Most species of laboratory animals are so well adapted that they live and breed in the most unfavorable conditions. Mice, for example, can tolerate everything from tropical conditions to sub-zero temperatures. They breed even in refrigerators, which store stocks of frozen meat. But at some temperature within this range, mice feel best, and it is at this temperature with slight fluctuations in both directions that they should be kept.

The freedom of movement of animals indoors is limited. They spend their lives in cells, i.e. under microclimatic conditions inside the cell. Therefore, the basis for designing a room for animals should be based on the principle of creating such environmental conditions that would provide the necessary microclimate for animals and such sizes and shapes of rooms that would be convenient for the attendants.

IN vivariums and nurseries there should also be office space necessary in the work of caring for animals. Practice shows that about half of the total area of the premises can be allocated to accommodate animals, and the other half can be used as a room for keeping all kinds of records, etc.

In small vivariums or nurseries, these ratios shift towards the predominance of utility rooms. The need to create maximum hygienic conditions in them, associated with the isolation of animals, the sterilization of materials coming from outside, and so on, also leads to a reduction in the usable area.

Vivarium(lat. Vivarium - game reserve, menagerie) - a room for keeping and breeding laboratory animals used for scientific purposes and in the practice of veterinary and health authorities. Vivariums are very different both in appearance and in the number of animals in them, which is determined by the characteristics of scientific research. Vivariums can be used not only for keeping, but also for increasing the number of laboratory animals.

Animal breeding in vivariums is usually practiced in those cases when it is necessary for the experiment to have animals of a certain type, size, weight, sex and age or grown under special conditions of feeding, lighting, etc. In addition, the presence in the vivariums of their own nursery will greatly facilitate the problem of uninterrupted supply of the laboratory with the necessary number of small animals. Some scientific research can be carried out in them, for example, to determine the results of the long-term influence of various diets, etc.

In laboratory practice, there are two concepts: vivarium - a facility for keeping animals in the experimental state in appropriate conditions, and nursery - a room for breeding and keeping animals until they are used. An uninterrupted supply of animals to the laboratory is possible only with the organization of large nurseries, led by qualified specialists and under strict veterinary and sanitary control.

The device of vivariums is determined primarily by the species composition of animals, in connection with which there are specialized vivariums (kennels, monkey houses, etc.) and general or complex types that provide for the maintenance of various animals - dogs, cats, rabbits, guinea pigs, white rats, mice. Vivarium

may also include aquariums for freshwater and marine animals, terrariums for amphibians and reptiles, aviaries and cages for birds and other adapted facilities for different types of mammals.

Distinguish premises winter And summer, permanent And temporary(the so-called walks). Each vivarium must have a quarantine room for newly arrived animals and an isolation room for sick animals (when working with artificially infected animals, the construction of specially designed isolation rooms is always provided). To care for animals after complex operations in modern vivariums, so-called clinics, where animals are given particularly favorable conditions and where they are under constant supervision.

Clinical premises can also be used for long-term keeping of animals requiring special care. If necessary, both general and single wards are provided for when setting up clinics. There must be specially equipped rooms that allow for the sanitation of both newly arrived animals and animals assigned for surgery or used in long-term experiments that require compliance with certain sanitary and hygienic conditions (conditioned reflex experiments, etc.).

In some cases, it is absolutely necessary to have a specially equipped disinfection chamber, allowing to quickly process infected cages, overalls of employees and auxiliary equipment. Should be provided room for autopsy of dead animals And storage of corpses. Vivariums are equipped with a kitchen with a distribution room, a sink and storage rooms for storing food and spare equipment (Fig. 87).

Great attention must be paid to the ventilation of vivariums. Conventional ventilation methods by simply sucking the air out of the animal room are generally not sufficient. To remove the gaseous decay products of animal secretions, supply and exhaust ventilation is arranged. Vivarium premises should have a waterproof, for example, stone or cement floor with gutters and reliable ladders, which will

(it allows you to quickly clean with a jet of water from a flexible hose connected to the water supply. In order to be able to wash and disinfect the walls, they are tiled.

Preference should be given to relatively small rooms for animals. They have less noise that disturbs animals, they are easier to ventilate and keep clean, and finally, they pose less of an infectious hazard. In terms of shape, animal rooms should be elongated rather than square in order to achieve a more economical use of space (Fig. 87).

However, in some cases, square rooms are more convenient, while in the center of the room there is enough space for work. So, for example, in a room measuring 2.5x5.0 m with one or two doors, it is convenient to place racks with cages along long walls. It is also possible to place shelving in the center of the room with access to it from two sides, however, such an arrangement of shelving is less economical.

^ 0

Rice. 87. Vivarium plan for laboratory animals. Pure office space: I- hallway, toilet, showers; 2 - office; 3- feed kitchen from a week's supply of feed; 4 - sterilization; five - warehouse bedding materials; 6 - warehouse of cells; 7- clean corridor. Accommodations for animals: 8 - experimental animals; 9 - animal breeding: a - racks. Dirty office space: 10 - dirty corridor; 11 washing; 12 - corpse and waste incinerator.

Practice shows that a vivarium should have four isolated sections. First section intended for animals not yet taken in experiments. This section is for breeding animals, perhaps for those coming from outside (quarantine), it is conveniently called section of healthy animals. Second - intended for on experimental animals. Animals from the first section arrive here and stay here for the entire duration of the experiment. They should not be returned to the healthy animal section. This section can be called test section animals. IN third sections stored feed, clean bedding, clean and spare cages and other equipment. Fourth - destined for cleaning cages, dirty bedding, corpses of dead animals, etc. Clean cages and other items of equipment are returned to the warehouse, from where they are delivered for use in one or another section for animals.

Great care should be taken to remove the message-links between the named sections. Ideally, the clean and dirty circulation paths of animals, feed and implements should not cross anywhere. It is advisable to arrange two doors in the animal room, one of them for the delivery of clean cages, feed, etc., the other for the removal of dirty cages. Thus, the flow of feed and inventory all the time goes in the same direction from clean to dirty. Cages and other items of equipment after use and cleaning are returned to clean sections only after sterilization. If the arrangement of separate clean and dirty crossings is not possible, then the same crossing can be used for different purposes at different times of the day. For example, for the movement of dirty materials - in the morning, and for clean materials - in the afternoon, after thorough washing and disinfection.

The most convenient for work elongated form of rooms for animals with near-wall (a) and central (b) placement of racks (C) for cages and a water sink (D). Wall placement of racks, in addition to convenience for work, is more economical than the central one (Fig. 88).

Rice. 88. Scheme of placement of shelving in the rooms: a-wall; b-central; B cells; G-plumbing sink.

So far, the discussion has dealt with animal rooms in the most general form, without regard to whether they are intended for mice, guinea pigs, rats, or other animals. With few exceptions, all such facilities should be suitable for all types of laboratory animals. An expensive and well-maintained nursery or vivarium is not built for 1-2 years, and the types of animals that it contains may be different in different years. A room that is used for mice this year may contain rabbits next year, and such a change should take place without major redevelopment.

RD-APK 3.10.07.02-09

MINISTRY OF AGRICULTURE
RUSSIAN FEDERATION

Moscow 2009

Developed by: Ph.D. s.-x. Sciences, Art. scientific collaborator P.N. Vinogradov, Ph.D. tech. Sciences S.S. Shevchenko, O.L. Sedov, E.S. Garafutdinova, M.F. Malygin (SPC "Giproniselkhoz"); dr vet. sciences, prof. V.G. Tyurin (GNU VNIIVSGE)

INTRODUCED: SPC "Giproniselkhoz".

APPROVED AND PUT INTO EFFECT: Deputy Minister of Agriculture of the Russian Federation A.I. Belyaev December 1, 2009

DESIGNED FOR THE FIRST TIME.

Introduction date 12/15/2009

1. General Provisions

1.1. These guidelines apply to all research organizations and educational institutions of the agro-industrial complex of Russia, regardless of their organizational and legal form, using laboratory (experimental, experimental) animals in their work.

In accordance with the Federal Law "On Technical Regulation" (adopted by the State Duma on December 15, 2002 and approved by the Federation Council on December 18, 2002), until the adoption of the relevant regulations, technical regulation in the field of taking veterinary and sanitary measures is carried out in accordance with the Law of the Russian Federation " On Veterinary Medicine (approved on May 14, 1993, No. 4979-1).

1.2. The guidelines apply both to newly designed facilities for keeping and working with laboratory animals - experimental biological clinics, vivariums, etc., and to existing and reconstructed ones.

1.3. Objects for keeping and working with laboratory animals are scientific support units of research organizations, educational institutions and are created for keeping and, if necessary, breeding laboratory animals used in experimental work and research. At these facilities, independent development of individual scientific issues can also be carried out.

1.4. The standards and requirements set out in these guidelines for the design, construction and operation of facilities for keeping laboratory animals are aimed at ensuring the safety of personnel working with animals and the general population from the occurrence of anthropozanoses and other diseases.

1.5. Development, coordination, approval and composition of project documentation for the construction of facilities for keeping laboratory animals are carried out in accordance with the requirements of SNiP 11.01-2003.

1.6. An object for keeping laboratory animals (hereinafter referred to as vivarium) is located in a separate building (complex of buildings) or on the upper floors of laboratory buildings of veterinary state scientific institutions, as well as on the territory of educational institutions.

1.7. Vivariums should be provided with drinking quality water, including hot water, electricity, equipped with sewerage (pipes with a diameter of at least 100 mm), supply and exhaust ventilation, heating, security and fire alarms, and have convenient access roads.

1.8. The distance between a separate building of the vivarium and the facilities of research institutions, which include this vivarium, must be at least the distance of fire breaks established by the current fire safety rules in the Russian Federation.

1.9. Separate buildings of vivariums should be fenced with a blank fence and separated from the residential area by a sanitary protection zone. The territory should be landscaped.

The dimensions of the sanitary protection zone are determined by the requirements of SaNPiN 2.2.1 / 2.1.1.1200-03.

1.10. The dimensions of the sanitary protection zone for vivariums located in separate administrative, industrial buildings and having an isolated exit are agreed in each case with the state sanitary and veterinary supervision authorities.

