Influenza and other acute respiratory viral infections. Is the influenza virus threatening the world? Characteristics of avian influenza in humans
Most of us perceive the flu as just one of the minor annoyances. But this is a misconception: the flu should not be underestimated. The infection spreads by airborne droplets so easily that every year it affects a significant part of the world's population. Influenza and other infectious diseases of the respiratory tract are the most common human viral diseases. They are the cause of death for many people. Due to mass morbidity, the economic damage from them is huge in all countries.
The flu virus changes so quickly that no one is immune to all of its varieties, and every year experts have to develop a new vaccine. So far, we have been talking about the usual types of influenza, but since December 2003, an unprecedented outbreak of avian influenza has occurred in the world, covering 38 countries. First of all, the countries of Southeast Asia were hit. At present, an epizootic of avian influenza caused by the H5N1 influenza A virus has been noted in many countries of Europe, Asia and Africa. Human cases have been reported in 7 countries. 3 countries out of this number border on Russia.
Despite emergency measures to prevent the spread of avian influenza, which resulted in the destruction of more than 100 million flocks of poultry, the H5N1 virus has gained a foothold in the natural population of wild birds and has acquired the ability to infect humans, which is the basis for considering it a possible precursor of a pandemic virus. As of March 21, 2006, 185 people fell ill in the world, of which 104 died.
It's not unusual for chickens to get the flu. There are many more varieties of bird flu than human flu. Avian influenza is a highly contagious viral infection that affects all types of birds. The most sensitive domestic species are chickens and turkeys. The natural reservoir for avian influenza viruses is waterfowl, which are most often responsible for bringing the infection into households.
Bird flu has always been around. In wild birds, the disease occurs in the form of enteritis (intestinal damage) without visible signs of a general disease. This indicates a high degree of adaptation of influenza A viruses to wild birds, which are their natural hosts. The virus persists in water for a long time (6-8 months), and the water-fecal route of infection of birds is the main mechanism for the persistence of the influenza virus in nature, from where it penetrates into populations of poultry and animals. A highly pathogenic virus can survive in the environment for a long time, especially at low temperatures. For example, it can survive in bird droppings for up to 35 days at 4 degrees C. At 37 degrees C, the virus remains viable in droppings for at least 6 days.
Avian influenza viruses can be transferred from farm to farm when moving live birds, as well as by people through shoes and clothes, contaminated transport wheels, equipment, and feed. For these reasons, poultry workers are advised not to keep poultry. These requirements must be taken seriously. When a disease occurs, the most important and effective infection control measures are the rapid destruction of the entire population of diseased or contact birds, the mandatory collection and burial or burning of bird carcasses, the introduction of quarantine and the thorough disinfection of all premises and equipment. It is also necessary to impose restrictions on the movement of live birds and poultry products both within a settlement or region, and on a wider scale, depending on the situation.
Strict restrictive measures are especially necessary for poultry farms and poultry farms, where the number of birds is kept in enclosed spaces. Avian influenza viruses can be transmitted to farms by living on their territory of various bird species: pigeons, crows, sparrows and others. In a number of cases, the routes of transmission remained unclear, which may indicate yet unknown sources of infection. In these cases, there is speculation about the possible role of birds or the use of bird droppings as fertilizer.
Infection control measures are much more difficult to implement on individual farms. In them, it is difficult to ensure the isolation of poultry from contact with wild birds, especially in water bodies. Indeed, in the summer, all poultry in the villages walks on the water or lawns, grazes around the dwelling in search of food. This is especially dangerous when grazing domestic ducks or geese. In addition, even with successful attempts to isolate poultry, there is a problem of feeding them.
In addition to the difficulties of control, outbreaks of influenza in households are fraught with a high risk of human exposure to the infection. Cases of infection of children playing in areas heavily contaminated with bird feces are described. Infection can occur through water contaminated with bird feces. Therefore, care is needed when bathing and consuming raw water. In Thailand, there have been cases of infection in owners of fighting cocks. In households, it is not uncommon for sick birds to be slaughtered for food. In this case, a dangerous situation arises during the slaughter of birds, removal of feathers, butchering of the carcass and cooking. For example, in Turkey in February of this year, 2 children were infected and died, who were instructed to slaughter sick chickens.
Many birds are known to breed in the northern territories and migrate south for the winter. Bird flights cannot be canceled or banned. The migration of many millions of birds can be compared to a giant pump, twice a year pumping pathogens of various diseases adapted to birds from continent to continent. With the beginning of spring, the birds moved north, and the list of countries involved in the influenza epic immediately expanded significantly. On February 21, it looked like this (in the order in which the H5N1 virus was detected): Iraq, Azerbaijan, Bulgaria, Greece, Italy, Slovenia, Iran, Austria, Germany, Egypt, India, France. Since then, this list has changed significantly.
Is the H5N1 virus easily transmitted from birds to humans? Fortunately, no. As already noted, the reported number of human cases is negligible compared to the number of birds affected by this virus. It was not clear why some people get infected and get sick, while others do not. Data has just emerged to explain this fact. It turned out that in humans, epithelial cells sensitive to the H5N1 influenza virus are located in the deepest parts of the lungs, almost around the alveoli, where oxygen exchange takes place. Therefore, coughing or sneezing is unlikely to shed the virus from an infected person. But in the future, as the virus adapts to the human body, it will acquire the ability to infect other parts of our respiratory system, which will contribute to its spread from person to person.
What is the risk of an influenza pandemic? It can start under three conditions. The first is the emergence of a new subtype of influenza virus. The second - cases of infection of a person with a severe course of the disease. The third is the ability of the virus to spread easily from person to person. The first two conditions are already in place. The H5N1 virus has never circulated in nature before, including in humans. Humans are not immune to this virus. Thus, the point is only the ability of the virus to quickly spread it from person to person. The risk of this virus acquiring this ability will remain whenever human cases are observed, which in turn depends on its circulation in poultry and wild birds.
