg-finding stick with a white flannel or towel fixed on the top. The fleas and other insects found are packaged and registered according to the method A2.1.
A2.3 Collection of nest fleas: Mainly obtained by digging the nests and surface soil in the nests. One nest must be packed in one bag, and then placed in a large white pond porcelain basin for egg inspection. The collected fleas are packaged and registered according to the method A2.1. A2.4 Collection of free eggs on the ground: usually collect with sticky flea paper, put the sticky flea paper at a certain position on the floor of the house as required, check it at dusk and in the morning, collect the eggs on the sticky egg paper, and pack and register it according to the method of A2.1. A2.5 Classify and identify the fleas collected above and conduct bacteriological examination. A3 Collection of human materials
Refer to Appendix A of GB15991.
Appendix B
(Standard Appendix)
Bacteriological examination methods for plague
B1 Microscopic examination
B1.1 Smear: Collect materials according to Appendix A, cut the glands, liver, spleen, lungs, and heart, and use the cut surface to press or smear on a clean glass slide; or take bone marrow, cerebrospinal fluid and various exudates, sputum, etc. and use an inoculation loop to directly smear. Fix with 95% ethanol or a fixative prepared with half ethanol and half ether for 10 minutes, take out and dry naturally.
B1.2 Staining: Gram staining must be performed on the smear. If there are multiple smears for one material, methylene blue staining or Weishen and Giemsa staining can be performed.
B1.3 Microscopic examination: If Gram-negative short coccobacilli with thick staining at both ends and blunt circles are found, it means that they meet the characteristics of plague bacteria. Attention should also be paid to the changes in the polymorphism of the bacteria in rotten materials or old materials. B2 Culture
GB16883—1997
B2.1 Culture medium: Fresh selective sensitive culture medium should be used, and gentian violet hemolytic trypsin digestion liquid agar is commonly used; gentian violet should contain 0.5×10-s for separating plague bacteria from general materials. The gentian violet content of contaminated materials should be 1×10-5. Gentian violet hemolytic trypsin digestion soup can be used for bacterial enrichment culture. The pH of the culture medium is required to be 6.9~~7.1.
B2.2 Inoculation method: Animal materials are collected according to Appendix A, and then the material to be tested is hooked in the center of the organ section with an inoculation loop and inoculated on a flat plate. If the material is fresh, the organ section can be directly pressed and then inoculated with a streak; bone marrow materials can be extracted from the broken end with a syringe or injected with physiological saline for flushing, and a drop of the flushing liquid can be drawn onto the flat plate and then streaked for inoculation; liquid materials can be directly hooked and streaked for culture with an inoculation loop. Flea materials can be processed according to the method in Appendix A and then grouped and ground, and the ground material can be hooked and inoculated. When grouping and culturing, the same flea species of one animal should be grouped together. When mixed groups are grouped, the same location, the same rat species, and the same flea species must be grouped together. When egg materials are scarce, single stomach inoculation and culture should be adopted. Other insects can be carried out in accordance with eggs. If necessary, dead rats or key materials can be enriched with liquid culture medium before isolation. Two flat plates should be inoculated for each material; one is used to isolate plague bacteria, and the other is used for rapid diagnosis of bacteriophages. B2.3 Culture: The inoculated culture medium should be cultured in a 28-30℃ incubator. Animal materials are generally cultured for 3 days, and insect materials are cultured for 4 days. B2.4 Observation: The culture is observed once a day, and animal materials are observed for 3 consecutive days. Insect materials are observed for 4 consecutive days. If suspected bacteria are found during the observation, they should be immediately purified and tested for bacteriophages. B3 Phage test
B3.1 Conventional method: Whenever suspected plague bacteria are found, they should be streaked on a hemolytic agar plate, and plague phages should be sucked with a syringe or capillary pipette and dropped on the upper part of the plate streaking line to make it flow vertically, and then cultured at 28℃ for 24 hours to observe the results. If plaques or phage bands appear, it can be judged that the plague phage test is positive. B3.2 Rapid phage diagnosis: For materials that require rapid phage diagnosis, the materials to be tested should be inoculated with two plates, one for isolating plague bacteria, and the other for dropping human plague phages for phage test immediately after inoculation. B4 Animal Test
B4.1 Preparation of Inoculation Material