2. Composition, mutual arrangement and area norms of vivarium premises

2.1. Each vivarium should include premises designed in accordance with the current building codes and regulations in compliance with the current veterinary and sanitary requirements and zoohygienic standards, including:

staff room with individual lockers for overalls;

premises for the reception and quarantine of animals newly entering the vivarium;

insulator;

premises for keeping experimental animals (separate for each species) or (in agreement with the state veterinary and sanitary supervision authorities) divided into sections according to animal species;

a sterilization room or a box for researchers to work with uninfected animals, with a room for dissection of animals and a refrigerator for temporary storage of corpses;

isolated rooms for keeping experimental animals infected with cultures of pathogens of especially dangerous infections or radioactive substances (separately) from the operating room at each isolated room, which has a refrigerator and the necessary equipment for infecting and dissecting animals;

feed kitchen for preparing feed (should be equipped with a stove and refrigerator);

disinfection and washing department for washing with hot water, disinfection and drying of cages and other equipment;

a warehouse of clean (decontaminated) spare inventory: cages, drinkers, etc.;

sanitary block (shower and toilet);

a room equipped with an oven for burning animal corpses;

a common cold store for storing animal corpses;

feed warehouse;

litter warehouse;

in a separate room or in a separate building - a technical unit for air conditioners, ventilation, electrical and other special installations.

2.2. Each vivarium should have a room to receive incoming animals. In vivariums where small rodents are kept, an insulated vestibule is installed in front of the receiving department, into which a car with arrived animals enters and unloads.

When the vivarium is located on the upper floors of the laboratory buildings, the reception area and the insulated vestibule are located on the first floor of the laboratory building and are connected to the vivarium by an elevator used only for lifting laboratory animals.

2.3. The reception room is a room of 12.5 - 18 m 2 with natural and artificial lighting in accordance with the requirements.

The height of all rooms of the vivarium is 3 - 3.5 m.

2.4. The quarantine room consists of several isolated rooms with an area of 12.5 - 18 m 2 and is isolated from the rooms that contain animals that have passed quarantine and come for experiments.

2.5. An isolation room for sick animals and animals with suspected diseases adjoins the quarantine premises. The areas of the isolation rooms are similar to those of the quarantine rooms.

2.6. Premises intended for keeping experimental animals may lead to one common corridor or be located between two corridors and have exits to each of them. With a single-corridor layout, "dirty" and "clean" services are located at different ends of the corridor.

With a two-corridor system, feed, quarantined animals arrive along one corridor (“clean”), employees enter in clean overalls and replaceable shoes before starting work with animals. In another (“dirty”) corridor, uneaten feed and manure are removed, animal corpses are taken out, employees come out after working with animals.

If it is impossible to isolate "dirty" and "clean" flows, it is allowed to use the same room for one or another purpose, provided that it is disinfected every time after the "dirty" flow passes through it.

2.7. The area of premises for keeping certain types of experimental animals is 12.5 - 18 m 2 ; the area of the premises for keeping experimental animals, divided into sections, is determined by calculation.

2.8. The area of the sterilization room or box for the work of researchers with uninfected animals is determined by calculation, based on the specifics of the proposed work.

2.9. The areas of isolated rooms for working with experimental animals infected with pathogens of especially dangerous infections, and for working with experimental animals contaminated with radioactive substances, as well as the areas of operating rooms in each isolated room, are determined by calculation, based on the conditions of the technological and special equipment used to carry out the necessary manipulations.

2.10. The total area of the premises occupied by the feed kitchen, the disinfection and washing department and the warehouse for clean spare equipment should be approximately 50% of the total area of the premises occupied by animals (in large vivariums this percentage may be slightly reduced).

The feed kitchen consists of two adjacent rooms intended for processing and preparing feed. Each room must have access to the corridor.

Disinfection-washing department (one or several) consists of two rooms connected by a walk-through autoclave or a walk-through dry-heat chamber.

The device of the disinfection-washing department should provide for a different sequence of its work:

in the presence of infected material - preliminary sterilization of inventory and bedding with further mechanical cleaning of the latter in another room;

sterilization after mechanical cleaning of cages and inventory, when there is no danger of contaminated material.

Regardless of the location of the vivarium (in a separate building or on the upper floor of the laboratory building), the disinfection and washing department should be provided with a garbage chute to remove dirty bedding and mechanized lifting of materials and equipment.

The warehouse for clean inventory and equipment is located next to the disinfection and washing department.

2.11. For storage of bedding (shavings, sawdust, peat, etc.), two rooms are allocated: one for sterilized and packed in containers used by this vivarium, the other for storing newly purchased bedding.

2.12. When designing the vivarium premises, it is necessary to provide maximum insulation:

all its premises from other divisions that are part of the research institution;

premises of the isolation ward and quarantine from other premises of the vivarium;

between the feed kitchen, animal rooms and the disinfection and washing department.

2.13. In each case, the area occupied by the feed kitchen, the disinfection and washing department and the warehouse for clean spare inventory is determined depending on the equipment used, the degree of mechanization of production processes and the types of feed for experimental animals.

The dimensions of the areas of the above premises are indicated in the design assignment.

3. Veterinary-sanitary and technological requirements for building solutions for vivarium premises and engineering equipment

Luminaires and lighting fixtures of a closed type must be accessible for wet cleaning.

3.5. The premises of the vivarium, in which laboratory animals are kept, are equipped with a forced supply and exhaust ventilation system that ensures the frequency of air exchange and temperature and humidity conditions in accordance with the data given in Table. .

Animal species	Temperature, °C		Relative humidity, %		Maximum allowable concentration in air
Animal species	fluctuations	average	fluctuations	average	ammonia, mg/l	carbon dioxide by volume, %	hood	inflow
Mice	18 - 22		50 - 65		0,01	0,15
Rats	18 - 22		50 - 65		0,01	0,15
Hamsters	18 - 22		50 - 65		0,01	0,15
Guinea pigs	15 - 18		50 - 65		0,01	0,15
rabbits	15 - 18		50 - 65		0,01	0,15
Dogs	18 - 22		50 - 65		0,01	0,15
cats	18 - 22		50 - 65		0,01	0,15

3.6. The temperature and humidity regime in other rooms of the vivarium should be provided in accordance with the data given in Table. .

room

Temperature in the cold and transition period of the year, ° С

Air exchange rate (volumes per hour)

inflow

hood

1. For staff

2. For receiving animals

3. For research

4. Washing-sterilizing

1 - 2

2 - 3

5. For euthanasia (euthanasia)

6. Opening

7. Recycling

By calculation

8. For keeping experimental farm animals

3.11. For the removal and collection of wastewater after washing and disinfection of process equipment and floors, it is necessary to provide for the installation of trays covered with removable perforated plates and ladders. The slope of the trays must be at least 0.02.

3.12. When designing the local sewerage system of vivariums, the following requirements should be observed:

wastewater from rooms for burning corpses is subject to sterilization in manzhus with live steam at a temperature of 120 ° C for 30 minutes or in a steam jet installation at a temperature of 110 ° C for 10 minutes; in the presence of especially dangerous infections, wastewater is sterilized at 140 ° and 130 ° C for 20 and 60 minutes, respectively;

industrial and domestic wastewater from washing floors and washing and disinfecting process equipment should be collected in a special receiver, and disinfected with chlorine-containing preparations before being discharged into the sewer;

storm drains from the territory of free-standing vivariums that are unfavorable in veterinary and sanitary terms are subject to disinfection with chemicals;

the resulting mechanical and biological sewage sludge is incinerated.

3.13. Supply and exhaust ventilation main ducts, power supply, water supply and sewerage pipes should be located in special niches in the corridors and have free access for inspection and repair.

3.14. Isolated rooms for keeping experimental animals infected with cultures of pathogens of especially dangerous infections or radioactive substances, and the isolation room are equipped with local ventilation systems with filters that provide 100% purification and disinfection of the emitted air. The ventilation system should provide in these rooms a reduced (by 3 - 5 mm Hg) atmospheric air pressure in relation to other rooms of the vivarium. Ventilation in these rooms by opening windows is prohibited.

4. Vivarium equipment and animal accommodation conditions

4.1. Mice, rats, hamsters, guinea pigs and rabbits are housed in cages mounted on metal racks.

4.2. Wall or other design racks should be with removable brackets and movable shelves, which allows them to be converted to cages of various overall dimensions with different types of laboratory animals.

4.3. To calculate the production area, it is necessary to proceed from the following standards for placing animals in cages (table).

Animal species	Minimum cage bottom area per animal, cm2	Number of animals
Animal species	Minimum cage bottom area per animal, cm2	maximum allowable in a cage	per 1 m 2 floor space
Mice			65 adults or 240 young animals
Rats			20 adults or 100 young animals
Hamsters			30 - 40
Guinea pigs			15 - 18
rabbits	2000		3 - 4

Notes.

1. For an approximate determination of the production area, one should proceed from the calculation that 1 cm 2 of the cage bottom area should account for 1 g of animal weight.

2. Racks are located mainly along the walls and should occupy approximately 40% of the production area.

4.4. Dogs are placed in separate cabins (boxes) strictly individually. The dimensions of the box must correspond to the length and height of the animals.

Boxing dimensions for large dogs weighing over 22.5 kg - 1.2 × 1.8 m = 2.2 m 2, medium weighing 16 - 22.5 kg - 1.2 × 1.5 m = 1.8 m 2, small weighing 4.5 - 16 kg - 0.9 × 1.2 m = 1.1 m 2. The gap between the bars is 4.5 - 5.5 cm, the diameter of the metal bars is 0.5 - 0.6 cm. The side walls are solid. Wooden shields are laid on the bottom wall (floor).

Feeding and watering - in boxing. Enclosures for walking are arranged individually, at the rate of up to 2 m 2 per animal. Walking time - at least 2 times a day, duration - at least 20 minutes. Separate sectional keeping of males from females, puppies and aggressive animals should be provided.

4.5. On the territory of the vivarium for dogs, special rooms are being built, equipped with cabins. Enclosures (walks) are attached to the cabins. Each dog should have its own enclosure.

Cabin dimensions, m: length - 2; width - 1.5; the height of the front wall - 2.5 and the back - 1.5 - 2; cabin door height - 1.7, width - 0.7. A glazed frame is installed above the cabin door. At the bottom of the door, installed in the rear wall of the cabin, which is the front wall of the enclosure, a hole is made into the enclosure measuring 40 × 50 cm, which is hung with a thick cloth for winter protection from the cold.

Enclosure dimensions, m: length - 3, width - 2, height - 2.2. In its front wall, a door is made with dimensions of 1.8 × 0.7 m.

4.6. Cats are housed in enclosures of five heads, where shelves (beds) are provided, sufficient in area to accommodate all animals. The area of the open-air cage for one cat is 0.5 m2. A mesh vestibule is equipped in front of the entrance to the aviary.

