METHODOLOGICAL INSTRUCTION

METHODOLOGICAL INSTRUCTION

FOR 3nd COURSE-STUDENTS

of MEDICAL FACULTY

 

LESSON № 17 (PRACTICAL  6 HOURS)

 

Themes:

I. Causative agents of anaerobic infections. Laboratory diagnosis of anaerobic infections

Ii. Causative agents of zoonose diseases. Laboratory  diagnosis of plague, other yersiniosis. Laboratory diagnosis of brucellosis, tularaemia, and anthrax.

 

Aim: To master the main principles of laboratory examination and prophylaxis of plague and other yersinioses. To take possession of techniques of serological examination for diagnosis of tularemia and brucellosis, to familiarize with biological preparation, which are used for diagnosis and specific prophylaxis of tularemia and brucellosis. To master the main principles of laboratory examination and prophylaxis of anthrax.

To master the main principles of laboratory examination of gas gangrene. To master the main principles of laboratory examination of tetanus and botulism, to familiarize with preparation which are used for diagnosis, specific prophylaxis and treatment of tetanus and botulism.

Professional motivation of students:In 1970, the genus Yersinia was created in commemoration of Yersin, the discoverer of the plague bacillus. Ithad been shown that yersiniae could conjugate with Escherichia coli and accept various plasmids such as resistancetransfer factors. In addition, Yersinia organisms have several antigens in common with some of the enterics and thus appear to be better positioned taxonomically as a members of the Enterobacteriaceae.

The genus Yersinia contains three species that are pathogenic for humans: Yersinia pestis, the etiologic agent of bubonic plague, and Yersinia enterocolitica and Yersinia pseudotuberculosis, which are primarily responsible‘ for a mild-to-severe gastroenteritis. All are pathogens that humans acquire directly or indirectly from infected animals, and all share a number of virulence factors.

Brucellosis and tularemia are dangerous infection disease. The knowledge obtained enables the students to understand the morphology of causative agents, epidemiology, pathogenesis and laboratory diagnosis of these nozological units, and help  to make a good treatment and prophylaxis ones.

Anthrax is primarily a disease of herbivorous sheep, cattle, horses, hogs, camels, and goats. It can occasionally be transmitted to people. The disease is caused by Bacillus anthracis, a Gram-positive, aerobic, endospore-forming rod. This bacterium produces a toxin that increases vascular permeability, leading to hemorrhage, necrosis, and pulmonary edema. Anthrax can be cutaneous from skin contiact, pulmonary from inhalation of endospores, or gastrointestinal from ingestion of B. anthracis. Although the pulmonary form is far less common than the cutaneous form, it generally is fatal.

There are a number of different species of the genus Clostridium that can infect wounds to produce the clinical syndrome known as gas gangrene (also called clostridial myonecrosis). All of these species are morphologically similar: they are gram–positive, spore–forming rods, and all arc obligate anaerobes. Furthermore, they are pervasive in soil and in feces, so that contamination of a wound with dirt may likely mean contamination with one or more of the gas gangrene clostridia. Clostridium tetani, the causative agent of tetanus, is widely distributed in the soil and feces of many animals, including humans. Clostridium botulinum is the  causative agent of a highly fatal food poisoning that usually follows the ingestion ofa preformed toxin produced by the organisms growing in the food.

 

 

Methodology of Practical Class. 9.00-12.00

 

THEME: I. Causative agents of zoonose diseases.

Microbiological diagnosis of plague, other yersiniosis.

Work 1. To examine under the microscope the smears of Y. pestis.

Work  2. To familiarize with Y. pestis growth outrient media.

Work  3. To carry out indirect hemagglutination test for serologic diagnosis of plague.

 

 

Test results are assessed after complete erythrocyte sedimentation in control (6 well) – markedly localized erythrocytes sediment (“rouleaus”), In the experimental wells rapid erythrocytes agglutination with starlike, marginally festooned sediment (“umbrella”) on the bottom are observed. The titer of serum is its maximum dilution, which causes hemagglutination. Diagnostic titre –   1:40.

Work  4. To familiarize with biological preparations, which are used for laboratory diagnosis and prophylaxis of a plague and others yersinioses

 

Microbiological diagnosis of brucellosis, TULARAEMIA, and anthrax.

Work 1. To study morphology of Brucella and Francisella in museum preparations.

Work 2.To carry out blood-drop agglutination reaction for rapid diagnosis of tularemia.