Grind the organs to be tested in a sterile mortar, and add 1mL of saline to every 100mg of the organ to make a suspension; insect materials can be grouped into 10~30 insects from the same location, the same host, and the same insect species. After washing with 1×10-5 gentian violet saline, grind them in a mortar, and add 1~2ml of saline to each group of insects to make a suspension; bone marrow can be diluted 2~3 times with saline; exudate, blood, etc. can be used directly, and blood that cannot be inoculated on site should be added with anticoagulants. Before absorbing the inoculation liquid, a small sterile cotton ball can be added to the suspension, and then the needle of the syringe can be inserted into the cotton ball to absorb the material to be tested and inoculate the animal. B4.2 Inoculation Method and Inoculation Dosage
B4.2.1 Intraperitoneal inoculation: Suitable for those who want to obtain results quickly but do not expect obvious pathological changes. Generally used for fresh materials to be tested, the method is to first cut off the abdominal hair on the side to be injected, disinfect with iodine and alcohol, wait until it is dry, lower the head of the test animal, raise the tail, extract the abdominal wall and slowly pierce it. After the needle is inserted, it should be inserted along the abdominal wall to prevent puncturing the internal organs, and slowly injected into the object to be tested. Guinea pigs are injected with 0.5-1.0ml., mice are injected with 0.2-0.4mL, then take out the syringe and gently press the needle eye with a dry cotton ball. B4.2.2 Subcutaneous inoculation: It is the most commonly used method. The course of disease is appropriate; animals can develop more typical lesions after disease induction; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: It is suitable for materials that are corrupt or seriously contaminated. The course of the disease is slow, and obvious pathological changes can be obtained. In general, animals will not be killed by other mixed infections, and it can play a good role in biological filtration. However, if the content of plague bacteria in the materials to be tested is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp object to scratch the skin until there is no bleeding. Dip a cotton swab in the material to be tested, apply it on the exposed skin and rub it to infect it percutaneously.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container, and fed and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs within 1 to 3 days. When the disease occurs, the animal wilts and rots, stops eating, has erect hair and hunched back, and usually dies within 3 to 7 days. After the animal dies, it should be dissected immediately to observe the lesions and isolate the plague bacteria. If the animal still does not get sick on the 7th day, it can be taken out and killed for dissection on the 8th day. If the material tested meets the characteristics of the plague bacteria through the above "four-step diagnosis", it can be determined on the spot that it is animal plague, and it will be reported step by step according to regulations for the epidemic area to be handled, and the strain will be sent to the higher-level business department for re-judgment and further inspection.4 Corrupted animals: bone marrow and brain tissue are often taken for examination. A2 Collection of insect materials
Insect materials include: fleas, ticks, mites, lice, etc., with fleas as the focus. Collection of fleas:
A2.1 Collection of animal fleas: Collected fleas and other insects are placed in a vial containing 0.5×10-5 gentian violet and 2% saline, and the host, collection location, and collection date are marked.
A2.2 Collection of tunnel fleas: Collect with an egg-finding stick with a white flannel or towel fixed on the top. The fleas and other insects found are packaged and registered according to the method A2.1.
A2.3 Collection of nest fleas: Mainly obtained by digging the nests and surface soil in the nests. One nest must be packed in one bag, and then placed in a large white pond porcelain basin to check for eggs. The collected fleas are packaged and registered according to the method A2.1. A2.4 Collection of free eggs on the ground: usually collect with sticky flea paper, put the sticky flea paper at a certain position on the floor of the house as required, check it at dusk and in the morning, collect the eggs on the sticky egg paper, and pack and register it according to the method of A2.1. A2.5 Classify and identify the fleas collected above and conduct bacteriological examination. A3 Collection of human materials
Refer to Appendix A of GB15991.
Appendix B
(Standard Appendix)
Bacteriological examination methods for plague
B1 Microscopic examination
B1.1 Smear: Collect materials according to Appendix A, cut the glands, liver, spleen, lungs, and heart, and use the cut surface to press or smear on a clean glass slide; or take bone marrow, cerebrospinal fluid and various exudates, sputum, etc. and use an inoculation loop to directly smear. Fix with 95% ethanol or a fixative prepared with half ethanol and half ether for 10 minutes, take out and dry naturally.
B1.2 Staining: Gram staining must be performed on the smear. If there are multiple smears for one material, methylene blue staining or Weishen and Giemsa staining can be performed.
