Biochemical properties of tularemia. The causative agent of tularemia

Tularemia is a primary disease of animals (usually rodents); in humans it occurs as an acute infectious disease with a varied clinical picture and slow recovery. The causative agent of tularemia - Francisella tularensis - was discovered by G. McCoy and S. Chapin in 1912 during an epizootic among ground squirrels in the area of ​​Tulare Lake (California), studied in detail by E. Francis, after whom the genus was named.

Small, 0.2-0.7 µm in size, coccoid or ellipsoidal polymorphic rods, which very often give a bipolar color with special staining methods; nonmotile, gram-negative, do not form spores; catalase-negative, form H2S, strict aerobes, optimum temperature for growth 37 °C, pH 6.7-7.2. Virulent strains have a capsule, form acid without gas during the fermentation of certain carbohydrates (glucose, maltose, mannose, fructose, dextrin), the degree of fermentation varies among different strains, the G + C content in DNA is 33-36 mol%. F. tularensis does not grow on regular media. G. McCoy and S. Chapin used coagulated yolk medium. On it, the tularemia bacillus grows in the form of delicate small colonies, reminiscent of dew drops, then the culture takes on the character of a delicate shagreen coating with a weakly expressed mucous consistency. E. Francis proposed a nutrient agar containing 0.05-0.1% cystine, 1% glucose and 5-10% blood for growing tularemia bacillus. On such a medium, growth is more lush and coarse: the colonies are round with a smooth surface, milky in color, moist, with a slimy consistency, surrounded by a characteristic green halo. Growth is slow, the maximum size of the colony is reached on the 3-5th day (1 - 4 mm). Tularemia bacteria multiply well in the yolk sac of a chicken embryo, causing its death on the 3-4th day.

For the growth of F. tularensis, the following amino acids are required: arginine, leucine, isoleucine, lysine, methionine, proline, threonine, histidine, valine, cystine, for some subspecies - series, tyrosine, aspartic acid; In addition, for growth they also need pantothenic acid, thiamine and Mg2 ions. Taking these features into account, synthetic media can be used for cultivating F. tularensis.

The genus Francisella is classified in the class Gammaproteobacteria, phylum Proteobacteria. F. novicida belongs to the same genus, the pathogenicity of which for humans has not been established.

F. tularensis in the S form (virulent) has two antigens - O and Vi (capsular antigen). The O antigen is related to Brucella antigens. Dissociation S->SR->R leads to loss of capsule, virulence and immunogenicity. The species F tularensis is divided into three geographical races (subspecies):

Holarctic (lowly pathogenic for domestic rabbits, does not ferment glycerol and does not have the enzyme citrulline ureidase, found in countries of the northern hemisphere);

Central Asian (low pathogenic for rabbits, has citrulline ureidase and ferments glycerol);

Nearctic (American), more pathogenic for rabbits, ferments glycerol, has citrulline ureidase.

In addition, strains of the American and Central Asian subspecies have phosphatase activity, which is absent in strains of the Holarctic subspecies.

Resistance of the causative agent of tularemia

F. tularensis is quite stable in the external environment, especially if contained in pathological material. In fodder and grain contaminated with secretions of sick rodents, survives up to 4 months; in water - up to 3 months; in ice - more than 1 month. Sensitive to direct sunlight (dies in 30 minutes), high temperature (at 60 ° C, dies in 10 minutes), under the influence of a 3% solution of Lysol, 50% alcohol, formaldehyde and other antiseptics, dies in 5-10 minutes.

Epidemiology of tularemia

The main reservoir of tularemia in nature is rodents, among which epizootics are observed in natural conditions. It is infected only from animals; the pathogen is not transmitted from person to person. The pathogen was found in 82 species of rodents and lagomorphs, most often found in representatives of 4 families: mouse-like (Muridae), hare-like (Leporidae), sciuridae (Sciuridae) and jerboa (Dipodidae). On the territory of Russia, the main carriers are mouse-like rodents: water rats, common voles, house mice and muskrats.

Based on sensitivity to tularemia, animals can be divided into four groups:

Group 1 - the most susceptible (voles, water rats, house mice, white mice, guinea pigs and some others). The minimum lethal dose is one microbial cell;

Group 2 - less sensitive (gray rats, gophers, etc.). The minimum lethal dose is 1 billion microbial cells, but one microbial cell is enough to infect some of them;

3rd group (predators - cats, foxes, ferrets). Resistant to high infectious doses, the disease occurs without visible manifestations;

Group 4 - immune to tularemia (ungulates, cold-blooded animals, birds).

For humans, the minimum infectious dose is one microbial cell. Human infection occurs in all possible ways: direct and indirect contact with sick rodents, their corpses or objects contaminated with rodents; nutritional (by consuming food and water infected with rodents), airborne dust and transmission. Infection with tularemia bacteria has been established in 77 species of blood-sucking arthropods. Ixodid ticks are especially important, in which the pathogen persists throughout life and is even transmitted transovarially to offspring. These circumstances contribute to the rooting of the disease in nature. Infection of a person with ticks does not occur through a bite, but as a result of the pathogen coming into contact with the skin along with tick feces.

On the territory of Russia, there are 7 main landscape types of natural foci of tularemia: swamp, meadow, steppe, forest, foothill stream, tundra and tugai (desert).

Symptoms of tularemia

The causative agent of tularemia enters the body through the outer integument (damaged and undamaged skin and mucous membranes). Ulcers often form at the injection site. Through the lymphatic vessels, bacteria enter the regional lymph node and multiply there unhindered; the inflammatory process leads to the formation of a bubo. From here the pathogen penetrates into the blood, bacteremia causes the generalization of the process, various organs and tissues are involved in it, the proliferation of bacteria in which leads to the formation of granulomas and necrotic ulcers. Allergic restructuring of the body is associated with bacteremia and generalization. The incubation period of tularemia varies from 2 to 8 days. The disease begins acutely: fever, headache, muscle pain, and facial flushing appear. The further course depends on the location of the entrance gate, according to which the following clinical forms of tularemia are distinguished: ulcerative-glandular (bubonic), oculoglandular, anginal-glandular, abdominal and pulmonary. The mortality rate for tularemia does not exceed 1-2%.