Work  3. To carry out agglutination test with patient’s serum for serologic diagnosis of tularemia.

Work  4. To carry out Wright’s test with patient’s serum for serological diagnosis of Brucellosis.

Work  5. To carry out Huddleson’s agglutination test for serological diagnosis of Brucellosis.

Work  6. To familiarize with biological preparation, which are used for laboratory diagnosis and specific prophylaxis of tularemia and brucellosis.

Work  7. To study the motility of Anthracoides, using wet-mount and hanging-drop technique

Work  8. To familiarize with cultivation of B. anthracis (growth in the MPB, gelatin, and on the surface of MPA).

Work  9. To examine the presence of B. anthracis antigen in tested material by Ascoli thermoprecipitation test.

Work  10.To familiarize with biological preparations using for laboratory diagnostics and prophylaxis of anthrax.

 

THEME II.Causative agents of anaerobic infections.

LAboratory diagnosis of anaerobic infections.

Work  1. To study morphology of gas gangrene Clostridia in smears from the culture and impression preparations from the tissues.

Work  2. To study character of the Clostridia growth on Kitt-Tarozzi medium, milk, Zeissler’s sugar blood agar. and in agar stab cultures.

Work  3. To inoculate a soil into Kitt-Tarozzi medium and  milk.

Work  4. To familiarize with slides and pictures about different properties of gas gangrene Clostridia.

Work  5. To familiarize with biological preparations which are used for laboratory diagnosis, active and passive prophylaxis and treatment of gas gangrene.

Work  6. To study morphological properties of Peptostreptococcus, Bacteroides, Prevotella. Porphyromonas, Fuzobacteria

Work  7. To make schemes of laboratory diagnosis of non-clostridial infections.

To fill in the table:

 

Microbiological diagnosis of tetanus and botulism.

Work  1. To study morphology of C. botulinum and C. tetani in smears.

Work  2. To study character of the C. tetani and C. botulinum nutrient media.

Work  3. To prepare the smears from microbial cultures on  Kitt-Tarozzi medium and  milk.

Work  4. To inoculate bacteria from Kitt-Tarozzi medium or milk by Veinberg’s technique.

Work  5. To perform neutralization test for determining of C. botulinum toxin type on white mice.

Work  6. To familiarize with biological preparations which are used for laboratory diagnosis, active and passive prophylaxis and treatment of tetanus and botulism.

 

Break 12.00-12.30

 

Individual Students Program.

  You should be  prepared for the practical class using the existing textbooks and lectures. Special attention should be paid to the following questions:

 

THEME I. Causative agents of zoonose diseases.

Microbiological diagnosis of plague, other yersiniosis.

1. Structure, tinctorial properties of causative agent of plague. Cultivation.

2. Biochemical and other differential diagnistic properties of Yersinia pestis.

3. Antigenic structure and toxon production of Y. pestis.

4. Reservoir of a plague iature, source of an infectious agents, mechanisms of transmission, vectors.

5. Main clinical forms of a plague.

6. Laboratory diagnosis of plague:

a  – features of collection of tested material a plague;

b – main stages of bacteriological diagnosis;

c – serological methods of diagnosis;

g – feature of biological method of diagnosis;

7. Quarantine measures and methods of specific prophylaxis of a plague.

8. Characteristic and biological properties of causative agents of yersiniosis (Y. pseudotuberculosis, Y. enterocolіtіca).

9. Diseases, which they cause. Methods of laboratory diagnosis and prophylaxis.

10. Morphology, tinctorial, biochemical properties of causative agent of  tularemia. Cultivation.

11. Antigenic structure of F. tularensis. Toxin production.

12. Environmental resistance of tularemia causative agent.

13. Epidemiology of disease and ecology of F. tularensis.

14. Pathogenesis of disease and immune defence.

15. Laboratory diagnosis of tularemia.

16. Specific and nonspecific prophylaxis of tularemia. Treatment.

17. Morphology, tinctorial, biochemical properties of causative agent of brucellosis. Cultivation.

18. Differential features of various species of brucellae.

19. Antigenic structure and virulence factors of brucellae.

20. Epidemiology of brucellosis. Pathogenesis of disease.

21. Laboratory diagnosis of brucellosis.

22. Prophylaxis and treatment of  brucellosis.

23. Structure, tinctorial properties of causative agent of anthrax. Cultivation.

24. Biological properties of Bacillus anthracis: fermentative features, toxin production, antigenic structure.

25. Resistance. Pathogenicity for animals

26. Differential signs of B. anthracis, anthracoides and soil bacilli.  Fill in the table:

 

 

27. Epidemiology of anthrax (reservoir of anthrax iature, source of an infectious agents, mechanisms of transmission, vectors). Pathogenesis. Clinical forms of disease.