B1.3 Microscopic examination: If Gram-negative short coccobacilli with thick staining at both ends and blunt circles are found, it means that they meet the characteristics of plague bacteria. Attention should also be paid to the changes in the polymorphism of the bacteria in rotten materials or old materials. B2 Culture
GB16883—1997
B2.1 Culture medium: Fresh selective sensitive culture medium should be used, and gentian violet hemolytic trypsin digestion liquid agar is commonly used; gentian violet should contain 0.5×10-s for separating plague bacteria from general materials. The gentian violet content of contaminated materials should be 1×10-5. Gentian violet hemolytic trypsin digestion soup can be used for bacterial enrichment culture. The pH of the culture medium is required to be 6.9~~7.1.
B2.2 Inoculation method: Animal materials are collected according to Appendix A, and then the material to be tested is hooked in the center of the organ section with an inoculation loop and inoculated on a flat plate. If the material is fresh, the organ section can be directly pressed and then inoculated with a streak; bone marrow materials can be extracted from the broken end with a syringe or injected with physiological saline for flushing, and a drop of the flushing liquid can be drawn onto the flat plate and then streaked for inoculation; liquid materials can be directly hooked and streaked for culture with an inoculation loop. Flea materials can be processed according to the method in Appendix A and then grouped and ground, and the ground material can be hooked and inoculated. When grouping and culturing, the same flea species of one animal should be grouped together. When mixed groups are grouped, the same location, the same rat species, and the same flea species must be grouped together. When egg materials are scarce, single stomach inoculation and culture should be adopted. Other insects can be carried out in accordance with eggs. If necessary, dead rats or key materials can be enriched with liquid culture medium before isolation. Two flat plates should be inoculated for each material; one is used to isolate plague bacteria, and the other is used for rapid diagnosis of bacteriophages. B2.3 Culture: The inoculated culture medium should be cultured in a 28-30℃ incubator. Animal materials are generally cultured for 3 days, and insect materials are cultured for 4 days. B2.4 Observation: The culture is observed once a day, and animal materials are observed for 3 consecutive days. Insect materials are observed for 4 consecutive days. If suspected bacteria are found during the observation, they should be immediately purified and tested for bacteriophages. B3 Phage test
B3.1 Conventional method: Whenever suspected plague bacteria are found, they should be streaked on a hemolytic agar plate, and plague phages should be sucked with a syringe or capillary pipette and dropped on the upper part of the plate streaking line to make it flow vertically, and then cultured at 28℃ for 24 hours to observe the results. If plaques or phage bands appear, it can be judged that the plague phage test is positive. B3.2 Rapid phage diagnosis: For materials that require rapid phage diagnosis, the materials to be tested should be inoculated with two plates, one for isolating plague bacteria, and the other for dropping human plague phages for phage test immediately after inoculation. B4 Animal Test
B4.1 Preparation of Inoculation Material
Grind the organs to be tested in a sterile mortar, and add 1mL of saline to every 100mg of the organ to make a suspension; insect materials can be grouped into 10~30 insects from the same location, the same host, and the same insect species. After washing with 1×10-5 gentian violet saline, grind them in a mortar, and add 1~2ml of saline to each group of insects to make a suspension; bone marrow can be diluted 2~3 times with saline; exudate, blood, etc. can be used directly, and blood that cannot be inoculated on site should be added with anticoagulants. Before absorbing the inoculation liquid, a small sterile cotton ball can be added to the suspension, and then the needle of the syringe can be inserted into the cotton ball to absorb the material to be tested and inoculate the animal. B4.2 Inoculation Method and Inoculation Dosage
B4.2.1 Intraperitoneal inoculation: Suitable for those who want to obtain results quickly but do not expect obvious pathological changes. Generally used for fresh materials to be tested, the method is to first cut off the abdominal hair on the side to be injected, disinfect with iodine and alcohol, wait until it is dry, lower the head of the test animal, raise the tail, extract the abdominal wall and slowly pierce it. After the needle is inserted, it should be inserted along the abdominal wall to prevent puncturing the internal organs, and slowly injected into the object to be tested. Guinea pigs are injected with 0.5-1.0ml., mice are injected with 0.2-0.4mL, then take out the syringe and gently press the needle eye with a dry cotton ball. B4.2.2 Subcutaneous inoculation: It is the most commonly used method. The course of disease is appropriate; animals can develop more typical lesions after disease induction; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: It is suitable for materials that are corrupt or seriously contaminated. The course of the disease is slow, and obvious pathological changes can be obtained. In general, animals will not be killed by other mixed infections, and it can play a good role in biological filtration. However, if the content of plague bacteria in the materials to be tested is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp object to scratch the skin until there is no bleeding. Dip a cotton swab in the material to be tested, apply it on the exposed skin and rub it to infect it percutaneously.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container, and fed and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs within 1 to 3 days. When the disease occurs, the animal wilts and rots, stops eating, has erect hair and hunched back, and usually dies within 3 to 7 days. After the animal dies, it should be dissected immediately to observe the lesions and isolate the plague bacteria. If the animal still does not get sick on the 7th day, it can be taken out and killed for dissection on the 8th day. If the material tested meets the characteristics of the plague bacteria through the above "four-step diagnosis", it can be determined on the spot that it is animal plague, and it will be reported step by step according to regulations for the epidemic area to be handled, and the strain will be sent to the higher-level business department for re-judgment and further inspection.4 Corrupted animals: bone marrow and brain tissue are often taken for examination. A2 Collection of insect materials
Insect materials include: fleas, ticks, mites, lice, etc., with fleas as the focus. Collection of fleas:
A2.1 Collection of animal fleas: Collected fleas and other insects are placed in a vial containing 0.5×10-5 gentian violet and 2% saline, and the host, collection location, and collection date are marked.