Post-infectious is durable, persistent, in most cases lifelong, has a cellular nature, is caused mainly by T-lymphocytes and macrophages, and to a lesser extent by antibodies. Phagocytosis in individuals with is complete.

Diagnosis of tularemia

All microbiological methods are used to diagnose tularemia. The research is carried out in secure laboratories. The material for research - blood, punctate from a bubo, scraping from an ulcer, conjunctival discharge, plaque from the pharynx, sputum, etc. - is determined by the clinical form of the disease. In addition, you can take water and food products for testing. In natural foci of tularemia, planned systematic studies are carried out to isolate the causative agent of tularemia from rodents.

The bacteriological method for diagnosing tularemia in humans rarely gives positive results. A pure culture is usually isolated after accumulation on susceptible laboratory animals. White mice and guinea pigs are used for bioassays. Mice are infected subcutaneously, guinea pigs - intraperitoneally; animals die on the 3rd-6th day, sometimes after frost. Infected animals are kept in special conditions (as when diagnosing plague) and observed for 6-14 days. Gels, experimental animals do not die for 7-15 days; they are killed on the 15-20th day and the corpses are opened. In the presence of tularemia, pathological and anatomical changes are detected in the form of a productive process with necrosis. A pure culture is isolated from internal organs on a yolk medium, a glucose-cysteine ​​blood focus, etc. Identification is based on the morphology and tinctorial properties of the pathogen, the lack of growth on MPA, and agglutination with homologous serum. pathogenicity for white mice and guinea pigs. A pure culture can be isolated by infecting 12-day-old chick embryos and the yolk sac. To isolate a pure culture of the pathogen from water, it is centrifuged or filtered through bacterial filters and the sediment is used to infect laboratory animals. When studying food products, they are washed with MP B, centrifuged, and laboratory animals are infected with the sediment.

Simultaneously with the bacteriological examination, fingerprint smears are prepared from the material being studied and stained according to Romanovsky-Giemsa. In smears from organs, small coccoid and rod-shaped bacteria can be found, which are located intracellularly and in the form of clusters, forming a delicate capsule.

For diagnosis, a detailed agglutination reaction, RPGA, and RIF are used.

Allergy tests are used for early diagnosis of tularemia (from the 5th day from the onset of the disease). Two types of tularin are used and, accordingly, two methods of their administration: cutaneous and intradermal. Since the concentration of the allergen in both types of tularin is different, it is unacceptable to use cutaneous tularin for an intradermal test and vice versa. The results of an allergic reaction are taken into account over time after 24, 36, 48 hours. An infiltrate with a diameter of at least 5 mm is taken as a positive result. In individuals who have been vaccinated or have recovered from tularemia, allergy tests remain positive for a number of years (anamnestic reaction).

Specific prevention of tularemia

For specific prevention, a vaccination against tularemia is used, obtained in 1930 by domestic military doctors B.Ya. Elbert and N.A. Gaisky from strain Me 15. The vaccine provides lasting immunity for 5-6 years when infected with the European and Holarctic subspecies and is effective against the American variety of the pathogen. Vaccination is carried out according to epidemiological indications, as well as to persons belonging to risk groups. Simultaneous vaccination against tularemia and brucellosis is allowed; tularemia and plague, and against tularemia and some other infections.

Nonspecific prevention of tularemia is the same as for other zoonoses and is aimed primarily at combating rodents.

Taxonomy. The causative agent of tularemia, Francisella tularensis, belongs to a genus with an unclear position in the taxonomy of bacteria. It was isolated in 1912 by J. McCoy and C. Chapin in the area of ​​Tulare Lake, located in California, and was comprehensively described by E. Francis.

Clinic and epidemiology. Depending on the entry point of infection, tularemia affects the skin, mucous membranes and lymph nodes (cutaneous-bubonic, anginal-bubonic forms) or internal organs (abdominal and pulmonary tularemia). There are also septic or generalized forms of the disease. Tularemia is a naturally occurring, particularly dangerous blood-borne zoonotic infection that produces a high mortality rate. The pathogen circulates in nature among 70 species of vertebrates. The main sources of infection are water rats, muskrats, hares, gray and black rats, and mice. Large and especially small cattle (sheep, cats and dogs) can get sick. A sick person is non-contagious and is a biological dead end for the causative agent of tularemia.

The mechanism of transmission of infection is varied. Tularemia is mainly transmitted transmissibly through blood-sucking arthropods (about 60 species in total). Among them, the main epidemiological significance is of ticks, which are not only carriers, but also reservoirs of tularemia in nature, since they are capable of transmitting the pathogen transovarially. People often become infected through contact, nutritional and aspiration routes.

Biological features of the causative agent of tularemia. Morphology. The causative agent of tularemia is small polymorphic microorganisms. In cultures, coccal forms (0.1-0.5 µm) are more often found, in animal organs - coccobacteria (0.7-1.0 µm). Virulent strains form a delicate capsule, while avirulent strains are often covered with a massive layer of mucus. They do not form spores and are motionless. In the smear, tularemia bacteria are located randomly, gram-negative (sometimes metachromasia is observed).

Cultural properties. Tularemia bacteria are grown aerobically at 37°C on coagulated egg yolk medium, glucose cystine blood agar, or regular agar with yeast lysates, gelatin, and fishmeal extracts. Colonies appear on them within a few days, sometimes after 2-3 weeks. They have point sizes (1-2 mm), round shape (Fig. 91), white or milky-white color, and with aging they form SR- and R-forms; mucus accumulates in cultures.

Antigens. The causative agent of tularemia contains two antigens: the somatic nucleoprotein O-antigen and the envelope protein-lipid Vi-antigen, the loss of which leads to dissociation of the microbe, loss of virulence and immunogenicity. Tularemia bacteria have sensitizing properties, have group antigens with the causative agent of brucellosis and are agglutinated in anti-RUcellosis serum.