28. Laboratory diagnosis of anthrax:

a – tested material, feature of its collection; b – bacteriological method; c – serological method; d – biological method; e – allergic method; f – determination of B. anthracis antigen using thermoprecipitation test (Ascoli’s thermoprecipitation reaction).

29. Nonspecific and specific prophylaxis of anthrax. Treatment.

 

THEME Ii. Causative agents of anaerobic infections.

Microbiological diagnosis of anaerobic infection.

1. Structure, properties of causative agents of gas gangrene. Cultivation.

2. Classification of causative agents of gas gangrene. Antigenic structure.

3. Biochemical activity causative agents of gas gangrene.

4. Toxin production and virulence factors of Clostridium perfringens.

5. Toxin production and virulence factors of C. novyi, C. septicum, C. hystolyticum.

6. Ecology of gas gangrene Clostridia. Resistance. Pathogenicity for animals.

7. Epidemiology of disease. Main conditions of disease development. Clinical findings. Forms of disease. 

8. Features of collection of tested material and stages of bacteriological examination of gas gangrene.

9. Value of neutralization test for identification of species of causative agents of gas gangrene.

10. Main preparations for specific prophylaxis and treatment of a gas gangrene. Methods of obtaining, units of activity.

11. Classification and morphology of causative agents non-clostridial anaerobic infections.

12. Cultivation of causative agents non-clostridial anaerobic infections.

13. Microecology of causative agents non-clostridial anaerobic infections.

14. Laboratory diagnosis, treatment.

 

Microbiological diagnosis of tetanus and botulism.

1. Structure, tinctorial, biochemical properties of causative agent of tetanus. Cultivation.

2. Toxin production and virulence factors of Clostridium tetani.

3. Ecology of C. tetani. Resistance.

4. Epidemiology of tetanus. Pathogenesis of tetanus in man and animals. Clinical pictures.

5. Main methods of  laboratory diagnosis of tetanus.

6. Active and passive prophylaxis of tetanus. Preparation, which are used for this purpose. Methods of their obtaining and determination of activity.

7. Structure, tinctorial, biochemical properties of causative agent of botulism. Cultivation.

8. Antigenic structure and toxin production of Clostridium botulinum. Types of toxin.

9. Ecology of C. botulinum. Resistance.

10. Epidemiology of botulism. Pathogenesis of botulism. Clinical findings.

11. Main methods of  laboratory diagnosis of botulism.

10. Specific prophylaxis and treatment of botulism.

 

Seminar discussion of theoretical issues (12.30 – 14.00 hour).

 

Break 14.00-14.15

 

Individial student’s work. 14.15-15.00

Checking practical skills. Checking with quizes  and  constructive questions to verify knowledge level of student who did not do it using “Moodle” system.

 

Test evaluation and situational tasks.

THEME I. Causative agents of zoonose diseases.

1. Microbiological diagnosis of plague, other yersiniosis.

Select the correct answers.

1.     Causative agent of a plague has such properties:

a – gram-positive; b – forms long chains in smears; c – ovoid-sharped form with tendency to bipolar staining; d – delicate capsule; e – forms spores.

2.     Cultivation of Y. pestis:

a – microbes are undemanding (unpretentious) to nutrient media; b – bacteria form colonies with turbid white centres, and scalloped boders resembling lace or crumpled lace handkerchief; c – grow slowly; d – anaerobe; e – on blood agar forms  hemolysis.

3.     Toxic properties of causative agents of a plague:

a – form endotoxin; b – form lethal, mouse toxin; c – have coagulase; d – form protein toxin; e – produce hyaluronidase.

4.     Antigenic structure of Y. pestis:

a – has themostable lipopolysaccharide O–antigen; b – has V–antigen  – protein; c – has H-antigen; d – has W-antigen – lipoprotein; e – has Vi–antigen.

5.     Resistance of Y. pestis:

a – boiling kills microbes within 1 min; b – temperature 22 °С kills bacteria; c – in 3 % phenol solution survive 10 min; d – alcohol kills bacteria in 3 – 5 min.

6.     Mechanism of plague transmission:

a – airborne; b – by the bites of flea; c – fecal–oral; d – direct contact; e – by the foodstuff.

