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Bohomolets Microbiology Lesson #12

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By Ms. Kostiuk from Microbiology department

By Ms. Kostiuk from Microbiology department

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  • 1. Lesson N12 LABORATORY DIAGNOSIS OF ZOONOSIS (PLAGUE, TULARAEMIA, BRUCELLOSISAND ANTHRAX)1.Scientifically methodical ground of themeThise group of diverse organisms is presented togerther as the mode of transmission iszoonotic. The resorvoir of infection is animals. Tramsmission may occur directly bycontact or bites of animals or through an insect vector where the insect feeds on ananimal and transmit the organisms to human.2.Educational purpose STUDENTS MUST KNOW: 1. Structure, tinctorial properties and cultivation of Y. pestis, F. tularensis, B. anthracis and causative agents of brucellosis 2. Antigenic structure of these microorganisms 3. Fermentative properties and toxin production of causative agents of zoonosis 4. Epidemiology and pathogenesis of plague, tularemia, brucellosis, anthrax. 5. Methods of laboratory diagnosis of diseases, main methods of prophylaxis and treatment.STUDENTS SHOULD BE ABLE TO: – make microscopical examination of the smears; – value the growth of bacteria on different nutrient media; – read the result of Wright’s test for serological diagnosis of brucellosis; – carry out an Huddleson’s agglutination test for accelerated diagnostics brucellosis. – make up conclusion about Huddleson’s and Wright’s tests – create scheme of diagnosis of plague, tularemia, brucellosis and anthrax – choose biological preparation, which are used for laboratory diagnosis andspecific prophylaxis of plague ,tularemia, brucellosis and anthrax3.Chart of topic content. Fig.N1:Yersinia Virulence Factors in Human Disease Factor Apparent Function Fraction 1 capsule Antiphagocytic V/W antigens Suppress granuloma formation Fibrinolysin Tissue invasion Low Ca2+ response YOP synthesis gene YOP H Protein tyrosine phosphatase YOP K & L Inhibit cell-mediated immune response Laboratory diagnosis. Examination is carried out in special laboratories and inantiplague protective clothing. A strict work regimen must be observed. Depending onthe clinical form of the disease and the location of the causative agent, test specimens arecollected from bubo content (in bubonic plague), ulcer secretions (in cutaneous plague),mucus from the pharynx and sputum (in pneumonic plague), and blood (in septicaemicplague). Test matter is also recovered from necropsy material (organs, blood, lungs,contents of lymph nodes), rodent cadavers, fleas, foodstuffs, water, air, etc. Examinationis performed in the following stages. 1
  • 2. 1. Microscopy of smears, fixed in Nikiforovs mixture and stained by the Grammethod or with methylene blue by Loefflers method. 2. Inoculation of the test material into nutrient media, isolation of a pure culture andits identification. To inhibit the growth of the accompanying microflora, 1 ml of a 2.5 percent sodium sulphite solution and 1 ml of a concentrated alcohol solution of gentianviolet, diluted in distilled water in a ratio of 1 :100, are added to 100 ml of meat-peptoneagar. Prior to inoculation 0.1 ml of antiphage serum is added to the culture to render theplague bacteriophage harmless. 3. Biological tests of the isolated pure culture and of material from which isolationof the organism is difficult are conducted on guinea pigs. In the latter case a thickemulsion prepared from the test material is rubbed into a shaven area of skin on theabdomen. If plague bacilli are present the animals die on the fifth-seventh day. To hastendiagnosis the infected guinea pigs are killed on the second-third day and the plaguebacillus is isolated from their organs. Y. pestis is identified by determining the morphological, cultural, fermentative,phagocytolytic, and agglutinative properties of the isolated culture. The growth isdifferentiated from the causative agent of rodent pseudotuberculosis (see Table 1). Thebiological test is decisive in the diagnosis of plague. Decomposed rodent cadavers are examined by the thermoprecipitin test.The importance of prompt diagnosis of plague has led 10 the elaboration of accelerateddiagnostic methods in recent times Laboratory diagnosis. The differential diagnosis of plague, anthrax, enteric fever,typhus fever, influenza, malaria, and brucellosis is difficult because these diseases havecommon symptoms. For differentiation of tularaemia from other diseases laboratory testsare the most effective. Those peculiarities of the disease which can be revealed easily andquickly by laboratory methods are taken into account. 