Morphology of Salmonella. Genus Salmonella - methods for detecting Salmonella in pathological material and products. Taking material for research

Purpose of the lesson: to study the main serotypes of Salmonella and substantiate the need for their typification, to work out methods for biochemical and serological typification of Salmonella.

Work plan:

Determine the genus of pathogens of food poisoning by studying crops on media of a short motley row and on three-sugar agar.

Consider the main serotypes of Salmonella and justify the need for their typification.

Carry out biochemical typification of Salmonella pathogens by studying crops on media of a long motley series.

Conduct serological typing of Salmonella using agglutinating Salmonella sera.

Consider the veterinary and sanitary assessment of foodborne pathogens.

Material support: medium with inoculations made by students in the previous lesson, 10 test tubes, pipettes, glass slides, a bacteriological loop, a bacteriological bridge, yogurt, a gas burner (alcohol), sets of diagnostic agglutinating Salmonella sera of complex and monoreceptor, tables (“Growth of pathogens of food , toxic infections on a long motley row", "Serological typing scheme for Salmonella", "Composition of the first set of diagnostic salmonella sera"), distilled water, hand disinfection solution, ethyl alcohol.

Main serotypes salmonella and their practical significance Currently, more than 2000 Salmonella serotypes are known. The most pathogenic for humans and many species of domestic animals are: S. typhi murium, for cattle S. Dublin, S. enteritidis; for pigs: S. cholerae suis, S. typhi suis; for birds: S. gallinarum, S. pullorum. All of the above Salmonella serotypes are pathogenic or conditionally pathogenic for humans. Salmonella are highly resistant to heat treatment and can persist in meat and other foods for a long time. In addition, it should be noted that bacteria of the genus Salmonella

have predominantly saccharolytic properties and practically do not secrete proteolytic enzymes, therefore, organoleptic examination of contaminated products usually does not reveal visible changes. These properties of Salmonella contribute to the widespread prevalence of this food poisoning.

Significance of typification of Salmonella and other foodborne pathogens

Typification of pathogens of food poisoning is necessary in order to establish the pathogenicity of this pathogen for humans and other animal species; to identify the source of food poisoning; to develop optimal methods for the treatment of food poisoning and the selection of the most effective antibiotics and therapeutic sera; for the prevention of salmonellosis and colibacillosis in farms and the optimal selection of vaccines and sera.

Biochemical typification of causative agents of food poisoning

on Wednesdays a long motley row

Biochemical typing is based on the differences in Salmonella certain enzymes. Due to enzymatic differences, some bacteria are able to decompose certain carbohydrates or alcohols, while others do not. For biochemical typification, mediums of a long motley series are used. To do this, it is necessary to take into account the growth of foodborne pathogens on a long motley row. If the pathogen under study breaks down a particular sugar, then during its decay, various acids are released, the pH of His medium becomes acidic, and Andrede's indicator turns the medium red. If the sugar is not broken down, then the medium remains yellow and the reaction is considered negative. After taking into account the reaction, the results obtained are compared with the biochemical properties of the causative agents of food poisoning (see Table 5) and a specific serotype of the bacterium is identified by the elimination method.

If, according to the results of the study, it turned out that two or more Salmonella serotypes have the same biochemical properties, then one of the following methods can be used to typify the pathogen: lengthen the variegated series (re-seeding on His media with those sugars that are cleaved differently by the compared Salmonella serotypes ); to carry out serological reactions with monoreceptor sera reacting with compared Salmonella serotypes. The establishment of the Salmonella serotype can be facilitated by logical analysis (the epizootic state of the settlement, the geography of the prevalence of the compared serotypes, their pathogenicity for this species of animals).

Biochemical typification of bacteria of the genera E. Coli and Proteus is carried out in a similar way (see Table 6).

In addition to Salmonella, the causative agents of typhoid fever and paratyphoid A and B, more than 2,400 pathogenic Salmonella serovars have been described that cause acute gastroenteritis in humans, typhoid-like and septicopyemic forms of the disease, united under the name salmonellosis. The main causative agents of salmonellosis are Salmonella serovars enteritidis, typhimurium, choleraesuis, haifa etc.

Bacteriological method is the leading method in the laboratory diagnosis of salmonellosis (Scheme 10). Salmonella cultures can most often be isolated from the feces of patients, somewhat less often from vomit and gastric lavage, and even more rarely from blood, urine and bile. Isolation of Salmonella from blood, bone marrow, cerebrospinal fluid, vomit and gastric lavage confirms the diagnosis of salmonellosis. Salmonella carriers can be found in feces, urine, and bile.