7.     Tested materials for plague diagnosis are:

a – blood; b – sputum; c – pus; d – puncture samples from a bubo; e – smear from stomatopharynx.

8.     For laboratory diagnosis of a plague use:

a – bacteriological method; b – biological method; c – serological method; d – allergic method; e – immunofluorescent test.

Real–life situation to be solved:

9.     The bubonic plague was suspected in a hunter. The doctor  collected a puncture sample from a bubo. There were revealed small, gram-negative coccobacilli with tendency to bipolar staining. On the surface of MPA there was not growth of bacteria in a day.

A.   How can we explain this result?

B.   How can we prevent bacteriophages action?

C.   What examinations must doctor do to confirm the diagnosis of plague?

10.  The patient has severe cough with foamy, blood-streaked sputum, chest pain, high temperature (40 °C). The smear was prepared from the sputum. There were revealed small, gram-negative bacilli with delicate capsule. On the MPA R-form colonies like crumpled lace handkerchief were grown.

A.   Can we make initial diagnosis of plague?

B.   Is it necessary to undertake urgent epidemiologic measures?

C.   What laboratory examinations is it necessary to do to determine the species of Y. pestis.

11.  The group of doctors has to go to Uganda. It is known there is unfavorable epidemic situation,  because hundreds of people have plague.

A.   What measures is it necessary to undertake for self-safety?

B.   Is it necessary to inoculate vaccine repeatedly if doctors should be in Uganda for 2 years?

 

2. Microbiological diagnosis of brucellosis,  tularaemia

Select the correct answers:  F. tularensis has such properties:

1.A – diplobacteria; b – coccobacteria; c – Gram-positive; d – produces spores; e – produces capsulae.

2. A – grows on simple media; b – grows on MacCou’s and Chepin’s media;  c – obligate aerobes; d – the isolated colonies don’t appear on the media after inoculation of material tested which was taken from the man;  e – grows onto media with a blood and cystine.

3. A – has an O-antigen; b – has a Vi-antigen;  c – forms an endotoxin; d – produces protein toxin; e – has a K-antigen.

4. A – resistant to low temperatures; b – resistant to high temperatures; c – sensitive to disinfectant solutions; d – resistant to alcohol; e – stands boiling for 30 minutes.

5. Epidemiology of a tularemia: a – the source of infection is rodents; b – the source of infection is sick persons; c – disease is transmitted by the bites of arthropods and insects;  d – the causative agent penetrates through an uninjured skin.

6. For diagnostics of tularemia are used: a – bacteriological method; b – blood-drop agglutination test; c – ELISA;  d – CFT;  e – allergic test.

7. For prophylaxis of tularemia are used:  a –  attenuated Gayskiy-Elbert’s vaccine; b  – tularin;  c – inactivated vaccine; d – anatoxin;  e – immunoglobulin.

8. Brucella have such properties: a – gram-negative;  b – small coccobacteria;  c – motile;  d – produce spores; e – produce capsula.

9. One can differentiate Brucella using: a – grows at presence of 5– 10 % of carbon dioxyde;  b – B. suis produces hydrogen sulphide; c – B. melitensis grows on media with thionine (1: 25000);  d – B. abortus is sensitively to specific phage;  e – B. suis ferments xylose.

10. The causative agents of brucellosis may be transmitted by: a – air–born mechanism; b – alimentary mechanism (through the milk, foodstuff; c – the bites of arthropods and insects;  d – from the bacterial carrier by a contact mechanism;  e – contact mechanism from the sick animals.

11. For bacteriological diagnosis of brucellosis are used:  a – blood; b – punctate of a marrow;  c – urine; d – faeces;  e – milk.

12. For diagnosis such methods are used:  a – bacterioscopic method; b – bacteriological method;  c – Huddleson’s agglutination test; d – IHA test; e – allergy test.

13. Prophylaxis and treatment of brucellosis: a – live attenuated vaccine; b – inactivated vaccine; c – anatoxin; d – live selective vaccine;  e – immunoglobulin.

Real-life situation to be solved:

14. After examination of the patient К., which complained of a high temperature, headache, pain in muscles, oedematous lids, a hyperaemia of conjunctiva, bubo on left paraauricularis region, the doctor made the clinical diagnosis of oculobubonic tularemia. The discharge from the conjunctiva were sent to the laboratory of dangerous infection. Tested material was inoculated onto solid yolk medium, and was injected subcutaneously to white mice. The smear was prepares too. There were small gram-negative bacteria in the smear, there were not any signs of growth outrient medium, and the mouse died in 5 day after inoculation of material tested.