A2.2 Collection of tunnel fleas: Collect with an egg-finding stick with a white flannel or towel fixed on the top. The fleas and other insects found are packaged and registered according to the method A2.1.
A2.3 Collection of nest fleas: Mainly obtained by digging the nests and surface soil in the nests. One nest must be packed in one bag, and then placed in a large white pond porcelain basin to check for eggs. The collected fleas are packaged and registered according to the method A2.1. A2.4 Collection of free eggs on the ground: usually collect with sticky flea paper, put the sticky flea paper at a certain position on the floor of the house as required, check it at dusk and in the morning, collect the eggs on the sticky egg paper, and pack and register it according to the method of A2.1. A2.5 Classify and identify the fleas collected above and conduct bacteriological examination. A3 Collection of human materials
Refer to Appendix A of GB15991.
Appendix B
(Standard Appendix)
Bacteriological examination methods for plague
B1 Microscopic examinationbZxz.net
B1.1 Smear: Collect materials according to Appendix A, cut the glands, liver, spleen, lungs, and heart, and use the cut surface to press or smear on a clean glass slide; or take bone marrow, cerebrospinal fluid and various exudates, sputum, etc. and use an inoculation loop to directly smear. Fix with 95% ethanol or a fixative prepared with half ethanol and half ether for 10 minutes, take out and dry naturally.
B1.2 Staining: Gram staining must be performed on the smear. If there are multiple smears for one material, methylene blue staining or Weishen and Giemsa staining can be performed.
B1.3 Microscopic examination: If Gram-negative short coccobacilli with thick staining at both ends and blunt circles are found, it means that they meet the characteristics of plague bacteria. Attention should also be paid to the changes in the polymorphism of the bacteria in rotten materials or old materials. B2 Culture
GB16883—1997
B2.1 Culture medium: Fresh selective sensitive culture medium should be used, and gentian violet hemolytic trypsin digestion liquid agar is commonly used; gentian violet should contain 0.5×10-s for separating plague bacteria from general materials. The gentian violet content of contaminated materials should be 1×10-5. Gentian violet hemolytic trypsin digestion soup can be used for bacterial enrichment culture. The pH of the culture medium is required to be 6.9~~7.1.