Enzymes. The biochemical properties of Francisella are variable. They constantly ferment glucose, maltose, mannose, and form hydrogen sulfide.

Pathogenicity. White mice are most sensitive to tularemia, and guinea pigs are less sensitive. When infected with material containing an infectious principle, animals die from a septic process within 4-10 days, often after 1 month.

Geographical options. Based on virulence and biochemical activity, three types of tularemia bacteria are distinguished: 1) American (nearctic) - highly pathogenic for rabbits (DCLM = 1 cell), fermenting glycerol and containing citrulline rheidase; 2) European (Holarctic) - low virulent for rabbits (DCLM = 1 billion bw) and biochemically inert; 3) Central Asian, which is similar in enzymatic properties to the American one, and similar in virulence to the European one.

Immunity. People's susceptibility to tularemia is high, but those who have had the disease develop a very intense and long-lasting immunity, which is reflected in an increase in the level of specific antibodies, activation of T-lymphocytes, phagocytes and allergic restructuring of the body, which persists for many years.

Laboratory diagnostics. The main microbiological methods confirming the diagnosis of tularemia are: positive allergy tests (5 days), full-blown RA (10-14 days) and RSC (5-7 days).

Allergic reaction. To identify it, an intradermal test with tularin is used or tularin is injected into the skin incisions. The results of allergic reactions are taken into account after 24-36-48 hours. Usually, severe hyperemia and an infiltrate with a diameter of 5 mm develop at the site of allergen injection. It should be taken into account that the same positive test can occur in convalescents and persons vaccinated against tularemia.

Deployed RA. Antibodies to the causative agent of tularemia appear in the second week of illness (after the 10th day). To diagnose full-blown RA, it is enough to take 2-3 ml of blood from the patient. The resulting serum is sequentially diluted from 1: 25 to 1: 400. Then 0.5 ml of tularemia diagnosticum is added to each test tube, as a result the serum is diluted from 1: 50 to 1: 800. Serum and diagnosticum are monitored as usual (Table 19 ).

Table 19. Scheme for setting up a deployed RA

---------						Control
Ingredients, ml		Tube number		serum	diagnosticum
Isotonic sodium chloride solution		0,5	0,5	0,5	0,5	0,5	0,5
Patient serum diluted 1:25	0,5	0,5	0,5	0,5	0,5	0,5
	1:25	1:50	1:100	1:200	1:400
Diagnostics!*!	0,5	0,5	0,5	0,5	0,5	-	0,5
Final serum dilution	1:50	1:100	1:200	1:400	1:800	1: 50
Accounting for results

The test tubes are placed in a thermostat for 2 hours and, after removal from it, kept at room temperature for another 18-20 hours, after which the final count is carried out. The diagnosis is confirmed by RA in a serum dilution of 1: 100. When RA is re-staged after 7-10 days, its titer can increase 4 times or more, which is very important when differentiating tularemia from brucellosis, the causative agents of which, having a common antigenic structure with F. tularensis, may agglutinate in the serum of patients with tularemia. RIGA and RSK are considered more sensitive in the cold.

During mass examinations, instead of full-blown RA, a blood-drop reaction can be used: a drop of blood is applied to a glass slide and hemolyzed in distilled water, followed by the addition of tularemia diagnosticum. Instantaneous agglutination of Francisella in a hemolyzed drop of blood corresponds to an antibody titer of 1: 100, and sticking of the diagnosticum after 2-3 minutes. – titer 1:50.

Isolation of culture. Obtain a pure culture of bactularemia from pus, bubo punctate, mucus from the throat

or the patient's blood by direct inoculation on nutrient media is not possible. To activate the growth potential of the pathogen, 3-5 white mice or 2 guinea pigs are infected with human material. Infected animals die after 1-2 weeks, and if they survive, then on the 15-25th day they are killed and a blind passage of organ emulsion is carried out into intact mice and pigs.

The F. tularensis culture is isolated by inoculating imprints of sections of the liver and spleen of infected animals into egg yolk medium and glucose cystine agar with 5-10% defibrinated rabbit blood or containing 2.5% yeast autolysate, 10% gelatin hydrolysate and 20% fish hydrolysate flour. It is also possible to grow the causative agent of tularemia in the yolk sac of a 12-day chicken embryo.

A pure culture is identified by the presence in smears of colonies of small, randomly located coccobacteria that agglutinate in the diagnostic serum.

To detect tularemia antigens in dead rodents in enzootic foci, the Ascoli thermo-colceprecipitation reaction is widely used.

Specific prevention and treatment. Specific prevention of tularemia is carried out with a live dry attenuated vaccine from the 15th line strain of NIIET. Persons at risk of infection with tularemia, who react negatively to tularin and who have low antibody titers are subject to vaccination. There are two methods of administering vaccines: cutaneous and intradermal. The first is used for routine vaccination of children aged 7 years and older, the second - for epidemic indications. Those vaccinated are revaccinated after 5 years. Tularemia is treated with aminoglycosides, tetracyclines, chloramphenicol, combining them with the administration of a killed vaccine for a torpid form of infection.

The causative agent of tularemia is Francisella - very small polymorphic bacteria. Included in the department Cracilicutes, section 4 (Gram-negative aerobic rods and cocci). Genus Francisella presented in two types, one of them is Francisella tularensis – pathogenic. This species causes a natural focal infectious disease of animals - tularemia - characterized by fever, paralysis in young animals, enlarged lymph nodes, and abortion.

The tularemia bacterium was isolated in 1912 by McCoy and Chapin while studying a plague-like disease in ground squirrels in Tulare County (California). Genus Francisella named after Francis, who first studied the biology of this microbe. Within a view F. tularensis There are three geographical races: Holarctic, Central Asian and Nearctic, differing in some biological features.