A. Whether do these results of bacteriological examination confirm the clinical diagnosis?

B. What tests are necessary to carry out?

15. In the patient with high temperature enlarged painless lymph node (bubo) was found. The physician carried out allergic test with a tularin and took a blood serum for agglutination test. In 48 hours he has made the diagnosis of tularemia.

А.  On what data has he made this conclusion?

16. Burnet’s intracutaneous allergic test was carried out for milkmaids. In five of 30 assays the tests were was positive. All persons were vaccinated against brucellosis before.

А. Is it possible according to positive Burnet’s test make conclusion, that these people have brucellosis?

B. How is Burnet’s test carried out? When one can read its results?

C. To what type of allergic responses does Burnet’s test belong to?

D. What is brucellin?

17. The patient К., has complaints of increased sweating, wavy fever, headache and muscle pain, joint pain. The initial diagnosis of brucellosis was made. Laboratory examination for confirmation of the diagnosis was assigned.

А. Enumerate laboratory methods, which allow to confirm the diagnosis of brucellosis;

B. From what day of disease is it possible to perform Wright reaction?

C. What criteria do testify about positive Wright reaction?

D. How is it possible to differentiate the sick persons from vaccinated ones according Wright reaction?

 

3. Microbiologic diagnosis of anthrax.

Select the correct answers.

1. B. anthracis coli has such properties:

A – gram-positive; b – produces spore; c – produce capsule outside organism; d – peritrichous, motile; e – streptobacillus, resembling bamboo canes with elbow-shaped articulation.

2. Cultivation of B. anthracis:

A – in MPB B. anthracis produces  flocculent growth resembling cotton wool which sinks to the bottom of the tube; b – on MPA B. anthracis produces colonies resembling crumpled lace handkerchiefs; c – on MPA colonies have uneven edges and resemble the head of medusa; d – in  gelatin stab-cultures growth resembles an inverted fir tree; e – on blood agar B. anthracis causes hemolysis.

3. B. anthracis produces:

A –oedema factor and protective antigen; b – endotoxins; c – lethal toxin; d – lecithinase; e – enterotoxin.

4. Antigenic structure of B. anthracis and tests for determination of antigen:

А – O–antigen, agglutination test; b – O–antigen, thermoprecipitation test; c – K–antigen, phagocytosis; d – protective antigen, precipitation test, e – Н–antigen, agglutination test.

5. Disinfection of pathologic material is spent by:

А – autoclaving at 110 °С 40 min; b – boiling at 100 °С 1 min; c – autoclaving at 120 °С 90 min; d – boiling in 1 % solution of soda for 2 hrs; e – burning or immersion in concentrated sulfuric acid.

6. What tested material are examined in various clinical forms of anthrax:

А – urine (septic form); b – feces (intestinal form); c – punctate of lymph nodes (cutaneous form); d – pus (cutaneous form); e – sputum (respiratory form).

7. Methods of laboratory diagnosis of anthrax:

А – biological;  b – ELISA; c – thermoprecipitation test; d – allergy test; e – bacteriological method.

8. Bacterial preparation which use for active and passive prophylaxis:

А – antianthrax immunoglobulin; b – anthrax toxin; c – live anthrax vaccine; d – inactivated anthrax vaccine; e – anthrax antitoxin.

Real-life situation to be solved:

9. A patient with initial diagnosis of anthrax was admitted to the infectious diseases hospital. There was  acute onset of disease. The patient had high temperature, and  small vesicle 2-3 mm in diameter with serous fluid was appeared on the skin of right forearm. In a few day anthracic carbuncle was formed. It is known that three weeks ago the patients bought a new sheepskin coat in the market.

A.   What disease may be in these patient?

B.   What microbiological methods can we use for examination?

C.   How can we determine the presence of B. anthracis on the surface of fur coat?

11.   The tested material from the carbuncle was delivered to the bacteriological laboratory. There were revealed gram-positive rods with capsules in pairs or in shot chains resembling bamboo canes with elbow-shaped articulation.

A.   Can we make diagnosis according these signs?

B.   What methods of examinations can confirm your diagnosis?

12. The patient complaints of high temperature (39 °C), headache, general weakness. On his forearm there is carbuncle of purple  colour outside and black spot in the centre. The doctor made allergic test with anthracin. In 48 hrs hyperaemia and infiltrate (4 mm in diameter) was appeared.

