Salmonella - pathogens of typhoid and paratyphoid

Typhoid fever is a severe acute infectious disease characterized by deep general intoxication, bacteremia and specific damage to the lymphatic apparatus of the small intestine. Intoxication is manifested by severe headache, confusion, delirium (typhus from Greek typhos - fog). Typhoid fever as an independent nosological unit was first attempted by the Russian physician AG Pyatnitsky as early as 1804, but finally it was done in 1822 by R. Bretonno, who differentiated this disease from intestinal tuberculosis and suggested the contagious nature of typhoid fever.

The causative agent of typhoid fever - Salmonella typhi - was discovered in 1880 by K. Ebert, and isolated in pure culture in 1884 by K. Gaffki. Soon, the causative agents of paratyphoid A and B - S. Paratyphi A and S. Paratyphi B. Were isolated and studied. The genus Salmonella includes a large group of bacteria, but only three of them are S. Typhi, S. Paratyphi A and S. Paratyphi B - cause disease in a person with a clinical picture of typhoid fever. Morphologically they are indistinguishable: short gram-negative rods with rounded ends, 1-3.5 microns long, 0.5-0.8 microns in diameter; spores and capsules do not form, have active mobility (peritrichi). The content of G + C in DNA is 50-52 mol%.

The causative agents of typhoid and paratyphoid are facultative anaerobes, a temperature optimum for growth of 37 ° C (but can grow in the range of 10 to 41 ° C), pH 6.8-7.2; are not exacting to nutrient media. Growth on the broth is accompanied by turbidity, on the MPA, tender round, smooth, translucent colonies with a diameter of 2-4 mm are formed. However, S. Typhi colonies having a Vi-antigen are cloudy. Colonies S. Paratyphi In more coarse, after a few days, they form along their periphery peculiar rollers. On Endomo environments, the colonies of all three salmonellae are colorless, on bismuth-sulfitagar - black. In the case of dissociation on dense media, R-form colonies grow. The selective environment for the causative agents of typhoid fever and paratyphoid is bile or bile broth.

[1], [2], [3], [4], [5], [6], [7], [8], [9]

Biochemical properties of pathogens of typhoid and paratyphoid

The causative agents of typhoid and paratyphoid give a positive reaction with MR, do not form indole, do not liquefy gelatin, reduce nitrates to nitrites, do not form acetoin. S. Typhi does not grow on hungry agar with citrate. The main biochemical differences between typhoid and paratyphoid pathogens are that S. Typhi ferments glucose and some other carbohydrates with the formation of only acid, and S. Paratyphi A and S. Paratyphi B - with the formation of both acid and gas.

S. Typhi on the ability to ferment xylose and arabinose are divided into four biochemical types: I, II, III, IV.

Xylose + - + -

Arabinose - - + +

[10], [11], [12], [13], [14], [15], [16], [17], [18]

Antigenic structure of pathogens of typhoid and paratyphoid

Salmonellae have O and H antigens. On O-antigens they are divided into a large number of serogroups, and according to H-antigens - into serotypes (for more details on the serological classification of salmonella, see the next section). S. Typhi, S. Paratyphi A and S. Paratyphi B differ from each other both in terms of O-antigens (belong to different serogroups) and H-antigens ..

In 1934, A. Felix and R. Pitt established that S. Typhi, in addition to the O and H antigens, has another surface antigen, which they called the virulence antigen (Vi antigen). According to its chemical nature, the Vi-antigen differs from O- and H-antigens, it consists of three different fractions, but its basis is the complex polymer N-acetylgalactosaminuronic acid with a mass of 10 MD. Vi-antigen is usually found in freshly isolated cultures, but it is easily lost under the influence of various factors (in particular, when grown at temperatures above 40 ° C and below 20 ° C, in media with carbolic acid, etc.) at long storage of cultures, is destroyed at temperature 100 ° С during 10 minutes. Since it is located more superficially than O-antigen, its presence inhibits agglutination of the culture of S. Typhi with O-specific serum, therefore such a culture must be checked in the agglutination reaction with Vi-serum. On the contrary, the loss of Vi antigen leads to the release of O-antigen and the restoration of O-agglutination, but Vi-agglutination is lost. The quantitative content of Vi-antigen in S. Typhi can vary greatly, therefore F. Kauffmann proposed to classify S. Typhi according to the content of Vi-antigen into three groups:

• pure v-forms (German viel - many);
• pure w-forms (German wenig - little);
• intermediate vw-forms.