4.7. If farm animals and birds are placed in vivariums for scientific purposes, the premises for them are constructed in accordance with the current technological design standards in compliance with the zoohygienic standards set forth in these standards.

5. Admission of animals to the vivarium

5.1. The replenishment of the vivarium with animals and birds is made from specialized nurseries that are free from infectious diseases.

The purchase of animals and birds in other organizations and individuals is allowed if it is not possible to purchase them in nurseries and if each purchase has a veterinary certificate of the well-being of the organization (farm, private person) for infectious diseases.

5.2. Animals are admitted to the vivarium with a veterinary certificate or accompanying documents from the nursery.

5.3. Animals received from the nursery are placed in isolated sections for a period of three days to adapt to new conditions. Subsequent periods of isolation or quarantine for these animals are determined depending on the conditions of keeping the animals, the nature of the upcoming experiments, distance, transportation conditions, etc.

5.4. For animals not obtained from nurseries, the following terms of their quarantine are established:

for mice and rats - 14 days, guinea pigs and rabbits - 21, dogs and cats - 30, for other animals and birds - 21 days.

In some cases, when pregnant females, newborns and young animals are used in experiments, as well as in short-term experiments, the duration of quarantine can be reduced if these animals are placed in isolated rooms and under appropriate supervision.

5.5. During the quarantine period, the animals are subject to daily clinical observation: thermometry and registration of the general condition of the animals in a special journal.

5.6. In quarantine and experimental sections, animals are placed in clean, pre-disinfected (autoclaved) cages.

5.7. Animals in the quarantine building are cared for by the personnel assigned to these premises.

5.8. It is forbidden to take out food, overalls and equipment from quarantine premises to other premises and sections for experimental animals.

5.9. During the quarantine period, a periodic change of cages is performed. At the end of the quarantine, the released cages and inventory are transferred to the disinfection and washing department.

Cleaning and washing of cells and other equipment from quarantine sections can be carried out in the general disinfection and washing department of the vivarium only after preliminary disinfection. Waste must also be decontaminated or incinerated. Methods of disinfection, disinfestation and autoclaving mode are established in each case, depending on the specifics of the institution.

5.10. During the period of adaptation or quarantine, animals with suspected infectious diseases are subjected to bacteriological examination. When an infectious disease is confirmed, mice, rats, hamsters, guinea pigs and rabbits of the entire incoming batch are destroyed, and for dogs, cats and other animals, the quarantine period is extended depending on the established disease.

5.11. Quarantine rooms after each batch of animals transferred for experiment and after each case of detection of infectious diseases are thoroughly disinfected.

5.12. In the event of mass diseases among animals observed in quarantine, or if individual cases of infectious diseases that are especially dangerous for laboratory animals and humans are detected during the experiments, the necessary set of preventive measures is carried out in the vivarium. In this case, experiments on animals are temporarily suspended.

5.13. At the end of the quarantine period, the animals are transferred to the experimental sections.

6. Mode of operation and basic rules for keeping animals

6.1. It is recommended to keep animals of only one species in each separate room. If, according to the conditions of the experiment, it is necessary to keep laboratory animals of different species in one section, then they should be placed on different racks.

6.2. Each cage (box, aviary, etc.) must have a label indicating the data on the animal and the timing of the experiment.

6.3. Laboratory animals and birds are kept in cages with a solid bottom on a litter or in cages with a mesh bottom - a floor. Wood chips, shavings or bedding peat are used as bedding. The litter is autoclaved in advance or kept in a dry-heat cabinet (at 150 - 180 ° C for 15 - 20 minutes). The thickness of the litter layer in the cage is 5 - 10 mm. When animals are kept in cages with a mesh bottom, the litter is poured into a tray (tray) located under the mesh floor.

6.4. All work on the care and maintenance of laboratory animals is built in accordance with the daily routine and work schedule approved by the head of this institution. The daily routine provides for time for sanitizing the premises and equipment, distributing feed and conducting experimental work and manipulations.

6.5. Feeding of laboratory animals is carried out in accordance with existing standards.

6.6. Feed and semi-finished products are stored in a room specially designated for this purpose. The distribution of feed is carried out in the prescribed manner.

In the feed kitchen of the vivarium, storage of no more than two to three days of food supply is allowed. When feeding animals with granular feed and if there are feed hoppers in the cages, advance receipt of feed from the warehouse for seven to ten days is allowed.

6.7. Special chests (metal or upholstered with tin on the inside) are equipped in the feed kitchen and in the pantry of the vivarium to store the supply of feed. Perishable foods are stored in the refrigerator. Delivery of feed from the warehouse is carried out by specially assigned personnel (workers who are not directly involved in caring for animals).

6.8. The distribution of feed in the rooms-sections is carried out by workers or kitchen staff specially allocated for this purpose in disinfected dishes (containers) assigned to each section. Write-off of feed is carried out in the prescribed manner according to the actual availability of animals for each day.

6.9. Entrance to the feed kitchen of personnel caring for laboratory animals and unauthorized persons is prohibited.

6.10. The supply of laboratory animals with drinking water is made from the water supply, the quality of the water must comply with SanPiN 2.1.4.1074-01.

6.11. Germination of grain on green mass for feeding laboratory animals is carried out in rooms specially designated for this purpose. It is allowed to feed the animals with the root mass of plants in the absence of mold in it.

6.12. Distribution of feed and watering of animals should be carried out only after cleaning the premises, cleaning or changing cages and removing dirty equipment, trays with bedding and other materials to be disinfected or disposed of from sections.

6.13. Cleaning of cages and cleaning of rooms is carried out with the help of inventory strictly assigned to each room.

6.14. With a periodic change of cages, animals are transplanted 1-2 times a week into pre-disinfected cages with prepared bedding, feeders and drinkers. Dirty cages, along with bedding, feeders and drinkers, are transferred to the disinfection and washing department for their subsequent processing.

6.15. Cells are cleaned daily. At the same time, contaminated bedding and other waste from the cages are collected in special metal tanks with lids. Tanks are tightly closed and transferred to the disinfection and washing department.

6.16. When using cages with a mesh bottom and trays isolated from cages, the latter are periodically (at least once a week) replaced with new ones. Dirty pallets with bedding are transferred to the disinfection and washing department for their further processing.

6.17. When one worker serves several types of laboratory animals, cages with guinea pigs are processed first, then cages with mice, rats and rabbits, and lastly, rooms where dogs and cats are kept.

6.18. Washing and disinfecting cages, feeders and drinkers directly in the sections is prohibited.

6.19. Before the end of the working day in the sections, the floor is wet cleaned using a 1% solution of chloramine or another disinfectant. At least once a month, a sanitary day is held, during which all premises are cleaned. The order of the sanitary day is determined by the head of the vivarium.

6.20. Disinfection, cleaning and washing of cages, feeders, drinkers and other equipment is carried out by workers specially assigned to the disinfection and washing department. Control over the effectiveness of cleaning and disinfection of inventory is assigned to the vivarium veterinarian.

6.21. The conditions for the collection, storage, removal (or disposal) of waste (litter, manure, feed leftovers, etc.) must be determined in each specific case in agreement with local authorities and institutions of Rospotrebnadzor. When working with infected material, it is necessary to decontaminate waste by autoclaving or treatment with disinfectant solutions.

6.22. In sections with experimental animals, constant monitoring of the temperature and humidity regime should be carried out. To control the quality of the air environment in rooms where animals are kept, it is recommended to periodically (2-3 times a month) determine the concentration of harmful gases (dioxide and ammonia).

6.23. The transfer of animals for experiments is carried out according to one-time requirements according to the annual application from the laboratories, approved by the head of the institution. Work with animals is allowed only during the hours provided for by the daily routine of the vivarium.

6.24. If sick animals are found in the sections, the latter, with the knowledge of the experimenter, are destroyed or transferred to an isolation ward. The issue of further use of diseased animals is resolved within no more than two days.

6.25. Animal corpses are stored in a special refrigerator for no more than one day before pathoanatomical autopsy, after which they are subject to disposal. Storage of animal corpses in cages and on the floor in the experimental sections is strictly prohibited.

6.26. Pathological anatomical autopsy of animals is performed by the experimenter. In the event of the death of an animal, regardless of the experiment, a vivarium veterinarian is present at the autopsy.

6.27. Each case of death or forced slaughter of animals must be recorded in a special journal.

6.28. It is forbidden to visit the vivarium by unauthorized persons without special permission. Employees of the institution performing work in the vivarium are required to:

observe the established rules of the daily routine and the vivarium work schedule;

conduct systematic observations of their experimental animals;

maintain primary documentation, timely filling in labels on cages with experimental animals;

visit only those premises of the vivarium in which there are animals assigned to this employee;

at the end of experiments or any other ongoing work with experimental animals, leave the workplace in the proper order;

monitor the timely write-off of experimental animals that have left the experiment, fallen or were forced to kill them;

inform the vivarium specialists about all observed cases of diseases of experimental animals, as well as timely notify the vivarium specialists about the alleged pathological conditions of animals in accordance with the conditions of the experiment.

6.29. Employees of the institution performing work in the vivarium with experimental animals are prohibited from giving any instructions to workers on changing the mode of keeping and feeding animals without the consent of the vivarium specialists.

6.30. When employees of this institution conduct joint research on animals in other institutions, it is prohibited for this time for these employees to work in the (clinic) vivarium of their institute (institution).

6.31. All actions that can cause pain to laboratory animals (surgeries, total bleeding, implantation of sensors, etc., as well as the forced slaughter of animals) must be carried out using anesthetics. If, according to the conditions of the experiment, the use of anesthesia is contraindicated, then all the above actions must be carried out as soon as possible.

6.32. During the experiment, the employee conducting this experiment must necessarily observe the following rules for the humane treatment of laboratory (experimental) animals.

In cases where surgical intervention or an experiment with pain stimulation is expected, anesthesia should be administered before the animal is tied to the machine.

The calculation of the amount of anesthetic should be carried out per 1 kg or 1 g of animal weight. The name of the substance and its quantity must be recorded not only in the protocol of the experiment, but also in a special map.

During the experiment, when it turns out to be longer than originally calculated, additional administration of anesthetics is mandatory.

If the acute experiment should end in the death of the animal, then the experimenter must kill the animal before the end of the effect of the anesthetic.

After the end of the surgical intervention, the animal should be transferred to the postoperative room on a special stretcher, which excludes the possibility of tissue displacement, suture divergence, etc.

The experimenter must foresee the possibility of the appearance of pain in the animal in the postoperative period and prescribe painkillers.