Morphology. IN In stained smears, the causative agent of tularemia has a coccoid or rod-shaped shape, 0.3-0.7 µm in length and 0.2-0.4 µm in width; there are smaller cells (0.15 µm or less) that can pass through bacterial filters. Coccoid forms are more often found in crops, rod-shaped ones - in animals. The bacterium is characterized by polymorphism, which is revealed during growth on nutrient media: in preparations from cultures, along with typical bacteria, spherical and filamentous forms can be found.

The microbe is immobile, does not form spores, and has a small capsule; in cultures produces mucus, which is easily detected when making smears.

The pathogen is stained with all aniline dyes, but is noticeably paler than other bacteria and is gram-negative. In smears-imprints from the organs of dead animals, it stains well according to Romanovsky-Giemsa, acquiring a lilac color. In tissues, bacteria do not stain bipolarly, which is why they differ from Pasteurella.

Cultivation. The bacterium does not grow on universal nutrient media. For its cultivation, folded McCoy's yolk medium (60% chicken egg yolk and 40% saline solution) is used. They also use Francis medium (2.5% meat peptone agar, 0.1% cystine, 1% glucose and 5-10% defibrinated rabbit blood), semi-liquid Drozhevkina yolk medium (10% chicken yolk and 90% sterile saline), blood fish -yeast agar with glucose and cystine, etc.

The tularemia bacterium is a strict aerobe, the optimum temperature is 36-37 0 C, the pH of the environment is 7.2-7.0. On a coagulated yolk medium with abundant growth, microbes grow in the form of a shiny thin coating with a tortuous (“shagreen”) surface; with sparse growth, small shiny convex colonies or groups of colonies grow. On Francis medium, the culture looks like small (1-2 mm), round, convex, smooth, shiny colonies with smooth edges, whitish in color with a bluish tint; growth is observed after 2-3 days. Colonies of pathogenic strains have an S-shape. In liquid nutrient media, the tularemia microbe grows much worse (only on the surface of the medium). The bacteria also multiply well in the yolk sac of the developing chick embryo.

Biochemical properties. The tularemia bacterium does not have pronounced biochemical activity. The ability to ferment carbohydrates and alcohols is limited and can only be reliably detected on special dense media with a reduced protein content and a certain pH. Hiss media is not suitable for this purpose. The microbe ferments glucose, maltose, and in some cases levulose and mannose with the formation of acid without gas; does not ferment lactose, sucrose, rhamnose, mannitol; forms hydrogen sulfide and reduces thionin, methylene blue, malachite green.

Antigenic structure. Pathogenic variants of the causative agent of tularemia (S-form) have two antigenic complexes localized on the cell surface. The first of them - Vi-antigen - contains lipids and proteins, determines the virulence and immunogenicity of the microbe; the second - O-antigen - is located in the cell wall and capsule-like layer of the bacterium, a thermostable glycoprotein. Both of these complexes have allergenic and antigenic properties, induce the formation of agglutinating, precipitating and complement-fixing antibodies, as well as delayed-type hypersensitivity. The function of the allergen in this bacterium is performed by a polysaccharide-polypeptide complex. The Vi antigen of pathogenic variants of the causative agent of tularemia is similar to the similar antigen of Brucella.

Sustainability. IN water or moist soil at 4 0 C remains without a decrease in virulence for over 4 months, in water at 20-25 0 C - 10-15 days, in grain and straw at temperatures below 0 0 C - up to 6 months, at 8-12 0 C – 56 days, at 20-30 0 C – no more than 20 days. The pathogen is viable in frozen meat for up to 93 days, in milk and cream at 8-10 0 C for at least 3 weeks, in frozen milk for up to 104 days. In frozen corpses of animals that died from tularemia - over 3 months, in their skins at 8-12 0 C - more than a month, at 32-33 0 C - 1 week. The microbe is resistant to drying.

Particularly sensitive to ethyl alcohol (dies in 0.5-1 minutes). Sensitive to disinfectants - Lysol, phenol, creolin, but most of all to bleach. Unresistant to many antibiotics - streptomycin, chloramphenicol, tetracycline, neomycin, kanamycin; resistant to penicillin.

Pathogenicity. The bacterium is pathogenic for hares, voles, house mice, gophers, and rats. Farm animals are relatively resistant to tularemia, they become ill sporadically, and the disease often occurs in a latent form. Lambs and piglets are most susceptible; horses and donkeys are affected. In cattle, the disease is accompanied by enlarged lymph nodes and mastitis. Buffaloes, camels, and reindeer are sensitive. Adult sheep are resistant to the disease, goats are even more resistant. Rabbits are susceptible; the disease occurs without characteristic signs and may be similar to pseudotuberculosis and the chronic form of pasteurellosis. Of the birds, chickens, especially chicks, are susceptible. Guinea pigs and white mice are susceptible to infection.

Humans also suffer from tularemia, but the disease is relatively benign and the patient does not pose a danger to others.

The true exotoxin of this microbe has not been isolated, but it synthesizes pathogenic enzymes: asparaginase, hyaluronidase, glutacinase, deaminase, transamidase, uronidase, fibrinolysins. Uronidase is found only in virulent strains. It is believed that the pathogenic effect of the tularemia microbe is mainly due to endotoxin.

Pathogenesis. Infection occurs through alimentary, air-dust and vector-borne routes. Bacteria can enter the body through intact skin, conjunctiva, and respiratory tract. The pathogen, multiplying at the site of penetration, first enters the lymph nodes, then penetrates the blood and causes septicemia. The symptom complex is determined by the species and age resistance of animals, as well as the ability of the pathogen to multiply in organs rich in reticuloendothelial elements.

Epizootological data. The disease is registered in America, Europe, Asia and Africa, as well as in Russia. Tularemia most often occurs during years of mass reproduction of rodents. Rodents (field mice, house mice, gray and water rats, muskrats, etc.), hares, rabbits, etc. are susceptible to tularemia; they create a reservoir of the pathogen in nature. Farm animals also get sick, especially sheep, and lambs are most seriously ill. Among laboratory animals, white mice and rats, guinea pigs and rabbits are susceptible. Dogs and cats are insensitive to the pathogen. A very sensitive person.