What changes are needed for the H5N1 virus to become pandemic? The virus can increase its transmissibility in humans through two mechanisms. The first is the exchange of genetic material with the simultaneous infection of a human or a pig with a human and avian virus. The second is a stepwise process of adaptive mutations that enhance the ability of the virus to infect human cells. Adaptive mutations appear initially as small outbreaks in humans with established human-to-human transmission of the virus. The registration of such cases will be a signal to actively prepare for a pandemic and to set in motion plans to reduce its devastating effects.
With the spread of the H5N1 virus outside of Southeast Asia, there has been an increase in human infections from domestic and wild birds. Each new human infection provides the virus with an opportunity to increase its transmissibility in humans, leading to the emergence of a pandemic strain. When and where this will happen is impossible to predict, but it will inevitably happen.
The spread of influenza viruses among dogs is dangerous because their level of genetic diversity is almost as high as among humans. This significantly increases the likelihood that the virus will learn to infect humans as well, adapting to new breeds of dogs.
It should be noted that outbreaks of bird (H5N1) and swine (H3N2) influenza at the end of the last decade caused serious concern among specialists.
American virologists learned about outbreaks of influenza among dogs in the Chinese provinces and asked colleagues to analyze samples with the source of the disease. It turned out that these viruses contain fragments of the genomes of three different strains of influenza, H1N1, H3N8 and H3N2, which until then only affected humans, birds and pigs, but not dogs.
Scientists believe that a new family of pathogens belonging to the H1N1 group is spread by airborne droplets and is capable of infecting both dogs and pigs. It is not yet clear whether this virus can also enter the human body - scientists are now figuring this out by conducting experiments on human cell cultures.
Experts believe that measures should be taken to limit the spread of influenza in dogs.
Previously, scientists have found that a fungus that is resistant to treatment can destroy people, animals and plants.
1.3. EPIDEMIOLOGY
Organization of monitoring of introductions and spread of avian influenza in natural conditions on the territory of the Russian Federation
Date of introduction: from the moment of approval
1. DEVELOPED by the Federal Service for Supervision of Consumer Rights Protection and Human Welfare (G.G. Onishchenko, E.B. Ezhlova, G.F. Lazikova); FGUN SSC VB "Vector" of Rospotrebnadzor (I.G. Drozdov, A.N. Sergeev, A.P. Agafonov, A.M. Shestopalov, E.A. Stavsky, A.Yu. Alekseev, O.K. Demina, E.B.Shemetova, A.A.Sergeev, Yu.V.Tumanov, V.A.Ternovoy); FGUZ Central Research Institute of Epidemiology of Rospotrebnadzor (G.A. Shipulin, A.T. Podkolzin, S.B. Yatsyshina); FGUZ RosNIPCHI "Microbe" of Rospotrebnadzor (A.V. Toporkov, S.A. Shcherbakova, N.V. Popov, V.P. Toporkov, A.A. Sludsky, I.N. Sharova, M.N. Lyapin, A .N.Matrosov, V.N.Chekashov, T.Yu.Krasovskaya); State Research Institute of Influenza of the Russian Academy of Medical Sciences (O.I. Kiselev, L.M. Tsybalova, T.G. Lobova).
3. APPROVED AND INTRODUCED by the Head of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare, Chief State Sanitary Doctor of the Russian Federation G.G. Onishchenko on December 26, 2008 N 01 / 15701-8-34
List of abbreviations
AVAI - avian influenza virus type A
Biosafety - biological safety
ELISA - enzyme immunoassay
IFA - method of immunofluorescent antibodies
RT-PCR - reverse transcription method - polymerase chain reaction
RTHA - hemagglutination inhibition reaction
WHO - World Health Organization
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1.2. These guidelines are intended for specialists of bodies and institutions of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare and other organizations, regardless of the legal form of ownership.
2. General provisions
2.1. The purpose of introducing these guidelines is to regulate measures for epizootological monitoring of avian influenza in natural conditions. The complex of monitoring measures includes organizing the collection, storage and transportation of material, conducting laboratory tests, as well as ensuring the biological safety of ongoing work. The main task of monitoring avian influenza is to identify the introduction of the pathogen and the spread of this infection in the populations of wild animals of the near-water complex in order to carry out adequate anti-epidemic and preventive measures among people.
2.2. Organization and implementation of activities for epizootological monitoring of avian influenza in natural conditions on the territory of the Russian Federation is carried out by bodies and institutions of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare in cooperation with bodies and institutions of the Ministry of Agriculture and Rosselkhoznadzor.
2.3. The volume, nature and direction of preventive measures among people are determined by the results of epizootic surveys and the forecast of the epizootic and epidemic situation for avian influenza in specific regions of the Russian Federation.
2.4. The Rospotrebnadzor departments for the constituent entities of the Russian Federation where avian influenza epizootics have been detected, together with the executive authorities of the constituent entities, Rosselkhoznadzor bodies, the Ministry of Emergency Situations and other interested services and departments, are planning to take measures aimed at preventing the spread of the virus among poultry, poultry farms and among people , as well as those aimed at minimizing the consequences of possible outbreaks, if they have already occurred, and their suppression. A comprehensive plan of preventive measures for avian influenza is drawn up by the departments of Rospotrebnadzor together with the health authorities of the constituent entities of the Russian Federation, Rosselkhoznadzor and other interested services and departments for a period of at least 2 years with annual adjustments.