B2.2 Inoculation method: Animal materials are collected according to Appendix A, and then the material to be tested is hooked in the center of the organ section with an inoculation loop and inoculated on a flat plate. If the material is fresh, the organ section can be directly pressed and then inoculated with a streak; bone marrow materials can be extracted from the broken end with a syringe or injected with physiological saline for flushing, and a drop of the flushing liquid can be drawn onto the flat plate and then streaked for inoculation; liquid materials can be directly hooked and streaked for culture with an inoculation loop. Flea materials can be processed according to the method in Appendix A and then grouped and ground, and the ground material can be hooked and inoculated. When grouping and culturing, the same flea species of one animal should be grouped together. When mixed groups are grouped, the same location, the same rat species, and the same flea species must be grouped together. When egg materials are scarce, single stomach inoculation and culture should be adopted. Other insects can be carried out in accordance with eggs. If necessary, dead rats or key materials can be enriched with liquid culture medium before isolation. Two flat plates should be inoculated for each material; one is used to isolate plague bacteria, and the other is used for rapid diagnosis of bacteriophages. B2.3 Culture: The inoculated culture medium should be cultured in a 28-30℃ incubator. Animal materials are generally cultured for 3 days, and insect materials are cultured for 4 days. B2.4 Observation: The culture is observed once a day, and animal materials are observed for 3 consecutive days. Insect materials are observed for 4 consecutive days. If suspected bacteria are found during the observation, they should be immediately purified and tested for bacteriophages. B3 Phage test
B3.1 Conventional method: Whenever suspected plague bacteria are found, they should be streaked on a hemolytic agar plate, and plague phages should be sucked with a syringe or capillary pipette and dropped on the upper part of the plate streaking line to make it flow vertically, and then cultured at 28℃ for 24 hours to observe the results. If plaques or phage bands appear, it can be judged that the plague phage test is positive. B3.2 Rapid phage diagnosis: For materials that require rapid phage diagnosis, the materials to be tested should be inoculated with two plates, one for isolating plague bacteria, and the other for dropping human plague phages for phage test immediately after inoculation. B4 Animal Test
B4.1 Preparation of Inoculation Material
Grind the organs to be tested in a sterile mortar, and add 1mL of saline to every 100mg of the organ to make a suspension; insect materials can be grouped into 10~30 insects from the same location, the same host, and the same insect species. After washing with 1×10-5 gentian violet saline, grind them in a mortar, and add 1~2ml of saline to each group of insects to make a suspension; bone marrow can be diluted 2~3 times with saline; exudate, blood, etc. can be used directly, and blood that cannot be inoculated on site should be added with anticoagulants. Before absorbing the inoculation liquid, a small sterile cotton ball can be added to the suspension, and then the needle of the syringe can be inserted into the cotton ball to absorb the material to be tested and inoculate the animal. B4.2 Inoculation Method and Inoculation Dosage
B4.2.1 Intraperitoneal inoculation: Suitable for those who want to obtain results quickly but do not expect obvious pathological changes. Generally used for fresh materials to be tested, the method is to first cut off the abdominal hair on the side to be injected, disinfect with iodine and alcohol, wait until it is dry, lower the head of the test animal, raise the tail, extract the abdominal wall and slowly pierce it. After the needle is inserted, it should be inserted along the abdominal wall to prevent puncturing the internal organs, and slowly injected into the object to be tested. Guinea pigs are injected with 0.5-1.0ml., mice are injected with 0.2-0.4mL, then take out the syringe and gently press the needle eye with a dry cotton ball. B4.2.2 Subcutaneous inoculation: It is the most commonly used method. The course of disease is appropriate; animals can develop more typical lesions after disease induction; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: It is suitable for materials that are corrupt or seriously contaminated. The course of the disease is slow, and obvious pathological changes can be obtained. In general, animals will not be killed by other mixed infections, and it can play a good role in biological filtration. However, if the content of plague bacteria in the materials to be tested is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp object to scratch the skin until there is no bleeding. Dip a cotton swab in the material to be tested, apply it on the exposed skin and rub it to infect it percutaneously.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container, and fed and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs within 1 to 3 days. When the disease occurs, the animal wilts and rots, stops eating, has erect hair and hunched back, and usually dies within 3 to 7 days. After the animal dies, it should be dissected immediately to observe the lesions and isolate the plague bacteria. If the animal still does not get sick on the 7th day, it can be taken out and killed for dissection on the 8th day. If the material tested meets the characteristics of the plague bacteria through the above "four-step diagnosis", it can be determined on the spot that it is animal plague, and it will be reported step by step according to regulations for the epidemic area to be handled, and the strain will be sent to the higher-level business department for re-judgment and further inspection.3 Microscopic examination: If Gram-negative short cocci with thick staining at both ends and blunt rounded ends are found, it means that they meet the characteristics of plague bacteria. Attention should also be paid to the polymorphic changes of bacteria in rotten materials or old materials. B2 Culture
GB16883—1997
B2.1 Culture medium: Fresh selective sensitive culture medium should be used, and gentian violet hemolytic trypsin digestion liquid agar is commonly used; gentian violet should contain 0.5×10-s for separating plague bacteria from general materials. Gentian violet should contain 1×10-5 for contaminated materials. Gentian violet hemolytic trypsin digestion soup can be used for bacterial enrichment culture. The pH of the culture medium is required to be 6.9~~7.1.