Three unusual mutants of S. Typhi were found: Vi-I-R-form, the cells lack H- and O-antigens, but they persistently retain the Vi-antigen; O-901 - devoid of H- and Vi-antigens; H-901 - contains O- and H-antigens, but is devoid of Vi-antigen. All three antigens: O-, H- and Vi- have pronounced immunogenic properties. The presence of Vi-antigens allows the culture of S. Typhi to be phage-typed. There are 2 types of phages that lyse only those cultures that contain the Vi antigen: Vi-I is the universal phage, lyses most of the Vi-containing S. Typhi cultures; and a set of Vi-II phage lysing the S. Typhi culture selectively. This was first shown in 1938 by J. Craig and K. Ian. With the help of Vi-phages of type II, they divided S. Typhi into 11 phage types. By 1987, 106 different Vi-phage types of S. Typhi had been identified. Their sensitivity to the corresponding phages is a stable sign, therefore phagotyping has an important epidemiological significance.

The schemes of phagotyping of S. Paratyphi A and S. Paratyphi B have also been developed, according to which they are divided into dozens of phagotypes. It is significant that the salmonella phagotypes can not differ from each other in any other way.

[19], [20], [21], [22], [23], [24], [25], [26]

Resistantness of pathogens of typhoid and paratyphoid

The causative agents of typhoid and paratyphoid in the external environment (water, soil, dust) persist, depending on the conditions, from several days to several months. In the running water can survive up to 10 days, in stagnant - up to 4 weeks, on vegetables and fruits - 5-10 days, on dishes - up to 2 weeks, in oil, cheese - up to 3 months, on ice - up to 3 months . And more; heating at a temperature of 60 ° C kills in 30 minutes, and boiling - instantly. Conventional chemical disinfectants kill them in a few minutes. The content of active chlorine in the tap water at a dose of 0.5-1.0 mg / l or ozonization of water ensures its reliable disinfection both from Salmonella and other pathogenic intestinal bacteria.

Factors of pathogenicity of pathogens of typhoid and paratyphoid

The most important biological feature of the causative agents of typhoid fever and paratyphoid A and B is their ability to withstand phagocytosis and multiply in the cells of the lymphoid system. They do not form exotoxins. The main factor of their pathogenicity, in addition to the Vi-antigen, is endotoxin, which is characterized by unusually high toxicity. Such pathogenicity factors as fibrinolysin, plasmocoagulase, hyaluronidase, lecithinase, etc., are found in pathogens of typhoid and paratyphoid infections very rarely. The highest frequency is found in DNA-ase (75-85% of the studied S. Typhi and S. Paratyphi B cultures). It has been established that strains of S. Typhi having a plasmid with 6 mM are more virulent. Therefore, the question of the pathogenicity factors of these salmonella remains to be studied.

[27], [28], [29], [30], [31], [32], [33]

Postinfectious immunity

Durable, prolonged, repeated diseases of typhoid and paratyphoid are rare. Immunity is caused by the appearance of antibodies to Vi-, O- and H-antigens, immune memory cells and increased activity of phagocytes. Postvaccinal immunity, unlike postinfection, is short-lived (about 12 months).

Epidemiology of typhoid and paratyphoid

The source of typhoid fever and paratyphoid A is only a person, a sick or a carrier. The source of paratyphoid B, in addition to humans, can be animals, including birds. The mechanism of infection is fecal-oral. The infecting dose of S. Typhi of 105 cells (causes a disease of 50% of volunteers), infecting doses of salmonella of paratyphoid A and B is much higher. Infection occurs mainly as a result of direct or indirect contact, as well as through water or food, especially milk. The largest epidemics caused infection with pathogens (water epidemics).