2.5. The forecast of the epidemiological and epizootological situation for avian influenza in the territory of the Russian Federation is compiled by the scientific and methodological center for reference diagnostics and study of highly pathogenic strains of the influenza virus - NMCG (FGUN SRC VB "Vector" of Rospotrebnadzor). The forecast is made on the basis of the conclusions on the epidemiological and epizootological situation for avian influenza in Russia (1-2 times a year), prepared by the Center for Ecology and Epidemiology of Influenza - CEEG (State Research Institute of Virology named after D.I. Ivanovsky RAMS) and the Federal Influenza Center - FCG (GU Research Institute of Influenza RAMS). These conclusions are formed on the basis of the information provided to the CEEG and the FCG from the respective institutions supervised by them (anti-plague stations and support bases determined by Order of Rospotrebnadzor dated 31.03.05 N 373). The formulated generalized forecast and conclusions on the epidemiological and epizootic situation in Russia are sent to the Federal Service for Supervision of Consumer Rights Protection and Human Welfare. Data on the forecast of the epidemiological and epizootic situation for avian influenza in the Russian Federation are transmitted to the WHO, to the information website of the NICH, to the National Influenza Centers of the CIS countries and development institutions (CEEG and FCG) after agreement with the Federal Service for Surveillance in the Field of Consumer Rights Protection and human well-being.
2.6. Advisory, methodological and practical assistance to the departments of Rospotrebnadzor in the constituent entities of the Russian Federation on the prevention and implementation of anti-epidemic measures among people in areas affected by an epizootic of avian influenza is provided by the NMCG, CEEG and FCG, the Anti-Plague Center, regional centers for monitoring infectious diseases I-II groups of pathogenicity and Centers for indication and diagnostics of pathogens of dangerous infectious diseases, created on the basis of anti-plague institutions.
3. Rationale for the need to monitor avian influenza
The need to monitor avian influenza is determined by the real risk of introducing the pathogen of this disease by migratory birds and the formation of foci of infection in many regions of the Russian Federation, as well as the occurrence of epizootics among domestic birds and the potential ability of the avian influenza virus to cause human infection.
Since 1997, there have been epizootics among wild and domestic birds caused by highly pathogenic influenza A viruses of the H5N1 subtype, which can also cause severe illness among people with high mortality. In recent years, there has been an expansion influenza virus range birds, an increase in the species spectrum of carriers, an increase in the virulence of circulating strains. The ability to survive in the environment for a long time, especially at low temperatures, determines the expansion of the virus distribution area and the need for monitoring activities in the vast territories of the Russian Federation.
Cases of transmission of the influenza A virus subtype H5N1 from person to person have not been registered, although familial foci of the disease have been noted repeatedly. However, co-circulation of human and avian influenza virus strains increases the likelihood of a reassortment event and the emergence of a pandemic influenza virus.
The Federal Service for Veterinary and Phytosanitary Surveillance (Rosselkhoznadzor) monitors the bird flu situation, primarily solving the problems of preventing epizootic outbreaks among domestic animals. At the same time, epizootological monitoring of avian influenza in natural biotopes is currently insufficient.
4. Organization of epizootological monitoring of avian influenza in natural conditions
4.1. Epizootological and epidemiological features of avian influenza
4.2. The purpose and objectives of epizootological monitoring of avian influenza in natural conditions
The main purpose of epizootological monitoring of avian influenza is the timely detection of cases of introduction of the avian influenza virus into natural biotopes and tracking the spread of this infection among wild animals of the near-aquatic complex.
To achieve the goal, it is necessary to solve the following tasks:
select geographic points for monitoring with the compilation of a cadastre of water bodies where a large number of birds of the limnophilic complex accumulate for rest, nesting and feeding;
organize field mobile teams to collect samples of material for research;
to establish the species composition, abundance, features of the placement of potential carriers of AIV in the biotopes of the near-water complex;
collect samples of field material for laboratory testing for the presence of avian influenza virus, process and promptly analyze the results;
to study the epizootological status of individual species and groups of birds and other animals of near-water biocenoses;
to study the parameters of the epizootic process in the foci of avian influenza (seasonal features, epizootic areas, species spectrum of infected animals, etc.);
assess the degree of risk of infection of various types of water bodies located in close proximity to rural settlements and large poultry farms;
compile a list of settlements where it is possible for domestic animals to become infected with avian influenza from wild near-water birds;
develop measures to prevent epizootic outbreaks and human diseases;
conduct sanitary-educational and explanatory work among the local population;
make forecasts of the development of the situation;
organize notification of health authorities and local executive authorities about the results of an epizootological survey of territories for the presence of foci of avian influenza virus and a forecast for the development of the epidemic situation.
4.3. Tactics and methods of epizootological monitoring of avian influenza in natural conditions
The basis of epizootological monitoring of avian influenza in natural conditions is the examination of aquatic and near-water biocenotic complexes, which is carried out in a planned manner. The search for the causative agent of avian influenza should be carried out, first of all, in near-water biotopes located both in places of concentration and nesting, and along and inside the intercontinental routes of seasonal flights or migrations of birds, primarily belonging to the orders of Anseriformes, Charadriiformes, Grebes, copepods, ankle-footed, crane-like, pigeon-like, chicken, passerine. At the same time, sites for collecting material are selected near and (or) on the territory of settlements and places of recreation, as well as in territories where the death of birds from influenza and cases of human disease were noted. Key sites are also selected here (points of long-term monitoring - PDM), where research will be carried out over several seasons. Each site is examined at least 3 times a year (during the spring migration period, during the nesting and post-nesting periods). According to epidemiological indications, emergency additional epizootological examinations are carried out.
A prerequisite for starting an epizootological survey of near-water biotopes is the beginning of spring migration of birds of the near-water complex, information on cases of mortality among marsh, lake, and river birds.
The epizootic situation is assessed on the basis of an epizootic survey, in which the state of the number of background animal species in near-aquatic biotopes is recorded, and on the basis of the results of laboratory studies confirming the presence of the avian influenza pathogen in various objects. Based on these data, a reasoned conclusion about the danger of epizootics is given.