B2.2 Inoculation method: Animal materials are collected according to Appendix A, and then the material to be tested is hooked in the center of the organ section with an inoculation loop and inoculated on a flat plate. If the material is fresh, the organ section can be directly pressed and then inoculated with a streak; bone marrow materials can be extracted from the broken end with a syringe or injected with physiological saline for flushing, and a drop of the flushing liquid can be drawn onto the flat plate and then streaked for inoculation; liquid materials can be directly hooked and streaked for culture with an inoculation loop. Flea materials can be processed according to the method in Appendix A and then grouped and ground, and the ground material can be hooked and inoculated. When grouping and culturing, the same flea species of one animal should be grouped together. When mixed groups are grouped, the same location, the same rat species, and the same flea species must be grouped together. When egg materials are scarce, single stomach inoculation and culture should be adopted. Other insects can be carried out in accordance with eggs. If necessary, dead rats or key materials can be enriched with liquid culture medium before isolation. Two flat plates should be inoculated for each material; one is used to isolate plague bacteria, and the other is used for rapid diagnosis of bacteriophages. B2.3 Culture: The inoculated culture medium should be cultured in a 28-30℃ incubator. Animal materials are generally cultured for 3 days, and insect materials are cultured for 4 days. B2.4 Observation: The culture is observed once a day, and animal materials are observed for 3 consecutive days. Insect materials are observed for 4 consecutive days. If suspected bacteria are found during the observation, they should be immediately purified and tested for bacteriophages. B3 Phage test
B3.1 Conventional method: Whenever suspected plague bacteria are found, they should be streaked on a hemolytic agar plate, and plague phages should be sucked with a syringe or capillary pipette and dropped on the upper part of the plate streaking line to make it flow vertically, and then cultured at 28℃ for 24 hours to observe the results. If plaques or phage bands appear, it can be judged that the plague phage test is positive. B3.2 Rapid phage diagnosis: For materials that require rapid phage diagnosis, the materials to be tested should be inoculated with two plates, one for isolating plague bacteria, and the other for dropping human plague phages for phage test immediately after inoculation. B4 Animal Test
B4.1 Preparation of Inoculation Material
Grind the organs to be tested in a sterile mortar, and add 1mL of saline to every 100mg of the organ to make a suspension; insect materials can be grouped into 10~30 insects from the same location, the same host, and the same insect species. After washing with 1×10-5 gentian violet saline, grind them in a mortar, and add 1~2ml of saline to each group of insects to make a suspension; bone marrow can be diluted 2~3 times with saline; exudate, blood, etc. can be used directly, and blood that cannot be inoculated on site should be added with anticoagulants. Before absorbing the inoculation liquid, a small sterile cotton ball can be added to the suspension, and then the needle of the syringe can be inserted into the cotton ball to absorb the material to be tested and inoculate the animal. B4.2 Inoculation Method and Inoculation Dosage
B4.2.1 Intraperitoneal inoculation: Suitable for those who want to obtain results quickly but do not expect obvious pathological changes. Generally used for fresh materials to be tested, the method is to first cut off the abdominal hair on the side to be injected, disinfect with iodine and alcohol, wait until it is dry, lower the head of the test animal, raise the tail, extract the abdominal wall and slowly pierce it. After the needle is inserted, it should be inserted along the abdominal wall to prevent puncturing the internal organs, and slowly injected into the object to be tested. Guinea pigs are injected with 0.5-1.0ml., mice are injected with 0.2-0.4mL, then take out the syringe and gently press the needle eye with a dry cotton ball. B4.2.2 Subcutaneous inoculation: It is the most commonly used method. The course of disease is appropriate; animals can develop more typical lesions after disease induction; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: It is suitable for materials that are corrupt or seriously contaminated. The course of the disease is slow, and obvious pathological changes can be obtained. In general, animals will not be killed by other mixed infections, and it can play a good role in biological filtration. However, if the content of plague bacteria in the materials to be tested is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp object to scratch the skin until there is no bleeding. Dip a cotton swab in the material to be tested, apply it on the exposed skin and rub it to infect it percutaneously.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container, and fed and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs within 1 to 3 days. When the disease occurs, the animal wilts and rots, stops eating, has erect hair and hunched back, and usually dies within 3 to 7 days. After the animal dies, it should be dissected immediately to observe the lesions and isolate the plague bacteria. If the animal still does not get sick on the 7th day, it can be taken out and killed for dissection on the 8th day. If the material tested meets the characteristics of the plague bacteria through the above "four-step diagnosis", it can be determined on the spot that it is animal plague, and it will be reported step by step according to regulations for the epidemic area to be handled, and the strain will be sent to the higher-level business department for re-judgment and further inspection.3 Microscopic examination: If Gram-negative short cocci with thick staining at both ends and blunt rounded ends are found, it means that they meet the characteristics of plague bacteria. Attention should also be paid to the polymorphic changes of bacteria in rotten materials or old materials. B2 Culture
GB16883—1997
B2.1 Culture medium: Fresh selective sensitive culture medium should be used, and gentian violet hemolytic trypsin digestion liquid agar is commonly used; gentian violet should contain 0.5×10-s for separating plague bacteria from general materials. Gentian violet should contain 1×10-5 for contaminated materials. Gentian violet hemolytic trypsin digestion soup can be used for bacterial enrichment culture. The pH of the culture medium is required to be 6.9~~7.1.