The disease in farm animals is recorded sporadically, and in rodents, especially muskrats, it manifests itself as an epizootic. Outbreaks of tularemia are recorded seasonally (usually in spring and autumn) during the period of mass reproduction and intensive migration of rodents, as well as during the period of activity of blood-sucking insects. Natural foci of tularemia, which remain active for up to 50 years or more, are confined to areas inhabited by rodents. Outbreaks of tularemia in sheep are preceded by epizootics of this disease in hares.

Pre-mortem diagnostics. Clinical manifestation is observed only in sheep. In other animals it proceeds latently. In sheep, a rise in temperature to 41.6 0 C, depression, unsteadiness of gait, and later dragging of the rear, paralysis, diarrhea, anemia of the mucous membranes, and coma are noted.

Post-mortem diagnostics. Phenomena of bacteremia with damage to the blood vessels of the lungs, spleen, heart, and lymph nodes. Lymph nodes are sharply enlarged, with necrotic nodules or abscesses.

Laboratory diagnostics. When collecting, delivering to the laboratory and testing material for tularemia, the precautions provided for in the rules for working with especially dangerous infections are observed. The material for the study is the liver, kidney, spleen, enlarged lymph nodes taken from the corpses of large animals; Rodent corpses are sent in their entirety.

The material examination scheme includes bacterioscopy, isolation of pure cultures, and biological testing.

Imprint smears from animal organs are stained according to Romanovsky-Giemsa; take into account large accumulations of lilac-colored coccobacteria. Bacterioscopy should be considered as an indicative method.

To indicate bacteria, a direct immunofluorescence reaction is used, however, this method is a signal one, and positive results must be confirmed by isolating a culture of the pathogen. For this purpose, the pathological material is inoculated on special nutrient media (McCoy's folded yolk medium, Drozhevkina and Emelyanova media). At the same time, control inoculations are made on MPA and in MPB, which are incubated under aerobic and anaerobic conditions at a temperature of 37 0 C. With abundant inoculation, the growth of tularemia bacteria on the coagulated yolk medium appears in the form of a continuous coating after 18-24 hours and reaches a maximum after 2-3 days ; with sparse inoculation, individual colonies are noticeable on the 3-5th day and later. Therefore, it is recommended to incubate the sown media for 10-14 days. On Drozhevkina's medium, the microbe grows diffusely and the presence of microbes is controlled by microscopic examination of smears. A freshly isolated culture is identified by morphological (non-motile coccobacteria), tinctorial (Gram-negative bacteria) properties, growth pattern on coagulated yolk medium, lack of growth on universal nutrient media, as well as by the results of test tube RA with specific agglutinating serum.

Biological sample. The most sensitive and reliable method for detecting tularemia bacteria in any material. They infect white mice, less often guinea pigs. A suspension of pieces of organs and lymph nodes is administered in a dose of 0.5 ml subcutaneously or intraperitoneally or rubbed into a freshly trimmed area of ​​skin. White mice die after 3-4 days, sometimes after 8-12 days, guinea pigs - on 4-6 days, with weakly infected material - within 8-20 days.

Serological diagnosis. Carry out using reactions of agglutination, precipitation, indirect hemagglutination and neutralization of antibodies.

RA is a fairly accurate method of testing for tularemia. The antigen is tularemia diagnosticum, prepared from microbial cells killed with formaldehyde. RA is diagnosed in two ways: test tube and blood-drop. Diagnostic titers for tularemia should be considered: for sheep – 1:25, for cattle and pigs – 1:100.

The indirect hemagglutination reaction (IRHA) is performed with red blood cells sensitized with tularemia antigen or with an antibody erythrocyte diagnosticum. In the first case, it is used to study the sera of agricultural and wild animals for the presence of specific antibodies, in the second - to determine the antigen in animal corpses. The precipitation reaction has relatively low sensitivity and is used mainly in the study of rodent corpses.

Allergic method. Delayed-type hypersensitivity in animals with tularemia develops early (until the fifth day of illness) and persists for a long time, so the allergic method can be used for early and retrospective diagnosis. The allergen is tularin; the drug is administered intradermally, the reaction is taken into account twice - after 24 and 48 hours.

Specific prevention. Animals that have recovered from the disease develop stable and long-lasting immunity, which is based on tissue and humoral mechanisms. Agglutinins are found in the sera of recovered animals, and cellular defense reactions are formed quite early.

For preventive immunization of humans, a dry live vaccine against tularemia is used, proposed in 1946 by N. A. Gaisky and B. Ya. Elbert.

No vaccine has been developed for farm animals.

Veterinary and sanitary assessment and measures. Sick animals are not allowed to be slaughtered. If tularemia is detected after slaughter, all slaughter products with skin are destroyed. Carcasses and organs suspected of being contaminated with tularemia pathogens are sent for disposal.

The veterinary service of the enterprise is obliged to report (in the prescribed manner) the veterinary department of the regional (territorial) department of agriculture, the Ministry of Agriculture of the Russian Federation or the main veterinary department of the ministry and local health authorities at the location of the enterprise about all cases of detection of animals with tularemia before or after slaughter. .

To disinfect slaughter sites, use a 2% solution of caustic soda or potassium (70 0 C), a 3% solution of xylonaphyte-5, and a 2% solution of formaldehyde.

Tularemia- zoonotic acute infection with natural focality. Characteristics of the pathogen. The pathogen was discovered in 1911. McCoy and Chapin in California during an earthquake near Lake Tulare, from where rodents began to move into human habitats and
A disease called tularemia emerged among humans. Francis studied the pathogen. The causative agent is named Francisella tularensis and belongs to the Gracilicutes department, genus Francisella.