The test material is inoculated on plates with bismuth-sulfite agar and in accumulation media (magnesium, selenite), from which, after 6-10 hours, re-seeding is done on bismuth-sulfite agar. The crops are grown at a temperature of 37 0 C, on the second day black colonies are selected and inoculated on the Olkenitsky (or Ressel) medium to accumulate a pure culture . On the 3rd day of the study, the isolated pure cultures are subcultured into the media of the "variegated" series and RA is placed with polyvalent and group (A, B, C, D, E) adsorbed salmonella sera . If a positive result is obtained with one of the groups of sera, RA is performed with adsorbed O-sera characteristic of this group, and then with monoreceptor H-sera (non-specific and specific phases) to determine the serogroup and serovar of Salmonella in accordance with the Kaufman-White scheme.

On the 4th day of the study, changes in the media of the “variegated” series are taken into account (Table 10). The causative agents of salmonellosis are the same as Salmonella paratyphoid B , do not ferment lactose and sucrose, break down glucose, mannitol and maltose with the formation of acid and gas, do not form indole and (with a few exceptions) emit hydrogen sulfide.

Scheme 10. Microbiological diagnosis of salmonellosis.

Bioassay. Material from the patient produce oral infection of white mice, which die in 1-2 days from septicemia. When sowing blood from the heart and material from the internal organs, a Salmonella culture is isolated.

Serological study - a study using RNGA of paired blood sera of patients taken at intervals of 7-10 days with salmonella polyvalent and group (groups A, B, C, D, E) diagnosticums. An increase in antibody titer by four or more times is of diagnostic value.

Independent work of students

To study the main stages of bacteriological examination of the method for diagnosing salmonellosis.

1. Accounting for Salmonella cultures on Levin and Olkenitsky's medium (demonstration).

2. Purity control of the endowed Salmonella culture(a smear was prepared from the environment of Olkenitsky, it was stained according to Gram). Microscopically and draw demonstration micropreparations of the pathogens of salmonellosis S. enterica spp. enterica ser. enteritidis , typhimurium , choleraesuis .The causative agents of salmonellosis are gram-negative rods identical in morphology with rounded ends.

3. Identification of the isolated pathogen culture:

- by antigenic properties- accounting for the approximate agglutination reaction on glass with diagnostic monoreceptor salmonella agglutinating O- and H sera in accordance with the Kaufman-White scheme;

- by biochemical properties- determination of the biochemical activity of the studied culture according to the "variegated" series of Giss or Peshkov (demonstration). Pay attention to the breakdown of glucose, the absence of breakdown of lactose and sucrose.

- by phagolizability(accounting for a sample with phage - demonstration)

4. Determination of the sensitivity of the isolated culture to antibiotics using the paper disc method(demonstration).

More than 2,000 different bacteria that cause diseases in humans and animals belong to this genus of the Enterobacteriaceae family. These diseases are called salmonellosis. Salmonella are similar in morphological, cultural and enzymatic properties, but differ in antigenic structure.

Salmonella is divided into monopathogenic and polypathogenic. The former include causative agents of typhoid fever, paratyphoid A and paratyphoid B. Only humans suffer from these diseases. The second group includes pathogens that affect humans and animals.

S. typhi were first discovered by Ebert (1880) in the organs of a person who died of typhoid fever. Ashar and Bansod (1886), in diseases similar to typhoid fever, isolated from the pus and urine of patients with bacteria that differ in biochemical and serological properties from the causative agents of typhoid fever. They were called S. paratyphi A and S. paratyphi B. Almost simultaneously, the American scientist D. Salmon (1885) first described the causative agents of swine cholera (S. choleraesuis). Subsequently, many similar bacteria were described, united in the genus Salmonella, named after the scientist who described them.

Morphology. All Salmonella are small, 1.0-3.0 × 0.6-0.8 µm sticks with rounded ends. Gram-negative. Mobile, peritrichous. Spores and capsules do not form.

cultivation. Salmonella are facultative anaerobes. They are not demanding on nutrient media. They grow well on MPA and MPB at 37°C (from 20 to 40°C) and pH 7.2-7.4 (from 5.0 to 8.0). On MPA they form tender, translucent, slightly convex, shiny colonies, on MPA - uniform turbidity.

