At present, what are the research results of highly pathogenic avian influenza?
Since the mid-1990s, China has done a lot of work on the epidemiology, diagnosis, immune control and basic research of avian influenza, and obtained many research results. The diagnosis technology of avian influenza has been established: ① agar diffusion (AGP) diagnosis technology. ② subtype typing technology of avian influenza virus. ③ Molecular diagnosis and detection technology of avian influenza virus. Avian influenza vaccine development: An inactivated vaccine of H5 subtype avian influenza has been developed, which can be used for emergency immunization of H5 subtype highly pathogenic avian influenza. The live carrier vaccine of H5N1 subtype avian influenza recombinant fowlpox virus has also entered the stage of environmental release and safety evaluation.
How long will it take to confirm the pathogen? Do what experiment?
Highly pathogenic avian influenza must be diagnosed by virus isolation and identification. It usually takes at least 3~5 days for virus isolation and subtype identification. The pathogenicity of the virus must be finally determined by artificial intravenous inoculation of non-specific pathogen chickens (SPF chickens).
What is the standard for diagnosing highly pathogenic avian influenza stipulated in the technical specification for prevention and control of highly pathogenic avian influenza issued by the Ministry of Agriculture? According to the Notice of the Ministry of Agriculture on Printing and Distributing Seven Technical Specifications for Prevention and Control of Highly Pathogenic Avian Influenza (Nongbanmu [2002] No.74), highly pathogenic avian influenza can be confirmed to have occurred under the following circumstances: ① There are typical clinical symptoms and pathological changes, acute onset and high mortality, and Newcastle disease and toxic diseases can be excluded, and serological tests are positive. ② Avian influenza serotypes H5 and H7 were seropositive in the poultry that were not immunized. ③ Avian influenza virus strains of H5 and H7 subtypes or other subtypes were isolated from poultry flocks.
According to the virulence, avian influenza is divided into several categories, how to identify it?
Influenza viruses are divided into highly pathogenic avian influenza virus (HP avian influenza virus), low pathogenic avian influenza virus (LP avian influenza virus) and non-pathogenic avian influenza virus (NP avian influenza virus) according to their pathogenicity. In view of the importance of HP avian influenza virus, the relevant identification standards and procedures are introduced as follows:
The World Organization for Animal Health classifies highly pathogenic avian influenza viruses as follows:
(1) Eight susceptible chickens aged 4-8 weeks were inoculated with 02 ml of 1∶10 diluted allantoic fluid infected with influenza virus by intravenous injection, which could cause 6-7 or 8 chickens to die within 10 days after inoculation. These influenza viruses should be highly pathogenic.
(2) The isolate can kill 1~5 chickens, but the virus is not H5 or H7 subtype, so the following experiments should be carried out: Inoculate the virus on the cell culture, and observe whether it causes cell pathological changes or forms plaque when trypsin is lacking. If the virus cannot grow on cells, the isolate should be considered as a non-highly pathogenic avian influenza virus.
(3) When all H5 and H7 strains with low pathogenicity and other viruses can grow on cells lacking trypsin, the amino acid sequence of peptide chain related to hemagglutinin should be analyzed. If the analysis results are similar to other highly pathogenic avian influenza viruses, this tested isolate should be considered as highly pathogenic avian influenza virus.
How to collect and separate the samples of the pathogen after the occurrence of avian influenza?
The diagnosis depends largely on the quality of the sample, the storage and transportation process of the sample before laboratory treatment. Samples of respiratory viruses used for cell culture, chicken embryo inoculation and direct detection of virus antigens or nucleic acids should be collected 3 days after the onset of flu symptoms. Avian influenza is mainly infected by respiratory tract and digestive tract. Therefore, the samples collected from the upper respiratory tract of mammals and birds are suitable for the identification and diagnosis of influenza virus. There are three types of upper respiratory tract swabs: nasal cotton swabs, throat cotton swabs and tracheal cotton swabs. Slaughtered or dead mammals should collect samples in the lower respiratory tract, which are divided into three types: tracheal cotton swab, bronchial cotton swab and lung tissue. Avian samples should be collected in the respiratory tract and most of the digestive tract. The virus samples collected include cloacal cotton swabs and feces, among which cloacal cotton swabs can be collected from live chickens or slaughtered chickens. Collecting feces samples from chicken coops or the environment is a common sampling method, but it has the disadvantage of not being able to determine the exact source of samples. If it is suspected that dead birds contain highly pathogenic avian influenza virus, representative internal organs such as brain, spleen, heart and lung should also be collected.
If you want to detect the virus directly by immunofluorescence staining of infected cells, then the collected disease materials should be placed in an ice bath and the samples should be processed within 1~2 hours. Samples used for virus isolation should be frozen in the refrigerator immediately after sampling, and inoculated into sensitive cells or chicken embryos as early as possible. If the sample cannot be processed within 48~72 hours, it should be frozen at -70℃ or below. If the test tube containing the sample is not sealed, or the plastic bag containing the sample is not tightly sealed, the sample cannot be transported or stored in dry ice. Because once the dry ice comes into contact with the diseased sample, it can quickly inactivate the influenza virus that may exist in it.
