Chicken embryo. Methods for experimental infection of chicken embryos
Purpose of the lesson: to familiarize students with methods of infecting chicken embryos for cultivating viruses.
Equipment and materials: chicken embryos 9-12 days of incubation, ovoscope, alcohol swabs, stands for embryos, tweezers, scissors, adhesive tape, needles, syringes, pencils, punches, needles, tables, diagrams, multimedia equipment, presentations MS Office Power Point on the topic of the lesson.
Teacher's explanation.
The method of infection significantly affects the reproduction of viruses in embryos. Before infection, incubated eggs are candled, and dead ones are separated. On the shells of eggs with live embryos, which are distinguished by the red color of the blood vessels and the movement of the embryo, the site of infection is marked with a pencil.
Embryos are infected in a sterile box with a work table, two stools, gas supply, water supply and vacuum. Infection into the amniotic cavity is carried out in a darkened room. Workplace should be well lit. Before work, the table is disinfected. To disinfect the surface of eggs, prepare an antiseptic solution (70% alcohol, 2% iodine solution) and a wooden stick wrapped in cotton wool, a bent dental probe or a drill for drilling. eggshells. Eggs are infected using sterile syringes for tuberculinization and special needles. The infection site on the egg is filled with paraffin. To do this, paraffin sticks are prepared: a glass rod is inserted into a test tube filled with molten paraffin, making sure that it is in the center of the test tube until the paraffin cools.
When a paraffin stick is needed, the walls of the test tube are heated on a burner and the paraffin is pulled out of the test tube using a glass rod. Holding the stick over the flame, melt the amount of paraffin required to close the hole in the egg. This method is very clean and does not produce paraffin fumes.
In addition, a glass with sterile cotton swabs, a glass with a 3% solution of caustic soda for used instruments, and a vessel with a solution of chloramine for used glass objects are placed on the work table in a box. If necessary, you can prepare an egg cup, a small enamel cup and sterile culture media. The work table prepared in this way is subjected to UV irradiation for 1-2 hours.
Considering the pathogenicity of the viruses being studied, work is carried out wearing a mask, rubber gloves and safety glasses.
There are six methods for infecting embryos. The most commonly used infection is allantoic cavity and on the chorioallantoic membrane, less often - into the amniotic cavity and into yolk sac and very rarely - into the body of the embryo and into the blood vessels of the CAO. The choice of method is determined by the tropism of the virus, as well as the purpose of infection. For any method of infection, 0.1-0.2 ml of infectious material is injected.
Infection in the allantoic cavity.
When infected by this method, influenza viruses, Newcastle disease, equine rhinopneumonia, vesicular stomatitis, etc. multiply well. There are several variants of the method.
First option. The embryo is fixed vertically with the blunt end up. A hole with a diameter of about 1 mm is made in the shell on the side of the embryo, and sometimes on the side opposite to the embryo, 5-6 mm above the border of the air chamber. The needle is inserted parallel to the longitudinal axis to a depth of 10-12 mm (Fig. 16). After injecting the virus-containing material, the needle is removed and the hole in the shell is closed with a drop of melted sterile paraffin.
Figure 16. Infection in the allantoic cavity (first option) (according to Nikolaou)
Second option. The hole made in the shell above the air chamber is used only to allow some of the air to escape. The hole for the infection itself is made in the area of the avascular zone of the chorioallantoic membrane (CAO) on the side of the embryo. The needle is inserted to a depth of no more than 2-3 mm. Inject an infectious liquid in a volume of 0.1-0.2 ml and close the hole with paraffin (see Fig. 17).
Infection of the chorioallantoic membrane.
This method of infecting chicken embryos is often used for cultivating epitheliotropic and pantropic viruses of smallpox, infectious laryngotracheitis of birds, distemper, Aujeszky's disease, bluetongue, etc.
Such infection can be accomplished through a natural or artificial air chamber.
For infection through a natural air chamber the embryo is placed vertically on a tripod with the blunt end up and cut out in the shell against the center of the air chamber round window with a diameter of 15-20 mm. Through this window, the subshell membrane is removed with tweezers. 0.2 mm of a virus-containing suspension is applied to the exposed area of CAO (Fig. 18), the hole is closed with an adhesive plaster or, less often, with a cover glass, strengthened with molten paraffin.
Infection through an artificial air chamber is used more often than the first, since it ensures contact of the virus-containing material with a larger surface of the CW and, therefore, leads to the formation of a larger amount of virus.
Figure 17. Infection of a chicken embryo in the allantoic cavity (second option) (according to Nikolaou)
Figure 18. Infection at KhAO through a natural air chamber (according to Nikolaou et al.)
Figure 19. Infection of a chicken embryo at KhAO through an artificial air chamber (according to Nikolaou et al.)
To infect an embryo using this method, it is placed horizontally in a stand with the embryo facing up. Two holes are made in the shell: one small above the center of the air chamber (designed to suck air from it), and the other with a diameter of 0.2-0.5 cm on the side, on the side of the embryo. The complexity of the method is that when making the second hole, you must first carefully remove a piece of the shell, then with a sliding movement, without damaging the chemical substance, move the shell to the side so that air can pass through the resulting defect. After this, air is sucked out of the natural air chamber using a rubber bulb through the first hole (Fig. 19, a). As a result, through the side hole outside air rushes inward, forming an artificial air chamber, the bottom of which is the XAO (Fig. 19, b).