1. Allergy develops on the third-fifth day of the disease. For this reason,intracutaneous and cutaneous tests with tularine are made for early diagnosis. Intularaemia patients the test gives a positive reaction 6-12 hours after inoculation oftularine. In distinguishing tularaemia from other infections one must bear in mind thatallergic tests may show positive reactions in convalescents and vaccinated individuals. 2. In the second week of the disease agglutinins begin to accumulate in the blood.They are detected by carrying out the agglutination reaction by the blood-drop andvolume methods. In some cases this test may give a positive reaction with materialcontaining brucella organisms, since they possess antigens common to tularaemiabacteria. 3. The tularaemia culture is isolated by the biological method as it is impossible torecover the pathogen directly from a tularaemia patient. For this purpose white mice orguinea pigs are infected by material obtained from people suffering from the disease(bubo punctate, scrapings from ulcers, conjunctiva! discharge, throat films, sputum, andblood). Biological tests are conducted in special laboratories where a standard regimen isobserved. The laboratory animals die in 4-12 days if tularaemia bacteria are present in thetest material. Autopsy is performed, smears from organs are made and organ specimensare inoculated onto coagulated egg medium for culture isolation. Microscopic,microbiological, and biological studies of the cultured organisms are made. If no culturecan be isolated from the first infected guinea pig, an emulsion, obtained from the lattersorgans, is inoculated into a second guinea pig, etc. 2
  • 3. 4. Laboratory diagnosis of rodent tularaemia is made by microscopy of smears fromorgans, precipitin ring reaction (thermoprecipitation), and biological tests. Water, foodstuffs, and blood-sucking arthropods are examined by biological tests. Aglutination in monospecific sera AM Growth on Metabolic tests Lysis under effect of T-b phage in TD media with dyes Requirements for CO2 thioni basic H2S production fuchsi glutami-nic acid n n Biotype arginine ribose lysine Species abc bcB. 1 – – –++ ++ –+ – + – – –melitensis 2 – – –++ ++ +– – + – – – 3 – – –++ ++ ++ – + – – –B. 1 +(–) + ––– ++ +– + + – + –abortus 2 +(–) + ––– –– +– + + – + – 3 +(–) + + + + ++ +– + + – + – 4 +(–) + ––– ++ –+ + + – + – 5 – – –++ ++ +– + + – + – 6 – –+ –++ ++ +– + + – + – 7 – +– –++ +–+ ++ + + – + – 8 + – –++ ++ –+ + + – + – 9 – + –++ ++ –+ + + – + +B. suis 1 – + +++ –– +– – – + + – 2 – – –++ –– +– – + + + + 3 – – +++ ++ +– – + + + + 4 – – +++ ++ ++ – + + +B. 1 – + ––+ –– +– – + – ± –neotomaeB. ovis + – +++ –– – + – – –B. canis – – – ++ – – – – + + + Laboratory diagnosis. The patients blood and urine (for isolation of the pathogen),serum (for detection of agglutinins), milk and dairy products (for detection of brucellaeor agglutinins in milk) are examined. The microbe is isolated in special laboratories. 1. Culture isolation. Since brucellosis is often accompanied by bacteraemia, blood isexamined during the first days of the disease (preferably when the patient has a hightemperature). For this purpose, 5-10 ml of blood is collected and transferred into two or 3
  • 4. three flasks (2-5 ml per flask) containing 100 ml of liver-extract or ascitic-fluid broth (pH6.8). The cultures are grown for 3-4 weeks or more. Five to ten per cent of carbondioxide is introduced into one of the flasks (for growth of the 23 bovine species of thebacteria). Inoculations on agar slants are made every 4-5 days for isolation andidentification of the pure culture. An antiphage serum is introduced into the cultures for neutralization of the phagewhich inhibits the growth of brucellae. The best results are obtained when the blood isinoculated into the yolk of an unfertilized egg or the yolk sac of a chick embryo. For this,0.1-0.2 ml of the tested blood diluted in citrate broth in a ratio of 1 : 3 is introduced intoeach egg. The infected eggs are placed in an incubation chamber for 5 days, after which0.3-0.5 ml of their contents is inoculated into the liquid nutrient media. Growth isexamined every 2-3 days. If the blood culture produces a negative result bone marrow obtained by sternumpuncture is inoculated onto solid and liquid media for isolation of myelocultures. The urine is also examined. It is obtained with a catheter, centrifuged, and 0.1 ml ofthe precipitate is seeded onto agar plates containing 1 :200000 gentian violet. In somecases faeces, cows and human milk, and amniotic fluid of sick humans and animals areexamined for the presence of Brucella organisms. Brucella cultures may be isolated by the biological method. For this purpose healthyguinea pigs or white mice are injected with 0.5 or 3 ml of the test material. A month laterthe guinea pigs blood is tested for agglutinins, the allergic test is carried out, and the pureculture is isolated. White mice are tested bacteriologically every three weeks. 