During the initial sowing of material from patients (feces, urine, vomit, blood, bile), a slow growth of Salmonella is often noted. For their accumulation, seeding is carried out on enrichment media: selenite broth, Muller's medium, Kaufman's medium. Elective (selective) media are also used: bile (10-20%) and Rappoport media.

On the differential diagnostic media of Endo, EMS, Ploskirev, Salmonella grow in the form of colorless colonies, since they do not break down lactose, which is part of the medium. On bismuth-sulfite agar, after 48 hours they form black colonies, leaving a trace after they are removed with a loop (except for Salmonella paratyphoid A).

In freshly isolated cultures of S. paratyphi B, after incubation in a thermostat for 18-20 hours and keeping at room temperature for 1-2 days, a mucous wall forms on the periphery of the colony.

Enzymatic properties. Salmonella break down glucose, mannitol, maltose with the formation of acid and gas. An exception is the causative agents of typhoid fever (S. typhi), which break down these sugars only to acid. Salmonella does not ferment lactose and sucrose. Proteolytic properties: most Salmonella breaks down protein media with the formation of hydrogen sulfide (the causative agents of paratyphoid A are distinguished by the absence of this property). Indole does not form. Gelatin is not liquefied.

Toxigenicity. Salmonella contains endotoxin - a lipopolysaccharide-protein complex.

Antigenic structure and classification. As early as the beginning of the 20th century, scientists noticed the different nature of Salmonella antigens. Kaufman (1934), based on the results of the agglutination reaction of various Salmonella with a set of sera, divided all Salmonella into groups and types and proposed a diagnostic scheme for their antigenic structure. In accordance with this scheme, Salmonella is currently being identified.

Salmonella contains two antigenic complexes: O and H, O-antigen - a lipopolysaccharide-protein complex; it is thermostable, inactivated by the action of formalin. The H-antigen is associated with flagella, has a protein nature; it is thermolabile, inactivated by alcohol and phenol, but resistant to formalin.

All Salmonella are divided into O-groups, each of which is characterized by the presence of certain O-antigens: the main one, indicated by an Arabic numeral (2, 4, 7, 8, 9, etc.), and additional ones common to several O-groups ( 1, 12). Currently, more than 60 O-groups are known, denoted by capital letters of the Latin alphabet (A, B, C, D, E, etc.).

S. typhi also contains the Vi-antigen, which is located in the microbial cell more superficially than the O-antigen, and prevents agglutination of the culture with O-serum. This antigen is thermolabile. Its presence was associated with the virulence of the pathogen. The Vi antigen is also contained in the cells of S. paratyphi C.

Salmonella H-antigens have two phases. Salmonella of different serovariants of the same O-group have a different first phase of the H-antigen, which is denoted by lowercase letters of the Latin alphabet: a, b, c, d, eh ... u, z, etc. The second phase of the H-antigen is usually denoted in Arabic numbers: 1, 2, 5, 6, 7 and lowercase Latin letters. The combination of various O- and H-antigens determines the antigenic structure of cultures and their name.

In practical work, to determine the antigenic structure of Salmonella, adsorbed monoreceptor agglutinating sera are used, which contain antibodies to one antigen. Put the agglutination reaction on the glass and the presence of agglutination with certain sera characterize the antigenic structure of the selected culture. For example, the culture is agglutinated with O-sera "9" and "12" and H-serum "d"; find in the scheme a serovar with such an antigenic composition (S. typhi), put an additional reaction with Vi-serum

There are sets of specific Salmonella phages that lyse only the Salmonella of the corresponding phage. To determine the fagovar of S. typhi cultures containing the Vi antigen, 45 phages are produced in our country; for S. paratyphi B-11 phages; S. paratyphi A - 6, etc. These studies are carried out to determine the source and route of transmission of the infection.

Resistance to environmental factors. Salmonella is quite resistant. At a temperature of 100 ° C, they die instantly, at 60-70 ° C - in 10-15 minutes. They tolerate low temperatures well, can be stored in clean water and ice for several months; in smoked and salted meat - up to 2 months. Resistant to drying, long-lasting in dust.

Under the action of disinfectants, they die within a few minutes (2-5% phenol solution, 1:1000 sublimate solution, 3-10% chloramine solution).

Animal susceptibility. Most salmonella causes disease in humans and many species of animals and birds (polypathogenic).

Typhoid fever, paratyphoid A and B

Source of infection. A sick person and a bacteriocarrier.