Through the side hole, an infectious liquid is applied to the surface of the CAO and the hole is closed with a piece of adhesive tape. There is no need to close the first hole, since the inner layer of the shell membrane is not damaged by this method of infection and continues to act as a barrier to microflora environment.
Further incubation of embryos infected by this method is carried out in a horizontal position with the side opening facing up.
Infection in the yolk sac.
For the most part, it is used for the propagation of chlamydia, as well as viruses of Marek's disease, equine rhinopneumonia, bluetongue of sheep, etc. They infect embryos of 5-7 days, and sometimes 2-3 days of age (RIF valley fever virus). Two infection options are used.
First option. Embryos are placed in a tripod in a vertical position. A hole is made in the shell above the center of the air chamber and a needle is inserted to a depth of 3.5-4 cm at an angle of 45° to the vertical axis in the direction opposite to the location of the embryo (Fig. 20).
Figure 20. Infection of a chick embryo into the yolk sac (according to Nicolaou et al.)
Second option. Sometimes a similar route of infection is carried out on an embryo horizontally mounted in a tripod; in this case, the embryo is at the bottom, and the yolk is above it. The hole in the shell is closed with a drop of molten paraffin.
Infection in the amniotic cavity.
For this purpose, embryos 6 to 10 days old are used. The method is used for cultivating influenza viruses, Newcastle disease, equine rhinopneumonia, etc. There are two methods of infection.
Closed method. Infection is carried out in a darkened box. The egg is placed on the ovoscope in a horizontal position with the embryo facing up. A needle with a blunt end is inserted through a hole in the shell above the air chamber towards the embryo. Proof that the needle has penetrated the amnion is the movement of the fetal body in the direction of movement.
Open method. The shell above the air chamber is cut so that a window with a diameter of 1.5-2.5 cm is formed. Through it, the subshell membrane is removed with tweezers under eye control. Then anatomical (14 cm) tweezers with closed jaws are guided, pushing the chorioallantoic membrane towards the embryo. When the tweezers reach it, the jaws open, grab the amniotic membrane along with the CAO and pull it to the window. Holding the tweezers with the amnion membrane fixed in it with your left hand, a virus-containing material is introduced (Fig. 21). Next, all the membranes are lowered, the window is closed with an adhesive plaster, and the embryo is incubated in a vertical position.
Figure 21. Infection of a chick embryo in the amnion open method(according to Nikolaou et al.)
Infection in the blood vessels of XAO.
When ovoscoping 11-13 day old embryos, a large blood vessel is noted. As it progresses, a section of the shell is removed, 1-2 drops of alcohol are applied, which makes the shell membrane transparent for a while. Under eye control using an ovoscope, the needle is inserted into the vessel, which is confirmed by its mobility with small lateral movements of the needle. The exposed area of the subshell membrane is covered with a piece of adhesive plaster.
The material can also be introduced into the vessels in a slightly different way: the shell above the air chamber is cut off, the subshell membrane is moistened with alcohol, and the material is introduced into the XAO vessels that have become visible. The hole is covered with a piece of sterile adhesive tape.
The described technical methods for experimental infection of chicken embryos are not the only ones, but have various options.
Infection in the body of the embryo.
Embryos 7-12 days old are used for infection. There are two known versions of the method.
First option. Infect in the same way as in the amnion in a closed way, with the only difference that they take a sharp needle and on an ovoscope, the indicator of the needle entering the body is the subordination of the embryo to the movements of the needle.
Second option. They infect the amnion in the same way as in the open way: through a window in the shell, the body of the embryo is pulled up with tweezers. The material is injected into the brain or specific areas of the body. With such infection methods, there is a significant percentage of nonspecific embryo death.
Virus accumulation in the chick embryo
Before further incubation, on the shell of chicken embryos infected by any method, they write with a simple (graphite) pencil what the embryo is infected with and when, and if necessary, other information. Infected chicken embryos are placed in a thermostat for further incubation, during which the introduced viruses are reproduced and accumulated in the corresponding structures. The incubation temperature of embryos varies from 33 to 38 °C depending on the properties of the virus with which the infection was carried out. The embryos are constantly monitored, examined on an ovoscope, and dead ones are selected.
The death of embryos in the first 24 hours after infection is most often due to the proliferation of fungi, bacterial microflora introduced into the embryo along with the inoculum, or injury during infection. This death is considered nonspecific. In more late dates embryos die as a result, as a rule, of virus multiplication in embryos. Having discovered dead embryos, they are immediately transferred to a refrigerator at a temperature of 4 °C. Such conditions, on the one hand, contribute to the preservation of the activity of the virus accumulated in the embryo, on the other hand, to the compaction of tissues and the emptiness of blood vessels, which greatly facilitates subsequent dissection.
Embryos are incubated until maximum virus accumulation occurs. For each virus and even strain, this period is specific and varies from 2 to 7-8 days. Thus, for the Newcastle disease virus strain H it is 2-3 days, for the same virus strain B - 5 days, for the avian infectious laryngotracheitis virus - 5 days, etc. Then all embryos are killed by cooling at 4 ° C for at least 3-4 hours and open.
Deembrinated eggs.