7. Number of vivarium attendants

7.1. The number of vivarium attendants is determined depending on the volume and nature of experimental studies, as well as on the number of laboratory animals. In this case, it is necessary to proceed from the following norms for the load of animals of the same species per care worker (taking into account the norms for placing animals in cages).

Animal species	Number
Animal species	animals	cells

Mice	800 - 1000	80 - 100
Rats	600 - 700	80 - 100
Hamsters		60 - 70
Guinea pigs		50 - 70
rabbits
Dogs	18 - 20	18 - 20
cats	35 - 40

When one person serves animals of several species, the calculation is carried out based on the above standards. In each specific case, when setting the norms for the animal care load per worker, it is necessary to take into account the type of cages, the degree of mechanization of production processes, the type of feeding (natural feed or granular feed), the frequency, nature and characteristics of the research, etc.

7.2. When working with radioactive substances or especially dangerous infections, as well as when keeping animal species not listed in Table. , service standards are established by the head of a scientific institution based on the timing of individual operations and taking into account the current standards for servicing farm animals.

8. Personal hygiene rules for vivarium employees

8.1. Vivarium personnel should be provided with overalls, safety shoes, soap and towels in accordance with current regulations.

8.2. In rooms with animals, in the feed kitchen, in the disinfection and washing department, it is necessary to have disinfectant solutions for disinfecting hands.

8.3. Vivarium staff must:

before starting work, remove outer clothing, shoes, put on overalls, safety shoes;

at the end of work (preferably before the start of work), undergo treatment in the sanitary block (take a shower or bath);

hang home clothes and overalls only in different sections of an individual closet;

periodically (but at least once a month) disinfect their individual cabinets;

at the end of each individual stage of work in accordance with the daily routine, as well as before eating, be sure to wash and disinfect hands.

8.4. It is strictly forbidden to eat and smoke in the production premises of the vivarium.

8.5. Persons newly hired to work with laboratory animals must undergo a medical examination, which includes tests for the presence of tuberculosis pathogens and the entire group of intestinal infections. Follow-up examinations are carried out at least once a year. Patients with tuberculosis, venereal, skin and other contagious diseases are not allowed to work in the vivarium.

8.6. When conducting experiments on animals with infectious pathogens that are dangerous to humans, the vivarium attendants are subjected to prophylactic immunization.

Fixing method

Cattle

The animal is tamed by squeezing the nasal septum with fingers, forceps of Garms, Nikolaev, nose rings or limiting movement by holding it by the horns with a rope, by the neck, head and the second loop around the nose. The hind limbs are fixed with a rope loop, which is applied to both limbs slightly above the hocks. When clearing and trimming hooves on the pelvic limbs of animals, you can put a twist on the lower leg.

Bulls are fixed with nose rings and a strong collar with a chain.

Breeding bulls, regardless of their disposition, are brought for examination only on a halter and they always use a carrier stick (carbine) about 2 m long, which is attached to the nose ring, which prevents the animal from suddenly attacking a person.

The calves are held by the hands by the neck, ears or with the help of a neck blind loop with a special knot and tied with a rope to the rack.

Pigs

Animals are fixed in a standing position by capturing the upper jaw with a metal cable and a handle holder or in a machine of a simple design.

It is convenient to hold fattening young animals and gilts with tongs proposed by K.P. Solovyov. Care is required when handling boars, old hogs and lactating sows, especially those fixed in pens.

Goats and sheep

Animals are held by the horns or neck. If necessary, fix in a supine position on the table.

Horses

Horses are fixed so that they cannot hit with their front and hind limbs or bite. Horses should be approached slightly from the side, in the direction of the shoulder and shoulder blade, preferably from the left side, as the horse gets used to this during operation. They approach the head, take the halter, bridle or mane with their left hand, and stroke and pat on the neck, withers, then on the shoulder blade and shoulder with the right hand. If the animal is kept without a leash in a stall, then it should be called out to draw attention to itself, call it up, uttering affectionate words. It is necessary that the horse must stand with its head towards the person.

An animal located in the machine or on a hitching post should not be approached from behind, but somewhat from the side from the side where it is looking.

During thermometry, rectal examination, various medical manipulations, in order to ensure the safety of the work of a veterinary specialist, it is necessary to raise the thoracic limb from the side from which the specialist manipulates, or put puts on one or both hind limbs.

The thoracic limb is fixed by lifting it by the brush or the putative part and bending at the carpal joint. At the same time, they stand on the side of the animal with their backs to its head. The raised limb is held with two hands, and during prolonged manipulations - with the help of a putty or rope thrown over the back. You can not put the raised limb of the animal on your knee, as the animal has a fourth point of support, which is not safe for humans. The rope should not be tied to any object or wrapped around the body of the animal, as in the event of an unexpected fall, the horse will not be able to quickly release the limb. When examining the posterior parts of the body, the pelvic limb is fixed. Standing at the croup of the horse facing the tail, with one hand they lean in the maklok, and with the other they lightly pat the leg from top to bottom, raise it, fasten the putty belt or put on a rope loop, which is then passed between the forelimbs, circled around the neck and tied with a non-stretching loop. In the study of obstinate and to tame restless horses, twists and lip pliers are used. To apply a twist, you need to insert your hand into the loop of the twist. Grab the upper lip, pull it forward, move the twist loop onto the lip with your left hand and twist it tightly. Animals can be securely fixed in special machines. In the machine, it is recommended to tie the horse to the stretch, and to the obstinate animal, so that it does not collapse, bring belts under the stomach.

camels

Camels are brought for research on a halter. It is necessary to approach camels carefully, preferably from the side (from the side of the chest limbs). The methods of taming these animals are the same as those of cattle and horses. The specific features of the behavior of these animals should be taken into account. It is desirable that personnel who constantly care for them be involved in fixing camels.

Bird

The bird is fixed, holding in a natural position by the limbs and wings, without squeezing the chest to avoid suffocation. When working with waterfowl (geese, ducks), you need to hold your head to avoid a blow to the eye, and carry out manipulations at arm's length.

fur animals

Animals are held with special tongs or hands in canvas (with cotton lining) mittens. They put it on the table and hold it with one hand by the neck, with the other by the torso. The oral cavity can be opened with the help of yawns designed by V.L. Berestov, it is recommended to use special muzzles. You can fix animals in mesh traps, use analgesic or tranquilizing agents with local anesthetics, as well as an anesthetic agent.

Dogs

With the help of the owner, a muzzle is put on the animals or their mouths are tied with a strong braid. For this purpose, a braid is applied to the jaws from above, tied with a simple knot under the lower jaw, then finally fixed on the back of the head with a sea knot. If rabies is suspected, as well as angry and restless dogs, it is better to place them in a special metal cage, one side of which moves and pinches it. To fix dogs in a supine position, an operating table for small animals is used, which allows them to be given any position convenient for work.

cats

During painful manipulations, animals are fixed in a special cloth sleeve or wrapped in a towel, leaving a free part of the body to be examined. The muzzle can be tied like a dog, and the legs can be fixed with hands, wearing leather or rubber gloves.

SNiP 11.01-2003. Instructions on the procedure for the development, approval, approval and composition of project documentation for the construction of enterprises, buildings and structures.

. SanPiN 2.2.1/2.1.1.1200-03. Sanitary protection zones and sanitary classification of enterprises, structures and other objects (New edition. Approved by the Decree of the Chief Sanitary Doctor of the Russian Federation No. 74 of September 25, 2007, registered by the Ministry of Justice of the Russian Federation No. 10995 of January 25, 2008).

Drinking water. Hygienic requirements for water quality of centralized drinking water supply systems. Quality control.

current

MUK 4.2.2939-11

METHODOLOGICAL INSTRUCTIONS

4.2. CONTROL METHODS. BIOLOGICAL AND MICROBIOLOGICAL FACTORS

The procedure for organizing and conducting laboratory diagnostics of tularemia for laboratories of the territorial, regional and federal levels

Date of introduction: from the moment of approval

1. Developed by the Federal State Healthcare Institution "Russian Research Anti-Plague Institute "Microbe" of Rospotrebnadzor (V.V. Kutyrev, I.N. Sharova, N.A. Osina, E.S. Kazakova, E.A. Plotnikova, S. A. Piontkovsky, T. Yu. Krasovskaya, D. V. Utkin, S. A. Shcherbakova); M.V. Chesnokova, A.V. Mazepa, S.A. Tatarnikov); Federal public health institution "Stavropol Research Anti-Plague Institute" of Rospotrebnadzor (A.N. Kulichenko, O.V. Maletskaya, T.V. Taran , A.P. Beyer, A.V. Taran); Federal State Healthcare Institution "Volgograd Research Anti-Plague Institute" of Rospotrebnadzor (V.V. Alekseev, A.V. Lipnitsky, V.A. Antonov, D.V. Viktorov); Federal State Healthcare Institution "Rostov-on-Don Research Anti-Plague Institute" of Rospotrebnadzor (N.V. Pavlovich, N.L. Pichurina, N.V. Aronova, N.N. Onoprienko, M.V. Tsimbalistova, A.S. Vodopyanov); Federal State Healthcare Institution "Anti-Plague Center" of Rospotrebnadzor (V.E. Bezsmertny, S.M. Ivanova); Federal budgetary health care institution "Federal Center for Hygiene and Epidemiology" of Rospotrebnadzor (V.G. Sennikova, M.V. Zarochentsev, V.V. Mordvinova); Federal State Institution of Science "State Scientific Center for Applied Microbiology and Biotechnology" of Rospotrebnadzor (I.A. Dyatlov, A.N. Mokrievich, S.F. Biketov, M.V. Khramov, N.I. Luneva); Federal State Budgetary Institution "GISK named after L.A. Tarasevich" of the Ministry of Health and Social Development (I.V. Borisevich, L.V. Sayapina).

3. Approved 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 July 14, 2011

1 area of use

1.1. These guidelines define the procedure for organizing and conducting laboratory diagnostics of tularemia for laboratories of the territorial, regional and federal levels, the forms and methods of their interaction, the nomenclature and scope of the study, the requirements for laboratories, specialists and personnel involved in the performance of studies, the logistics of research, to the biological safety of work.

1.2. These guidelines are intended for specialists of bacteriological laboratories of institutions exercising state sanitary and epidemiological surveillance of tularemia in the Russian Federation, treatment-and-prophylactic and anti-plague institutions.

2. Regulatory references

2.1. Federal Law of 03/3/1999 N 52-FZ "On the sanitary and epidemiological well-being of the population" .
______________
Federal Law of March 30, 1999 N 52-FZ "On the sanitary and epidemiological well-being of the population" . - Database manufacturer's note.

