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The structure of the chicken embryo microbiology. Infection of chick embryos

The structure of the chicken embryo microbiology. Infection of chick embryos

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. Contents can be used as virus-containing material yolk sac, allantoic and amniotic fluid, 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.

Prepared by: teacher Ugysheva Sh.E.

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Structure of viruses

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Structure of viruses A – simple virus B – complex virus

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Based on their degree of danger, viruses are divided into four groups: Group I: pathogens of Ebola, Lassa, Marburg, Machupo fever, smallpox, and hepatitis B virus (monkeys). Group II: arboviruses, some arenaviruses, rabies viruses, human hepatitis C and B viruses, HIV. Group III - influenza viruses, polio, encephalomyocarditis, vaccinia. Group IV - adenoviruses, coronaviruses, herpesviruses, reoviruses, oncoviruses.

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Principles of classification of viruses Type of nucleic acid, its structure, replication strategy Size, morphology, symmetry of the virion, number of capsomeres, presence of supercapsid. Presence of specific enzymes, especially RNA and DNA POLYMERASES, neuraminidases Sensitivity to physical and chemical agents, especially ether Immunological properties Natural transmission mechanisms Tropism to the host, its tissues and cells Pathology, formation of inclusions Symptomatology of diseases.

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Classification of viruses (RNA-containing)

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Classification of viruses (DNA-containing)

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Chemical composition of viruses Viruses include nucleic acid, protein, lipids, glycolipids, and glycoproteins. They always contain one type of nucleic acid (DNA or RNA), which makes up from 1% to 40% of the virion's mass. Viral genomes contain information sufficient for the synthesis of only a few proteins. Their mass reaches 10-15 mg, which is 1 million times less than that of cells, and their length is up to 0.093 mm. The number of nucleotide pairs ranges from 3150 (hepatitis B virus) to 230,000 (variola virus). Viral proteins (70-90%) are divided into structural and non-structural. Structural - proteins that are part of mature extracellular virions. They perform a number of important functions: - protect the nucleic acid from external damage; interact with the membranes of sensitive cells; ensure the penetration of the virus into the cell; - have RNA and DNA polymerase activity, etc. Non-structural proteins are not part of mature virions, but are formed during their reproductions. They: - provide regulation of the expression of the viral genome - are precursors of viral proteins that can suppress cellular biosynthesis. Depending on their location in the virion, proteins are divided into capsid, supercapsid, matrix, core proteins and associated proteins. nucleic acid. Lipids (15-35%) are contained in complex viruses and are part of the supercapsid shell, forming its double lipid layer. They: - stabilize the viral envelope - provide protection to the inner layers of virions from hydrophilic substances in the external environment - take part in the deproteinization of virions.

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Reproduction of viruses. Its features are that the genomes are represented by both RNA and DNA, they are diverse in structure and form, almost all viral RNAs are capable of replicating independently of the DNA of the cell. Viruses have an inherent disjunctive method of reproduction, which consists in the fact that the synthesis of the genome and Virus proteins are separated in space and time: nucleic acids are replicated in the cell nucleus, proteins in the cytoplasm, and the collection of entire virions can occur on the inner surface of the cytoplasmic membrane. Viral reproduction is a unique system for recreating foreign information in eukaryotic cells and ensures the absolute subordination of cellular structures to the needs of viruses. There are a number of stages in the reproduction of viruses. The early ones include the adsorption of viruses on the cell surface, their penetration (penetration) into the cell and their undressing (deproteinization). Late stages (viral genome strategy) include viral nucleic acid synthesis, protein synthesis, virion assembly, and release of viral particles from the cell.

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Attachment of viruses to the cell surface is ensured by two mechanisms: nonspecific and specific. Nonspecific is determined by the forces of electrostatic interaction that arises between chemical groups on the surface of viruses and cells that carry different charges. The specific mechanism (reverse and non-reversible adsorption) is determined by complementary viral and cellular receptors. They can be of protein, carbohydrate, or lipid nature. For example, the receptor for influenza viruses is sialic acid. The number of receptors at adsorption sites can reach 3000. On the surface of viruses, receptors are usually located at the bottom of recesses and crevices. The penetration of viruses into the cell occurs through the mechanism of receptor endocytosis (a variant of viropexis) in special areas of cell membranes that contain special block with high molecular weight - clathrin. The membranes invaginate and clathrin-coated intracellular vacuoles form. their number can reach 2000. Vacuoles unite to form receptosomes, and the latter merge with lysosomes. The surface proteins of viruses interact with the membranes of lysosomes, and their nucleoprotein is released into the cytoplasm. However, there is another mechanism for the penetration of viruses into the cell - induction of membrane fusion. It occurs thanks to a special viral fusion protein (F- from fusion - fusion). As a result of this process, the viral lipoprotein envelope integrates with cell membrane, and its genome penetrates the cell. Such a protein has been identified in influenza viruses, parainfluenza, rhabdoviruses, etc.