The method is based on removing the embryo from the egg during the period when the chorioallantoic membrane is completely adjacent to its shell from the inside. If a nutrient medium is added inside such an egg, a kind of tissue culture is formed in which the virus can multiply. The method has a number of advantages: it allows one to obtain a purer virus than in allantoic-amniotic fluid, which is important for reducing the allergenic properties of vaccines prepared from this material; The absence of the yolk sac and, therefore, the specific antibodies contained in it allows some viruses to multiply better. In some cases, this method gives good results in diagnostic work, since it can introduce large quantities of the test material, which increases the possibility of virus isolation.
The method was proposed by Bernkopf back in 1949, but due to difficulties in application it is not widely used. These difficulties are associated with the property of the chorioallantoic membrane to separate from the shell during incubation, which reduces the value of the culture and complicates the manipulation of obtaining the virus and replacing the nutrient medium.
E. Groiel (1963) introduced a number of modifications that eliminated these difficulties. He suggested using 15-day-old or even older eggs, which should be turned frequently during incubation to ensure uniform development of the shell on the egg shell. When transilluminated, the boundaries of the air chamber are indicated. The shell is sawed off 0.5 cm above this line, molten paraffin (56-58 C°) is poured onto the edge of the shell, which hardens and fixes the edge of the egg. Then cut round hole in the shell above the embryo, leaving a narrow rim around. The next step is to carefully remove the embryo from the egg. In this case, the egg should be held in a horizontal position and slowly rotated. In this way, the vessels connecting the embryo with the CAO are found and cut with scissors. If the egg is held vertically, then the embryo with its mass pulls back the shell, usually tearing it off in the area of the sharp end of the egg. The entry of even a small amount of liquid between the chemical substances and the shell causes complete detachment of the shell within 1-2 days.
After removing the embryo, the membrane lining the egg is washed several times with saline solution cooled to 4-0°C until the liquid becomes clear. Then 20 ml of a nutrient medium heated to 37 ° C is introduced into the egg cavity, and the egg is closed with a sterile rubber cap, having previously immersed it in paraffin. A tube with a rubber stopper embedded in the egg allows both the collection and administration of liquid without the risk of bacterial contamination. Such a biological object remains viable for several days.
According to the method of Yoshino and others, the cavity of deembrinated eggs is filled with a solution of agar in Hanks' solution. Deembrinated eggs are infected through a tube in the cap.
1. The structure of a chicken embryo.
2. Why are chicken embryos used in virology?
3. What is the structure of a developing chicken embryo?
Purpose of the lesson
To familiarize students with methods for selecting chicken embryos for cultivating viruses.
Equipment and materials
Chicken embryos 9-12 days of incubation, ovoscope, alcohol swabs, embryo stands, tweezers, scissors, adhesive tape, needles, syringes, pencils, punches, needles, tables, diagrams, multimedia equipment, presentations MS Office Power Point on the topic of the lesson.
Methodology of conducting the lesson and guidelines on this topic
Teacher's explanation
Cultivation of viruses on chicken embryos is the most accessible and convenient method for the primary isolation of the virus. The practice of using this method of infection has shown a number of advantages over other methods. For infection, embryos of 5-12 days of incubation are used.
ADVANTAGES AND DISADVANTAGES OF CHICKEN EMBRYOS AS BIOLOGICAL OBJECTS
Cultivation of viruses in chicken embryos is the most accessible and convenient method for the primary isolation of viruses from sick animals and objects external environment, and for subsequent cultivation of viruses in the laboratory. This method is widely used for the identification of viruses and antibodies, as well as for the preparation of vaccines and diagnostics. Practice has shown a number of advantages of this method over cultivating viruses in laboratory animals. It is known that white mice, which are widely used in virological studies, can be spontaneously infected with a number of viral infections: ectromelia, lymphocytic choriomeningitis, Taylor encephalitis, viral pneumonia, Sendai virus and others, which extremely complicates the work and often leads to erroneous conclusions when assessing the results obtained. results. Cultivation of viruses on chicken embryos largely eliminates the above difficulties. Along with this, a significantly larger amount of virus can be obtained from a chicken embryo than from laboratory animals. The chicken embryo has greater viability and resistance to various types of influences that are inevitable when the test material is introduced. With a known skill in working with embryos and observing the rules of asepsis, their death is insignificant. The greatest waste of embryos occurs when the material is introduced into the amniotic cavity and yolk sac, but even in these cases it does not exceed 10–15% if the necessary incubation conditions are met.
Chicken embryos as a living system entered virological practice in the 30s of the 20th century. Their use has expanded the range of viruses cultivated in the laboratory, making it possible to more successfully solve the problems facing virology due to the fact that chicken embryos have a number of advantages over laboratory animals: 1) the shell and subshell membrane reliably protect the embryo from bacterial infection from the external environment; 2) an important advantage of embryos is also their high sensitivity to a wide range of viruses, which is explained by the insufficient development of protective mechanisms; 3) chicken embryos are an easily accessible object due to the development of a wide network of poultry farms and hatcheries; 4) chicken embryos are economical and do not require care or feeding.
The main disadvantages are: 1) the inability to fully guarantee the sterility of this living system, since embryos can carry viruses and other pathogenic agents in their contents (viruses of chicken infectious bronchitis, Newcastle disease, influenza, leukemia, chlamydia and mycoplasma). Their presence may distort the results of the study; 2) chicken embryos are not sensitive to all viruses.