2. Serological test. From the tenth-twelfth day of the disease onwards, theagglutinins accumulate in the blood in an amount sufficient for their detection by theagglutination tests. The Wright (in test tubes) and Huddleson (on glass) reactions arecarried out. The Wright reaction is valued highly positive in a 1 800 serum dilution,positive in a 1 :400-1 :200 dilution, weakly positive in 1 :100 dilution, and doubtful at atitre of 1 :50. The Huddleson reaction is used mainly in mass examinations for brucellosis. However, there is a disadvantage of this reaction in that it sometimes shows positiveresults with sera of healthy individuals who have normal antibodies in their blood. 3. Skin allergic test. To determine allergy, Burnes test is made beginning from thefifteenth-twentieth day of the disease. A 0.1 ml sample of the filtrate of a 3- or 4-week-old broth culture (brucellin) is injected intracutaneously into the forearm. The test isconsidered positive if a painful red swelling 4 by 6 cm in size appears within 24 hours(Plate III). 4. Opsono-phagocytic test. This test detects changes in the phagocytic reaction. Theindex of healthy individuals averages 0-1 and occasionally 3-5. In sick persons thereaction is considered high if the index is 50-70, mild, if it is 25-49, and low, if it is10-24. For detecting brucellae in the external environment the reaction for demonstrating arise in bacteriophage titre is carried out. 5. In some cases the complement-fixation test,the indirect haemagglutination reaction, and the immunofluorescence reaction are used. Components of Anthrax Toxin Compone Function nt EE Inactive adenylate cyclase activated by calmodulin 4
  • 5. LF Causes pulmonary edema and death in rats; cytolytic for macrophages PA Required for the binding of both EF and LF to host cellEF, edema factor, LF, lethal factor, PA, protective antigen Laboratory diagnosis. In cases of cutaneous anthrax the malignant pustularexudate is examined; it is obtained from the deep layers of the oedematous area where itborders with the healthy tissues. Sputum is examined in cases of the respiratory form,faeces and urine, in intestinal form, and blood is examined in cases of septicaemia. 1. The specimens are examined under the microscope, the smears are Gram-stained,or stained by the Romanowsky-Giemsa method. The presence of morphologicallycharacteristic capsulated bacilli, arranged in chains, allows a preliminary diagnosis. 2. For isolation of the pure culture the specimens are inoculated into meat-peptoneagar and meat-peptone broth. The isolated culture is differentiated from othermorphologically similar bacteria by its morphological and biochemical properties. 3. Laboratory animals (white mice, guinea pigs and rabbits) are inoculated with thepathological material and with the pure culture de-rived from it. B. anthracis causes thedeath of white mice in 24-48 hours and of guinea pigs in 2-3 days following inoculation.Microscopic examination of smears made from blood and internal organs reveals anthraxbacilli which are surrounded by a capsule. A rapid biological test is also employed. The culture obtained which has to beidentified is introduced intraperitoneally into white mice. Several hours after inoculationsmears are prepared from the peritoneal contents. Detection of typical capsulated bacilligives a basis for con-firming the final result of the biological test. The allergic test with anthracin (a purified anthrax allergen) is employed when aretrospective diagnosis is required in cases which have yielded negative results withmicroscopical and bacteriological examination. Postmortem material as well as leather and fur used as raw materials are examinedserologically by the thermoprecipitin reaction (Ascolis test) since isolation of the bacilliis a matter of difficulty in such cases. The result in the first test tube (containing the test material) may be either positiveor negative, in the second test tube (control) it must be only positive, and in the third,fourth, fifth, and sixth control test tubes the results must always be negative. When employing laboratory diagnosis of anthrax, one must bear in mind thepossibility of the presence of bacteria identical with B. anthracis in their biologicalproperties (see Table 1). These sporing aerobes are widely distributed in nature and arenormally sporeforming saprophytes. They include B. cereus, B. subtilis, B. megaterium,etc. The anthrax bacilli may be differentiated from anthracoids (false anthraxorganisms) and other similar sporing aerobes by phagodiagnosis. The specificbacteriophage only causes lysis of the B. anthracis culture.4. Student’s independent study program 1. Structure, staining properties of causative agent of plague, tularemia, brucellosisand anthrax . Cultivation. 5