Transmission routes. Infectious agents are transmitted through objects contaminated with human secretions, through hands, water, food. Often pathogens are carried by flies. Depending on the ways of transmission, household, water, food outbreaks of typhoid fever and paratyphoid fever are distinguished.

Pathogenesis. Infection occurs through the mouth. From the oral cavity, microorganisms enter the stomach, where they are partially destroyed under the influence of gastric juice and enzymes. The remaining Salmonella enter the small intestine, penetrate into the lymphoid tissue of the small intestine (group lymphatic and solitary follicles), in which they multiply during the incubation period (10-14 days). By the end of this period, pathogens enter the lymph and blood (bacteremia) and spread throughout the body. During this period, they are localized in the lymphoid tissue of internal organs, the macrophage system, the liver, spleen, and bone marrow. Salmonella accumulate in the gallbladder, where they find favorable conditions for reproduction, since bile is a good breeding ground for these bacteria. At the same time, they again enter the small intestine and, affecting the already sensitized lymphoid tissue (group lymphatic and solitary follicles), cause the formation of specific typhoid ulcers (Fig. 42).

During the period of bacteremia, part of the microorganisms is destroyed, while endotoxin is released and intoxication occurs: the temperature rises, general malaise, weakness, headache, etc. appear. , urine, saliva, etc.

The period of convalescence (recovery) is characterized by the cleansing of the body from the pathogen, increased phagocytic activity of cells, and the accumulation of antibodies in the blood.

However, in typhoid fever and paratyphoids, bacterial excretion often does not end with the patient's recovery - a bacteriocarrier is formed. Chronic inflammatory phenomena in the gallbladder contribute to the survival of Salmonella in bile and their long-term excretion from the body (sometimes up to several years).

Immunity. Post-infectious immunity is quite tense and long. Recurrences are rare. During the course of the disease, antibodies are produced: at the end of the 1st week, agglutinins, precipitins and other types of antibodies appear. Their number increases, reaching a maximum on the 14-15th day of illness. Antibodies remain in the blood serum of the ill person for a long time.

The activity of phagocytes and other cellular protective factors are also important in the formation of the body's immune state.

Prevention. Compliance with personal hygiene and carrying out all sanitary and hygienic measures: supervision of water sources, control of food and catering establishments.

Specific prophylaxis. A chemical vaccine containing full antigens of typhoid, paratyphoid A and B pathogens and tetanus toxoid (TAB "te). There is also a typhoid alcohol vaccine enriched with the Vi antigen, the introduction of which for prophylactic purposes has a good effect. In the focus of the disease, people who have been in contact with patients are given a typhoid bacteriophage.

Treatment. Antibiotics: chloramphenicol, tetracycline, etc.

Food poisoning

When eating foods contaminated with Salmonella of various serovars (except for S. typhi, S. paratyphi A and B), food toxic infections occur.

Sources of infection. Animals and birds sick with salmonellosis, or healthy, in the body of which, without harming them, there are salmonella.

Transmission routes. Infection occurs when eating meat, meat products, eggs, milk, dairy products infected with salmonella. The most dangerous is the use of food in which the reproduction and death of Salmonella and the accumulation of endotoxin occur.

Pathogenesis. Once in the body through the mouth, salmonella penetrate the digestive tract. At the same time, a significant part of the bacteria dies and endotoxin is released, which can penetrate into the blood. There are symptoms of damage to the gastrointestinal tract and general toxicosis. The disease lasts no more than 4-5 days; sometimes those who have been ill become carriers of salmonella.

Immunity short. Various antibodies accumulate in the blood of patients and convalescents: agglutinins, precipitins, etc. There are a lot of Salmonella serovars, and immunity is specific, that is, directed against only one pathogen, so a person can get sick with salmonellosis again.

Prevention. Constant strict veterinary and sanitary control over livestock, slaughter and cutting of carcasses, storage and processing of meat and meat products. It is necessary to strictly observe the sanitary and hygienic regime and personal hygiene in catering establishments.

Specific prophylaxis. People who are in the foci of food poisoning should be given a salmonella polyvalent bacteriophage.

Treatment. The main therapeutic agent is detoxification of the body - the introduction of a large amount of liquid, gastric lavage. Antibiotics are also used.

Nosocomial Salmonella Infection

The causative agent of nosocomial Salmonella infection is most often S. typhimurim. There are also "hospital" outbreaks caused by S. heidelberg, S. derby, etc. Although the morphological and cultural properties of these pathogens do not differ from those of other Salmonella, there are some biological features characteristic of them. So, for example, pathogens of nosocomial infections belong to certain biovars, they are more pathogenic for white mice, etc.