[34], [35], [36], [37], [38], [39]

Symptoms of typhoid and paratyphoid

The incubation period for typhoid fever is 15 days, but it can vary from 7 to 25 days. It depends on the infecting dose, the virulence of the pathogen and the immune status of the patient. The pathogenesis and clinical picture of typhoid fever and paratyphoid A and B are very similar. In the development of the disease, the following stages are clearly revealed:

• invasion stage. The causative agent penetrates through the mouth into the small intestine;
• through the lymphatic pathways of salmonella penetrate into the lymphoid formations of the submucosa of the small intestine (Peyer's plaques and solitary follicles) and multiply in them, cause lymphangitis and lymphadenitis (peculiar typhoid granules);
• bacteremia - the exit of the pathogen in large quantities into the blood. The stage of bacteremia begins at the end of the incubation period and can (in the absence of effective treatment) continue throughout the entire disease;
• the stage of intoxication occurs due to the decay of bacteria under the action of bactericidal properties of the blood and the release of endotoxins;
• stage of parenchymal diffusion. From the blood, salmonella is absorbed by macrophages of the bone marrow, spleen, lymph nodes, liver and other organs. In a large number of cases, the causative agent of typhoid fever accumulates in the bile ducts of the liver and in the gall bladder, where it finds favorable conditions for its reproduction and where the bactericidal properties of blood are weakened by the influence of bile;
• excretory-allergic stage. As the formation of immunity begins the process of release from the pathogen. This process is performed by all glands: salivary, intestines, sweat, dairy (during the feeding of the baby), urinary system and especially actively - the liver and gall bladder. Isolated from the gall bladder, Salmonella again enter the small intestine, from which part of it is excreted with feces, and some invade again into the lymph nodes. Secondary introduction into the already sensitized nodes causes a hyperergic reaction in them, which manifests itself in the form of necrosis and the formation of ulcers. This stage is dangerous by the possibility of perforation of the intestinal wall (ulcers), internal bleeding and the development of peritonitis;
• stage of recovery. The healing process of ulcers occurs without the appearance of disfiguring scars on the ground, cleared of necrotic raids.

In turn, in the clinical picture of the disease, the following periods are distinguished:

• I initial stage - stadium incrementi (1st week): a gradual increase in temperature to 40-42 ° C, an increase in intoxication and other manifestations of the disease.
• II - the stage of maximum development of all symptoms - stadium acme (2-3 weeks of illness): the temperature is kept at a high level;
• III - the stage of recession of the disease - the fourth decrementi (4th week of the disease): a gradual decrease in temperature and a decrease in the manifestation of other symptoms;
• IV - stage of recovery.

On the 8th-9th day of the disease, and sometimes even later, many patients develop a roseolous rash on the skin of the abdomen, chest and back. The appearance of a rash (small red spots) is a consequence of local inflammatory processes of an allergic nature in the superficial layers of the skin near the lymphatic vessels, which contain the causative agent of the disease in abundance. Clinical recovery does not always coincide with bacteriological. Approximately 5% of patients become chronic carriers of salmonella typhus or paratyphoid. The reasons underlying long-term (more than 3 months, and sometimes many years) carriage of salmonella remain unclear. The local inflammatory processes in the biliary tract (sometimes in the urinary tract), which often arise in connection with typhoid paratyphoid infections or become aggravated as a result of these infections, play a significant role in the formation of carriage. However, the L-transformation of them plays an equally important role in the formation of the long-term carrier of salmonella of typhoid fever and paratyphoid A and B. L-forms of Salmonella lose H-, partially 0-and Vi-antigens, are located, as a rule, intracellularly (inside the macrophages of the bone marrow), so they become inaccessible to either chemotherapy or antibodies and can persist for a long time in the body of a sick person. Returning to the original forms and completely restoring its antigenic structure, the salmonella again become virulent, re-enter the bile ducts, exacerbate the bacteriocarrier process, stand out with feces, and such carrier becomes a source of infection for others. It is also possible that the formation of bacterial transport depends on some deficit of the immune system.

Laboratory diagnosis of typhoid and paratyphoid

The earliest and basic method of diagnosing typhoid and paratyphoid is bacteriological - obtaining blood culture or myeloculture. To this end, examine the blood or punctate bone marrow. Blood is best sowed on the medium Rapoport (bile broth with the addition of glucose, indicator and a glass float) in a ratio of 1:10 (per 10 ml of 1 ml of blood). Sowing should be incubated at 37 C for at least 8 days, and taking into account the possible presence of L-forms - up to 3-4 weeks. To identify the isolated Salmonella culture, diagnostic adsorbed sera containing antibodies to 02 antigens (S. Paratyphi A), 04 (S. Paratyphi B) and 09 (S. Typhi) are used (taking into account their biochemical properties). If the isolated culture of S. Typhi is not agglutinated with 09 serum, it must be checked with Vi-serum.