B2.2 Inoculation method: Animal materials are collected according to Appendix A, and then the material to be tested is hooked in the center of the organ section with an inoculation loop and inoculated on a flat plate. If the material is fresh, the organ section can be directly pressed and then inoculated with a streak; bone marrow materials can be extracted from the broken end with a syringe or injected with physiological saline for flushing, and a drop of the flushing liquid can be drawn onto the flat plate and then streaked for inoculation; liquid materials can be directly hooked and streaked for culture with an inoculation loop. Flea materials can be processed according to the method in Appendix A and then grouped and ground, and the ground material can be hooked and inoculated. When grouping and culturing, the same flea species of one animal should be grouped together. When mixed groups are grouped, the same location, the same rat species, and the same flea species must be grouped together. When egg materials are scarce, single stomach inoculation and culture should be adopted. Other insects can be carried out in accordance with eggs. If necessary, dead rats or key materials can be enriched with liquid culture medium before isolation. Two flat plates should be inoculated for each material; one is used to isolate plague bacteria, and the other is used for rapid diagnosis of bacteriophages. B2.3 Culture: The inoculated culture medium should be cultured in a 28-30℃ incubator. Animal materials are generally cultured for 3 days, and insect materials are cultured for 4 days. B2.4 Observation: The culture is observed once a day, and animal materials are observed for 3 consecutive days. Insect materials are observed for 4 consecutive days. If suspected bacteria are found during the observation, they should be immediately purified and tested for bacteriophages. B3 Phage test
B3.1 Conventional method: Whenever suspected plague bacteria are found, they should be streaked on a hemolytic agar plate, and plague phages should be sucked with a syringe or capillary pipette and dropped on the upper part of the plate streaking line to make it flow vertically, and then cultured at 28℃ for 24 hours to observe the results. If plaques or phage bands appear, it can be judged that the plague phage test is positive. B3.2 Rapid phage diagnosis: For materials that require rapid phage diagnosis, the materials to be tested should be inoculated with two plates, one for isolating plague bacteria, and the other for dropping human plague phages for phage test immediately after inoculation. B4 Animal Test
B4.1 Preparation of Inoculation Material
Grind the organs to be tested in a sterile mortar, and add 1mL of saline to every 100mg of the organ to make a suspension; insect materials can be grouped into 10~30 insects from the same location, the same host, and the same insect species. After washing with 1×10-5 gentian violet saline, grind them in a mortar, and add 1~2ml of saline to each group of insects to make a suspension; bone marrow can be diluted 2~3 times with saline; exudate, blood, etc. can be used directly, and blood that cannot be inoculated on site should be added with anticoagulants. Before absorbing the inoculation liquid, a small sterile cotton ball can be added to the suspension, and then the needle of the syringe can be inserted into the cotton ball to absorb the material to be tested and inoculate the animal. B4.2 Inoculation Method and Inoculation Dosage
B4.2.1 Intraperitoneal inoculation: Suitable for those who want to obtain results quickly but do not expect obvious pathological changes. Generally used for fresh materials to be tested, the method is to first cut off the abdominal hair on the side to be injected, disinfect with iodine and alcohol, wait until it is dry, lower the head of the test animal, raise the tail, extract the abdominal wall and slowly pierce it. After the needle is inserted, it should be inserted along the abdominal wall to prevent puncturing the internal organs, and slowly injected into the object to be tested. Guinea pigs are injected with 0.5-1.0ml., mice are injected with 0.2-0.4mL, then take out the syringe and gently press the needle eye with a dry cotton ball. B4.2.2 Subcutaneous inoculation: It is the most commonly used method. The course of disease is appropriate; animals can develop more typical lesions after disease induction; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: It is suitable for materials that are corrupt or seriously contaminated. The course of the disease is slow, and obvious pathological changes can be obtained. In general, animals will not be killed by other mixed infections, and it can play a good role in biological filtration. However, if the content of plague bacteria in the materials to be tested is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp object to scratch the skin until there is no bleeding. Dip a cotton swab in the material to be tested, apply it on the exposed skin and rub it to infect it percutaneously.