  • 6. 2. Biochemical and other differential diagnistic properties of causative agent ofplague, tularemia, brucellosis and anthrax 3. Antigenic structure and toxon production and virulence factors. 4. Reservoir of these diseases in nature, source of infectious agents, mechanisms oftransmission, vectors. 5. Main clinical forms of diseases(plague, tularemia, brucellosis and anthrax ). 6. Laboratory diagnosis of plague, tularemia, brucellosis and anthrax a – collection of tested material b – main methods of diagnosis c-bacterioscopy d - stages of bacteriological diagnosis; e – serological method h – feature of biological method of diagnosis; g-allergic method 7. Methods of zoonosis prophylaxis .5. Students’ practical activities: 1.To examine under the microscope the smears of causative agents of zoonosis inmuseum preparations. Drow your observation. Y.pestis stains with ordinary aniline dyes and given bipolar appearance, its endsstaining more intensively. It is Gram negative. Y.pestis is characterized by markedindividual variability (pleomorphism). In smears from organs and in young cultures it hasan ovoid shape, while in cultures on solid media it is elongated and sometimes thread-like. The tularaemia bacteria (F. tularensis) are short coccal-shaped or rod-like coccimeasuring 0.2-0.7 mcm. In old cultures the organisms retain the coccal form. They arenon-motile, and Gram-negative. In the animal body they are sometimes surrounded by afine capsule. Brucellae are small, coccal, ovoid-shaped micro-organisms 0.5-0.7 mcm in size.Elongated forms are b.6-1.5 mem in length and 0.4 mcm in breadth. Under the electronmicroscope Brucella organisms of cattle, sheep and goats appear as coccal andcoccobacilary forms, while those of pigs are rod-shaped. They are Gram-negative, non-motile, and do not form spores or capsules (in some strains capsules are sometimespresent). B. anthracis is among the largest of all bacterial pathogen, composed of block-shaped, angular nonmotile rod 3-5 mkm long and 1-1,2mkm wide. Division begins beforethe cells have separated after the previous division, which leads to the formation ofstreptobacilli. Outside the hosts body they produce oval-shaped central spores which aresmaller in diameter than the bacillus. In the bodies of man and animals the bacilli producecapsules which surround a single organism or are continuous over the whole chain. 2.Read the result of Wright’s test with patient’s serum for serological diagnosis ofbrucellosis. Make up conclusion and record your observation. To carry out a standard agglutination test, on the second week of the disease (the10th–15th day) withdraw 2-3 ml of blood from the patients cubital vein. Dilutesuccessively the obtained serum (Table 2) with isotonic saline in titres from 1:25 to1:400. 6
  • 7. Schematic Representation of the Agglutination Reaction Number of the test tube 1 2 3 4 5 6 7 Ingredient antigen serum control control Isotonic sodium 1 1 1 1 1 1 – chloride solution, ml The diagnostic 1→ 1→ 1→ 1→ – 1 serum in a 1:50 1 dilution, ml The obtained 1:100 1:200 1:400 1:800 1:1600 – 1:50 dilution of the serum Bacterial 2 2 2 2 2 2 – suspension, drops Incubation at 37 0C for 2 hrs, then at room temperature for 18-20 hrs Wright’s reaction is performed at the beginning of the second week of the disease.Suspension of killed Brucella organisms stained with gentian violet or methylene violetand containing 1 mird of brucellae (per ml) killed with formalin or phenol is used as adiagnosticum. Currently a uniform colour brucellar diagnosticum for Wrights andHuddlesons tests is commercially available. The procedure of performing the Wright test is similar to that used in otheragglutination tests, with the exception of the fact that the serum is diluted in a multipleorder with isotonic NaCI solution containing 0.5 per cent of phenol. The test tubes areplaced in the incubator for 18–20 hrs and then left at room temperature for 2 hrs. Theresults of agglutination are denoted with pluses. The reaction is considered positive if thetitre is 1:200 and doubtful if it is 1:50. Wrights reaction may be positive in vaccinatedsubjects and patients with brucellosis. So, it is to be repeated in the course of the diseaseto look for increase in the antibody litre. 