Sources of infection. More often a bacteriocarrier, less often a patient.

Transmission routes. Indirect contact predominates (toys, underwear, patient care items). Less common are airborne and foodborne transmission routes.

Pathogenesis. The disease develops against the background of a weakening of the body and a decrease in its immune activity. The pathogen enters the body orally or through the respiratory tract, which determines the development of the pathological process: dysfunction of the gastrointestinal tract with dehydration or damage to the respiratory system, bacteremia, septic complications. First of all, young children get sick.

Immunity. It is produced only in relation to one serovar of Salmonella.

Prevention. Strict observance of the sanitary and hygienic regime in medical institutions.

Specific prophylaxis. If nosocomial Salmonella infection occurs, children who have been in contact with the patient should be given a Salmonella polyvalent bacteriophage.

Treatment. Symptomatic.

Control questions

1. What are the morphological, cultural and enzymatic properties of Salmonella?

2. What is the classification of Salmonella based on?

3. What diseases are caused by salmonella?

Microbiological research

The purpose of the study: the isolation of pathogens and the determination of the serovar of Salmonella.

Research material

2. Bowel movements.

4. Duodenal contents.

Depending on the stage of the disease, different materials are examined.

The contents of roseola, bone marrow, sputum and the material obtained at the autopsy - pieces of organs can also be subjected to research.

In case of toxic infections, gastric lavage, vomit, food residues can serve as research material.

Regardless of the nature of the material taken for the study, from the moment the pure culture was isolated, the study is carried out according to the general scheme.

Basic research methods

1. Bacteriological (Fig. 43).

2. Serological.

Research progress

Second day of research

Remove the cups from the thermostat (incubation 18-24 hours) and view the grown colonies with the naked eye and with a magnifying glass. Several (5-6) suspicious colonies are isolated on Olkenitsky's or Russell's medium. Sowing is carried out as follows: the removed colony is carefully, without touching the edges of the test tube, introduced into the condensation liquid, then the entire sloping surface of the medium is inoculated with strokes and an injection is made into the depth of the column to detect gas formation. The injection should be made in the center of the agar column.

Test tubes with crops are placed in a thermostat. If the test material was sown on the enrichment medium, then after 18-24 hours, the enrichment medium is sown on plates with differential media. Further research is carried out according to the general scheme.

1 (A black trace remains in place of the removed colonies (the color of the medium changes).)

Third day of research

Take out test tubes with crops from the thermostat and view the nature of growth.

Combination media contains lactose, glucose, sometimes urea, and an indicator. The breakdown of glucose occurs only under conditions of anaerobiosis. Therefore, the sloping surface of the medium does not change during the breakdown of glucose, and the column turns into a color corresponding to the indicator. Bacteria that break down lactose and urea change the color of the entire medium.

If isolated cultures ferment lactose or break down urea, changing the color of the entire medium, then they are not Salmonella and a negative answer can be given.

A culture that degrades only glucose is subjected to further study: smears are made, they are Gram-stained and microscoped. If there are gram-negative rods in the smears, their mobility and enzymatic properties are studied.

Motility can be determined in a hanging drop or a crushed drop, and by growth pattern in semi-liquid Hiss medium or 0.2% agar. In the presence of mobility during sowing by injection, growth on the medium is diffuse, the medium becomes cloudy.

To detect enzymatic activity, inoculation is carried out on Hiss media, MPB, peptone water. Indicator papers are lowered (under the cork) into test tubes with the last media for the determination of indole and hydrogen sulfide. Do also sowing on litmus milk.

Fourth day of research

Biochemical activity is taken into account according to the result of fermentation of carbohydrate and other media (see Table 33).

Note. to - the formation of acid; kg - formation of acid and gas; u - alkalization; + presence of property; - lack of property.

Having determined the morphological, cultural and enzymatic properties of the isolated culture, it is necessary to analyze the antigenic structure (Table 34).

Serological identification of Salmonella begins with an agglutination test on glass with polyvalent O-serum A, B, C, D, E. In the absence of agglutination, the isolated culture is tested with polyvalent O-serum to rare groups of Salmonella. In case of a positive reaction with one of the sera, the culture is tested with each O-serum, which is part of the polyvalent one, to determine the O-serogroup. Having established that the culture belongs to the O-group, its H-antigens are determined with the sera of the first, and then the second phase (Table 35).