PURPOSES OF USING CHICKEN EMBRYOS
Chicken embryos are used in virology mainly for the same purposes as laboratory animals, namely:
– detection of active virus by bioassay in the pathological material;
– primary isolation of the virus. Viruses that cause diseases in birds, as well as some mammalian viruses, are effectively isolated and cultivated on chicken embryos;
– maintaining viruses in the laboratory;
– titration of viruses;
– accumulation of the virus for laboratory research and obtaining vaccines;
– as a test object in the neutralization reaction.
REQUIREMENTS FOR CHICKEN EMBRYOS
Eggs must be obtained from farms that are free from viral diseases. Fertilized eggs, even from clinically healthy chickens, may contain various viruses found in these birds: Newcastle disease, infectious bronchitis, infectious laryngotracheitis, encephalomyelitis, parainfluenza-2, polyarthritis, smallpox, arboviruses, adenoviruses, etc. The presence of these viruses can, on the one hand , lead to diagnostic errors, and on the other hand, based on the phenomenon of interference, to the suppression of the reproduction of the virus located in the test sample. Embryos that do not contain a virus, but obtained from chickens asymptomatically infected with certain viruses, may also be less sensitive or completely insensitive to the effects of a given virus due to the presence of specific antibodies obtained from the mother with the yolk. For successful isolation of the virus, it is necessary that the chickens whose embryos are used in the work are not vaccinated against the disease whose causative agent is being sought. Egg shells must be unpigmented and clean (cannot be washed). The age of the embryo must correspond chosen method infection.
To ensure normal development of embryos in fertilized eggs during the incubation period, it is necessary to maintain a certain temperature and humidity. Developing embryos tolerate overheating much worse than cooling. Therefore, a short-term (within a few hours) breakdown in the heating system does not cause much harm. For a longer break, heating is necessary.
Hatching eggs need free access fresh air, which enters through vents. They must always be open. One egg, as is commonly believed, consumes 1 liter of oxygen per day, which is not surprising if we recall the rapid development of the embryo during 21 days of incubation. That is why eggs should not be placed close together, or one on top of another. Conventional thermostats are not suitable for incubating eggs. Only in cases of extreme necessity can they be used for these purposes, but at the same time ventilate them frequently and place a vessel with water inside to ensure the required humidity.
Eggs laid for incubation are examined after 3–5 days using a special tabletop or hand-held (if the eggs are not removed from the incubator tray) lamp to select unfertilized ones. Leghorn eggs are most convenient for virological work, since their thin white shell allows a better view of the contents. Eggs that are unfertilized or contain dead embryos are removed from the incubator. The proportion of fertilized eggs varies widely depending on many factors, including the time of year: in spring it is highest, in winter it is lowest.
The embryos are scanned for the second time on the day of planned infection. This period depends on the type of virus, as well as the route of its introduction. The site of infection is marked on the shell with a regular (not ink) pencil.
STRUCTURE OF CHICKEN EMBRYO
Typically, a chicken lays a fertilized egg in which the embryo is at the blastula or early gastrula stage. When an egg is heated to a temperature close to the chicken's body temperature, further development embryo (Fig. 15). During the period from the 5th to the 12th day of incubation, chicken embryos can be used for infection with viruses.
Figure 15 - Schematic section of a chicken embryo on the 8th day of incubation: 1 – shell; 2 – subshell membrane; 3 – chorioallantoic membrane; 4 – allantoic cavity; 5 – yolk sac; 6 – protein; 7 – air chamber; 8 – body of the embryo; 9 – amniotic cavity
An egg with a developing chicken embryo is covered on the outside with a hard, porous shell, to which the subshell membrane fits tightly. The latter, at the blunt end of the egg, is divided into two leaves, between which an air chamber is formed. The body of the embryo lies eccentrically in the egg, with its back closer to the shell, and its head directed towards the air chamber. The embryo is immersed in the amniotic fluid that fills the amniotic cavity and is connected to the yolk by an umbilical cord. The yolk is also located eccentrically and relative to the embryo, as if on the other side of the longitudinal axis.
Directly under the shell membrane there is an allantoic cavity, covering the amnion and yolk sac, and by the 10-11th day it is closed in the sharp end of the egg. During development, the allantoic membrane fuses with the chorion, forming a single chorioallantoic membrane (CAO). At the sharp end of the egg is the rest of the protein.
Infection in one or another part of the embryo is carried out during the period of its maximum development, when the number of sensitive cells is greatest.
During the incubation process, the size of the embryonic structures changes, which is largely explained by their functional purpose and determines the optimal age of the embryo for infection.
So, the yolk sac is like a reservoir nutrients has the greatest volume at the beginning of incubation, and then (after the 12th day) as the embryo develops it decreases. Infect the yolk sac from the 5th to 7th day of incubation.
The amniotic cavity, being a buffer environment for the development of the embryo, covers it already on the 5th day of incubation. The average amount of liquid by the middle of the incubation period is about 1 ml.
For infection into the amniotic cavity, embryos aged 6-10 days are used.
The allantoic cavity serves to collect metabolic products; uric acid salts, phosphorus and nitrogen compounds accumulate in it. During the growth and development of the embryo, the allantoic fluid becomes acidic. Maximum sizes the allantoic cavity reaches on the 9-12th day of embryo development, therefore infection in the allantoic cavity is carried out mainly on the 9-11th day of incubation.