A.   How can you value the results of this test?

B.   Can we refute the diagnosis of anthrax according this test?

C.   What another methods of examination can you propose to confirm the diagnosis of anthrax?

 

THEME II. Causative agents of anaerobic infections.

4. Microbiological diagnosis of gas gangrene.

Select the correct answers.

1. The causative agents of gas gangrene have such properties:

А – C. perfringens is motile; b – C. perfringens has a capsule; c – C. novyі forms spores; d – C. novyі has a capsule; e – C. septicum is motile.

2. Features of growth of gas gangrene Clostridia:

А – grow on simple media; b – on blood agar form hemolysis zones; c – on sugar broth form pellicle; d – grow on Kitt-Tarozzi medium; e –colonies form in agar stab cultures.

3. Toxin production of C perfringens:

А – produces an alpha – hemolysin; b – do not produces a leukocidin; c – produces an enterotoxin; d – has no a necrotic factor; e – has virulence enzymes.

4. Toxin production of another gas gangrene Clostridia:

А – an alpha – toxin of C. novyі has lethal action; b – an alpha – toxin of С. hystolytіcum evokes a cytopathogenic effect; c – C. septicum has no hemolytic properties; d – C. septicum produces aggression enzymes; e – C. sordellіі produces hemolysin.

5. Methods of laboratory diagnosis of gas gangrene:

А – allergic test; b – bacteriological; c – neutralization test; d – CFT; e – biological.

Real-life situation to be solved:

6. A patient injured the femur during transport accident. In the deep wound were left the pieces of clothes and soil. In 2 hours after the accident the primary surgical treatment of the wound was made and surgeon placed the sutures. In a day patient’s condition became grave. The femur increased in its size, the skin became pale and lucid. Muscles look like boiled meat. There are foamy discharge from the wound with unpleasant smell. During palpation of the tissues near the wound the crepitaion was determined.

A.   What disease may be in this patient?

B.   What bacteria cause the disease?

C.   What laboratory examinations can confirm the diagnosis?

D.   What specific preparation is used for treatment?

 

5. Microbiological diagnosis of tetanus and botulism

Select the correct answers.

1. The causative agent of tetanus has such morphological and tinctorial properties:

А – rod; b – produces capsule; c – has flagella; d – produces spores; e – Gram-negative; f – Gram-positive.

2. Cultivation of C. tetani:

А – aerobe; b – anaerobe; c – grows on simple nutrient media; d – growth on Kitt-Tarozzi medium;  e – zones of haemolysis are produced around  the colonies on blood agar.

3. Toxin production of C tetani:

А – produces enterotoxins; b – produces  tetanospasmin; c – has necrotic factor; d – produces tetanolysin; e – produces virulence enzymes.

4. Bacteria preparations used for passive and active immunization:

А – Pertussis-diphtheria-tetanus vaccine; b – chemical associated adsorbed vaccine which contains O- and Vi-antigens of typhoid, paratyphoid B, and a concentrated purified and sorbed tetanus anatoxin; c – antitoxic antitetanus serum; d – BCG vaccine; e – associated diphtheria-tetanus toxoid.

5. The causative agent of botulism has such morphological and tinctorial properties:

А – is motile; b – has a capsule; c – produces spores; d – in stained preparation occurs in chains; e  – Gram-negative.

6. Cultivation of C. botulinum:

А – in aerobic conditions; b – it grows outrient media with casein; c – on blood agar it produces zones of hemolysis around colonies; d – it grows on Kitt-Tarozzi medium; e – it grows on Ploskirev’s medium.

7. Toxin production of C. botulinum:

А – produces exotoxins; b – does not produce hemolysin; c – produces neurotoxin; d – produces enterotoxin; e – does not produce virulence enzymes.

8. Methods of laboratory diagnosis of botulism:

А – bacteriological; b – allergic; c – biological; d – CFT; e – neutralization test.

9. What specific preparations are used for treatment and prophylaxis of botulism:

А – anatoxin (toxoid); b – botulinus vaccine; c – botulinum antitoxin; d – bacteriophage; e – antibotulinum immunoglobulin.

Real-life situation to be solved:

10. A patient was injured during transport accident. After the primary surgical treatment of the wound 3000 IU of antitetanic serum was injected to the patient.

