Salmonellae - causative agents of typhoid fever and paratyphoid fever

Typhoid fever is a severe acute infectious disease characterized by profound general intoxication, bacteremia, and specific damage to the lymphatic apparatus of the small intestine. Intoxication is manifested by severe headache, clouding of consciousness, and delirium (typhoid from the Greek typhos - fog). Typhoid fever as an independent nosological entity was first attempted to be identified by the Russian physician A. G. Pyatnitsky in 1804, but it was finally done in 1822 by R. Bretonneau, 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. Gaffky. Soon, the causative agents of paratyphoid fever 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 - S. typhi, S. paratyphi A and S. paratyphi B - cause disease in humans with a clinical picture of typhoid fever. Morphologically, they are indistinguishable - short gram-negative rods with rounded ends, 1-3.5 μm long, 0.5-0.8 μm in diameter; they do not form spores or capsules, and have active mobility (peritrichous). The G + C content in DNA is 50-52 mol %.

The causative agents of typhoid and paratyphoid fever are facultative anaerobes, the temperature optimum for growth is 37 °C (but can grow in the range from 10 to 41 °C), pH 6.8-7.2; they are not demanding to nutrient media. Growth on broth is accompanied by turbidity, on MPA delicate round, smooth, translucent colonies 2-4 mm in diameter are formed. However, S. typhi colonies with Vi antigen are turbid. S. paratyphi B colonies are coarser, after a few days peculiar ridges are formed along their periphery. On Endo media, colonies of all three salmonella are colorless, on bismuth sulfite agar they are black. In case of dissociation on dense media, R-form colonies grow. The selective environment for the pathogens of typhoid and paratyphoid fever is bile or bile broth.

[ 1 ], [ 2 ], [ 3 ], [ 4 ], [ 5 ], [ 6 ]

Biochemical properties of typhoid and paratyphoid pathogens

Typhoid and paratyphoid pathogens 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 starvation agar with citrate. The main biochemical differences between the 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 is divided into four biochemical types according to its ability to ferment xylose and arabinose: I, II, III, IV.

Xylose + - + -

Arabinose - - + +

[ 7 ], [ 8 ], [ 9 ], [ 10 ]

Antigenic structure of typhoid and paratyphoid pathogens

Salmonella have O- and H-antigens. They are divided into a large number of serogroups by O-antigens, and into serotypes by H-antigens (for more information 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 O-antigens (belong to different serogroups) and in H-antigens.

In 1934, A. Felix and R. Pitt established that S. typhi, in addition to O- and H-antigens, has another surface antigen, which they called the virulence antigen (Vi-antigen). The Vi-antigen differs from the O- and H-antigens in its chemical nature; it consists of three different fractions, but its basis is a complex polymer of N-acetylgalactosaminouronic acid with a molecular weight of 10 MD. The 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, on media with carbolic acid, etc.), and during long-term storage of cultures, it is destroyed at a temperature of 100 °C for 10 min. Since it is located more superficially than the O-antigen, its presence prevents agglutination of the S. typhi culture with O-specific serum, so such a culture must be tested in an agglutination reaction with Vi-serum. On the contrary, the loss of Vi-antigen leads to the release of O-antigen and restoration of O-agglutination, but Vi-agglutination is lost. The quantitative content of Vi-antigen in S. typhi can vary greatly, so F. Kauffmann proposed to classify S. typhi into three groups according to the content of Vi-antigen:

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

Three unusual mutants of S. typhi have been discovered: Vi-I, an R-form in which the cells lack H and O antigens but persistently retain the Vi antigen; O-901, lacks H and Vi antigens; H-901, contains O and H antigens but lacks the Vi antigen. All three antigens: O, H and Vi, have pronounced immunogenic properties. The presence of Vi antigens allows S. typhi cultures to be subjected to phage typing. There are two types of phages that lyse only those cultures that contain the Vi antigen: Vi-I, a universal phage that lyses most Vi-containing S. typhi cultures; and a set of Vi-II phages that selectively lyse S. typhi cultures. This was first shown in 1938 by J. Craige and K. Ian. Using type II Vi phages, 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 feature, so phage typing is of great epidemiological importance.

Phage typing schemes for S. paratyphi A and S. paratyphi B have also been developed, according to which they are divided into dozens of phage types. It is significant that salmonella phage types may not differ from each other by any other features.