The chorioallantoic membrane is rich in blood vessels, which, closely adjacent to the inner surface of the porous shell, are saturated with oxygen and supply it to the body of the embryo, performing the function of the embryo's respiratory organ. CAO reaches its maximum development on the 11-13th day. Infection of the chorioallantoic membrane is carried out on the 10-12th day of incubation.
PREPARATION OF CHICKEN EMBRYOS FOR INFECTION
Embryos are transported from the hatchery without being cooled en route. In the laboratory, embryos are incubated in a thermostat at a temperature of 37 °C and a humidity of 60-70%, which is achieved by placing open wide-necked vessels with water in the thermostat. Ventilation holes thermostats must be open. Embryos are placed with the air chamber facing up in special stands. It is recommended to allow the embryos to adapt to new conditions within 24 hours before infection and normalize their functions after transport stress. If the laboratory has its own hatchery, then the fertilized eggs laid by the chicken are suitable for laying there within 10 days.
Preparation of chicken embryos for infection includes ovoscoping and disinfection of the shell, as well as appropriate preparation of the workplace. Ovoscoping involves viewing eggs against a sufficiently bright light source (ovoscope), as a result of which shadows from internal structures are formed on the unlit side of the shell (Fig. 16). Ovoscoping is carried out in a darkened room. At the same time, on the shell, with a graphite pencil, the border of the air chamber, the location of the embryo and a section of the avascular zone measuring 0.5x0.5 cm are marked. These marks serve as a guide when choosing the site for introducing the virus-containing material. During ovoscopy, it is also determined whether the embryo is alive or dead. Embryos showing active movements with good blood supply to the vessels of the CAO, they are considered alive.
Figure 16 - Ovoscoping of a chicken embryo on the 10th day of incubation. Shadows are visible: 1 – embryo; 2 – yolk sac; 3 – blood vessels XAO; 4 – air chamber; 5 – squirrel
Chicken embryos are infected under aseptic conditions (preferably in a box). In the prebox, the embryo shells are treated with iodized alcohol, then in the box they are wiped again, and sometimes they are also flambéed - treated with the flame of a swab moistened with alcohol.
Embryos are fixed in special stands installed in an enamel cuvette on a 3-4-layer gauze cloth moistened with a disinfectant solution.
The work uses tools sterilized by boiling. They are placed in a jar of alcohol and burned with a burner flame before each reuse.
Demonstration
a) clinical signs of disease in infected laboratory animals; b) methods of killing laboratory animals; c) autopsy techniques (draw students’ attention to the state of internal organs) and methods for obtaining virus-containing material; d) techniques for making brain prints; e) actions to disinfect the workplace and the corpse after autopsy of an infected animal.
Tasks
1.Study the structure of a chicken embryo.
2. Conduct an ovoscopy of a chicken embryo, determine its viability and mark the boundaries of the shadows of natural formations.
3. Prepare chicken embryos for infection.
Independent work students
a) recognition of mice infected by each student by color mark, analysis of their clinical condition, killing, fixation in a cuvette with a wax (paraffin) bottom, autopsy; b) analysis of pathological changes, obtaining virus-containing material (parenchymal organs), preparing layer-by-layer brain prints.
Students perform an ovoscopy of a chicken embryo, determine its viability and mark the boundaries of the shadows of natural formations.
Summing up the lesson
Assignment for the next lesson
Control questions
1. The structure of a chicken embryo.
2. Why are chicken embryos used in virology?
3. What is the structure of a developing chicken embryo?
Embryos are used at the age of 8 to 14 days, depending on the type of virus and method of infection. Reproduction in chicken embryos occurs in different parts of the embryo. Methods of infection: on the chorioallantoic membrane, in the allotonic and amniotic membrane, yolk sac, embryo body.
29.Tissue cultures.
Depending on the preparation technique, there are 3 types of cells:
Single-layer glass can reproduce on the surface of chemically neutral glass in the form of a monolayer;
Suspension, spreading throughout the entire volume of the nutrient medium;
Organs are whole pieces of organs and tissues that retain their original structure.
The preparation of a primary cell culture consists of several stages: grinding the tissue, separating the cells, and washing the resulting suspension from trypsin.
Indication:
- cytopathic effect - morphological changes in cells visible under a microscope, up to rejection from the glass.
Viral inclusions are an accumulation of viral particles or separation of viral components in the cytoplasm or nucleus of cells.
Plaques are limited areas consisting of degenerative cells. They are visible as light spots against the background of colored cells.
Color test
Hemadsorption is the ability of cell cultures to adsorb red blood cells on their surface.
Interference.
30. Classification, chemical composition of bacteriophages.
Bacteriophages are bacterial viruses that have the ability to penetrate bacterial cells and cause their dissolution. They have a tadpole shape, some are cubic thread-like. They consist of an icosahedral head and a tail. Inside the caudal process there is a cylindrical rod, outside there is a sheath, the process ends in a hexagonal basal plate with short spines. Phages are composed of nucleic acid and protein. In sperm-shaped phages, double-stranded DNA is tightly packed in a helix inside the head. Proteins are part of the shell. In addition to structural proteins, internal proteins associated with nucleic acid and enzyme proteins involved in the interaction of the phage with the cell.
31. The process of interaction between virulent phages and a bacterial cell sensitive to them
Virulent phages can only increase in number through the lytic cycle. The process of interaction between a virulent bacteriophage and a cell consists of several stages: adsorption of the bacteriophage on the cell, penetration into the cell, biosynthesis of phage components and their assembly, and release of bacteriophages from the cell.