During an epizootological examination for avian influenza in natural biocenoses, it is necessary to pay attention to water bodies where solitary, flocking and colonial birds accumulate. Especially on stagnant fresh or slightly mineralized water bodies with an abundance of coastal aquatic and shrub vegetation, where there are optimal conditions for shelters, rest, feeding and nesting of birds. When choosing survey sites and determining the composition and number of samples, they are guided by the features of the hydrographic network of the surveyed area: the location of reservoirs, their size. First of all, lakes, swampy lowlands, ponds, bays, estuaries, eriki, floodplains, etc., located in close proximity to rural settlements, are controlled.
In the process of epizootological examination, observations are made of weather conditions, phenological phenomena, bird counts, the nature of their distribution, abundance and activity are carried out. It is necessary to identify and monitor the timing, duration and routes of their mass seasonal flights and forage migrations. When searching for foci of avian influenza, attention is paid to the external signs of an epizootic in wild birds, especially noting a sharp decrease in the number and activity of birds, changes in their behavior in water bodies, the appearance of lethargic individuals, ruffled feathers, immobility, etc. Considering the high sensitivity of chicks to the influenza virus, sick individuals are most likely to be detected during the brood period.
The main objects in the collection of samples for laboratory analysis are waterfowl and near-water birds: geese, swans, ducks, sandpipers, gulls and terns, herons and shepherds. Other water birds, including diurnal birds of prey (falconiformes) and passerines, should also be hunted for complete information. It is obligatory to study birds of synanthropic species: pigeons, magpies, crows and sparrows. All dead birds found on a reservoir or in a coastal zone are necessarily objects of collection and laboratory analysis. It is also necessary to catch small mammals that live along the banks of water bodies: water voles, muskrats, mice, shrews, etc.
The terms and duration of expeditionary field work are determined by the weather regime, phenological phenomena of the year, and peculiarities of bird ecology. The optimal periods for collecting material in the study of avian influenza should be considered the periods of mass seasonal migrations of migratory birds in spring and autumn (April, September), as well as the nesting and brood periods from the appearance of chicks to their rise to the wing (May-July).
At the preliminary stage, before leaving for the field, the study of cartographic materials is carried out, topographic, hydrographic, geobotanical or landscape maps and schemes of scales 1:25000-1:200000 are acquired. On the basis of these documents, calendar-territorial plans and work schedules are drawn up, parking places and routes of movement of zoological groups are outlined.
Epizootological examination is carried out by sequential radial detour of the territory. Routes, sequence of transfers, places, number and duration of stops are determined depending on the situation, the nature of the terrain, working conditions, convenience of access and transportation of the collected samples to the laboratory.
The duties of the expeditionary group are also charged with monitoring the population: its size, economic activity and the nature of its stay on the reservoirs. Particular attention is paid to the movement of agricultural workers, hunters, fishermen, tourists and vacationers during the period of possible deterioration of the situation with avian influenza. It is necessary to carry out active explanatory and sanitary-educational work among the local and temporary population, in close contact with representatives of local health authorities, authorities, police, and representatives of the veterinary service.
The minimum composition of the field team: an ornithologist, a teriologist, a virologist, an epidemiologist, a laboratory assistant, a driver, a cook. Shooting of birds can be carried out on a contractual basis by a special team of hunters.
During the epizootological examination, generally accepted zoological and ecological methods are used, regulated by the current regulatory documents (for example, MU 3.1.1029-01).
4.4. Rules for the collection, storage and transportation of material for laboratory research
All work on the collection, storage and transportation of field material suspected of containing type A avian influenza virus (subtypes H5 and H7) is carried out in accordance with the current SP 1.2.036-95 and MU 3.1.1027-01. Work on collecting field material is carried out in seasonal protective clothing, supplemented with a respirator, goggles and rubber gloves (Appendix 6 to SP 1.3.1285-03).
For laboratory research from natural biotopes take:
birds, chicks;
bird eggs;
bird faeces and (or) smear from the cloaca and trachea;
small mammals of semi-aquatic biotopes;
water and silt at nesting sites.
Harvested chicks, small birds and mammals(living and agonizing individuals are first killed with a forceps) are placed in bags of dense white fabric (each animal in a separate bag), the edges of the bags are folded twice and tightly tied. Use the pouches with the scar out. They are provided with labels indicating the date, exact address, station, type of animal, and the name of the picker. For transportation, cloth bags with animal carcasses are placed in an oilcloth bag.
In large birds take a smear from the cloaca and cut off the head with a section of the neck. The head is placed in a separate oilcloth bag, which is provided with a label.
Storage conditions. At a temperature of 2 to 8 °C - during the day, if long-term storage is necessary, the animals are opened, organs and tissues are frozen at a temperature below minus 40 °C.
Transportation conditions. Animal carcasses and heads - during the day at a temperature of 2 to 8 °C. Organs - frozen in a Dewar vessel or thermal container with dry ice.
Smears from the cloaca take dry sterile probes with cotton swabs. After taking the material, the swab (the working part of the probe) is placed in a sterile disposable microtube with 500 µl of a sterile 0.9% sodium chloride solution or phosphate buffer. The end of the probe is broken off or cut off with the expectation that it will allow you to close the lid of the tube. The test tube with the solution and the working part of the probe is closed and placed in a tripod, which is then placed in a thermal container with cooling elements.
Storage conditions. At a temperature of 2 to 8 ° C - for 3 days. If long-term storage is necessary, freeze the material at a temperature below minus 40 °C.
Transportation conditions. At a temperature of 2 to 8 ° C - for 3 days. Frozen - in a Dewar vessel or thermal container with dry ice.
If the bird must be left alive ( representatives of rare species), swabs are taken from her cloaca after capture.
bird eggs taken from the nest (no more than 50% of the clutch), marked and placed in plastic containers with holes for eggs, shifting with cotton wool. The containers are placed in a metal container and delivered to the laboratory.
Storage conditions. Store at 2 to 8°C for 3 days. If long-term storage is required, the contents of the eggs are transferred to sterile plastic bottles with screw caps and frozen at a temperature below minus 40 °C.