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The undressing of virions is a multi-step process, during which their nucleic apparatus is released, the protective shells that inhibit genome expression disappear. It occurs in specialized areas - lysosomes and the Golgi apparatus. The later stages of reproduction are aimed at the synthesis of viral nucleic acids and protein. The mechanism of replication (the formation of viral genomes that are an exact copy of the predecessor) depends on the characteristics of the nucleic acid. U different types viruses are not the same. Replication in viruses that contain RNA occurs according to similar patterns. mRNA is synthesized on the mother RNA, and intermediate forms of RNA serve as the template for the synthesis of the viral genome. Transcription is the process of formation of messenger RNA. It occurs with the help of special enzymes called DNA- or RNA-dependent RNA polymerases. DNA viruses have these enzymes of cellular origin, while RNA viruses have their own virus-specific transcriptases. At the translation stage, genetic information is read from messenger RNA and translated into an amino acid sequence. The process occurs in ribosomes. RNA molecules move into ribosomes according to a triplet code sequence that is recognized by transfer RNAs. The latter carry amino acids in special areas.

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Cultivation of viruses Chicken embryos 6-12 days of age. Methods of infection - open, closed

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Cultivation of viruses Cell cultures: - primary trypsinized cultures of human embryos, monkey kidneys, chicken embryo fibroblasts, etc.; capable of growing over several passages as secondary cultures; transplantable cells; they are cell cultures that have acquired the ability for unlimited growth and reproduction; They are obtained from tumors or from normal human or animal tissues that have an altered karyotype. HeLa (cervical carcinoma) Hep-2 (human laryngeal carcinoma), CV (human oral carcinoma), RD (human rhabdomyosarcoma), RH (human embryonic kidney), Vero (green monkey kidney), SPEV (pig embryonic kidney), VNK-32 (Syrian hamster kidney). Cell culture diploid cells; They are cultures of cells of the same type, have a diploid set of chromosomes and are capable of withstanding up to 100 subcultures in laboratory conditions. They are a convenient model for obtaining vaccine preparations of viruses, since they are free from contamination by foreign viruses, retain the original karyotype during passages, and do not have oncogenic activity. Most often, they use culture lines that are obtained from human embryonic fibroblasts (WI-38, MRC-5, MRC-9, IMR-90), cows, pigs, sheep, and the like. Cell cultures are stored frozen.

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Nutrient media that are used to support cell cultures or their growth can be natural or synthetic (artificial). Natural media - cattle blood serum, fluids from serous cavities, milk hydrolysis products, various hydrolysates (5% hemohydrolysate, 0.5% lactoalbumin hydrolysate) or tissue extracts. Their chemical composition helps create conditions that are similar to those that exist in the human body. A significant disadvantage of such media is their non-standard nature, since the qualitative and quantitative composition of the components that make up them can vary. Synthetic nutrient media do not have this drawback, because their chemical composition is standard, because they are obtained by combining various salt solutions (vitamins, amino acids) under artificial conditions. These most commonly used solutions include medium 199 (cultivation of primary trypsinized and continuous cell cultures), Eagle medium (contains a minimum set of amino acids and vitamins and is used for the cultivation of diploid cell lines and continuous ones), EagleMEM medium (cultivation of especially demanding cell lines), solution Hanks, which is used for making culture media, washing cells, etc.

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Infection of laboratory animals. Numerous laboratory animals are widely used in virology to isolate and identify viruses, obtain specific antiviral sera, and study various aspects of pathogenesis viral diseases, developing ways to combat diseases and prevent them. White mice are most often used of different ages(two days old), white rats, Guinea pigs, rabbits, ground squirrels, cottonmouth rats, monkeys and others.

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There are various ways to infect animals, depending on the tropism of the viruses and the clinical picture of the disease. The test material can be administered: - through the mouth - into the respiratory tract (inhalation, through the nose) - cutaneous - intradermal - subcutaneous, intramuscular - intravenous - intraperitoneal - intracardiac - onto the scarified cornea - into the anterior chamber of the eye - into the brain.