[ 11 ], [ 12 ], [ 13 ], [ 14 ]

Resistance of typhoid and paratyphoid pathogens

Typhoid and paratyphoid pathogens survive in the external environment (water, soil, dust), depending on conditions, from several days to several months. They can survive in running water for up to 10 days, in stagnant water - up to 4 weeks, on vegetables and fruits - 5-10 days, on dishes - up to 2 weeks, in butter, cheese - up to 3 months, in 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 tap water at a dose of 0.5-1.0 mg / l or ozonation of water ensure its reliable disinfection from both salmonella and other pathogenic intestinal bacteria.

Pathogenicity factors of typhoid and paratyphoid pathogens

The most important biological feature of the causative agents of typhoid and paratyphoid A and B is their ability to resist 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, plasma coagulase, hyaluronidase, lecithinase, etc., are very rarely found in the causative agents of typhoid and paratyphoid. DNAase is found most frequently (in 75-85% of the studied cultures of S. typhi and S. paratyphi B). It has been established that S. typhi strains with a plasmid with m. m. 6 MD have higher virulence. Therefore, the question of the pathogenicity factors of these salmonella remains poorly understood.

[ 15 ], [ 16 ], [ 17 ]

Post-infectious immunity

Durable, long-lasting, repeated typhoid and paratyphoid fevers are rare. Immunity is due to the appearance of antibodies to Vi-, O- and H-antigens, immune memory cells and increased phagocyte activity. Post-vaccination immunity, unlike post-infection, is short-lived (about 12 months).

Epidemiology of typhoid fever and paratyphoid fever

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

[ 18 ], [ 19 ], [ 20 ], [ 21 ], [ 22 ]

Symptoms of typhoid and paratyphoid fever

The incubation period for typhoid fever is 15 days, but it can vary from 7 to 25 days. This 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. The following stages are clearly identified in the development of the disease:

• invasion stage. The pathogen penetrates the small intestine through the mouth;
• through the lymphatic pathways, salmonella penetrates the lymphoid formations of the submucosa of the small intestine (Peyer's patches and solitary follicles) and, multiplying in them, causes lymphangitis and lymphadenitis (a kind of typhoid granules);
• bacteremia - the release of the pathogen in large quantities into the blood. The bacteremia stage begins at the end of the incubation period and can (in the absence of effective treatment) continue throughout the disease;
• the stage of intoxication occurs as a result of the breakdown of bacteria under the influence of the bactericidal properties of the blood and the release of endotoxins;
• stage of parenchymatous diffusion. Salmonella are absorbed from the blood by macrophages of the bone marrow, spleen, lymph nodes, liver and other organs. The typhoid fever pathogen accumulates in large quantities 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 the blood are weakened by the influence of bile;
• excretory-allergic stage. As immunity develops, the process of release from the pathogen begins. This process is carried out by all glands: salivary, intestinal, sweat, milk (during breastfeeding), urinary system and especially actively - the liver and gall bladder. Salmonella released from the gall bladder again enter the small intestine, from where some of them are excreted with feces, and some invade the lymph nodes again. Secondary penetration into already sensitized nodes causes a hyperergic reaction in them, which manifests itself in the form of necrosis and ulceration. This stage is dangerous due to the possibility of perforation of the intestinal wall (ulcers), internal bleeding and the development of peritonitis;
• recovery stage. The process of ulcer healing occurs without the formation of disfiguring scars in areas cleared of necrotic deposits.

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

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

On the 8th-9th day of the disease, and sometimes later, many patients develop a roseola rash on the skin of the abdomen, chest and back. The appearance of the rash (small red spots) is a consequence of local productive-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 recovery. About 5% of those who have recovered become chronic carriers of salmonella typhoid or paratyphoid. The reasons underlying long-term (more than 3 months, and sometimes many years) carriage of salmonella remain unclear. Local inflammatory processes in the biliary (sometimes in the urinary) tract, which often arise in connection with typhoid-paratyphoid infections or are exacerbated as a result of these infections, play a certain role in the formation of carriage. However, their L-transformation plays an equally important role in the formation of long-term carriage of typhoid and paratyphoid salmonella A and B. L-forms of salmonella lose H-, partially 0- and Vi-antigens, are located, as a rule, intracellularly (inside bone marrow macrophages), therefore they become inaccessible to either chemotherapy drugs or antibodies and can persist in the body of a recovered person for a long time. Returning to their original forms and completely restoring their antigen structure, salmonella again become virulent, again penetrate the bile ducts, cause an exacerbation of the process of carriage, are excreted with feces, and such a carrier becomes a source of infection for others. It is also possible that the formation of carriage depends on some deficiency of the immune system.