Transportation conditions. Within a few hours after collection - at ambient temperature. Within 3 days - at a temperature of 2 to 8 °C. The contents of the eggs are frozen at a temperature below minus 40 ° C in a thermal container with dry ice.
bird feces(4-5 g) are collected with disposable spatulas (spatulas) in sterile plastic containers (plastic bottles with screw caps).
Storage conditions. At a temperature of 2 to 8 °C - within 3 days, at a temperature of minus 40 °C - 30 days.
Transportation conditions. At a temperature of 2 to 8 ° C - for 3 days. Frozen material - in a thermal container with cooling elements at a temperature of minus 70 °C.
water and silt collected in nesting sites in the coastal zone. Sludge (5-10 g) is collected with scoops and transferred to sterile plastic bottles with screw caps. Water in a volume of 1 liter is collected in sterile plastic bottles with screw caps. Containers and bottles are labeled and placed in a metal container with absorbent material, the amount of which should be sufficient to absorb the contents in case of violation of the integrity of the shipping container.
Storage conditions. At temperatures from 2 to 8 °C.
Transportation conditions
When sampling organs, a sterile surgical instrument (scissors, scalpels, tweezers) and sterile dishes are used.
Internal organs(fragments of the trachea, lungs, spleen, brain, sinuses, air sacs, intestines) from slaughtered or dead birds, chicks, and small mammals are obtained by autopsy of animals. Before opening, the carcass is immersed in a disinfectant solution (5% chloramine B) for 20-30 seconds. When taking samples of animal organs, the site of the future incision is treated with a 5% iodine solution or 70% ethanol solution and the skin, muscles of the abdominal wall or skull bones are cut with sterile instruments. An incision in the wall of the abdominal cavity is made with an "apron", bringing the side lines of the incision along the ribs above the level of the heart, and the resulting flap is folded back to expose the internal organs. Before taking the brain, the entire occipital part of the skull is cut off. Using the second set of instruments, pieces of internal organs are cut off in size from a small pea to a hazelnut, the samples are placed over the burner flame in sterile disposable plastic test tubes or containers, hermetically sealed.
Storage conditions. Freeze at temperatures below minus 40 °C.
Transportation conditions. Frozen - in a Dewar vessel or thermal container with dry ice.
Smears-imprints, obtained from the mucous membrane of the upper respiratory tract (better) and internal organs, are prepared on clean glass slides defatted with ether, to which mucous or fresh sections of organs are pressed. The preparations are dried in air and fixed for 20 min in chemically pure acetone chilled from 2 to 8 °C. Placed in stands for glass slides (on the edge). Make a note that the strokes are fixed.
Storage conditions. At a temperature of 2 to 8 ° C for a week, at a temperature of minus 20 ° C - up to 6 months.
Transportation conditions. At temperatures from 2 to 8 °C.
The temperature below minus 40 °С is provided in a Dewar vessel filled with liquid nitrogen (minus 196 °С) or in a thermal container with dry ice (minus 70 °С).
Transportation of samples is carried out in accordance with SP 1.2.036-95. The thermal container and the Dewar vessel are wrapped with paper (sheathed with material), laced, sealed and transported to the laboratory by courier. The delivered material is accompanied by a cover letter, an act of packaging. The thermal container and the Dewar must have a special sign (label with a mark) "Danger! Do not open during transportation." If the material is to be stored and transported in a Dewar vessel or in a thermal container with dry ice, sealed plastic containers resistant to low temperatures or cryovials are used to collect the material. Dewars and dry ice containers must not be hermetically sealed so as not to prevent the release of slowly evaporating nitrogen or carbon dioxide.
Before leaving for the field, Dewar vessels must be checked in the laboratory for compliance with passport data and suitability for operation and transportation. When filling, loading, unloading and carrying Dewar vessels, it is necessary to have ordinary overalls, shoes and canvas gloves, so that in the event of a spill or splashing of nitrogen, it is impossible to get it on open parts of the body. During transport, the Dewars must be carefully secured to prevent tipping, splashing or spilling of nitrogen.
Only one-time freezing and thawing of any material is allowed.
5. Methods of laboratory research
Laboratory studies are carried out in accordance with the current sanitary and epidemiological rules SP 1.3.1285-03, regulating the work with microorganisms of I-II groups of pathogenicity (danger), MUK 4.2.2136-06 "Organization and conduct of laboratory diagnostics of diseases caused by highly virulent strains of the influenza virus type A birds (VGPA).
Fluorescent antibody method (MFA)
For MFA, fixed smears-imprints of animal organs and mucous membranes are used. The reaction is carried out in accordance with the instructions for the diagnostic preparation "Fluorescent immunoglobulins for the early differential diagnosis of influenza A (H5)", manufactured by LLC "Enterprise for the production of diagnostic products" State Research Institute of Influenza RAMS (St. Petersburg).
In each smear, at least 20-25 fields of view are examined.
The assessment of the degree of brightness of the luminescence of viral antigens stained with luminescent immunoglobulins is carried out according to the generally accepted scale:
++++ (4+) - bright fluorescence inside tissue cells;
+++ (3+) - moderate fluorescence inside tissue cells;
++ and + (2+ and 1+) - weak fluorescence inside (or outside) tissue cells.
A positive result of the immunofluorescent study is the detection in the preparation of at least 5-8 cells of the organ tissue, which have characteristic inclusions with specific fluorescence for three and four pluses.
Polymerase chain reaction (PCR)
Molecular genetic studies are carried out in accordance with the current regulatory documents: MU 1.3.1794-03 "Organization of work during PCR studies of material infected with microorganisms of pathogenicity groups I-II"; WHO guidelines for the detection of H5N1 avian influenza virus in samples from people with suspected disease (WHO, Geneva, August 2007); Instructions for using the test system for detecting influenza A virus RNA and identifying subtypes H5 and H7 by reverse transcription and polymerase chain reaction (for example, AmpliSense kits Influenza virus A H5N1-FL" or "FLU", produced by the Central Research Institute of Epidemiology of Rospotrebnadzor).