Salmonella typhoid culture is also tested with Vi-serum. The causative agents of typhoid fever containing the Vi-antigen are tested with Vi-phages (there are 86 of them). Determination of the phage type is of great epidemiological importance (see Fig. 43).

Phage typing technique. 1st method. 20-25 ml of agar are poured into Petri dishes and dried with open lids in a thermostat. The bottom of the cup is divided into sectors. The name of the phage is written on each sector. A 4-6 hour broth culture is studied as it contains more Vi antigen. 8-10 drops of broth culture are applied to the surface of the agar and rubbed over the surface of the agar with a glass spatula. Cups with crops are dried with open lids in a thermostat. A drop of the corresponding typical phage is applied to each sector. After the drops have dried, the cups are placed in a thermostat for 18-24 hours. The result is taken into account with the naked eye or with a magnifying glass through the bottom of the cup.

The presence of culture lysis by one or several typical phages makes it possible to determine whether the isolated strain belongs to a particular phage type.

2nd method. The culture is applied dropwise to the nutrient medium. After drying the culture in a thermostat, a drop of a typical phage is applied to each drop. Put in a thermostat.

The degree of lysis is expressed by the four-cross system.

Control questions

1. What material is examined for typhoid, paratyphoid and toxic infections?

2. In what period of the disease is the blood culture isolation method used?

3. In what period of the disease with typhoid fever and paratyphoid fever are feces and urine examined?

4. On what differential diagnostic media is the test material inoculated?

5. What media are used for the accumulation of Salmonella?

6. What is determined by monoreceptor O-sera and what by monoreceptor H-sera?

1. Study according to the table. 32 the nature of the growth of Salmonella on differential media. Look at the teacher's cups with the inoculation of typhoid Salmonella on the Endo, Ploskirev media, bismuth-sulfite agar. Sketch the colonies with colored pencils and show the teacher.

2. Take Salmonella culture, O- and H-monoreptor sera from the teacher. Put the agglutination reaction on the glass. Consider the reaction and show the teacher.

The isolated culture gave a positive agglutination test with O-serum 4. What H-sera should be tested with agglutination if you think it is a culture of Salmonella paratyphoid B?

Serological diagnosis of typhoid and paratyphoid fever

Vidal reaction. From the second week of the disease, antibodies against the infectious agent accumulate in the blood of patients. To identify them, the patient's blood serum is examined in the agglutination reaction. Killed Salmonella cultures - diagnosticums - are used as an antigen.

To set up the Vidal reaction, the patient's serum, a set of diagnostic kits, and isotonic sodium chloride solution are used.

Blood (2-3 ml) from the pulp of the finger or cubital vein is collected in a sterile tube and delivered to the laboratory. In the laboratory, the test tube is placed in a thermostat for 20-30 minutes to form a clot, then the clot is circled with a Pasteur pipette to separate it from the wall of the test tube, and placed in the cold for 30-40 minutes. The separated serum is sucked off and used to set up an agglutination reaction with diagnosticums from salmonella typhoid and paratyphoid. To obtain serum, blood can be centrifuged.

When an infectious process occurs - typhoid fever or paratyphoid fever - O- and H-antibodies to the pathogen antigens of the same name are produced in the body.

O-antibodies appear first and disappear rather quickly. H-antibodies persist for a long time. The same thing happens during vaccination, therefore, a positive Vidal reaction with O- and H-antigens indicates the presence of a disease, and a reaction with only H-antigens can be both in those who have been ill (anamnestic reaction) and in those who have been vaccinated (vaccination). Based on this, the Vidal reaction is placed separately with O- and H-antigens (diagnosticums).

Since clinically typhoid fever and paratyphoid fever A and B are similar, to identify the nature of the disease, the patient's serum is tested simultaneously with diagnosticums from salmonella typhoid and paratyphoid A and B.

The Vidal reaction is widely used because it is simple and does not require special conditions.

There are two ways to set up the reaction: drop and volume (see Chapter 12). In practice, the volumetric method is more often used. When setting up a linear agglutination reaction, the number of rows should correspond to the number of antigens (diagnosticum). The causative agent of the disease is considered to be a microorganism, the diagnosticum from which was agglutinated by the patient's serum. Sometimes group agglutination is noted, since the causative agents of typhoid and paratyphoid fever have common group antigens. In this case, the result of the reaction in the series in which agglutination is noted in a larger dilution of serum is considered positive (Table 36).