Initially, bacteriophages attach to phage-specific receptors on the surface of the bacterial cell. The phage tail, with the help of enzymes located at its end (mainly lysozyme), locally dissolves the cell membrane, contracts and the DNA contained in the head is injected into the cell, while the protein shell of the bacteriophage remains outside. Injected DNA causes a complete restructuring of the cell's metabolism: the synthesis of bacterial DNA, RNA and proteins stops. The bacteriophage's DNA begins to be transcribed using its own transcriptase enzyme, which is activated after entering the bacterial cell. First, early and then late mRNAs are synthesized, which enter the ribosomes of the host cell, where early (DNA polymerases, nucleases) and late (capsid and tail proteins, enzymes lysozyme, ATPase and transcriptase) bacteriophage proteins are synthesized. Bacteriophage DNA replication occurs according to a semi-conservative mechanism and is carried out with the participation of its own DNA polymerases. After the synthesis of late proteins and the completion of DNA replication, the final process begins - the maturation of phage particles or the combination of phage DNA with the envelope protein and the formation of mature infectious phage particles.
Isolation of viruses in laboratory animals.
The choice of laboratory animals depends on the type of virus. Laboratory animals are a biological model. Sometimes it is necessary to carry out 3-5 “blind”, asymptomatic passages before it is possible to adapt the virus to laboratory conditions. However, laboratory animals are not sensitive to some viruses, in which case it is necessary to use naturally susceptible animals. As, for example, with swine fever and infectious anemia of horses.
The purpose of infecting laboratory animals:
1. Study the pathogenesis of the disease;
2. Isolate the virus from the pathogenic material;
3. Production of immune and hyperimmune serums;
4. Production of vaccines;
5. Maintenance of viruses in laboratory conditions;
6. Titration, in order to determine the amount of virus per unit volume;
7. Biological model for staging the neutralization reaction;
The choice of method for infecting laboratory animals depends on the tropism of the virus. Thus, when cultivating neurotropic viruses, animals are infected into the brain; respiratory intranasally, intratracheally; dermatropic - subcutaneously and intradermally.
Infection is carried out in compliance with the rules of asepsis and antiseptics.
There are many ways to introduce virus-containing material into the body of animals:
Subcutaneous; - Intracerebral; - Intradermal;
Intraperitoneal; - Intramuscular; - Intraocular;
Intravenous; - Intranasal; - Nutritional;
After infection, the animals are marked, placed in an isolated box and monitored for 10 days. The death of an animal on the first day after infection is considered nonspecific and is not taken into account later.
3 signs indicate the effectiveness of infection:
Presence of clinical signs
Death of an animal
Pathoanatomical changes (size, shape, color and consistency of the organ).
A chicken embryo is a fertilized egg, in which the embryo (embryo) develops. Cultivation of viruses on chicken and quail embryos in Lately has become widespread as one of the simplest and most reliable methods for cultivating and diagnosing many viruses and some bacteria - Brucella, Rickettsia, Vibrio.
Many human and animal viruses can be cultured in developing chicken embryos. Embryonic tissue, especially the membranes of the embryo, rich in germinal epithelial tissues, is a favorable environment for the proliferation of many viruses. Viruses with epitheliotropic properties (smallpox, ILT, etc.) successfully develop on the chorioallantoic membrane, causing macroscopically visible changes. Various representatives myxoviruses (influenza, Newcastle disease, canine distemper, etc.), infectious bronchitis viruses, duckling hepatitis, arboviruses, etc. multiply well in the embryo when the material is introduced into the allantoic cavity. Some viruses can be successfully cultured in the yolk sac.
Advantages:
1. Economically profitable, in addition, eggs are easily available;
2. Developing chicken embryos do not have defense mechanisms, because the immune system is not yet developed;
3. Egg shells prevent bacteria and viruses from the environment from penetrating through it;
To cultivate and isolate viruses on chicken embryos, very simple equipment is required - a regular thermostat or incubator.
Conditions:
1. Eggs are obtained from farms known to be safe infectious diseases;
2. It is better to obtain chicken embryos from white breeds of chickens (Leghorn, Russian White), because they are more resistant to manipulation and do not die from minor injuries. In addition, their shell is white and more transparent than that of other breeds, and is easier to see through, which is convenient for viewing and observation while working with them;
3. For incubation, fertilized eggs laid no more than 10 days ago are selected;
4. Take uncontaminated eggs, because they cannot be washed before incubation, and dirty eggs are less visible when viewed (ovoscopy) and when working with them, the embryo can be infected during the manipulation process;
The eggs are incubated in an incubator or in a thermostat with water heating and air access, and during incubation in the thermostat, the eggs need to be turned 2-3 times a day and, for better gas exchange, taken out for 5-10 minutes into the air. To maintain a certain humidity, a vessel with water is placed in the thermostat for evaporation; the temperature in the thermostat should be 38°.
The development of the embryo occurs already on the first day of incubation, the laying of the brain and skeleton occurs. The structure of a chicken embryo at the age of 7-9 days(see notebook).
For infection, embryos 7-12 days old are most often used. Work with chicken embryos is carried out in a sterile room-box with the strictest adherence to asepsis.