The "FLU" kit allows you to detect influenza A virus RNA and identify H5 and H7 subtypes in material from dead and sick animals and environmental objects. The material for the study is: litter, smears from the cloaca and trachea, internal organs (fragments of the trachea and lungs, spleen, brain), water, washings from eggs and egg white. The test system includes reagent kits: for RNA extraction, for obtaining cDNA on an RNA matrix, for amplifying cDNA regions (PCR) and detecting amplification fragments in electrophoretic analysis and hybridization-fluorescence detection formats (FEP and FRT), and also contains control samples .
Set "AmpliSense" Influenza virus A H5N1-FL" allows you to detect influenza A virus RNA and identify the H5N1 subtype in the material from dead and sick animals and environmental objects. The material for the study is: droppings, smears from the cloaca and trachea, internal organs (fragments of the trachea and lungs, spleen, brain), water, washings from eggs and egg white.The test system includes kits of reagents: for RNA extraction, for obtaining cDNA on an RNA template, for amplification of cDNA regions (PCR) and detection of amplification fragments in hybridization-fluorescence detection formats (FEP and FRT) and also contains control samples.
Enzyme immunoassay (ELISA)
For the study, individual blood sera of birds without signs of hemolysis and bacterial contamination in the volume of 0.3-0.5 ml are used. The reaction is set up according to the temporary instruction on the use of a kit for the detection of antibodies to the avian influenza virus (AIV) by enzyme immunoassay (for example, "Kit for the detection of antibodies to the avian influenza virus by ELISA" produced by NPP "AVIVAC").
The results are recorded on a spectrophotometer at a wavelength of 492 nm (when using OFD) or 450 nm (when using TMB).
All stages of incubation are carried out for 30 minutes at a temperature of 20-30 °C.
Haemagglutination inhibition reaction (HITA)
The detection of specific antibodies to the avian influenza virus in the blood sera of birds is carried out by a micromethod in accordance with the instructions for use of the "Set of antigens and sera for the diagnosis of avian influenza in the hemagglutination inhibition test (HITA)".
Accounting for the reaction is carried out visually after complete sedimentation of erythrocytes in the control wells (in the form of "buttons"). Serum antibody titer is considered to be the highest dilution, in which agglutination of erythrocytes by the influenza virus antigen is completely absent.
Positive samples identified during the laboratory test are sent for virus isolation and identification to the FGUN SRC VB "Vector" of Rospotrebnadzor.
6. Ensuring the requirements of biological safety during epizootological monitoring of avian influenza in natural conditions
To ensure the biological safety of work during epidemiological monitoring in potential natural foci of avian influenza, the following should be taken into account:
employees of anti-plague institutions participate in the survey, and employees of other medical and biological organizations and institutions that have access to work with PBA of I-II pathogenicity groups can also be involved. Auxiliary personnel (drivers, shooters, etc.) are allowed to work after the briefing;
the entire composition of the detachment or expedition must be familiar with the requirements of biological safety when working with pathogens of natural focal infections circulating in the area. The head (chief) of the epidemiological detachment (expedition) is responsible for compliance with these requirements when trapping wild animals and collecting field material;
any material is considered potentially dangerous in relation to the possible content of pathogens of natural focal diseases, characteristic of the landscape zone within which it was collected;
reconnaissance survey of the area, the installation of fishing gear is carried out in special clothing (overalls or anti-encephalitis suit, boots);
inspection of the exposed fishing gear and collection of field material is carried out in working clothes, supplemented with aprons and sleeves made of waterproof fabric (film), rubber gloves (2 pairs) [at the end of work, aprons, sleeves and gloves are disinfected];
to protect the respiratory organs, use disposable cotton-gauze bandages or anti-dust respirators (preference is given to respirators "Petal" or respirators of a class not lower than FFP2);
protection of the organs of vision is carried out with tight-fitting glasses;
disinfection of fishing gear and other tools is carried out daily at the end of work by warming up in the sun (in summer), boiling, treating with disinfectant solutions, followed by ventilation, boxes and jigger are treated with disinfectant solutions;
analysis of field material, autopsy of animals is carried out in a type I anti-plague suit (respiratory protection is similar to clause 6 of this section, the features of taking material and preparing samples for transportation to the laboratory are described in section 4.4 "Collecting field material for laboratory research");
upon completion of the work specified in clause 9, tools and protective clothing are disinfected (see Appendix 4), used tips, pipettes are disinfected by immersion in a 6% hydrogen peroxide solution for 60 minutes, dispensers are disinfected by wiping twice with an interval of 15 minutes 6 % hydrogen peroxide solution (exposure 120 min);
the remains of field material not subject to laboratory research are burned or disinfected by autoclaving, the resulting waste is placed in specially dug pits, which are then buried;
transportation of the material to the diagnostic laboratory is carried out by the transport of the expedition;
members of the expedition are subjected to daily thermometry, after the completion of work, an observation is established for a period of 7 days;
the first aid kit for emergency prevention must be completed in accordance with SP 1.3.1285-03
7. Ensuring biological safety during laboratory diagnostic studies
7.1. Carrying out work not related to the accumulation of the virus, the formation of aerosols of infected material (staining smears, setting serological reactions with non-disinfected diagnostic material, serological studies with non-disinfected material, RNA isolation) is carried out in a type IV anti-plague suit, supplemented with a cotton-gauze bandage (respirator) and two pairs of rubber gloves. The work is carried out in a class II biological safety cabinet*.
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7.2. Carrying out work on infection of cell cultures or chicken embryos, as well as work related to the possibility of aerosol formation, is carried out in class III safety boxes. The work is carried out in a type IV anti-plague suit, a cotton-gauze bandage (respirator) and rubber gloves (two pairs)*.
