The causative agent of tularemia

Tularemia is a primary disease of animals (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 with Tulare Lake (California), studied in detail by E. Francis, in whose honor the genus is named.

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

The following amino acids are necessary for the growth of F. tularensis: arginine, leucine, isoleucine, lysine, methionine, proline, threonine, histidine, valine, cystine, for some subspecies - serine, 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 the cultivation of F. tularensis.

The genus Francisella belongs to the class Gammaproteobacteria, phylum Proteobacteria. This genus also includes F. novicida, whose pathogenicity for humans has not been established.

The causative agent of tularemia is an intracellular parasite. Its virulence is due to a capsule that inhibits phagocytosis; neuraminidase, which promotes adhesion; endotoxin; allergenic properties of the cell wall, as well as the ability to reproduce in phagocytes and suppress their killer effect. The mechanisms of virulence have not yet been deciphered. In addition, receptors capable of interacting with Fc fragments of IgG immunoglobulins have been found in the tularemia bacillus. As a result of such binding, the activity of the complement systems and macrophages is disrupted.

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

• Holarctic (low 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.

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Resistance of the causative agent of tularemia

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

Epidemiology of tularemia

The main reservoir of tularemia in nature are rodents, among which epizootics are observed in natural conditions. Humans become infected only from animals; the pathogen is not transmitted from person to person. The pathogen has been found in 82 species of rodents and lagomorphs, and is most often found in representatives of 4 families: mouse-like rodents (Muridae), hares (Leporidae), squirrels (Sciuridae) and jerboas (Dipodidae). In Russia, the main carriers are mouse-like rodents: water rats, common voles, house mice and muskrats.

According to their 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;
• 2nd group - less sensitive (gray rats, gophers, etc.). The minimum lethal dose is 1 billion microbial cells, however, one microbial cell is enough to infect some of them;
• 3rd group (predators - cats, foxes, ferrets). Resistant to high infectious doses, the disease proceeds without visible manifestations;
• Group 4 - immune to tularemia (ungulates, cold-blooded animals, birds).

For humans, the minimum infectious dose is one microbial cell. Humans can become infected in all possible ways: direct and indirect contact with sick rodents, their corpses, or objects infected with rodents; alimentary (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. Of particular importance are ixodid ticks, in which the pathogen persists throughout life and is even transmitted transovarially to offspring. These circumstances contribute to the establishment of the disease in nature. Humans become infected with ticks not by bites, but as a result of the pathogen getting on the skin along with the tick's excrement.

In Russia, there are 7 main landscape types of natural foci of tularemia: marsh, meadow-field, steppe, forest, foothill-stream, tundra and tugai (desert).

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Symptoms of tularemia

The causative agent of tularemia penetrates the body through the outer coverings (damaged and intact skin and mucous membranes). Ulcers often form at the site of penetration. Through the lymphatic vessels, the bacteria enter the regional lymph node and freely multiply in it; the inflammatory process leads to the formation of a bubo. From here, the pathogen penetrates 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, facial hyperemia appear. The further course depends on the site of the entry gate, according to which the following clinical forms of tularemia are distinguished: ulcerative-glandular (bubonic), ocular-glandular, anginal-glandular, abdominal and pulmonary. Mortality in tularemia does not exceed 1-2%.

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

Laboratory diagnostics of tularemia

All microbiological methods are used to diagnose tularemia. The study is conducted in secure laboratories. The material for the study - blood, bubo puncture, ulcer scraping, conjunctival discharge, pharyngeal plaque, sputum, etc. - is determined by the clinical form of the disease. In addition, water and food products can be taken for the study. In natural foci of tularemia, planned systematic studies are conducted to isolate the causative agent of tularemia from rodents.

The bacteriological method of diagnosing tularemia in humans rarely gives positive results. A pure culture is usually isolated after accumulating it on susceptible laboratory animals. White mice and guinea pigs are used for bioassays. Mice are infected subcutaneously, guinea pigs - intraperitoneally; the animals die on the 3rd-6th day, sometimes after hoarfrost. Infected animals are kept in special conditions (as in diagnosing plague) and observed for 6-14 days. Gels experimental animals do not die for 7-15 days, they are slaughtered on the 15th-20th day and the corpses are autopsied. 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, glucose-cysteine blood focus, etc. Identification is based on the morphology and tinctorial properties of the pathogen, the absence 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 chicken 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 the sediment is used to infect laboratory animals.

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

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

Allergic tests are used for early diagnostics of tularemia (from the 5th day after the onset of the disease). Two types of tularin are used and, accordingly, two methods of their administration are used: 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 the allergic reaction are taken into account dynamically after 24, 36, 48 hours. An infiltrate with a diameter of at least 5 mm is considered a positive result. In vaccinated persons or those who have had tularemia, allergic tests remain positive for a number of years (anamnestic reaction).

Specific prevention of tularemia

For specific prevention, a vaccine against tularemia is used, obtained in 1930 by Russian military doctors B. Ya. Elbert and N. A. Gaisky from the Me 15 strain. The vaccine provides strong 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 for people belonging to risk groups. Simultaneous vaccination against tularemia and brucellosis; tularemia and plague, as well as against tularemia and some other infections is allowed.

Non-specific prevention of tularemia is the same as for other zoonoses and is aimed primarily at controlling rodents.