Note. In practice, the Vidal reaction is put with four diagnosticums: typhoid fever "O" and "H", and paratyphoid A and B - with diagnosticums "OH".

If agglutination occurs only in small dilutions of serum - 1:100, 1:200, then to distinguish the reaction in case of a disease from a vaccination or anamnestic, they resort to re-staging the agglutination reaction after 5-7 days. In a patient, the antibody titer rises, but in a vaccinated or recovered patient it does not change. Thus, an increase in the titer of antibodies in the blood serum serves as an indicator of the disease.

Vi-agglutinins appear in the patient's blood in response to the introduction of typhoid pathogens possessing the Vi-antigen into the body. They are determined from the 2nd week of illness, but their titer usually does not exceed 1:10. The detection of Vi-antibodies is associated with the presence of typhoid pathogens in the body; therefore, the determination of these antibodies is of great epidemiological importance, since it makes it possible to identify bacterial carriers.

Vi-hemagglutination reaction. This is the most sensitive reaction for detecting antibodies.

The principle of the reaction is that human (group I) or sheep erythrocytes, after special treatment, can adsorb the Vi-antigen on their surface and acquire the ability to agglutinate with the corresponding Vi-antibodies.

Erythrocytes with antigens adsorbed on the surface are called erythrocyte diagnosticums.

To set up the Vi-hemagglutination reaction, take:

1) patient's blood serum (1-2 ml); 2) erythrocyte Salmonella Vi diagnosticum; H) Vi-serum; 4) O-serum; 5) isotonic sodium chloride solution.

The reaction is placed in agglutination test tubes or in plastic plates with wells.

Blood is taken from the patient in the same way as for the Vidal reaction. Get the serum. Two-fold serial dilutions are prepared from serum, starting from 1:10 to 1:160.

0.5 ml of each dilution is added to the well and 0.25 ml of erythrocyte diagnosticum is added. The reaction is put in a volume of 0.75 ml.

The controls are: 1) standard agglutinating monoreceptor serum + diagnosticum - the reaction must be positive up to the serum titer; 2) diagnosticum in isotonic sodium chloride solution (control) - the reaction should be negative.

The contents of the wells are thoroughly mixed, placed in a thermostat for 2 hours and left at room temperature until the next day (for 18-24 hours).

Accounting begins with control. The reaction is evaluated depending on the degree of diagnosticum agglutination.

The results are taken into account according to the four-cross system:

Erythrocytes are completely agglutinated - sediment at the bottom of the hole in the form of an "umbrella";

+++ "umbrella" is smaller, not all erythrocytes were agglutinated;

++ "umbrella" is small, at the bottom of the hole there is a sediment of non-agglutinated erythrocytes;

The reaction is negative; erythrocytes did not agglutinate and settled on the bottom of the well in the form of a button.

Control questions

1. In what period of the disease is the Vidal reaction diagnosed?

2. What ingredients are needed to perform the Vidal reaction?

3. What diagnosticums are used for Vidal's reaction?

4. Which of the serological reactions is the most sensitive in diagnosing typhoid and paratyphoid infections?

5. What diagnosticum is used in the formulation of the Vi-hemagglutination reaction?

6. What serum is used to determine the presence of Vi-antigen in the studied culture?

7. What is the significance of the definition of the Vi-phage type?

Take from the teacher O- and H-diagnosticums from salmonella typhoid, paratyphoid A and paratyphoid B and the patient's serum. Put Vidal's reaction.

Nutrient media

Environments EMS, Ploskirev, bismuth-sulfite agar are produced by the medical industry in the form of a dry powder. They are prepared according to the instructions on the label: a certain amount of powder is weighed, the appropriate amount of water is poured, boiled and poured into sterile Petri dishes.

Wednesday Russell. In 950 ml of distilled water add 40 g of dry nutrient medium and add 5 g of nutrient agar. Heat to boil and dissolve the powders. Dissolve 1 g x in 50 ml of distilled water. hours of glucose and added to the prepared mixture. The medium is poured into sterile test tubes of 5-7 ml, sterilized with flowing steam (2 days for 2 minutes) and beveled so that a column remains. Russell's medium with mannitol and sucrose is prepared in the same way.

Olkenitsky's medium from dry agar. 2.5 g of dry nutrient agar is melted in 100 ml of distilled water. All the ingredients indicated in the recipe (label) are added to the agar cooled to 50 ° C. The medium poured into test tubes is sterilized with flowing steam (3 days for 20 minutes) and then bevelled. The finished medium should be a pale pink color.