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* In the absence of biological safety cabinets, work is carried out in a type I anti-plague suit, supplemented with a waterproof apron and a second pair of gloves.
7.3. Working with inactivated material, carrying out the reverse transcription reaction and PCR, electrophoretic detection of the results of the study is carried out in a type IV anti-plague suit, supplemented with rubber gloves (two pairs).
7.4. Before starting work, personnel should be instructed on how to proceed in the event of an accident, including the following scenarios: an accident in a biological safety cabinet; accident outside the biological safety cabinet; aerosol accident.
7.5. The modes of disinfection of various objects in the laboratory diagnosis of avian influenza virus are carried out in accordance with SP 1.3.1285-03:
7.5.1. Disinfection of room surfaces (floor, walls, doors), equipment, desktops and other by double wiping with an interval of 15 minutes with a 6% hydrogen peroxide solution or a 3% chloramine solution (exposure 120 minutes) followed by UV treatment for 30 minutes .
7.5.2. Disinfection of protective clothing is carried out:
a) boiling in a 2% soda solution for 30 minutes from the moment of boiling;
b) soaking for 30 minutes at 50 °C in a 3% hydrogen peroxide solution with the addition of 0.5% detergent.
7.5.3. Disinfection of gloves - by soaking for 60 minutes in a 6% hydrogen peroxide solution with the addition of 0.5% detergent or in a 3% chloramine solution.
7.5.4. Disinfection of laboratory glassware, autoclavable dispensers, tips, virus-containing liquids, agarose gel, metal instruments is carried out by autoclaving - pressure 2.0 kgf / cm (0.2 MPa), temperature (132 ± 2) ° C, time 45 min.
7.5.5. Disinfection of dispensers - double wiping with an interval of 15 minutes with a 6% hydrogen peroxide solution (exposure 120 minutes), followed by UV treatment for 30 minutes.
7.6. The first aid kit for emergency prevention must be completed in accordance with SP 1.3.1285-03 and supplemented with two of the following antiviral drugs: arbidol, rimantadine, algirem, oseltamivir, zanamivir.
8. Regulatory references
1. Fundamentals of the legislation of the Russian Federation on the protection of the health of citizens. M., 1993 .. - Note of the database manufacturer.
6. SP 1.2.036-95 "Procedure for accounting, storage, transfer and transportation of microorganisms of I-IV pathogenicity groups".
7. The procedure for the development, examination, approval, publication and distribution of regulatory and methodological documents of the system of sanitary and epidemiological regulation: Collection R 1.1.001-1.1.005-96. M., 1998.
8. SP 3.1.097-96 "Prevention and control of infectious diseases common to humans and animals: Collection of sanitary and veterinary rules".
9. MU 3.1.1029-01 "Guidelines for catching, accounting and forecasting the number of small mammals and birds in natural foci of zoonoses".
10. MU 1.3.1794-03 "Organization of work during PCR studies of material infected with microorganisms of pathogenicity groups I-II".
11. MU 4.2.2039-05 "Technique for collecting and transporting biomaterials to microbiological laboratories".
12. MUK 4.2.2136-06 "Organization and conduct of laboratory diagnostics of diseases caused by highly virulent strains of avian influenza A virus (AVAI) in humans".
16. Guidelines "Rapid diagnosis of influenza and other acute respiratory viral infections by immunofluorescent method". St. Petersburg, 2006, approved. Head of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare G.G. Onishchenko April 25, 2006
17. Guidelines "Isolation of influenza viruses in cell cultures and their identification". St. Petersburg, 2006, approved. Head of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare G.G. Onishchenko April 25, 2006
18. SP 1.2.1318-03 "The procedure for issuing a sanitary-epidemiological conclusion on the possibility of working with pathogens of human infectious diseases of I-IV pathogenicity (danger) groups, genetically modified microorganisms, poisons of biological origin and helminths."
19. Interstate standard GOST 25581-91 "Agricultural, synanthropic, wild, exotic birds". Date of introduction 01.01.93. Methods of laboratory diagnostics of influenza.
20. Order of the Ministry of Health and Social Development of Russia dated May 31, 2005 N 376 "On the provision of extraordinary reports on emergency situations of a sanitary and epidemiological nature".
21. Order of the Federal Service for Surveillance on Consumer Rights Protection and Human Welfare No. 373 of March 31, 2005 "On Improving the System of Epidemiological Surveillance and Control of Influenza and Acute Respiratory Virus Infections".
22. Order of the Federal Service for Surveillance on Consumer Rights Protection and Human Welfare dated May 10, 2007 N 144 "On the Establishment of a Scientific and Methodological Center for Reference Diagnostics and Study of Highly Pathogenic Influenza Virus Strains".
23. Order of the Ministry of Agriculture of Russia dated March 27, 2006 N 90 (registration number 7756) "On approval of the Rules for the fight against avian influenza".
24. Recommendations for the protection of people in contact with infected birds and involved in the mass slaughter of animals potentially infected with avian influenza viruses, approved. Chief State Sanitary Doctor of the Russian Federation 05.08.05 N 0100/6198-0523.
25. WHO guidelines for the diagnosis and control of animal influenza (WHO/CDC/CSR/NSC/2002.5).
26. WHO guidelines for the detection of H5N1 avian influenza virus in specimens from suspected individuals. WHO, Geneva, August 2007. (Recommendations for laboratory procedures to detect avian influenza A H5N1 virus in specimens from suspected human cases. WHO Geneva, August 2007).
27. Onishchenko G.G., Kiselev O.I., Sominina A.A. Strengthening Influenza Surveillance and Control as a Critical Element in Preparedness for Seasonal Epidemics and the Next Pandemic (guidance). Moscow-St. Petersburg, 2004.
28. Bird flu. Clinical features, standardized principles for diagnosis, treatment and prevention, approved. Head of the Federal Service for Supervision of Consumer Rights Protection and Human Welfare G.G. Onishchenko 02.09.05.