Morphology: small rods or cocci, sometimes in the form of dumbbells (polymorphic), do not have spores or flagella; may form a capsule. Tintorial properties: gram"-", in smears-imprints from organs stained according to Romanovsky - Giemsa, have a soft purple color. Cultural properties: facultative anaerobes, do not grow on simple media, but only in media with the addition of yolk or cystine. On solid nutrient media they form whitish shiny colonies with a smooth edge; in liquid media - surface growth. Biochemical properties: low activity, break down carbohydrates into acid; ferment glycerol, sometimes forming hydrogen sulfide. Antigenic structure: contain envelope Vi- and somatic O-antigens. The Vi antigen determines virulent properties. Pathogenicity factors: virulent properties are due to Vi-antigen and toxic substances such as endotoxin. Resistance: The causative agent of tularemia is well preserved in the external environment, especially at low temperatures. Epidemiology of the disease. The source of this disease is rodents, most often water voles, muskrats, house mice, and hares. Transmission ways: transmission(main route of transmission) - through the bites of insects, ticks, mosquitoes, less often fleas; contact-household- through damaged skin or mucous membrane of the eyes; food- when consuming contaminated water or food products; airborne dust- when inhaling airborne dust or droplets contaminated with rodent secretions. The disease is not transmitted from person to person. Rural residents and people of certain professions who are constantly in contact with infected animals are more likely to get sick.

Pathogenesis and clinical picture of the disease. Entry gate: damaged skin and mucous membranes of the respiratory tract and digestive tract. Once in the body, the pathogen spreads throughout the body through the lymphatic vessels. The toxins of the pathogen affect the lymph nodes, resulting in the formation of buboes. Depending on the route of penetration, there are bubonic form, cutaneous bubonic, ulcerative bubonic, ocular, intestinal, pulmonary and septic forms of tularemia. The incubation period is 3-7 days. As a result of inflammation of the lymph nodes, they increase in size (from a hazelnut to a chicken egg). Buboes often fester and take a long time to heal. In the generalized form, when the pathogen enters the blood, there is a high temperature (38-40°C), chills, muscle pain in the back, legs, damage to the spleen, liver, respiratory tract, and digestive tract. Based on severity, they distinguish between mild, moderate and severe forms of tularemia, and based on duration - acute and protracted forms. On average, the disease lasts 16-30 days and, as a rule, ends with recovery. With the disease, allergization to pathogen antigens develops. Immunity. After the disease, strong immunity remains. Laboratory diagnostics. Material studied: swabs, blood, contents of buboes. Diagnostic methods: skin-allergic test with tularin; serological method with tularemia diagnosticum;

These 2 methods are used most often: bacteriological- isolation of a pure culture (carried out only in special laboratories): for this purpose, guinea pigs are infected intraperitoneally with the test material (it is not possible to isolate a pure culture directly from a patient), which die after 5-7 days, they are opened, fingerprint smears are made from the organs (bacterioscopic method), and the material is inoculated onto a coagulated yolk medium; bioassay- infection with pathological material of white mice or guinea pigs; guinea pigs die on the 4th-6th day. To detect the pathogen in dead animals (in cadaveric material), microscopy of fingerprint smears from organs, isolation of a pure culture, a bioassay, as well as a thermoring precipitation reaction with a pre-boiled suspension from the liver or spleen of animals are used.

Treatment. Aminoglycosides are used - streptomycin, kanamycin, as well as chloramphenicol, erythromycin. The pathogen is not sensitive to penicillin and sulfonamides.

Prevention.

General prevention- rodent control, protection of water sources, sanitary education, protection of food warehouses from mice and rodents.

Specific prevention- for epidemic indications, a live vaccine (strain N15) obtained by Gaisky and Elbert is used. Immunity lasts 5-6 years.

Tularemia is a particularly dangerous infection. The disease is included in the group of acute zoonotic infections that have a natural focality. The causative agent of tularemia is a small bacterium resistant to low temperatures and high humidity.

In nature, bacteria affect hares, rabbits, water rats, and voles. When in contact with a sick animal, microbes are transmitted to humans. The source of infection can be contaminated food and water. Pathogens can be acquired by inhaling infected dust, which is formed during the grinding of bread and the processing of grain products. The infection is carried by horseflies, ticks and mosquitoes.

The symptoms of tularemia are vivid. The disease is severe in the form of bubonic, intestinal, pulmonary and septic forms. The lymph nodes of the axillary, inguinal and femoral areas are most often affected.

Rice. 1. The photo shows the sites of rodent bites due to tularemia.

The causative agents of tularemia are highly sensitive to antibiotics of the aminoglycoside and tetracycline group. The suppurating lymph nodes are opened surgically.

Vaccination against tularemia protects against the disease for a period of 5 - 7 years. Disease surveillance activities are aimed at preventing the introduction and spread of infection. Timely identification of natural foci of disease among animals and implementation of deratization and disinfestation measures prevent diseases among people.

Tularemia is a highly contagious disease. It is included in the list of products subject to regional (national) supervision.

The causative agent of tularemia

The disease received its name “Tularemia” in honor of Tulare Lake (California), where a disease similar in clinical picture to plague was discovered in ground squirrels. Bacterium named after researcher E. Francis, who established the fact of transmission of the disease to humans.

It is a gram-negative rod (Gram stained pink), which means the bacterium has a capsule. The causative agent of tularemia is an aerobe. Does not create a dispute.

Rice. 2. The photo shows the bacteria Francisella tularensis under a microscope (left, Gram stain) and computer visualization of pathogens (right). The causative agent of tularemia has the form of a coccobacilli, but may have the appearance of filaments.

Tularemia bacteria have the following abilities that determine their pathogenicity:

• adhesion (sticking to cells);
• invasion (penetration into tissue);
• intracellular reproduction in phagocytes with subsequent suppression of their killer effect;
• the presence in bacteria of receptors for the Fc fragments of IgG (class G immunoglobulins), which leads to disruption of the activity of the complement system;
• when microbes are destroyed, they are released endotoxins. They play a leading role in the pathogenesis of the disease and determine its clinical manifestations;
• toxins and components of microbial cells have strong allergenic properties, which contributes to even greater tissue damage.

Antigenic structure of bacteria

The O and Vi antigens were found in virulent forms of tularemia bacteria.