2.2. Decree of the Government of the Russian Federation of October 29, 2007 N 720 * "On Amendments to Clause 5 of the Regulation on Licensing Activities Related to the Use of Infectious Disease Agents", approved by Decree of the Government of the Russian Federation of January 22, 2007 N 31 *.
________________
* The document became invalid on the basis of the Decree of the Government of the Russian Federation of April 16, 2012 N 317

2.3. Decree of the Chief State Sanitary Doctor of the Russian Federation dated February 24, 2009 N 11 "On the submission of extraordinary reports on emergency situations in the field of public health of a sanitary and epidemiological nature" (registered with the Ministry of Justice of the Russian Federation on April 10, 2009 N 13745).

2.4. Order of the Ministry of Health and Social Development of the Russian Federation of July 7, 2009 N 415n "On approval of qualification requirements for specialists with higher and postgraduate medical and pharmaceutical education in the field of healthcare" (registered in the Ministry of Justice of the Russian Federation on July 9, 2009 N 14292).

2.6. SP 1.2.036-95 "Procedure for accounting, storage, transfer and transportation of microorganisms of pathogenicity groups I-IV" (approved by the Decree of the State Committee for Sanitary and Epidemiological Supervision of the Russian Federation of August 28, 1995 N 14).

2.7. SP 3.1.7.2642-10 "Prevention of tularemia" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of May 31, 2010 N 61 "On Approval of SP 3.1.7.2642-10". Registered in the Ministry of Justice of the Russian Federation on July 7, 2010 N 7745).

2.8. SP 1.3.1285-03 "Safety of working with microorganisms of groups I-II of pathogenicity (danger)" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation dated April 15, 2003 N 42 "On the Enactment of Sanitary and Epidemiological Rules SP 1.3.1285- 03" . Registered in the Ministry of Justice of the Russian Federation on May 10, 2003 N 4545).

2.9. SP 1.3.1318-03* "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" (approved by the decision of the Chief State Sanitary Doctor of the Russian Federation dated April 30, 2003 N 85 "On the Enactment of Sanitary and Epidemiological Rules SP 1.2.1318-03" . Registered in the Ministry of Justice of the Russian Federation on May 19, 2003 N 4558).
______________
*Probably an original error. Should read: SP 1.2.1318-03. - Database manufacturer's note.

2.12. SP 3.4.2318-08 "Sanitary protection of the territory of the Russian Federation" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation dated January 22, 2008 N 3 "On approval of the sanitary and epidemiological rules SP 3.4.2318-08" . Registered in the Ministry of Justice of the Russian Federation 3.04 .2008 N 11459).

2.13. SanPiN 2.1.7.2790-10 "Sanitary and epidemiological requirements for the handling of medical waste" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of December 9, 2010 N 163. Registered in the Ministry of Justice of the Russian Federation on February 17, 2011 N 19871).

2.14. SanPiN 2.1.3.2630-10 "Sanitary and epidemiological requirements for organizations engaged in medical activities" (approved by the Decree of the Chief State Sanitary Doctor of the Russian Federation of May 18, 2010 N 58. Registered in the Ministry of Justice of the Russian Federation on August 9, 2010 N 18094).

2.15. Sanitary rules for the arrangement, equipment and maintenance of experimental biological clinics (vivariums) (approved by the Chief State Sanitary Doctor of the USSR dated April 6, 1973 N 1045-73).

2.16. MU 3.1.2007-05 "Epidemiological surveillance of tularemia".

2.17. MU 3.3.2.2124-06 "Control of diagnostic nutrient media for biological indicators for pathogens of plague, cholera, anthrax, tularemia".

2.18. MUK 4.2.2316-08 "Methods for monitoring bacteriological nutrient media".

2.19. MU 1.3.2569-09 "Organization of the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV".

2.20. MU 4.2.2495-09 "Determination of the sensitivity of pathogens of dangerous bacterial infections (plague, anthrax, cholera, tularemia, brucellosis, glanders and melioidosis) to antibacterial drugs."

3. List of abbreviations

LPS - lipopolysaccharide

MPU - medical and preventive institution

OOI - especially dangerous infections

SP - sanitary and epidemiological rules

SanPiN - sanitary and epidemiological rules and regulations

MU - guidelines

PBA - pathogenic biological agent

MFA - method of fluorescent antibodies

ELISA - enzyme immunoassay

PCR - polymerase chain reaction

RA - agglutination reaction

RNGA - reaction of indirect hemagglutination

RTNHA - reactions of inhibition of indirect hemagglutination

RNAt - antibody neutralization reaction

MIS - magnoimmunosorbents

RLA - latex agglutination reaction

IC - immunochromatography

IC test - immunochromatographic test

4. General provisions

Characteristics of the disease and the causative agent of tularemia

Tularemia is a zoonotic systemic natural focal bacterial infectious disease characterized by symptoms of general intoxication, fever, inflammatory changes in the area of the infection gate, regional lymphadenitis, and a tendency to a protracted course.

The main reservoirs and sources of the causative agent of tularemia in natural conditions are wild animals (about 50 species), mainly various types of rodents, and hares. On the territory of natural foci of tularemia, sheep, pigs, and cattle can become infected. The reservoir and carriers of the pathogen are also blood-sucking arthropods: ixodid and gamasid mites, mosquitoes, horseflies, fleas. A sick person does not represent an epidemiological danger.

As with all zoonoses, tularemia is characterized by a plurality of mechanisms (aspiration, contact, fecal-oral, transmissible), as well as transmission routes and factors. In accordance with the International Statistical Classification of Diseases and Related Health Problems (Tenth Revision. Geneva, 2003, (ICD-10), and according to the localization of the main pathological process, the following forms of tularemia are distinguished:

A21.0 - ulceroglandular (ulcer-bubonic);

A21.1 - oculo-glandular (oculo-bubonic);

A21.2 - pulmonary;

A21.3 - gastrointestinal (abdominal);

A21.7 - generalized;

A21.8 - other forms of tularemia (anginous-bubonic);

5.1.1. Requirements for laboratories of medical institutions that carry out research on tularemia

Medical institutions whose laboratories carry out diagnostic tests for tularemia must have a license to carry out activities related to the use of pathogens of pathogenicity (danger) groups III-IV.

Laboratories of healthcare facilities must have a sanitary and epidemiological conclusion on the possibility of working with microorganisms of pathogenicity (danger) groups III-IV in accordance with the current SP on the procedure for issuing a sanitary and 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.

Accounting, storage, transfer and transportation of isolated cultures of cholera vibrios (suspicious) should be carried out in accordance with the current regulatory documents on the procedure for accounting, storage, transfer and transportation of microorganisms of pathogenicity groups I-IV.

Conducting research at all stages - sampling, storage, delivery to the laboratory, registration, research procedure, issuance of results, interaction with Rospotrebnadzor institutions - must comply with the requirements of current regulatory and administrative documents.

Tests for tularemia can be performed by specialists not younger than 18 years of age with higher and secondary medical, biological education, who have completed training courses in the specialty "Bacteriology" with the development of methods for safe work with pathogens of infectious diseases of III-IV pathogenicity (danger) groups, who have a permit to work with PBA III-IV pathogenicity groups on the basis of the order of the head of the institution. Specialists conducting diagnostic tests for tularemia must have the necessary professional skills in accordance with the nomenclature of the tests performed (Appendix 8).

Specialists engaged in activities related to the use of pathogens of infectious diseases must improve their qualifications at least once every five years and have a specialist certificate.

Quality control of diagnostic studies for tularemia in the laboratories of medical facilities includes:

Documentation rules

To take material and conduct diagnostic tests for tularemia in bacteriological laboratories, healthcare facilities must have:

Personnel should be provided with overalls and personal protective equipment (for sampling clinical material and conducting immunoserological reactions).

5.1.2. Nomenclature and scope of research

Clinical facilities select clinical material from persons suspected of having tularemia, patients with various forms of tularemia and vaccinated, as well as sectional material from deceased persons.

In the bacteriological laboratories of health care facilities, blood sera from patients with tularemia and vaccinated against tularemia are examined by immunoserological and allergological methods:

1) detection of antibodies in paired sera;

2) carrying out the reaction of lysis of leukocytes.

The infectious diseases doctor of the health facility assesses the allergological status of patients by staging an allergic test with tularin.

5.1.3. The order of laboratory diagnosis of tularemia in the laboratories of medical institutions

Sampling and transportation of samples of clinical material

The material from the patients is taken by the medical staff of the health facility upon admission of the patient, before the start of treatment with antibacterial drugs. Sampling is carried out by two medical workers, one of whom is an infectious disease specialist or a general practitioner (surgeon) trained in the diagnosis of especially dangerous infections and compliance with biological safety requirements when working with clinical material suspected of containing pathogens of infectious diseases of I-II pathogenicity groups. Material from vaccinated persons is taken by the medical staff of health facilities

Sectional material is taken by medical workers of the pathological and anatomical departments (or BSME) in the presence of a specialist in especially dangerous infections, guided by the current methodological guidelines for the organization and implementation of primary anti-epidemic measures in cases of detection of a patient (corpse) suspected of infectious diseases that cause emergency situations in areas of sanitary and epidemiological welfare of the population, in compliance with the regulated biological safety requirements when working with pathogenic biological agents of groups I-II.

To be sent to bacteriological laboratories of Rospotrebnadzor institutions, they take:

from sick people, depending on the clinical form of the disease: the contents of the bubo, material from the pharynx, from the conjunctiva of the eye, ulcer discharge, sputum, blood;

from dead people: enlarged lymph nodes, altered areas of the lungs and spleen, trachea;

from vaccinated people: blood.

Sampling of all types of material is carried out in sterile glass or plastic dishes corresponding to the volume of samples.

Punctate from bubo take up to 14-20 days of illness with a syringe with a capacity of at least 5 ml. The skin at the site intended for puncture is treated with 70% alcohol, and then lubricated with 5% iodine solution and again wiped with 70% alcohol. The needle is inserted in such a way that its tip reaches the central part of the bubo, after which, pulling the piston to failure, the needle is slowly removed. The contents are transferred into a sterile tube with a screw cap. It is possible to introduce 0.3-0.5 ml of sterile 0.9% sodium chloride solution into the bubo before taking the material and then select the contents. When the bubo is opened, the material is taken separately from the peripheral dense part and the detachable fistula.