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Bacteriophages (phages) (from ancient Greek φᾰγω - “I devour”) are viruses that selectively infect bacterial cells. Most often, bacteriophages multiply inside bacteria and cause their lysis. Typically, a bacteriophage consists of a protein coat and genetic material of single- or double-stranded nucleic acid (DNA or, less commonly, RNA). The particle size is approximately 20 to 200 nm. Independently of Frederick Twort, the French-Canadian microbiologist D'Herelle, Felix reported the discovery of bacteriophages on September 3, 1917. Along with this, it is known that the Russian microbiologist Nikolai Fedorovich Gamaleya, back in 1898, first observed the phenomenon of lysis of bacteria (anthrax bacillus) under the influence of a transplantable agent. Life cycle Temperate and virulent bacteriophages on initial stages interactions with a bacterial cell have the same cycle. Adsorption of bacteriophage on phage-specific cell receptors. Injection of phage nucleic acid into a host cell. Co-replication of phage and bacterial nucleic acid. Cell division. Further, the bacteriophage can develop according to two models: lysogenic or lytic path. Temperate bacteriophages after cell division are in the prophage state (Lysogenic pathway). Virulent bacteriophages develop according to the Lytic model: The nucleic acid of the phage directs the synthesis of phage enzymes, using the bacterial protein-synthesizing apparatus for this. The phage in one way or another inactivates the host DNA and RNA, and the phage enzymes completely break it down; The RNA of the phage “subordinates” the cellular apparatus for protein synthesis. The phage nucleic acid replicates and directs the synthesis of new envelope proteins. New phage particles are formed as a result of spontaneous self-assembly of the protein shell (capsids) around the phage nucleic acid; Lysozyme is synthesized under the control of phage RNA. Cell lysis: the cell bursts under the influence of lysozyme; about 200-1000 new phages are released; phages infect other bacteria. 1 - head, 2 - tail, 3 - nucleic acid, 4 - capsid, 5 - “collar”, 6 - protein sheath of the tail, 7 - tail fibril, 8 - spines, 9 - basal plate

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Infection of the chicken embryo The chicken embryo is used for cultivating viruses and mycoplasmas. Embryos are used at the age of 8-14 days, depending on the type of virus and method of infection; to the chorion-allantoic membrane, into the allantoic and amniotic cavity, into the yolk sac. Before infection, the viability of the embryo is determined in an ovoscope and the boundaries of the air sac are marked with a pencil on the shell. Infection of chicken embryos is carried out in a box under strictly aseptic conditions, using an instrument sterilized by boiling. The shell above the air space is wiped with alcohol, burned in a flame, smeared with a 2% iodine solution, wiped again with alcohol and burned. Viral material in an amount of 0.05 - 0.2 ml is applied to the chorion-allantoic membrane with a tuberculin syringe or Pasteur pipette. The embryos are dissected after 48 - 72 hours of incubation in a thermostat. The presence of the virus in the chrolantoic membrane is determined: 1. By whitish opaque spots of various shapes; 2. In the hemagglutination reaction.

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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 were discovered.

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.

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 the most accessible and convenient method for primary virus isolation. 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 both for the primary isolation of viruses from sick animals and from environmental objects, 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.

Thus, the yolk sac as a reservoir of nutrients has the largest 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 that exhibit active movements with good blood supply to the CAO vessels 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, dissection; 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?

Chicken embryos are a very cheap and convenient system for cultivating influenza viruses. Almost all strains of viruses of both human and animal origin multiply in embryos to one degree or another. The best way laboratory strains adapted to this system give fairly high titers in embryos, or 10-10 PFU per 1 embryo). In addition, the embryo is isolated from the external environment, and therefore the virus does not need to multiply in it. create special sterile conditions.

The virus is usually injected into the fluid-filled allantoic cavity of the egg, from where it can penetrate the chorioallantoic membrane and infect the cells that form it. The viral progeny is released into the allantoic fluid and can be easily removed from the egg by suctioning out this fluid. Some strains V in particular new natural isolates, as well as viruses like WITH do not multiply well on the chorio-callantoic membrane, but multiply when injected directly into the amniotic cavity; in this case, the virus gains access to various tissues of the embryo, and the resulting offspring are released into the amniotic fluid, which can be extracted separately.

The virus multiplies best in 10- to 12-day-old embryos. Fresh fertilized eggs should be purchased and incubated for the required time in the laboratory. For this you do not need to have special equipment, however, the incubator is very convenient. However, eggs can be successfully incubated in a regular thermostat in a humid atmosphere at 37 ° C if they are turned regularly. The grade of eggs is not particularly important, but in some cases it is important to exclude the possibility of infection of the egg with other microorganisms; some suppliers sell special uncontaminated eggs.

Before infection, you should make sure that the eggs are actually fertilized. To do this, just look at the egg near a bright light source in a dark room. 4-5 days after fertilization, the embryo should be clearly visible.