• Vi-antigen (envelope). The virulence of bacteria and immunogenicity depend on it.
• O-antigen (somatic). In tularemia bacteria, the somatic antigen is endotoxin.

Resistance of bacteria in the external environment

The causative agents of tularemia exhibit high resistance in the external environment:

• they remain viable in water and moist soil at a temperature of 4°C for up to 4 months, and for up to 2 months at a temperature of 20–30°C;
• in straw and grain crops, bacteria persist for up to 6 months at a temperature of 0°C;
• bacteria remain in the skins of killed animals for up to 20 days, and in their excrement for up to 120 days;
• Bacteria can be stored in frozen meat for up to 6 months, and in milk for up to 8 days.

When boiled, bacteria die instantly; when exposed to sunlight, they die after 30 minutes. Solutions of sublimate, chloramine and 50% alcohol have a detrimental effect on bacteria.

Rice. 3. The photo shows a colony of tularemia pathogens.

When growing on solid nutrient media, they are white with a bluish tint.

Epidemiology of tularemia

In the Russian Federation, 50 to 380 cases of human tularemia are registered annually. These are mainly small or isolated outbreaks of the disease in the summer-autumn periods, caused by tick attacks, handling of muskrat and hare carcasses, and consumption of infected food and water. The mechanization of agriculture has minimized cases of mass accumulation of small rodents and mice in agricultural fields. Persons with summer cottages and garden plots, hunters and fishermen, geologists and agricultural workers are at risk.

Places where rodents actively breed are particularly dangerous for tularemia.

Rice. 4. The photo shows carriers of tularemia pathogens.

Reservoir of infection

• In nature in the Russian Federation, tularemia bacteria most often affect hares, rabbits, hamsters, water rats and voles. Their disease progresses rapidly and always ends in death. Black rats, gophers and ferrets also suffer from tularemia. The second place for tularemia is in cattle, pigs and sheep.
• The source of infection can be contaminated food products.
• Water can become a source of infection. Voles living along the banks of rivers, lakes and ponds pollute the water. The source of infection can be water from random abandoned wells. The causative agents of tularemia make water bodies long-term reservoirs of infection.
• Infected dust particles that are formed during grain threshing, dust from straw and mixed feed can also become a source of tularemia pathogens. In this case, the respiratory organs are most often affected.

A sick person does not pose a danger to others.

Tularemia vectors

The infection is carried by mosquitoes, horseflies and ixodid and gamas ticks.

Rice. 5. In the photo there is a male ixodes taiga tick (Ixodes persulcatus) on the left and a gamasid tick on the right.

Routes of transmission

• Contact (involves contact with sick animals and their biological material).
• Nutritional (consumption of contaminated food and water).
• Transmissible (bites from infected bloodsuckers).
• Aerogenic (inhalation of infected dust).

Rice. 6. Contact with the skins of killed infected animals and bites of blood-sucking animals are the main routes of transmission of infection.

Mechanism of transmission of infection

Tularemia has multiple routes of transmission:

• through damaged skin,
• through the mucous membrane of the oropharynx and tonsils,
• through the mucous membrane of the eyes,
• through the respiratory tract,
• through the digestive tract.

One microbial cell is enough to become infected with tularemia.

How does tularemia develop (pathogenesis of the disease)

• The skin, mucous membranes, upper respiratory tract and gastrointestinal tract are entry points for infection. This fact is decisive in the development of the clinical form of tularemia: ulcerative-bubonic, bubonic, oculobubonic, anginal-bubonic, pulmonary, abdominal or generalized.
• During the incubation period, tularemia pathogens are fixed and multiply in the area of ​​the entrance gate. Once the number of bacteria reaches a certain amount, the period of clinical manifestations begins.
• At the end of the incubation period, the bacteria penetrate through the lymphatic tract into the regional lymph nodes, where they multiply intensively. This is how primary buboes are formed.
• The death of bacteria is accompanied by the release of endotoxin, which increases local inflammation, and the entry of endotoxins into the blood causes intoxication.
• Intracellular proliferation of bacteria in phagocytes with subsequent suppression of their killer effect leads to the formation of granulomas in primary buboes, which leads to their suppuration. Primary buboes take a long time to heal. The suppurating lymph nodes are opened surgically.
• A generalized infection occurs with severe toxicosis and allergic reactions, the appearance of secondary buboes, and damage to various organs: lungs, liver and spleen. Secondary buboes do not suppurate.

The natural susceptibility of a person to the disease reaches 100%.

Rice. 7. The photo shows an ulcer at the site of a tularemia bite.

Signs and symptoms of tularemia

Incubation period

The incubation period for tularemia lasts on average a week. Sometimes it takes up to one month. During the incubation period, pathogens are fixed and multiply in the area of ​​the entrance gate. Once the number of bacteria reaches a certain amount, the period of clinical manifestations begins.

Symptoms of tularemia in the initial period of clinical manifestations

Intoxication syndrome

During the initial period of clinical manifestations, body temperature rises to 39 - 40 ° C, headache and muscle pain appear, weakness develops, appetite disappears, bradycardia develops, and blood pressure drops.

Local changes

An inflammatory-necrotic reaction develops at the sites of infection. An ulcer develops on the skin, which in its development passes through the stage of papules, vesicles and pustules. When bacteria settle in the tonsil area, necrotizing tonsillitis develops. When bacteria enter the lungs, necrotizing pneumonia develops. When bacteria enter the mucous membrane of the eyes, conjunctivitis develops.

Rice. 8. The photo shows an enlarged lymph node due to tularemia.

Symptoms of tularemia during clinical manifestations

The period of the height of the disease is characterized by a long (up to 1 month) febrile period and the manifestation of one of the clinical forms of the disease (depending on the entrance gate): ulcerative-bubonic, bubonic, oculobubonic, anginal-bubonic, pulmonary, abdominal or generalized.

The patient's appearance has characteristic features: the face is puffy and hyperemic, sometimes with a bluish tint, the sclera is injected, and pinpoint hemorrhages appear on the mucous membrane of the oropharynx. Lymph nodes enlarge.