The purpose of infecting chicken embryos is:
1. Isolate the lining from the patent material;
2. Production of vaccines;
3. Maintaining the virus in a laboratory;
4. Titration of viruses;
5. Biological model for staging the neutralization reaction;
6. Study of virus interference and interferon production; Infection of chicken embryos:
For infection, viable embryos with well-defined motility must be selected. Before infection, all embryos are carefully examined in a darkened room using an ovoscope.
During candling of embryos before infection, a puga (air cavity), the course of large blood vessels and the place of presentation of the embryo are outlined on the shell with a simple pencil, i.e. the area on the shell where the embryo lies closest to it. The marking of the pug, the place of presentation of the embryo and the course of large blood vessels then serves as a guide when choosing the site for introducing the virus-containing material at the time of infection.
Chicken embryos selected for infection are transferred to a box, where they are worked on. Before infection, the shell at the site of introduction of the material is treated twice with iodized alcohol and burned. The dose of infection is 0.1-0.2 cm.
Depending on the type of virus and the purpose of infection, there are different methods of introducing virus-containing material:
1) Infection of the chorioallantoic membrane , embryos of 7-12 days of age are used for the isolation and cultivation of neurotropic, dermatropic and some pantropic viruses (smallpox, encephalomyelitis, ILT, foot-and-mouth disease, rabies, plague, etc.). There are 3 infection options:
a) open the puga and cut it off with scissors, separate the subshell membrane and apply material to the chorioallantoic membrane (CAO). The hole in the egg is then sealed with a sterile glass cap and the edges of the cap are waxed;
b) cut out a triangle with a side length of about 1 cm in the shell with a needle file (file) or a serrated scalpel at the border of the puja on the side of presentation of the embryo, remove the section of the shell and substellar membrane with tweezers and introduce the material. The hole is covered with a sterile cover glass and the edges are paraffinized or sealed with a sterile adhesive tape.
c) removed with a scalpel small area shell with an area of about 0.5 cm at the place where the embryo is presented, then the subshell membrane is removed in this area using tweezers or a needle and the material is injected. If the material does not fit well into the cavity of the egg, you can use a rubber bulb to pump out the air from the egg through the hole in the shell on the egg, and as a result, an artificial egg is formed at the place where the material is introduced, and then the material is easily inserted. The hole in the shell is covered with an adhesive plaster or waxed.
2) Infection in the allantoic cavity. This infection method is very simple and is used to isolate many viruses. For infection, 10-11 day old embryos are taken. There are two infection options:
a) infection is carried out through the puga without cutting it off. Using a needle, measure the distance from the top of the puga to the border of the puga, marked with a pencil on the shell, and insert the needle to the marked depth and deepen another 0.5 cm to pierce the chorioallantoic membrane;
b) the material is inserted with a needle through a puncture in the shell at the place of presentation of the embryo to a depth of 3-5 mm in an avascular area. The hole in the shell is covered with an adhesive plaster or waxed.
3) Infection in the yolk sac. 5-8 day old embryos are used for infection. There are two infection options:
a) the needle is inserted from the side of the puja into the yolk sac at an angle of 45° to the place of presentation of the embryo under the control of an ovoscope;
b) the egg is placed on the stand with the embryo’s presentation point downwards and the needle is inserted from top to bottom towards the embryo to a depth of about 1 cm.
The injection site is sealed with adhesive tape and paraffinized. 4) Infection in the amniotic cavity. With this method of infection, the virus can penetrate and multiply in various cells that are in contact with the amniotic fluid. For ease of infection, it is recommended to incubate the embryos 2-3 days before infection with the pug facing up. Then the embryo and amnion move upward and it is more convenient to infect. There are two infection options:
a) open and cut off the puga, remove the subshell membrane with tweezers and grab the amnion, pull up the amnion with tweezers and introduce material in a dose of 0.1 ml into the amniotic cavity. The hole in the shell is then sealed with a sterile glass cap and the edges are waxed;
b) infection using a long needle through a puga in a dark room under eye control. The needle tip is first bent at a right angle to create a small area. The needle is inserted under the control of the eye through the protuberance of the pre-embryo; in this case, under the pressure of a blunt needle, the embryo will move, then the amnion is pierced with a slight push and the needle is slightly pulled back. In this case, the embryo should move upward behind the needle. Then the material is introduced.
5) Infection into the body of the embryo and infection into the brain. 7-12 day old embryos are used, infection is carried out by introducing the material into various parts of the body or directly into the brain. For infection, the puga is opened and the embryo is pulled up with tweezers. With these methods of infection, up to 30% or more of the number of infected embryos may die from injury.
6) Infection in large blood vessels of the chorioallantoic membrane. This method of infection, like the previous one, is used very rarely. The material is injected with a thin needle after removing the shell along the blood vessel directly into the vessel along the blood flow.
After infection, chicken embryos must be marked with a simple pencil and placed in a thermostat. They are monitored daily by viewing, observation is carried out for up to 7-8 days, depending on the type of virus. If embryos die, they are immediately removed from the thermostat and placed in the refrigerator until opening. If the embryo dies within the first 14-18 hours, it may be due to injury or toxicity of the pathological material. Therefore, just as when infecting laboratory animals, in doubtful cases it is recommended to make several passages and take several embryos for each material.