A.   How is it necessary to inject antitoxic serum to prevent complications.

B.   How much time does this immunity last?

11. A patient with diagnosis of tetanus was admitted to the infectious diseases hospital. Two months ago after the trauma he was injected with antitetanic serum. There were not any traumas during last months.

A.   Could tetanus develop in this patient?

B.   What clinical findings testify about tetanus?

C.   Why tetanus was developed in patient?

D.   What mistakes did the doctor make?

E.    What immune response does antitetanic serum create?

12. One family (3 persons) was hospitalized to the infectious disease department with complaints of aphonia, disturbance of swallowing, double vision, ptosis of the upper lips, difficult breathing, weakness in arms and legs. In a day they died. To determine the cause of intoxication the examination of canned fish was perfumed.

A.   What microbes can cause this disease?

B.   What methods of diagnosis must be used for examination of food remains?

C.   How canned fish was infected with these causative agents?

 

 

Students should know:

1.     Biological properties of Y. pestis,  Y. enterocolitica, Y. pseudotuberculosis, Brucella, Francisella and anthrax bacilli.

2.     Epidemiology and pathogenesis of plague, yersinioses, brucellosis, tularemia, anthrax.

3.     Methods of laboratory diagnosis of diseases, main methods of prophylaxis and treatment.

4.     Structure, tinctorial properties and cultivation of gas gangrene Clostridia.

5.     Antigenic structure of Clostridia.

6.     Fermentative properties  and toxin production of Clostridia.

7.     Ecology of gas gangrene Clostridia. Resistance. Pathogenicity.

8.     Epidemiology and pathogenesis of gas gangrene.

9.     Methods of laboratory diagnosis of gas gangrene, main methods of prophylaxis and treatment.

10.  Structure, tinctorial properties and cultivation of C. tetani and C. botulinum.

11.  Antigenic structure of Clostridia tetani and C. botulinum.

12.  Fermentative properties  and toxin production of Clostridia tetani and C. botulinum.

13.  Ecology of C. tetani and C. botulinum. Resistance. Pathogenicity for animals.

14.  Epidemiology and pathogenesis of tetanus and botulism.

15.  Methods of laboratory diagnosis of tetanus and botulism, main methods of prophylaxis and treatment.

 

Students should be able to:

1. make microscopical examination of the smears;

2. value the growth of bacteria on different nutrient media;

3. carry out blood-drop agglutination reaction for serological diagnosis of tularemia;

4. carry out an agglutination test for serological diagnosis of  tularemia;

5. carry out an Wright’s test for serological diagnosis of  brucellosis;

6. carry out an Huddleson’s agglutination test for accelerated diagnostics brucellosis.

7. carry out Ascoli’s thermoprecipitation test for revealing of B. anthracis antigen;

8. draw the schemes of laboratory diagnosis of diseases.

9. inoculate a soil onto Kitt-Tarozzi medium and milk;

10.  draw the scheme of laboratory diagnosis of gas gangrene.

11.  inoculate microbes from Kitt–Tarozzi medium or milk by Veinberg technique;

12.  select preparations for passive and active prophylaxis of tetanus and botulism.

 

Answers to the Self-Assessment:

 

THEME: I. Causative agents of zoonose diseases.

1. Microbiological diagnosis of plague, other yersiniosis.

1. C, d. 2. A, b, e. 3. A, b, c, d, e. 4. A, b, d. 5. A, c, d. 6. A, b, c, d, e. 7. A, b, c, d. 8. A, b, c, d,  e. 9. A. There is lizogenic culture of yersiniae; B. It is necessary to place antiphage serum on the surface of agar; C. immunofluorescent test, to isolate and identify the pure culture of causative agent, biological test. 10. A. Yes, we can; B. Yes, it is necessary; C. to isolate the pure culture of causative agent, to determine motility of bacteria, lysis by plague phage, abscence of fermentation of rhamnose, agglutination test with palgue serum, pathiogenicity for laboratory animals. 11. A. To immunize the doctors with live EV vaccine; B. The immunity lasts for a year, it is necessary repeated  vaccination in 8-12 months.