In standard passaging of laboratory strains, the viral inoculum is usually a sample of infected allantoic or culture fluid. Typically, samples are diluted with Hanks' saline solution to a virus concentration of 10-10 PFU/ml before use, since introduction into the egg large quantity virus promotes the formation of DIC. If the diluted virus must be stored before infection, gelatin should be added to the dilution solution. Naturally, it is necessary to ensure the sterility of the virus used for infection; in cases where this is difficult, the antibiotic gentamicin is added to the inoculum.

These methods involve the use of homogenization to grind the samples, low-speed centrifugation to clarify them, and injection of undiluted supernatant into the amniotic cavity.

Infection of embryos.

I. Introduction of the virus into the allantoic cavity

  • 1. Place the eggs on their side and wipe the top with alcohol to sterilization area around the point of infection.
  • 2. A small hole is made in the shell using a dental drill equipped with cutting disc. The hole must be deep enough to allow the needle to be inserted. For injections, but it is advisable not to damage the inner membrane of the shell.
  • 3. Using a syringe with a No. 25 needle, 0.1 ml of virus is inoculated into the allantoic cavity, inserting the needle directly under the shell.
  • 4. The hole in the shell is sealed with a small amount of melted wax.
  • 5. Infected eggs are incubated in a thermostat in a humid atmosphere with the pointed end down. There is no need to turn the eggs during incubation.

II. Injection of the virus into the amniotic cavity

  • 1. The blunt end of the egg is sterilized by wiping with alcohol and a small hole is made, as shown in the diagram.
  • 2. The egg is placed near a bright light source in a dark room so that the embryo is visible.
  • 3. A needle No. 23, 4 cm long, is inserted into the hole made, and then with a sharp movement in the direction of the embryo - into the amniotic cavity. In this case, 0.1 ml of virus is added and the needle is carefully removed.
  • 4. The hole in the shell is sealed with melted wax and the eggs are incubated as above.

Incubation time.

The optimal incubation time required to obtain maximum virus yield depends on the specific strain and must be determined experimentally. For some strains of influenza A virus, for example fowl distemper virus, the maximum yield is achieved after 24-26 hours, while most strains of human influenza A virus, as well as viruses of types B and WITH require 48-72 hours of incubation.

Virus collection.

Before attempting to remove virus-containing liquid from an egg, it is recommended to cool the egg for 4-18 hours at 4°C or for 30 minutes at -20°C. When cooled, the embryo dies and the surrounding blood vessels narrow. Thus, the possibility of contamination of virus-containing liquid with red blood cells, which can bind part of the virus and thereby reduce its yield, is significantly reduced. If contamination with red blood cells cannot be avoided, virus loss can be minimized by incubating the material for 30 minutes at 37°C. Under these conditions, virion neuraminidase destroys the erythrocyte receptors to which the virus binds, and it is released.

I. Collection of allantoic fluid

  • 1. Place the eggs on a stand with the blunt end up and sterilize their surface with alcohol.
  • 2. The blunt end of the egg is opened using either sterile tweezers or special device. The shell around the air sac is removed, allowing access to the allantoic cavity.
  • 3. Using sterile blunt-tipped tweezers, tear the chorioallantoic membrane and move it to the edge of the egg.
  • 4. The embryo and yolk sac are carefully moved away with tweezers and the allantoic fluid is sucked out with a wide-ended pipette. From a medium-sized egg, 7-10 ml of pale yellow liquid is obtained. The admixture of yolk in the allantoic fluid interferes with further purification of the virus, therefore, when selection Damage to the yolk sac should be avoided. If the virus is intended for biochemical studies, for example, to analyze the activity of virion enzymes, allantoic fluid should be collected at 0 °C.
  • 5. Allantoic fluid is clarified by centrifugation for 10 minutes at 10,000 g and store the supernatant at 4 or -70 °C.

II. Collection of amniotic fluid

  • 1. The surface of the egg is sterilized and the blunt end is opened as described above. Remove as much of the shell as possible so that the contents of the egg can be easily removed.
  • 2. The chorioallantoic membrane is ruptured and the contents of the egg are poured into a sterile Petri dish. In this case, the amniotic cavity should remain intact.
  • 3. WITH Using a syringe with a No. 25 needle, the contents are removed from the amniotic cavity as completely as possible.
  • 4. Amniotic fluid is clarified and stored in the same way as allantoic fluid.

Expected output.

The intensity of virus reproduction in embryos depends on the specific strain. Wild strains initially give very low titers, but after several passages in embryos the titer increases significantly. For different laboratory strains adapted for reproduction on embryos, the yield also varies significantly, but in most cases 2000-5000, and sometimes up to 20,000 HAE/ml of virus accumulate in the allantoic fluid. It is obvious that adaptation to reproduction in embryos is associated With selection of genetic variants, so you should have V Keep in mind that the results of detailed genetic and biochemical studies of strains passaged on embryos do not necessarily characterize the original isolate.

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