The collected samples should be placed in a suitable transport buffer to ensure the isolation of the virus. At present, there are some transport buffers suitable for different virus samples, such as Hanks balanced salt solution, cell culture medium, phosphate buffer, tryptone-phosphate broth, calf beef soup and sucrose phosphate buffer, etc. In these transport buffers, 0.5% ~ 1% protein, such as bovine serum albumin and gelatin, should be added, and antibiotics should be added to prevent the growth of bacteria.
How to collect and transport pathological samples?
Pathological samples should generally be taken from dead or live birds in the early stage of infection or acute stage of onset. Dead birds collected tissue samples such as trachea, lung, liver, kidney, spleen and cloaca. Live birds should be smeared with sterilized cotton swabs of different sizes on throat, trachea or cloaca. Cotton swabs with secretions should be put into broth containing 1 000 international units of penicillin, 2 000 micrograms of streptomycin and pH 72 ~ 76 per ml. When there is no broth, Hanks solution or 25%~50% glycerol saline can be used. The concentration of antibiotics used in feces and cloaca cotton swabs should be increased.
Transportation and storage of samples: if the collected samples are inspected within 48 hours, they can be stored at 4℃; Otherwise, it should be stored at low temperature (-70℃ is the best).
What are the diagnostic methods and procedures for highly pathogenic avian influenza?
(1) Epidemiology Avian influenza virus has a wide range of hosts, such as chickens, turkeys, ducks, geese, quails and pheasants, and wild birds, waterfowl and seabirds. Among them, chickens and turkeys are the most harmful after being infected with avian influenza virus, and more viruses are isolated from ducks than other poultry. Birds of all ages can be infected.
Avian influenza virus is mainly transmitted through direct contact and indirect contact between infected birds (including equipment in contact with infected birds). In addition, poisonous birds or waterfowl often become the source of infection, causing a large number of diseases and deaths of poultry.
(2) The incubation period of clinical symptoms of avian influenza varies from several hours to three days. The incubation period depends on the virulence and dose of the infected virus, the route of infection, the species of infected birds and the state of the birds. Acute infection of avian influenza has no specific clinical symptoms. In a short time, loss of appetite, sudden rise of body temperature, respiratory symptoms, diarrhea and neurological symptoms can be seen. With a large number of deaths.
(3) Pathological changes The pathological changes of avian influenza vary with the virulence of the infected strain, the duration of the disease and the species of birds. The main manifestations are head swelling, fleshy beard, crown bleeding, subcutaneous bleeding and edema of legs and toes, glandular stomach nipple bleeding and salpingitis.
(4) Etiological diagnosis-virus isolation needs to be completed by the national laboratory.
(5) Serodiagnosis At present, the methods used for avian influenza detection include avian influenza virus isolation, agar diffusion (AGP) test, hemagglutination inhibition (HI) test, neuraminidase inhibition (NI) test, enzyme-linked immunosorbent assay (ELISA), virus neutralization test (SN), reverse transcription polymerase chain reaction (RT-PCR), immunofluorescence technology (IF) and nucleic acid probe technology.
Avian influenza virus hemagglutinin typing antigen and standard typing serum were provided by Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, and operated according to the instructions.
In a word, the diagnosis of avian influenza, the isolation and identification of laboratory viruses and the pathogenicity test all take a long time and cost a lot. Only a few laboratories can carry out this inspection, and they can’t make a diagnosis within a short time (48 hours) after receiving the inspection samples, and the diagnosis lags behind the requirements of taking measures on the spot. The faster and earlier the diagnosis of avian influenza is made, the smaller the loss will be. When bird flu is suspected, it is very important to report the epidemic situation in time so as to obtain timely diagnosis from relevant experts.
How to make a differential diagnosis of avian influenza?
Because the epidemic characteristics, symptoms and pathological changes caused by avian influenza infection are similar to some infectious diseases of poultry, it is necessary to make differential diagnosis in time, such as Newcastle disease, infectious bronchitis, infectious laryngotracheitis, infectious rhinitis, mycoplasma disease, chlamydia disease, egg drop syndrome, etc. Especially the mixed infection or secondary infection of some diseases makes the condition more complicated, which makes diagnosis difficult or easy to be misdiagnosed. Therefore, the differential diagnosis is very important.
The first is the differential diagnosis with Newcastle disease. The epidemic characteristics, symptoms and pathological changes of avian influenza are very similar to those of Newcastle disease in chickens. Generally speaking, the incubation period and course of highly pathogenic avian influenza are shorter than those of Newcastle disease in China at present. The symptoms such as dyspnea, hydrops in crop and mouth, cooing when dyspnea, typical neurological symptoms and conventional typical pathological changes are all more obvious and characteristic than avian influenza. In addition, the emergency immune effect of Newcastle disease on the spot is all related to avian influenza. However, some of them are not obviously different. The accurate differential diagnosis of the two diseases can only rely on laboratory diagnosis. The simplest and most practical methods are virus isolation and hemagglutination inhibition test (HI). Newcastle disease antiserum can not inhibit the hemagglutination of avian influenza virus, and vice versa.
According to the comprehensive analysis of epidemic characteristics, symptoms, pathological autopsy and laboratory examination (etiology, serology), avian influenza can be distinguished from other epidemics. Similarly, secondary or complicated bacterial or viral diseases can be determined by pathogen isolation and some detection methods.
Excerpted from "Questions and Answers on Prevention and Control of Highly Pathogenic Avian Influenza" by China Agricultural Publishing House.
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