2. Microbiological diagnosis of brucellosis,  tularaemia

1. B, e. 2. B, c, d, e. 3. A, b, c. 4. A, c, d. 5. A, c, d, e. 6. A, b, c, e. 7. A. 8. A, b, e. 9. A, b, c, d, e. 10. B, e. 11. A, b, c, d, e. 12. B, c, d, e. 13. D. 14. A. Yes; B. The blood, bone marrow, and pieces of the internal organs and lymph nodes from the animal corpse are inoculated by rubbing them into the surface of one of the media such as yolk, glucose-cystine agar with blood, Emelyanova’s medium, etc. with following identification of the causative agents. 15. A. Positive allergic test (presence of marked infiltrate and hyperaemia more than 0,5 cm), positive agglutination test, titer 1:100. 16. A. No; B. A total of 0.1 ml of brucellin is injected intracutaneously into the middle area of the upper arm. The reaction is con­sidered positive if 24 hrs after the injection there is tenderness, hyperaemia, and infiltration of the skin some 3 X 3 cm in size, weakly positive if the area of hyperaemia and infiltration is 1 X 1 cm in size, and sharply positive if the area of hyperaemia and infiltration is 6 X 6 cm in size; C – to delayed hypersensitivity; D. Brucellin is filtrate of 3-4 week heat-killed broth culture of three species of brucella. 17. Wright’s test, Huddleson’s test, opsonophagocytic test, CFT, IHA test, allergic test; B. From a beginning of the second week of disease; C. The reaction is considered positive if the titre of patient’s serum is 1:200, doubtful if it is  1:50; D. Repeat Wright test with the purpose of revealing of increase of antibodies  titer.

3. Microbiological diagnosis of anthrax.

1. A, b, e. 2. A, c, e. 3. A, c. 4. B, c. 5. C, d, e. 6. B, d, e. 7. A, c, d. 8. A, c. 9. A.. Anthrax. B. Bacterioscopic, bacteriological and biological methods; C. Ascoli’s thermoprecipitation test. 10. A. No, because there are another bacteria resembling B. anthracis; B. Bacteriological and biological methods. 12.  A. The test is negative. The test is considered positive if patient develops hyperemia over 16 mm in diameter and an infiltrate; B. No, as there may be negative result of such test in people with anthrax; C. Bacteriological method. 

 

THEME II. Causative agents of anaerobic infections.

4. Microbiological diagnosis of gas gangrene.

1. B, c, e. 2. B, d, e. 3. A, c, e. 4. A, b, d, e. 5. B, c, e.  6. A. Gas gangrene; B. Clostridium perfringens, C. novyi, C. septicum, C. hystolyticum; C. Bacteriologic and biologic methods; D. Polyvalent purified and concentrated antitoxin “Diaferm”.

5. Microbiological diagnosis of tetanus and botulism

1. A, c, d, f. 2. B, d, e. 3. B, d, e. 4. A, b, c, e. 5. A, c. 6. B, c, d. 7. A, c. 8. A, c, e. 9. C, e. 10. A. To make special test for determination of human susceptibility to heterologous protein. A preliminary desensitization according to Bezredka’s method is necessary; B. The length of protective action of serum is for 2-3 weeks. 11. A. Yes; B. Spasmodic contraction of the masseter muscles, tonic spasms of face (risus sardonicus) and occipital muscles, opisthotonus; C. It was injected only antitetanic serum to the patient; D. The patient was not immunized  with tetanus toxoid; E. Passive, antitoxic immunity which lasts for 2-3 weeks. 12. C. botulinum; B. Bacteriological, neutralization test using white mice to determine toxin type;  C. The fish could be contaminated with endospores of C. botulinum and bad sterilization was made.

 

References:

Basic:

Materials for practical class 17

1. Hadbook on Microbiology. Laboratory diagnosis of Infectious Disease/ Ed. by Yu.S. Krivoshein, 1989, P. 127-136.

2. Medical Microbiology and Immunology: Examination and Board Rewiew /W. Levinson, E. Jawetz.– 2003.– P. 138-131, 107-112.

3. http://intranet.tdmu.edu.ua/data/kafedra/theacher/micbio/klymjuk/English/Lectures/Microbiology,%20virology%20and%20immunology/medical/3%20year/Causative%20agents%20of%20%D0%B0naerobic%20infection.ppt

Additional:

1.    Review of Medical Microbiology /E. Jawetz, J. Melnick, E. A. Adelberg/ Lange Medical Publication, Los Altos, California, 2002. – P.180-187, 250-254, 246-249

2. Wesley A.Volk et al. Essentials of Medical Microbiology. Lippincott – Raven Publishers, Inc., Philadelphia–New York.

 

 

The methodical instruction has been done

by prof. Sergey I. Klymnyuk, ass. prof. Olena V. Pokryshko

 

Methodical instruction was discussed at the Department sitting

24.09.2013. Minute № 3