To isolate S. Typhi, it is possible to use exudate, obtained by scarifying roseol - growing roseolocultures.

Bacteriological study of feces, urine and bile is carried out to confirm the diagnosis, control of bacteriological recovery at discharge of convalescent and for the diagnosis of bacterial transport. In this case, the material is pre-seeded on enrichment media (media containing chemicals, for example selenite, which inhibit the growth of E. Coli and other intestinal microflora, but do not inhibit the growth of salmonella), and then from the enrichment medium to differential diagnostic media ( Endo, bismuth sulfitagar) in order to isolate isolated colonies and obtain from them pure cultures identified by the above scheme. To detect O- and Vi-antigens in the blood serum and feces of patients, DSC, RPGA with an antigen diagnosticum, coagglutination reactions, hemagglutination aggregate, IFM can be used. For the accelerated identification of S. Typhi, it is promising to use as a probe a DNA fragment carrying the Vi-antigen gene (identification time 3-4 h).

Since the end of the first week of the disease, antibodies appear in the serum of patients, therefore, in order to diagnose typhoid fever in 1896, F. Vidal proposed the reaction of expanded tube agglutination. The dynamics of antibodies to S. Typhi is unique: antibodies to O-antigen appear first of all, but their titer decreases rapidly after recovery; H-antibodies appear later, but they persist after illness and vaccinations for years. In view of this circumstance, Vidal's reaction is put simultaneously with separate O- and H-diagnosticums (as well as with paratyphoid A and B diagnostics) to exclude possible errors associated with vaccination or previous disease. However, the specificity of the Vidal's reaction is not high enough, therefore, the use of RPGA, in which the erythrocyte diagnosticum is sensitized either O- (for detecting O-antibodies) or Vi-antigen (for detecting Vi-anti-bodies), is more preferable. The most reliable and specific is the last reaction (Vi-hemagglutination).

Diagnosis of bacterial carry of typhoid and paratyphoid

The only evidence of bacteriocarrier is the isolation of S. Typhi, S. Paratyphi A, S. Paratyphi B. Cultures from the carrier. The material for the study is duodenal contents, feces and urine. Complexity of the problem lies in the fact that carriers do not always excrete with these substrates, there are pauses, and quite long. As ancillary methods that allow to narrow the range of subjects, serological reactions are used (simultaneous detection of O-, H-, Vi- or O-, Vi-antibodies indicates the possible presence of an agent in the body) and an allergic skin test with Vi-typhin. The latter contains Vi-antigen, which when interacting with Vi-antibodies gives a local allergic reaction in the form of reddening and swelling for 20-30 minutes. A positive reaction with Vi-typhin indicates the presence of Vi-antibodies in the body and the possible presence of S. Typhi. To identify the L-forms of S. Typhi, special immunofluorescent antibodies are proposed (to L-form antigens of the pathogen). V.Murom offered an original method for the detection of bacterial carriers. It consists in the study of tampons, simultaneously thrown into the sewer hatches all over the sewer network of the settlement.

[40], [41], [42], [43], [44], [45], [46], [47], [48]

Treatment of typhoid and paratyphoid

Treatment of typhoid fever is based on the use of various antibiotics, to which pathogens show high sensitivity (levomycetin, ampicillin, tetracyclines, etc.). Antibiotics reduce the severity of the course of the disease and shorten its duration. However, the transfer of R-plasmids to salmonella from E. Coli or other enterobacteria can lead to the emergence of dangerous epidemic clones among them.

Specific prophylaxis of typhoid and paratyphoid

Instead of seven different typhoid vaccines, previously used, since 1978, only one has been produced in our country - a chemical sorbed abdominal-typhoid monovalent. However, due to the fact that typhoid fever from epidemic disease has become sporadic (and this became possible primarily due to improved water supply and sewerage systems and improved sanitary culture of the population), the need for mass immunization against it has disappeared. Therefore, inoculation from typhoid fever is carried out only in the case of epidemic indications.