The autopsy of dead infected chicken embryos, or those removed after the observation period has expired, is carried out with all the rules of asepsis in sterile boxing conditions. When opened, the shell is treated with alcohol and burned, then the puga is cut off. From the opened embryo, the allantoic fluid is first carefully sucked out (its amount is about 7 ml), then the amniotic membrane is pulled back with tweezers, pierced with a Pasteur pipette and the amniotic fluid is sucked out (its amount is 1.0-1.5 ml), then the yolk is collected, the membranes are removed and embryo. The fluid, membranes and the embryo itself are carefully examined for changes. Amniotic fluid is normally completely clear, but when infected it can be cloudy and bloody. Characteristic changes caused by the virus are most pronounced on the chorioallantoic membrane: inflammatory foci appear, opaque, round in shape and hemorrhages. Hemorrhages may occur on the body of the embryo. All material is collected in sterile containers.
In virology, chicken embryos are widely used not only for isolating viruses, but also for accumulating and obtaining antigens, for preparing live and killed vaccines, titrating viruses, for staging a virus neutralization reaction, for attenuating (weakening) viruses, for studying the interference of viruses and obtaining interferon .
Isolation and cultivation of viruses
Isolation and identification of the pathogen is the “gold standard” in the diagnosis of viral infections.
Cell culture
Viruses reproduce only in living cells, and isolation of the pathogen in an infected cell culture is one of the main methods for diagnosing viral infections. Since most pathogenic viruses are distinguished by tissue and type specificity, it is possible to select appropriate cell or tissue cultures for almost every virus, as well as create standard cultivation conditions (the presence of cells of the same type). Reproduction of the virus is ensured by sensitive (permissive) cells. Therefore, when an unknown pathogen is isolated, 3-4 cell cultures are simultaneously infected, assuming that one of them may be permissive. Cell cultures are obtained by dispersing the corresponding organs and tissues, but more often they use embryonic tissues (human and animal) or transformed tumor cells. When placed on an appropriate flat surface, cell cultures typically grow as a monolayer.
Primary trypsinized cultures. Cell suspensions are obtained by homogenizing the corresponding tissues, pre-treated with trypsin. Cultures are often represented by cells mixed type and cannot be re-cultivated. The viability of such crops is 2-3 weeks.
Semi-continuous cell lines represented by diploid cells of humans and animals. Cultures have limited suitability for re-dispersion and growth (usually no more than 20-30 reseedings), while maintaining viability and not undergoing spontaneous transformation.
Continuous cell lines(heteroploid cultures) are represented by cells subjected to long-term cultivation and spontaneous transformations. Cultures are capable of repeated dispersion and transplantation. Working with them is less labor-intensive compared to preparing primary crops; The transplanted cells are relatively identical in their morphology and stable in properties.
Organ cultures
Not all types of cells are capable of growing as a monolayer; in some cases, maintaining differentiated cells is possible only in organ culture. It is usually a suspension of tissue with a specialized function, also referred to as culture of experiencing tissue.
Chicken embryos (Fig. 1-20) - almost ideal models for cultivating certain viruses (for example, influenza and measles). The closed cavity of the embryo prevents the penetration of microorganisms from the outside, as well as the development of spontaneous viral infections. Embryos are used for the primary isolation of viruses from pathological material; for passivating and preserving them, as well as for obtaining required quantities virus. Some pathogens (for example, herpes viruses) cause characteristic changes (the disease can be recognized by them). Infection is carried out on the chorion-allantoic membrane, in the amniotic or allantoic cavity, or in the yolk sac.
Infection of the chorion-allantoic membrane. Usually 10-12 day old embryos are used. The eggs are viewed in transmitted light, the location of the air sac is noted, and an area without blood vessels is selected. Carefully remove the shell fragment, release the outer shell and peel it off with gentle pressure. Then a hole is made at the edge of the air sac. When suctioning through this hole, the chorion-allantoic membrane is peeled off from the outer membrane. The test material, free of bacteria and protozoa, is applied to it (passed through bacterial filters and treated with bactericides).
Infection in the amniotic cavity. Typically, 7-14-day-old embryos are used, in which, after detachment of the chorionic-allantoic membrane (see above), the opening is expanded, the amniotic membrane is grabbed with tweezers and removed through the chorionic-allantoic membrane. Through it, the test material is introduced into the amniotic cavity.
Infection in the allantoic cavity. 10-day-old embryos are infected through holes made in the shell and underlying membranes (see above).
Infection in the yolk sac. 3-8 day old embryos are used, in which at this age the yolk sac occupies almost the entire egg cavity. Infection is carried out through a hole made in the air sac
Observation and recording of results. As virus-containing material, you can use the contents of the yolk sac, allantoic and amniotic fluids, or the entire embryo, cut together with surrounding
fabrics into pieces. To identify Fig. 1-20.Schematic illustration
characteristic lesions on the chorion of the developing chick embryo.
the shell is removed from the allantoic membrane
and the outer shell. The membrane is then removed and placed in sterile water. The nature of the lesions is studied against a dark background.
Animal models
If it is impossible to isolate and identify the virus using standard methods in vitro infectious material is administered to animals sensitive to the pathogen, and after the development of a typical infectious process, sensitive cell cultures are re-infected. The most commonly used are mice, rabbits and monkeys; To isolate some viruses (for example, Coxsackie viruses), suckling mice are infected. Due to the high cost and complexity of keeping laboratory animals, they have been replaced almost everywhere by cell cultures. Nevertheless, animal models are actively used to study the characteristics of pathogenesis and the formation of immune responses during viral infections.