Signs and symptoms of tularemia in the bubonic form of the disease

The lymph node appears on the 3rd day of clinical manifestations of the disease and reaches its maximum size by the end of the first week of the disease. Its location depends on the location of the entrance gate.

The primary lymph node in tularemia is large - from the size of a walnut to 10 cm in diameter. Most often, the femoral, inguinal, elbow and axillary lymph nodes are enlarged. The phenomena of periadenitis are clearly expressed. The skin on the lymph node turns red. The node itself becomes painful on palpation.

The lymph node either resolves or suppurates. A suppurated lymph node takes a long time to heal. A scar forms in its place.

Rice. 9. The photo shows the bubonic form of tularemia.

Signs and symptoms of tularemia in the ulcerative-bubonic form of the disease

With the transmissible and contact-household mechanism of transmission of infection, in addition to the bubo, a primary affect develops. Damage to the skin begins with the appearance of hyperemia (spot), the skin over which quickly thickens (papule). Next, in place of the papule, a pustule appears, when opened, a painless ulcer up to 7 mm in diameter is exposed. The edges of the ulcer are undermined. The discharge is scanty. Healing occurs with a scar after 2 - 3 weeks. Ulcers usually appear on exposed parts of the body - the neck, forearm and lower leg.

Rice. 10. The photo shows an ulcer due to tularemia.

Signs and symptoms of tularemia in the oculobubonic form of the disease

When pathogens enter the mucous membrane of the eyes, conjunctivitis develops. Bacteria enter the eyes with dust and dirty hands. Conjunctivitis most often develops on one side. The patient is bothered by severe lacrimation. The eyelids swell. Suppuration is noted. Yellowish-white nodules appear on the mucous membrane of the lower eyelid. The cornea is rarely affected. The disease lasts a long time and is very serious. Lymph nodes most often enlarge in the area behind the ear, anterior cervical and submandibular.

Rice. 11. The photo shows conjunctivitis due to tularemia.

Signs and symptoms of tularemia in the anginal-bubonic form of the disease

When pathogens enter the mucous membrane of the oropharynx, a sore throat develops. Bacteria enter the oropharynx through contaminated food or water. In the future, the abdominal form of the disease may develop. The tonsils quickly acquire a bluish tint. Swelling of the tonsils, uvula and palatine arches develops.

A grayish coating forms on their surface. The film, as with diphtheria, is difficult to remove, but, unlike diphtheria, it never goes beyond the organ. Ulcers form under the film and take a long time to heal. Buboes often develop on the side of the tonsils - submandibular, cervical and parotid.

Rice. 12. The photo shows a sore throat due to tularemia.

Signs and symptoms of tularemia in the abdominal form of the disease

The gastrointestinal form of tularemia is rare, but in terms of the severity of clinical manifestations it is the most severe. The patient experiences severe abdominal pain, nausea, vomiting and lack of appetite. The stool is often loose, but there may also be constipation. The mesenteric lymph nodes are enlarged.

Signs and symptoms of tularemia in the pulmonary form of the disease

When pathogens enter the respiratory tract, bronchitis or pneumonia develops. With bronchitis, the patient is bothered by a strong dry cough, and with pneumonia, a high, debilitating body temperature. With bronchitis, dry wheezing is heard. After 2 weeks, recovery occurs. When lung tissue is damaged, focal pneumonia develops, which is prone to complications in the form of bronchiectasis, abscess, gangrene and pleurisy. The bronchopulmonary, paratracheal and mediastinal lymph nodes are involved in the process.

Signs and symptoms of tularemia in the generalized form of the disease

The generalized form of the disease occurs as sepsis. The patient is worried about prolonged high temperature. Symptoms of intoxication are pronounced. The liver and spleen enlarge. A rash appears on symmetrical areas of the body. Bacteria, spreading through the blood, cause the development of secondary buboes.

Tularemia lasts from 2 to 4 weeks and almost always ends in recovery. After suffering from the disease, a person develops lifelong, strong immunity.

Diagnosis of tularemia

When diagnosing tularemia, the following methods are used:

• allergy,
• serological,
• biological.

Allergological method

The allergological method is strictly specific and the earliest among all methods for diagnosing tularemia. Tularin is a suspension of killed tularemia bacteria in an isotonic solution of sodium chloride with glycerol. A skin allergy test is carried out from the 3rd day of the disease. Tularin is injected intradermally into the middle third of the forearm. Infiltration is measured after a day, two and three. If the infiltrate diameter is 0.5 cm, the test is considered positive. If the redness disappears by the end of the first day, the test is considered negative.

Rice. 13. An allergy test with tularin (allergen) is a diagnostic method for identifying sensitization of the body.

The test with tularin is strictly specific. Once a positive reaction occurs, it lasts for years.

Serological methods

The agglutination reaction (RA) for tularemia gives a positive result from the 2nd week of the disease. An antibody titer of 1:100 and an increase in antibody titer (RPGA) after 7 - 10 days confirm the diagnosis.

Enzyme-linked immunosorbent assay (ELISA) allows you to determine the presence of immunoglobulins of class G and M. The analysis is highly sensitive. Used from the 6th day of illness. The presence of IgM indicates the severity of the disease, IgG indicates later stages of the disease and indicates the presence of a good immune response in the patient.

Bacteriological diagnosis of tularemia

Bacteriological diagnosis of tularemia does not always give a positive result due to the difficulty of isolating tularemia pathogens from the patient’s biological material.

does not grow when sown on conventional nutrient media. A pure culture is obtained by infecting an animal with subsequent inoculation of pathogens from the biological material of the animal onto nutrient media. This process is carried out only in specialized high-security laboratories, since tularemia is a particularly dangerous infection.

Rice. 14. Francisella tularensis colonies appear within a few days after sowing. They are white with a bluish tint.

Polymerase chain reaction (PCR)

Polymerase chain reaction is a genetic method for diagnosing tularemia. Information about the presence of pathogens can be obtained already in the febrile period.

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