29. Neklyudova L.I., Gumennik A.E., Fedorova Yu.B. et al. Practical Virology (Part III). M., 1981.
30. Detection of circulation of arboviruses. Methods of virological and serological studies. Clinical and epidemiological characteristics of little-studied arbovirus infections. Approaches to monitoring natural foci of arboviruses / Ed. acad. RAMS D.K.Lvova //Itogi science and technology. Ser. Virology. T.25. M., 1991.
31. Syurin V.N., Famuilenko A.Ya., Soloviev B.V. etc. Viral diseases of animals. M.: VNITIBL, 1998.
Appendix 1. List of documents authorizing the collection of field biological material as part of avian influenza monitoring within the borders of the Russian Federation
Attachment 1
1. Permission to shoot birds.
A) The permit is issued by the regional hunting inspection. An organization planning to shoot birds in order to detect individuals affected by the avian influenza virus writes a letter of justification addressed to the head of the regional hunting inspection. The letter is written on company letterhead in the prescribed form.
B) Shooting of birds can only be carried out by a member of the regional society of hunters, endowed with the relevant documents. It is advisable to involve from among the employees of the organization conducting the epizootological survey, persons who have the right to conduct hunting. Acquisition of consumables (cartridges) in sufficient quantities should be carried out from the item of travel expenses.
2. Permission to conduct epizootological research in near-water habitats with the right to set up a temporary camp in a water protection area.
The permit is issued by the regional fish inspection. It is necessary to provide explanations about the purpose and objectives of the planned research in the territories controlled by the fish inspection, which are written on letterhead in the prescribed form.
3. Coordination with regional environmental authorities.
A clear explanation of the planned activities is necessary in connection with the real threat of complication of the epidemiological situation. An explanatory letter is written to the head of the regional committee on ecology on letterhead in the prescribed form.
4. Coordination with the border service of the Russian Federation.
Coordination is carried out only in cases of epidemiological studies in the border areas. The request for permission to work in the border zone is written in the name of the head of the border service of the given region on a letterhead in the prescribed form.
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» » Influenza Virus Natural Reservoir + Survival
Natural reservoir of influenza virus + survival
2603
Publication date: December 23, 2012
Influenza viruses have a wide range of reservoirs, mainly birds, in particular aquatic ones, in which the infection is mainly intestinal in nature and asymptomatic. Domestic ducks in Southeast Asia are the main host of the influenza A virus, these birds also play a central role in the creation and maintenance of the H5N1 virus. In Thailand, there was a strong association between the H5N1 virus and wild ducks and, to a lesser extent, little association with chickens and roosters. Wetlands that are used to grow and double-crop rice are always associated with free-range ducks all year round, apparently a critical factor in the spread of the virus. Highly pathogenic avian virus can survive in the environment for a long time, especially at low temperatures. In water, the virus can survive for four days at 22°C, and more than 30 days at 0°C. In frozen material, the virus is likely to survive, but the duration is uncertain. Recent studies show that H5N1 viruses isolated in 2004 are more stable and survive at 37°C, in the 1997 outbreak the viruses survived only 2 days. The virus is killed by heat (56°C for 3 hours or 60°C for 30 minutes) and a common disinfectant such as formalin and iodine compounds.
Naturally, the question arises: where is the virus stored, where is the reservoir, where do its new varieties come from? This question is very important and scientists are making a lot of efforts to find the answer to it.
The identification of reservoirs of infection made it possible to find ways to significantly reduce or even eliminate a number of diseases. For example, it turned out that the main reservoir of infections in plague, tularemia, and rabies are wild animals and rodents. The elimination of natural foci of these infections, the creation of effective cordons against the importation of sick animals proved to be sufficient to significantly reduce or completely eliminate these infectious diseases.
Aren't animals also reservoirs for influenza? This idea arose as early as 1931, when a virus similar to the human influenza virus was isolated from sick pigs. Scientists returned to this idea after 1957. In the study of influenza-like diseases of domestic animals and birds, viruses were again isolated from horses, pigs, sheep and ducks, in some properties related to type A influenza viruses. But they all differed significantly from each other and could not be completely identified with any of the human influenza viruses .
Further observations showed that influenza-like diseases in animals and birds are quite rare and animals are not the source of influenza in humans. Science has data showing that the opposite can happen - the transfer of the influenza virus from humans to pigs and its further spread among them. Thus, some animals are a kind of piggy bank of the virus.
However, there is every reason to believe that only the person himself is the source of infection and the reservoir of viruses in influenza.
Systematically conducted studies have shown that in large cities and towns, influenza A and B diseases are observed all year round, although in inter-epidemic times, especially in summer, they make up a small percentage of the total number of observed acute respiratory diseases.
These individual diseases, stretching in a chain from case to case, retain the virus in the period between individual epidemic waves. Moreover, it is during these outwardly calm inter-epidemic periods that new varieties of the virus are formed.
How does the flu virus change? Is it infinite, or does it have a periodicity, and pre-existing varieties can reappear? Recently discovered phenomena have shed light on these questions. As mentioned earlier, after a disease, antibodies to the type of virus that caused the disease appear in the human blood. These antibodies are like traces of the virus. They can be used to determine which type or variety of it caused the disease. It was generally believed that antibodies persisted in the blood for no more than a year. However, it has now been established that antibodies produced in response to the first flu in a person's life persist until old age. At the same time, the number of original antibodies will always be greater than antibodies to any other type of influenza that a person has encountered in subsequent years.
Knowing the year a person was born and the type of virus to which he has more antibodies, you can determine which type of flu caused the disease in childhood.
The systematic conduct of this kind of research allowed scientists to establish the frequency of occurrence of various varieties of the virus and the duration of their circulation among the population. These observations give grounds to assert that the variability of the influenza virus is not chaotic, not unlimited, but has its own patterns that can be revealed and used to combat the disease.