6. To carry out Huddleson’s agglutination test for serological diagnosis of brucellosis. Read the result of test. Make up conclusion and record your observation. The plate agglutination test proposed by Huddleson (Table 3) is most frequentlyused in brucellosis foci (in field conditions) since it is simple to perform. Using a waxpencil, divide a glass plate into six squares, then pipette on them undiluted serum to bestudied in 0.08, 0.04, 0.02, 0.01, and 0.02-ml portions, and add 0.03 ml of the samediagnosticum that is used in Wrights reaction to each dose of the serum but the last (thefifth square) into which 0.03 ml of isotonic sodium chloride solution (control of theserum) is placed. In the sixth square, place 0.03 ml of the antigen and 0.03 ml of isotonicsaline (control of the diagnosticum). Mix drops of the serum and antigen with a glass rodand after slight rocking of the plate read the results of the reaction which is manifestedalready in the first minutes by the appearance of microgranular stained agglutinate. Theabsence of agglutination in all doses of the serum is assessed as a negative reaction, thepresence of agglutination in the first dose (0.08 ml of the serum) as a doubtful one, in the 7
  • 8. second dose (0.04 ml), as a weakly positive one, in the third or fourth doses (0.02–0.01ml), as a positive one; the agglutination reaction expressed by 4 pluses in all doses isevaluated as a drastically positive one. The value of Huddlesons agglutination test isdiminished by the fact that not infrequently it produces positive results with healthypeoples sera due to the presence of normal antibodies. Hence, sera showing positiveHuddlesons reaction should be subjected to the tube agglutination test. Table 3 Schematic Representation of Huddlesons Agglutination Test Number of square Control Ingredient, ml of of 1 2 3 4 serum antigen 5 6 Isotonic saline – – – – 0.03 0.03 Patients serum 0.08 0.04 0.02 0.01 0.02 – Diagnosticum 0.03 0.03 0.03 0.03 – 0.03 6. To familiarize with biological preparation, which are used for laboratorydiagnosis and specific prophylaxis of plague ,tularemia, brucellosis and anthrax6. Control questions and tests: 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 formcolonies with turbid white centres, and scalloped boders resembling lace or crumpled lacehandkerchief; 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 fromstomatopharynx 8. For laboratory diagnosis of a plague use: a – bacteriological method; b – biological method; c – serological method; d – allergic method; e – immunofluorescent test. 9.F. tularensis has such properties: 8
  • 9. 1.A – diplobacteria; b – coccobacteria; c – Gram-positive; d – produces spores; e –produces capsulae. 10. 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 ofmaterial tested which was taken from the man; e – grows onto media with a blood andcystine. 11. A - has an O-antigen; b – has a Vi-antigen; c – forms an endotoxin; d –produces protein toxin; e – has a K-antigen. 12 A – resistant to low temperatures; b – resistant to high temperatures; c – sensitiveto disinfectant solutions; d – resistant to alcohol; e – stands boiling for 30 minutes. 13. Epidemiology of a tularemia: a – the source of infection is rodents; b – thesource of infection is sick persons; c – disease is transmitted by the bites of arthropodsand insects; d – the causative agent penetrates through an uninjured skin. 14. For diagnostics of tularemia are used: a – bacteriological method; b – blood-drop agglutination test; c – ELISA; d – CFT; e – allergic test. 15. For prophylaxis of tularemia are used: a – attenuated Gayskiy-Elbert’s vaccine;b – tularin; c – inactivated vaccine; d – anatoxin; e – immunoglobulin. 16. Brucella have such properties: a – gram-negative; b – small coccobacteria; c –motile; d – produce spores; e – produce capsula. 17. One can differentiate Brucella using: a – grows at presence of 5– 10 % ofcarbon dioxyde; b – B. suis produces hydrogen sulphide; c – B. melitensis grows onmedia with thionine (1: 25000); d – B. abortus is sensitively to specific phage; e – B.suis ferments xylose. 18. The causative agents of brucellosis may be transmitted by: a – air–bornmechanism; b – alimentary mechanism (through the milk, foodstuff; c – the bites ofarthropods and insects; d – from the bacterial carrier by a contact mechanism; e –contact mechanism from the sick animals. 19. For bacteriological diagnosis of brucellosis are used: a – blood; b – punctate ofa marrow; c – urine; d – faeces; e – milk. 20. For diagnosis such methods are used: a – bacterioscopic method; b –bacteriological method; c – Huddleson’s agglutination test; d – IHA test; e – allergy test. 21. Prophylaxis and treatment of brucellosis: a – live attenuated vaccine; b – inactivated vaccine; c – anatoxin; d – live selective vaccine; e – immunoglobulin 22. B. anthracis has such properties: A – gram-positive; b – produces spore; c – produce capsule outside organism; d –peritrichous, motile; e – streptobacillus, resembling bamboo canes with elbow-shapedarticulation. 