Before taking detachable ulcer, papules, vesicles, or sloughed eschar with a pre-injection disinfectant wipe, carefully clean the skin around the affected area, if necessary, remove necrotic masses and pus with a sterile gauze wipe. By rolling the swab over the wound surface from the center to the periphery, the material is absorbed onto the swab for 5-10 s. The swab with the material is placed in a test tube or transport medium. When using a syringe, the needle is inserted at the edge of the vesicle (pustule) and then advanced towards the middle. In ulcers, a dense edge is punctured.

Phlegm collected in special wide-mouthed containers with a screw-top lid.

Detachable pharyngeal mucosa taken on an empty stomach or 3-4 hours after eating. Gently pressing the tongue with a spatula, insert a tampon between the tonsil arches and the tongue (do not touch the lips, cheeks, tongue with the tampon) and collect material from the posterior surface of the pharynx, tonsils and areas of inflammation or ulceration of the mucosa. The swab with the material is placed in a sterile test tube or in a test tube with a transport or nutrient medium.

Blood for research, they are taken in compliance with the rules of asepsis and personal protective measures. Blood is taken from the cubital vein in the amount of 10-20 ml with a disposable syringe and transferred into a test tube for inoculation on nutrient media and infection of bioassay animals, into a test tube with an anticoagulant (4% sodium citrate solution in a ratio of 1:10 to the blood volume or 6% -th solution of EDTA in the ratio of 1:20 to the volume of blood) for PCR analysis, in a test tube to obtain serum for immunoserological reactions.

To set up a blood-drop agglutination reaction and a leukocytolysis reaction, blood is taken from a finger.

Discharge of the conjunctiva of the eye should be taken up to the 17th day of illness using a sterile swab, pre-moistened with a 0.9% sodium chloride solution. Samples from each eye are collected with separate swabs with two or three circular movements along the mucous membrane of the eye. The swab with the material is placed in a sterile test tube or transport medium. In the presence of abundant purulent discharge with a sterile dry cotton swab, pus is taken from the inner surface of the lower eyelid by moving towards the inner corner of the palpebral fissure. It is necessary to ensure that the eyelashes do not touch the swab (hold the eyelid with your hand). Delivery of the material to the laboratory within 1 hour, if special transport media are used - within a day.

Containers with samples are labeled, treated on the outside with a disinfectant solution, packed in a plastic bag with a zipper and placed in a container for transporting biological material for research. The container with the packed material is sealed and sent to the laboratory by courier on a specially designated transport. The surface of the table after packing the samples is treated with a disinfectant solution.

For samples delivered to the laboratory, fill out the direction (Appendix 1), which indicates: the address of the institution to which the sample (samples) is sent; surname, name, patronymic of the patient (deceased); gender, age, place of residence, date of illness, date of seeking medical help, date of hospitalization, preliminary diagnosis; features of the epidemiological history; whether the patient was given antibacterial therapy before taking the material (when, which drugs were used, at what dose); type of material taken for bacteriological examination; purpose of the study; date and hour of material collection; the address to which the results of the bacteriological examination should be reported; name of the institution, position, surname and initials of the person sending the sample (samples), signature; sample delivery time; position, surname and initials of the person who took the samples.

The material is transported to the laboratory in a cooler bag. In the absence of conditions for storing the material in the cold, the time from the moment of taking the material to the start of the study should not exceed 5-6 hours.

The setting and recording of immunoserological reactions is carried out in the bacteriological laboratory of the health facility in accordance with the instructions for the use of diagnostic preparations. In the dynamics of the disease, paired sera are examined with an interval of 7-10 days. A 4-fold or more increase in antibody titer is diagnostically reliable.

Hypersensitivity in sick and vaccinated individuals is determined by in vitro

The formulation and recording of the results of an allergic test with tularin (tularemia liquid allergen, suspension for skin scarification application) in persons infected or suspected of being infected with tularemia is carried out by an infectious disease specialist at a healthcare facility in accordance with the instructions for using the drug.

It should be remembered that the allergic test remains positive in people who have had tularemia.

5.1.4. Registration of research results

Registration of the results of serological and allergic testing of sera for tularemia in the bacteriological laboratories of health care facilities is carried out in accordance with the accounting forms established in the institution. Issuance of answers for case histories - according to unified forms.

5.1.5. The procedure for interaction of medical institutions with organizations of Rospotrebnadzor

5.2. The procedure for organizing and conducting laboratory diagnostics of tularemia for the branches of the FBUZ "Center for Hygiene and Epidemiology" in the municipality (the city and administrative districts of the subject, united on a territorial basis) in the subject of the Russian Federation

5.2.1. Requirements for laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation that carry out research on tularemia

Availability of permits and regulatory documents

FBUZ "Center for Hygiene and Epidemiology" in the constituent entity of the Russian Federation, on the basis of whose branches bacteriological laboratories operate, must have a license to carry out activities related to the use of pathogens of II-IV (or III-IV) pathogenicity (danger) groups.

Laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation that perform research on tularemia must have a sanitary and epidemiological conclusion on the possibility of working with microorganisms of III-IV groups of pathogenicity (danger) in accordance with the current SP on the procedure for issuing sanitary and 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.

Laboratories of branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation must be accredited for technical competence in the prescribed manner in accordance with the current legislative framework of the Russian Federation.

Accounting, storage, transfer and transportation of samples of clinical material should be carried out in accordance with the current SP on the procedure for recording, storage, transfer and transportation of microorganisms of pathogenicity groups I-IV.

Waste disposal must be carried out in accordance with the current sanitary and epidemiological requirements for the treatment of medical waste.

Requirements for specialists and personnel involved in the performance of research on tularemia

Tests for tularemia can be performed by specialists not younger than 18 years of age with higher and secondary medical, biological education, who have completed training courses in the specialty "Bacteriology" with the development of methods for safe work with pathogens of infectious diseases of III-IV pathogenicity (danger) groups, who have a permit to work with PBA III-IV groups on the basis of the order of the head of the institution. Specialists conducting diagnostic tests for tularemia must have the necessary professional skills in accordance with the nomenclature of the tests performed (Appendix 8).

Specialists carrying out activities related to the use of pathogens of infectious diseases must have a specialist certificate and improve their skills at least once every five years.

Requirements for ensuring the safety of personnel

Each laboratory performing tests for tularemia must have a package of documents that determine the safe work regime for employees, taking into account the nature of work, technology features, and properties of microorganisms. The documents must be coordinated with the commission for monitoring compliance with biological safety requirements, specialists in labor protection, fire prevention measures and approved by the head of the institution. The results of checking the knowledge of personnel safety rules during work are recorded in a special journal.

All employees must comply with the requirements for ensuring the safety of working with material suspicious or infected with pathogens of infectious diseases of III-IV pathogenicity (danger) groups, in accordance with current regulatory documents.

Employees of the institution involved in the epizootological examination of the enzootic territory should be vaccinated against tularemia, followed by monitoring the level of immunity and recording the results in a special journal.

The procedure for organizing internal quality control of laboratory tests

Quality control of diagnostic tests for tularemia in laboratories includes:

quality control of diagnostic preparations and test systems, distilled water, chemical reagents and disinfectants;

timely verification of measuring instruments, certification of test equipment;

quality control of sterilization of laboratory glassware;

control of the operation of steam and dry-air sterilizers;

control of the operation of germicidal lamps;

temperature control of refrigerators;

temperature control of thermostats;

checking the air condition of industrial premises and boxes, temperature, humidity;

inspection of the sanitary condition of the premises, including the conditions of cleaning, disinfection, control of flushing from surfaces and equipment.

The control results are recorded in special journals.

Documentation rules

Maintaining laboratory documentation, including registration and work logs, is carried out in accordance with the requirements of the current regulatory and methodological documents.

Requirements for material resources necessary to perform diagnostic tests for tularemia

To conduct diagnostic tests for tularemia in bacteriological laboratories of the branches of the Center for Hygiene and Epidemiology, the following must be available:

diagnostic preparations, test systems registered in accordance with the established procedure (Appendix 3);

chemical reagents (Appendix 4);

devices, equipment, consumables (Appendix 5, 6).

It is recommended to have a medical kit for taking material (universal packing for taking material from people and from environmental objects for testing for especially dangerous infectious diseases).

Personnel must be provided with overalls and personal protective equipment.

5.2.2. Nomenclature and scope of research

Laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in municipalities in the constituent entity of the Russian Federation, when exercising epidemiological surveillance, monitor the state of anti-tularemia immunity in vaccinated people.

Research is carried out in the following scope:

1) detection of antibodies;

2) staging the reaction of lysis of leukocytes.

If the laboratory of the healthcare facility does not perform serological tests for tularemia, the serum of patients or those with suspicion of this disease is examined at the branch of the FBUZ "Center for Hygiene and Epidemiology" in the subject of the Russian Federation (by agreement).

5.2.3. The procedure for laboratory diagnosis of tularemia in the laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation

The state of immunity in vaccinated people is checked 5 years after vaccination and subsequently - 1 time in 2 years.

Control of the state of anti-tularemia immunity is carried out using an allergological (leukocytolysis reaction) or one of the serological research methods (RA, volumetric agglomeration reaction, RNHA, ELISA). In this case, it is preferable to use serological methods of research. The material for the study is the blood and blood serum of the vaccinated. If necessary, you can use a blood-drop reaction, which allows you to give a response within 5 minutes and can be delivered with a dry drop of blood.

From patients or persons with suspected tularemia in the dynamics of the disease, paired sera are examined with an interval of 7-10 days. A 4-fold or more increase in antibody titer is diagnostically reliable.

Hypersensitivity in vaccinated and sick individuals is determined by in vitro in the leukocytolysis reaction in accordance with the current guidelines for the epidemiological surveillance of tularemia.

5.2.4. Registration of research results

Registration of research results in the laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation is carried out in accordance with the accounting forms established in the institution. Issuance of answers - according to unified forms.

5.2.5. The procedure for the interaction of branches of the FBUZ "Center for Hygiene and Epidemiology" in a municipality in a constituent entity of the Russian Federation with other organizations of Rospotrebnadzor

Information on the results of laboratory diagnosis of tularemia in the laboratory of the branch of the FBUZ Center for Hygiene and Epidemiology in the constituent entity of the Russian Federation is transmitted in accordance with the current regulatory documents.