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container, and fed and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs within 1 to 3 days. When the disease occurs, the animal wilts and rots, stops eating, has erect hair and hunched back, and usually dies within 3 to 7 days. After the animal dies, it should be dissected immediately to observe the lesions and isolate the plague bacteria. If the animal still does not get sick on the 7th day, it can be taken out and killed for dissection on the 8th day. If the material tested meets the characteristics of the plague bacteria through the above "four-step diagnosis", it can be determined on the spot that it is animal plague, and it will be reported step by step according to regulations for the epidemic area to be handled, and the strain will be sent to the higher-level business department for re-judgment and further inspection., inject 0.2-0.4mL into the mouse, then take out the syringe and gently press the needle hole with a dry cotton ball. B4.2.2 Subcutaneous inoculation: the most commonly used method. The course of disease is appropriate; the animal can show typical lesions after the disease; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: suitable for corrupt or seriously contaminated materials to be inspected, with a slow course of disease, obvious pathological changes can be obtained, and generally animals will not be killed by other mixed infections, and can play a good role in biological filtration. However, if the content of plague bacteria in the inspected material is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp tool to scratch the skin until there is no bleeding. Use a cotton swab to soak the material to be tested and apply it on the exposed skin and rub it to make it 50.4
transdermally infected.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container and kept and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs in 1 to 3 days. When the disease occurs, the animal will wilt, not eat, stand up the hair, and arch its back. Death usually occurs in 3 to 7 days. After the death of the animal, the lesions should be observed immediately and the plague bacteria should be isolated. If the animal still does not get sick on the 7th day, it can be taken out and killed on the 8th day for dissection and examination. If the materials tested meet the characteristics of plague bacteria through the above “four-step diagnosis”, they can be diagnosed as animal plague on the spot, reported to higher authorities for epidemic area treatment, and the strains are sent to higher-level business departments for re-judgment and further inspection., inject 0.2-0.4mL into the mouse, then take out the syringe and gently press the needle hole with a dry cotton ball. B4.2.2 Subcutaneous inoculation: the most commonly used method. The course of disease is appropriate; the animal can show typical lesions after the disease; the detection rate is high. This method can be used for fresh materials or slightly contaminated materials. The inoculation method is similar to intraperitoneal inoculation, except that the needle is only inserted into the subcutaneous part; it can also be inserted from the inner thigh to the subcutaneous part and injected into the upper thigh. The inoculation dose is the same as intraperitoneal inoculation. B4.2.3 Percutaneous inoculation: suitable for corrupt or seriously contaminated materials to be inspected, with a slow course of disease, obvious pathological changes can be obtained, and generally animals will not be killed by other mixed infections, and can play a good role in biological filtration. However, if the content of plague bacteria in the inspected material is small or the virulence is weak, it may not be infected and cause omissions. The method is to shave or cut off 4cm2 of hair on the lower abdomen of guinea pigs; shave or cut off 1~2cm of hair on the abdomen of mice, and use a sharp tool to scratch the skin until there is no bleeding. Use a cotton swab to soak the material to be tested and apply it on the exposed skin and rub it to make it 50.4
transdermally infected.
B4.3 Feeding and Observation
GB16883--~1997
The inoculated animals should be placed in a safe and easy-to-observe container and kept and observed in the experimental animal room of the plague strong toxin system. Generally, the disease occurs in 1 to 3 days. When the disease occurs, the animal will wilt, not eat, stand up the hair, and arch its back. Death usually occurs in 3 to 7 days. After the death of the animal, the lesions should be observed immediately and the plague bacteria should be isolated. If the animal still does not get sick on the 7th day, it can be taken out and killed on the 8th day for dissection and examination. If the materials tested meet the characteristics of plague bacteria through the above “four-step diagnosis”, they can be diagnosed as animal plague on the spot, reported to higher authorities for epidemic area treatment, and the strains are sent to higher-level business departments for re-judgment and further inspection.
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