Laboratory diagnostics of typhoid fever and paratyphoid fever

The earliest and main method of diagnosing typhoid and paratyphoid fever is bacteriological - obtaining a blood culture or myeloculture. For this purpose, blood or bone marrow puncture is examined. It is better to inoculate blood on Rapoport medium (bile broth with the addition of glucose, indicator and glass float) in a ratio of 1:10 (1 ml of blood per 10 ml of medium). The culture should be incubated at a temperature of 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 antigens 02 (S. paratyphi A), 04 (S. paratyphi B) and 09 (S. typhi) are used (taking into account their biochemical properties). If the isolated S. typhi culture is not agglutinated by 09 serum, it must be tested with Vi serum.

To isolate S. typhi, one can use exudate obtained by scarification of roseola - roseola cultures grow.

Bacteriological examination of feces, urine and bile is carried out to confirm the diagnosis, monitor bacteriological recovery when convalescents are discharged and to diagnose bacterial carriage. In this case, the material is preliminarily inoculated onto enrichment media (media containing chemicals, such as selenite, which inhibit the growth of E. coli and other representatives of the intestinal microflora, but do not inhibit the growth of salmonella), and then from the enrichment medium onto differential diagnostic media (Endo, bismuth sulfite agar) in order to isolate isolated colonies and obtain pure cultures from them, identified according to the above scheme. To detect O- and Vi-antigens in the blood serum and feces of patients, RSC, RPGA with antibody diagnosticum, coagglutination reactions, aggregate-hemagglutination, and IFM can be used. For accelerated identification of S. typhi, it is promising to use a DNA fragment carrying the Vi-antigen gene as a probe (identification time 3-4 hours).

From the end of the first week of the disease, antibodies appear in the serum of patients, therefore, in 1896, F. Widal proposed the reaction of expanded test tube agglutination for the diagnosis of typhoid fever. The dynamics of the content of antibodies to S. typhi is peculiar: antibodies to the O-antigen appear first, but their titer quickly decreases after recovery; H-antibodies appear later, but they persist after the disease and vaccinations for years. Taking this circumstance into account, the Widal reaction is carried out simultaneously with separate O- and H-diagnosticums (as well as with paratyphoid A- and B-diagnosticums) to exclude possible errors associated with vaccinations or a previously suffered disease. However, the specificity of the Widal reaction is not high enough, therefore, the use of RPGA, in which the erythrocyte diagnosticum is sensitized either with O- (to detect O-antibodies) or Vi-antigen (to detect Vi-antibodies), turned out to be more preferable. The most reliable and specific is the last reaction (Vi-hemagglutination).

Diagnosis of carriage of typhoid fever and paratyphoid fever

The only proof of carriage of bacteria is the isolation of S. typhi, S. paratyphi A, S. paratyphi B cultures from the carrier. The material for the study is the duodenal contents, feces and urine. The complexity of the problem is that the carriers do not always excrete the pathogen with these substrates; there are pauses, and quite long ones. As auxiliary methods that allow narrowing the circle of people to be examined, serological reactions are used (the simultaneous detection of O-, H-, Vi- or O-, Vi-antibodies indicates the possible presence of the pathogen 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 redness 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. Special immunofluorescent antibodies (to the antigens of the L-forms of the pathogen) have been proposed to identify L-forms of S. typhi. An original method for identifying carriers of the bacteria was proposed by V. Moore. It involves examining tampons that are simultaneously thrown into manholes along the entire length of the sewer network of a populated area.

[ 23 ], [ 24 ], [ 25 ], [ 26 ], [ 27 ], [ 28 ]

Treatment of typhoid fever and paratyphoid fever

Treatment of typhoid fever is based on the use of various antibiotics, to which the pathogens are highly sensitive (levomycetin, ampicillin, tetracyclines, etc.). Antibiotics reduce the severity 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 prevention of typhoid fever and paratyphoid fever

Instead of seven different typhoid vaccines used previously, since 1978 our country has produced only one - a chemically sorbed typhoid monovaccine. However, due to the fact that typhoid fever has moved from an epidemic to a sporadic disease (and this became possible, first of all, due to the improvement of water supply and sewerage systems and the increase in sanitary culture of the population), the need for mass immunization against it has disappeared. Therefore, vaccination against typhoid fever is carried out only in case of epidemic indications.