No. 2 Salmonella causative agents. Taxonomy. Feature. Microbiological diagnosis of salmonellosis. Treatment.
Acute intestinal zoonotic infection caused by Salmonella serovars, characterized by lesions of the gastrointestinal tract.
Morphological properties: mobile, gram "-" sticks, no capsules. They grow well on simple nutrient and bile-containing media. On dense ones, they form colonies in R- and S-forms, on liquid ones - turbidity. On lactose-containing media form colorless colonies.
Biochemical activity: glitch fermentation. to acid and gas, no lactose fermentation, hydrogen sulfide production, no indole formation.
Antigenic structure: somatic O-antigen, flagellar H-antigen, Some - K-antigen. Genus Salmonella consists of two types - type S.enterica, which includes all Salmonella that are pathogens of humans and warm-blooded animals, and the species S.bongori, which is subdivided into 10 serovars.
View S.enterica divided into 6 subspecies, which are subdivided into serovars. Some Salmonella serovars, in particular S. typhi, have a polysaccharide Vi antigen, which is a type of K antigen.
Epidemiology. The causative agents of salmonellosis are a large group of Salmonella, which is part of the subspecies enterica. The most common causative agents of salmonellosis in humans are serovars S. Typhimurium, S. Dublin, S. Choleraesuis. The main transmission factors are meat, milk, eggs, water.
Pathogenesis and clinic. The disease proceeds in a local form of gastroenteritis, the leading syndrome is diarrheal. Having invaded the mucosa of the small intestine through M-cells and penetrating into the submucosa, Salmonella are captured by macrophages, being transferred by them to Peyer's patches, where they form the primary focus of infection. In this case, endotoxin and protein enterotoxin are released. Enterotoxin activates the entry into the intestinal lumen of a large amount of fluid, K, Na. Diarrhea, vomiting.
Immunity: Non-stressed, serovar-specific, secretory IgA mediated, which prevents Salmonella from penetrating the small intestinal mucosa. Antibodies can be detected in the blood.
Microbiological diagnostics. Bacteriological examination is subjected to vomit, gastric lavage, feces, bile, urine, blood. When identifying isolated cultures, a wide range of diagnostic O- and H-sera is required.
For serological studies, RNGA, ELISA are used. An important diagnostic value is the increase in antibody titer in the dynamics of the disease.
Treatment. Pathogenetic therapy is used, aimed at normalizing water-salt metabolism. In generalized forms - etiotropic antibiotic therapy.
Salmonella group, adsorbed O- and H-agglutinating sera. They are used to establish serogroups and serovars of Salmonella in the agglutination reaction.
Salmonella O- and H-monodiagnosticums are suspensions of Salmonella killed by heating (O-diagnosticum) or formalin treatment (H-diagnosticum). They are used for serodiagnosis of typhoid fever.
Prevention . Specific prevention of salmonellosis in agricultural animals and birds. Nonspecific prevention - carrying out veterinary and sanitary measures.

Table of contents of the subject "Shigella. Dysentery. Salmonella. Salmonellosis.":

Morphology of Salmonella. Cultural properties of Salmonella. Biochemical signs of salmonella.

Genus Salmonella It is represented by small elongated bacteria with rounded ends, 0.7-1.5x2-5 µm in size. Bacteria do not have capsules.

Most salmonella isolates mobile (peritrichous), but there are immobile mutants and serovars. chemoorganotrophs, oxidase-negative, catalase-positive.

Temperature Optimum Salmonella is 35-37 ° C, the optimum pH is 7.2-7.4. growth of salmonella suppress or limit high concentrations of sodium chloride and sugar. On nutrient media, Salmonella form small (2-4 mm) transparent S-colonies typical of most enterobacteria. They also form rough and dry R-colonies. On Endo agar, S-colonies are pinkish and transparent, on Plbskirev agar they are colorless and look more dense and cloudy, on bismuth-sulfite agar they are black-brown, with a metallic sheen, surrounded by a black halo, the medium under the colonies turns black. The exceptions are S. paratyphi A, S. choleraesuis and some others, which form brown-greenish colonies on bismuth-sulfite agar (Fig. 25, see color insert). On broth, S-forms give a uniform turbidity of the medium; R-forms - sediment.

Biochemical signs of salmonella

Biochemical signs of salmonella are presented in the table below. The characteristic properties of Salmonella are the formation of H 2 S and the absence of indole formation (excluding some serovars).