5.3. The procedure for organizing and conducting laboratory diagnostics of tularemia for the FBUZ "Center for Hygiene and Epidemiology" in a constituent entity of the Russian Federation

5.3.1. The procedure for organizing and conducting laboratory diagnostics of tularemia for the FBUZ "Center for Hygiene and Epidemiology" in the subject of the Russian Federation, in the structure of which there are no departments and laboratories of especially dangerous infections

The procedure for organizing and conducting laboratory diagnostics of tularemia for the laboratories of the FBUZ "Center for Hygiene and Epidemiology" in the constituent entities of the Russian Federation, in the structure of which there are no departments or laboratories of especially dangerous infections, corresponds to the procedure for organizing and conducting laboratory diagnostics of tularemia for laboratories of the branches of the FBUZ "Center for Hygiene and Epidemiology" in a constituent entity of the Russian Federation (Section 5.2).

5.3.2. The procedure for organizing and conducting laboratory diagnostics of tularemia for laboratories of especially dangerous infections of the FBUZ "Center for Hygiene and Epidemiology" in the subject of the Russian Federation

5.3.2.1. Requirements for laboratories of especially dangerous infections FBUZ "Center for Hygiene and Epidemiology" in the subject of the Russian Federation, carrying out research on tularemia.

Availability of permits and regulatory documents

FBUZ "Center for Hygiene and Epidemiology" in the constituent entity of the Russian Federation, on the basis of which laboratories of especially dangerous infections operate, performing research on tularemia, must have a license to carry out activities related to the use of pathogens of II-IV pathogenicity (danger) groups.

Laboratories of the OOI FBUZ "Center for Hygiene and Epidemiology" in a constituent entity of the Russian Federation that carry out research on tularemia must have a sanitary and epidemiological conclusion on the possibility of working with microorganisms of II-IV pathogenicity (danger) groups in accordance with the current SP on the procedure for issuing sanitary and epidemiological conclusions on the possibility of carrying out work with pathogens of human infectious diseases of I-IV pathogenicity (danger) groups, genetically modified microorganisms, poisons of biological origin and helminths.

Laboratories of the FBUZ "Center for Hygiene and Epidemiology" in a constituent entity of the Russian Federation must be accredited for technical competence in the prescribed manner in accordance with the current legislative framework of the Russian Federation.

Accounting, storage, transfer and transportation of isolated suspicious cultures of the causative agent of tularemia and / or samples of clinical material should be carried out in accordance with the current SP on the procedure for recording, storage, transfer and transportation of microorganisms of pathogenicity groups I-IV.

Waste disposal must be carried out in accordance with the regulated sanitary and epidemiological requirements for the treatment of medical waste.

Conducting research at all stages: sampling, storage, delivery to the laboratory, registration, research procedure, issuance of results, interaction with Rospotrebnadzor organizations must comply with the requirements of current regulatory documents.

Requirements for specialists and personnel involved in the performance of research on tularemia

Studies for tularemia can be performed by specialists not younger than 18 years old with higher and secondary medical, biological education, who have completed training courses in the specialty "Bacteriology" with the basics of safe work with pathogenic biological agents (PBA) of groups I-II, who have a permit to work with PBA II - IV groups on the basis of the order of the head of the institution. Specialists conducting research on tularemia must have the necessary professional skills (Appendix 8).

Specialists carrying out activities related to the use of pathogens of infectious diseases must have certificates and improve their skills at least once every five years.

Requirements for ensuring the safety of personnel

Each laboratory conducting research on tularemia must have a package of documents that determine the safe work regime for employees, taking into account the nature of work, technology features, and properties of microorganisms. The documents must be coordinated with the commission for monitoring compliance with biological safety requirements, specialists in labor protection, fire prevention measures and approved by the head of the institution. The results of checking the knowledge of personnel safety rules during work are recorded in a special journal.

Specialists who carry out epizootological examination of a territory enzootic for tularemia and its laboratory support should be vaccinated against tularemia, followed by monitoring the level of immunity and recording the results in a special journal.

All employees must comply with the requirements for ensuring the safety of working with material suspicious or infected with pathogens of infectious diseases of I-II pathogenicity (danger) groups, in accordance with current regulatory documents.

The procedure for organizing internal quality control of laboratory tests

Quality control of diagnostic studies for tularemia in the laboratories of the OOI FBUZ "Center for Hygiene and Epidemiology" includes:

quality control of nutrient media, diagnostic preparations and test systems, disks with antibacterial preparations, distilled water, chemical reagents and disinfectants;

timely verification of measuring instruments, certification of test equipment;

quality control of sterilization of laboratory glassware;

control of the operation of steam and dry-air sterilizers;

control of the operation of germicidal lamps;

temperature control of refrigerators;

temperature control of thermostats;

checking the air condition of industrial premises and boxes, temperature, humidity;

inspection of the sanitary condition of the premises, including the conditions of cleaning, disinfection, control of flushing from surfaces and equipment.

The control results are recorded in special journals.

Documentation rules

Maintaining laboratory documentation, including registration and work logs, is carried out daily in accordance with the requirements of the current methodological documents.

Requirements for material resources necessary to perform diagnostic tests for tularemia

To conduct diagnostic tests for tularemia, laboratories must have:

nutrient media registered in the prescribed manner (Appendix 2);

diagnostic preparations, test systems, antibacterial preparations registered in accordance with the established procedure (Appendix 3, 7);

chemical reagents (Appendix 4);

instruments, equipment, consumables (Appendix 5, 6);

medical kit (universal packing for taking material from people and from environmental objects for testing for especially dangerous infectious diseases).

Nutrient media are subject to mandatory control in accordance with the current guidelines for the control of diagnostic nutrient media for biological indicators (for the causative agent of tularemia).

5.3.2.2. Nomenclature and scope of research.

Laboratories of the OOI FBUZ "Center for Hygiene and Epidemiology" in the constituent entities of the Russian Federation, carry out:

study of material from patients and deceased with suspected tularemia;

study of material from persons subject to examination for tularemia in accordance with the requirements of epidemiological surveillance (as agreed);

study of samples collected during the epizootological survey of the territory;

study of samples from environmental objects;

identification of isolated cultures of the causative agent of tularemia according to an abbreviated scheme;

quality control and inhibitory properties of nutrient media.

Diagnostic studies of the material are carried out in the following volume:

a) indication of the pathogen in native material by methods of express and accelerated diagnostics (MFA, PCR, ELISA, RA, RNHA, RNAb, selective concentration on MIS followed by ELISA);

b) setting up a biological sample;

c) sowing on nutrient media in order to isolate a pure culture of the pathogen;

d) detection of antibodies to the causative agent of tularemia;

e) identification of the selected culture according to the reduced scheme.

5.3.2.3. The order of diagnostic studies for tularemia in the laboratories of especially dangerous infections of the FBUZ "Center for Hygiene and Epidemiology" in the subject of the Russian Federation.

Procedure for the study of clinical material

The selection of material is carried out in accordance with paragraph 5.1.

To identify the causative agent of tularemia, diagnostic preparations and complex agar or yolk media with the addition of cysteine, tissue extracts, defibrinated blood, glucose, registered in the prescribed manner, are used. Each batch of agar should be tested for sensitivity to the growth of the tularemia microbe in accordance with the current regulatory and methodological documents. To suppress the growth of foreign microflora, penicillin (100 units/ml), ampicillin (100 units/ml), polymyxin B (50-100 µg/ml), kefzol (or cephalexin), amphotericin B (or amphoglucamine), ristomycin sulfate are used. and some other antibacterial drugs.

Objects with crops are incubated at a temperature of 37 °C. The crops are viewed after 24-48 hours (hereinafter - daily for 10 days from the moment of sowing).

Preparation of samples for PCR is carried out in accordance with the requirements of the guidelines for organizing the work of laboratories using nucleic acid amplification methods when working with material containing microorganisms of pathogenicity groups I-IV.

Examination of material from a sick person (corpse)

Stage I:

preparation of smears, staining of fixed smears according to Gram, Romanovsky-Giemsa, fluorescent tularemia immunoglobulins;

PCR setting;

setting immunoserological reactions to detect antigens and antibodies to the causative agent of tularemia (RA, MFA, RNGA, RNAt, ELISA, etc.);

setting the reaction of leukocytolysis (blood of the patient);

infection of bioassay animals (guinea pigs intraperitoneally; white mice intraperitoneally or subcutaneously (blood, bubo punctate), subcutaneously (sputum, throat swab, opened bubo, ulcer discharge, conjunctiva);

sowing on dense nutrient media (blood, bubo punctate);

sowing on solid nutrient media with inhibitors of foreign flora (sputum, throat swab, substrate from an opened bubo, discharge from an ulcer, conjunctiva).

II stage(2-6 hours from the start of the study):

accounting for the results of MFA, ELISA, PCR;

accounting for the results of RA, RPGA and RNAt after 18-24 hours;

extradition preliminary positive response based on the presence in smears of small coccoid rods of gram-negative or lilac color when stained according to Romanovsky-Giemsa, their specific luminescence when stained with fluorescent tularemia immunoglobulins, a positive PCR result, positive immunoserological reactions with negative controls.

Stage III(48-72 hours from the start of the study):

viewing crops of native material on agar plates;

bacterioscopy of smears from suspicious colonies (Gram stain);

setting up an IC test for express identification of a tularemia microbe with material from suspicious colonies;

screening of suspicious colonies of the tularemia microbe on nutrient agar to isolate a pure culture;

extradition confirmation of a preliminary positive answer based on the presence of characteristic growth on dense nutrient media, the presence of small gram-negative coccal rods in smears from colonies, a positive IC test for rapid identification of the tularemia microbe.

IV stage(3-5 days from the start of the study):

after the accumulation of a pure culture, setting tests for its identification. Identification of the isolated culture is carried out according to the following tests:

cell morphology, the nature of the Gram stain and fluorescent tularemia immunoglobulins;

the nature of growth on FT-arape nutrient media or on McCoy's folded yolk medium;

lack of growth on simple nutrient media (meat peptone agar and/or broth);

agglutination of cultures with specific tularemia serum or RLA with an isolated culture;

express identification of the tularemia microbe using the IC test;

identification of species-specific DNA targets by PCR;

autopsy of dead bioassay animals, inoculation of organs and blood on solid nutrient media, preparation and examination of smears-imprints of organs, PCR with organ suspensions.

Stage V(5-15 days from the start of the study):

accounting for crop identification results;

viewing of crops of material from dead bioassay animals;

autopsy and examination of slaughtered biotest animals;

extradition final positive answer is carried out on the basis of isolation of a pure culture of the tularemia microbe from crops of native material, its identification by morphological, cultural properties, positive results of immunoserological reactions, the presence of pathogen DNA, as well as on the basis of isolation of identical cultures from dead or slaughtered laboratory animals.

Second group. Highly susceptible, but insensitive mammals (they become infected when single microbial cells of the causative agent of tularemia enter the body, get sick seriously, but quickly get rid of the microbe, acquiring stable immunity). This group includes field mice, all kinds of rats and ground squirrels, squirrels, chipmunks, beavers, hedgehogs, muskrat, water shrew, shrew and some other species of mammals.

Third group. Malosusceptible and practically insensitive mammals. These include most predatory mammals and farm animals.

Scheme of the study of field material

Laboratory research of field material begins immediately after its receipt. Its short-term storage (no more than 20 hours) is allowed at a temperature of 4 to 6 °C. When the animals are autopsied at the collection site, organs can be stored and delivered to the laboratory in a preservative. Vaseline-paraffin mixture can serve as preservatives (1 part of paraffin and 10 parts of vaseline oil are mixed and sterilized for 45 minutes by heating in a boiling water bath), 5% sodium chloride solution, in addition, deep freezing in liquid nitrogen, etc. is used. In preservatives and at low temperature, animal organs can be preserved for one month.

The study is carried out by biological, bacterioscopic (light and luminescent microscopy), bacteriological (inoculation on nutrient media, isolation of pure cultures and their identification), molecular genetic (PCR analysis) and immunoserological (RA, RLA, RNHA, RNAt, RNAg, ELISA) methods. The scheme for studying the material depends on the sensitivity group of the animals and on the form in which the material was delivered.

small mammals, taken in nature by fishing gear or alive, are examined by a group method, combining in one sample the organs of several animals (5-10) of the same species and caught in one place.

For research, pieces of the spleen, liver, lymph nodes, blood or "washouts" from the chest cavity are taken. The material is examined by biological, molecular genetic and immunoserological methods.

The organ suspension is used to infect bioassay animals and to detect antigens and DNA of the causative agent of tularemia. Blood serum or "washes" from the chest cavity are examined for the presence of antibodies to the causative agent of tularemia.

Corpses of animals that died in nature, died in the laboratory, or animals in which, at autopsy, pathological and anatomical changes characteristic of tularemia were found, are subjected to individual research. Pieces of the spleen, liver, kidneys, lymph nodes, bone marrow are examined by biological, bacteriological, molecular genetic and immunoserological methods.

Under conditions of an established epizootic, when studying animals of the first group, one can limit oneself to sowing organs on nutrient media and bacterioscopy of smears from organs, keeping some of them in the cold until the results of the studies are obtained. In doubtful cases resort to the biological method. Animals of the second and third groups are examined by the biological method without fail.

The probability of detecting the causative agent of tularemia in the organs of animals of the first group during microscopic examination (it is better to use fluorescent microscopy) is much higher than during bacterioscopy of smears from the organs of the corpses of animals of the second group.

Pets(cattle, pigs, sheep, reindeer) are species that are insensitive to tularemia (third group). In their study, mainly immunoserological methods are used (RA, RNGA, ELISA), less often - an intradermal test with tularin. Bacteriological and biological methods are used only when examining dead, slaughtered or sick animals. Examine primarily the lymph nodes and spleen. In a serological study, the possibility of detecting cross-reactions with Brucella and the microbial flora of the intestines of animals should be taken into account. It is advisable to test the sera of domestic animals in at least two serological tests. Positive reactions in RNGA should be monitored in RTGA.

Birds of prey pellets and dung of predatory mammals it is recommended to study individually. The death of the tularemia microbe in pellets and droppings occurs quickly (on the first day; at negative temperatures, perhaps more slowly), and therefore biological and bacteriological studies of this material are inappropriate. Samples of pellets and litter are used to search for the antigen of the causative agent of tularemia by immunoserological methods and DNA by PCR.

Blood-sucking insects and other invertebrates are examined by a group method, insects or invertebrates of the same species (genus) and taken from the same place are combined into one sample.

Adult ixodid ticks combine up to 50 individuals.

Larvae are united in 100-200 specimens, nymphs - in 50-100 specimens, depending on the degree of their fatness. Washing of larvae and nymphs of ixodid ticks in alcohol is not carried out, because. this can damage the analysis.

Fleas, gamasid mites, lice are sorted by species (genus), as well as by the species of animals from which they were collected, placed in sterile test tubes and then subjected to processing in the same manner as the larvae and nymphs of ixodid ticks.

Blood-sucking dipteran insects are euthanized with ether vapors to limit mobility. In horseflies, limbs and wings are preliminarily cut off, mosquitoes and midges are examined as a whole. Up to 25-50 horseflies or up to 100 mosquitoes or up to 250 midges are included in one analysis.

Hydrobionts - caddisflies, amphipods, daphnia, cyclops and others before the study, they are washed in several portions of water and 1-2 portions of sterile distilled water. In animals with covers or shells, the latter are removed if possible. Animals are combined into groups of 5-10-50 specimens, depending on the size of individual species.

Detection of the tularemia microbe or its DNA in invertebrates is most effective when using the biological method and PCR. It is also possible to detect the specific antigen of tularemia LPS using the IC test.

Water samples(100-200 ml) are taken from various reservoirs: rivers, streams, ponds, lakes, swamps, wells, etc. The most effective study of water in the names of swamp foci of tularemia in winter. Samples are taken in a shaded place, at a depth of 10-20 cm from the surface of stagnant or low-flowing water. 2 samples should be taken from each point. Samples are taken in the habitats of animals (near feeding tables, burrows, huts of beavers or muskrats). Filtration, centrifugation, magnetic sorbents and other methods are used to concentrate the pathogen. For the study, a biological method is used (a white mouse is injected subcutaneously up to 1 ml, and a guinea pig - up to 5 ml of water), molecular genetic and immunoserological methods aimed at detecting DNA and antigens of the causative agent of tularemia.

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Rice. 1. The operation of isolating and cutting the branches of the recurrent nerves to create silent barking in dogs.
1-left lobe of the thyroid gland; 2 - age branch of the vagus nerve; 3 - left sternohyoid muscle (drawn with a hook); 4 - esophagus; 5-trachea.

The room where the cages with animals are located should be well ventilated, the relative humidity of the air should be in the range of 40-45%. Even in the presence of supply and exhaust ventilation, in order to reduce the content of ammonia and animal waste products in the air, it is recommended to use peat bedding or sawdust with the addition of superphosphate. The cell harvesting process can be automated; cleaning is carried out 1-2 times a day.
On the front wall of the cage, a feeder, an autodrinker are installed and a sign is hung on which the basic data about the animal, the type of surgical intervention, etc. are entered.
Rabbits and guinea pigs are often kept outside the vivarium. For this, cells are used, placing them in several tiers, under a common roof. Outdoor housing contributes to the cultivation of healthy, more resistant to various diseases of rabbits and is mostly common in nurseries.

In experimental studies, the correct maintenance and feeding of laboratory animals both before and during the experiment is of great importance.

Violation of the regimen and diet, non-compliance with hygienic measures during feeding contribute to the weakening of the body of animals and increase their susceptibility to various infectious and somatic diseases. Their occurrence during the experiment can lead to a distortion of the results of the study and, consequently, to incorrect conclusions. All substances necessary for the animal organism (proteins, carbohydrates, fats, minerals, vitamins and water) must be introduced into the feed ration. From concentrates, rabbits and guinea pigs receive millet, oats, wheat, barley, peas, lentils, vetch corn. The daily rate of concentrates: for adult rabbits - 80 g, for young animals - 60 g, for guinea pigs - 20-25 g. It is advisable to give animals a mixture of seeds of several (2-3) cultures in the daily rate. Rabbits and pigs can be fed with food industry waste (flaxseed, sunflower and hemp cake, wheat bran). Cake is fed in a steamed and crushed form, and bran is fed with root crops or cake. The daily norms of waste and bran are 15-20 g for rabbits and 5-10 g for guinea pigs.

From succulent feed, rabbits and pigs are given well-washed and sliced red carrots (vitamin A), turnips, beets, turnips and rutabaga. The introduction of succulent feed into the diet in the autumn-winter period is mandatory. The daily norm of root crops for rabbits is 100-120 g, for guinea pigs - 80-100 g. Fresh grass and vegetable greens are an indispensable food for rabbits and pigs in summer, and sprouted grain in winter. From roughage they give good quality hay, and for rabbits also tree food (branches of linden, birch, aspen, maple, poplar). Animals are fed with natural (pasteurized or boiled or acidophilic) milk and clean, non-cold water. Rabbits need milk not only for young animals, but also for pregnant and lactating females.

White mice and rats are given oats, wheat, millet, barley, linseed, hemp, sunflower seeds. In order to avoid obesity in animals, oilseeds are introduced into the diet in small quantities. The daily norm of concentrates: 3-5 g for mice and 12-15 g for rats. Wheat bread is also fed (sour rye bread can cause diarrhea), crackers, cereals (oatmeal, pearl barley, millet), young semolina porridge in milk. In addition, the diet includes carrots, Antonovka apples (the latter are especially necessary for gastrointestinal diseases) and green foods (lettuce, spinach, carrot tops).

Kovalevsky recommends introducing boiled beets and mashed potatoes into the diet of rats. The daily norm of bread and cereals for mice is 3-3.5 g, for rats - 15-20 g, succulent feed - 0.5-1 and 2-3 g, respectively, green feed, washed with boiled water and chopped, -2-3 and 4-6 g, natural milk (pasteurized or boiled for 4-5 minutes) or acidophilic - in the amount of 4-5 g for mice and 6-8 g for rats (part of the milk is used to make cereals).

In addition, every day all animals should receive table salt: mice - 0.01 g each, rats - 0.07 g each (per porridge), pigs - 0.1 g, rabbits - 0.5 g each (in the form of a solution with concentrates) and bone meal, in approximately the same quantities. In each cage for mice and rats put a piece of 1-5 g) of chalk for 3-4 days. To enrich the feed with vitamins in the autumn-winter period, they give fish oil, dry irradiated yeast, tomato juice, and pigs, in addition, blackcurrant juice, rosehip tincture (vitamin C), etc.

Daily norm of fish oil: 0.5 g for rabbits, 0.3 g for pigs, 0.2 g for rats and 0.1 g for mice (with concentrates or on bread). Straw, peat are used as bedding for rabbits; for mice and rats - fine hay.

Animals are fed and watered twice, and lactating females three times a day, at exactly the right time. Feeding troughs are best made of clay (made of baked clay), of sufficient weight to prevent them from turning over by animals and are easy to clean (wash). Feeders are cleaned daily and washed with hot water. Each cage should have two feeders: for concentrates and for swill. Feed is given only fresh.