23. Cultivation of B. anthracis: A – in MPB B. anthracis produces flocculent growth resembling cotton wool whichsinks to the bottom of the tube; b – on MPA B. anthracis produces colonies resemblingcrumpled lace handkerchiefs; c – on MPA colonies have uneven edges and resemble thehead of medusa; d – in gelatin stab-cultures growth resembles an inverted fir tree; e – onblood agar B. anthracis causes hemolysis. 24. B. anthracis produces: A –oedema factor and protective antigen; b – endotoxins; c – lethal toxin; d – lecithinase; e – enterotoxin. 25. Antigenic structure of B. anthracis and tests for determination of antigen: 9
  • 10. А – O–antigen, agglutination test; b – O–antigen, thermoprecipitation test; c – K– antigen, phagocytosis; d – protective antigen, precipitation test, e – Н–antigen, agglutination test. 26. Disinfection of pathologic material is spent by: А – autoclaving at 110 °С 40 min; b – boiling at 100 °С 1 min; c – autoclaving at120 °С 90 min; d – boiling in 1 % solution of soda for 2 hrs; e – burning or immersion inconcentrated sulfuric acid. 27. 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). 28. Methods of laboratory diagnosis of anthrax: А – biological; b – ELISA; c – thermoprecipitation test; d – allergy test; e –bacteriological method. 29. 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. 30. Each of following statements concerning plague is correct EXCEPT: A. Plague is caused by gram-negative rods which can be caltivated on blood agar B. Plague is transmitted to humans by flea bite C. The main resorvoir in nature are small rodents D. Plague occurs only in underdeveloped countries 31. Which one of following statements concerning the organisms that causebrucellosis is CORRECT? A. Brucellae are transmitted primery by tick bite B. The principal resorvoir in nature are small rodents C. Brucellae are found in reticuloendothelial cells and often cause graanulomatous lesions D. Brucellae are obligate intracellular parasites that are usually identified by grow in human cell culture 32. A patient with brucellosis is found to have a positive skin test (brucelin test).Which of the following factors can induce such reaction? A. IgА. B. T-cells C. IgE. D. IgG. E. IgD. 33. The territory of old tomb cattle yard, which was not used for more than 50 years,is planned for house building. However, soil investigation has shown the presence ofviable spores of the causative agent of especially dangerous disease. Namemicroorganisms, which could be kept in soil during such a long time? A. Yersinia pestis. B. Francisella tularensis. C. Brucella abortus. D. Bacillus anthracis. E. Mycobacterium bovis. 10
  • 11. 34. A 34 year-old- patient complained of carbuncle on his face. His examinationrevealed subcutaneous painful edema with black eschar in the center, vesicular eruptionon circumference. Microbiologic examination revealed nonmotile, capsule-, formingstreptobacillli. What microorganisms are causative agents of this disease? A. Bacillus anthracis. B. Staphylococcus aureus. C. Bacillus anthracoides. D. Bacillus subtilis. E. Bacillus megaterium. 35. In laboratory for examination of animal skin the precipitation test is used (Askolytest). Result: in some minutes after adding immune serum to skinr extract albescent ringhas been formed. What does this result say about? A. Presence of Bacillus anthracis antigens. B. Presence of Clostridium perfringens toxin. C. Presence of the brucellosis pathogen. D. Presence of escherichia antigen. E. Presence of salmonella Vi-antigen. 36. The woman during epidemic of flu has addressed the doctor with complaints ofhigh body temperature, weakness, absence of appetite, pain in joints. For 10 days she wastreated for flu. But infectionist has suspected brucellosis. Using what reaction is itpossible to diagnose brucellosis? A. Right. B. Wassermann test. C. Coombs test. D. Widal test. E. Ouhterlony. 37. There was used biological test for diagnostics. In touch smear from organs of ananimal streptobacilli surrounded with a capsule were revealed. It gives the basis todiagnose: A. Tularemia. B. Anthrax. C. Plague. D. Brucellosis. E. Streptococci pneumonia 38. There is the patient with the presumptive diagnosis of acute pneumonia. In thesmear of sputum there are revealed chaotically located microorganisms of oval forms inlength up to 2 microns bipolar stained. What the most likely diagnosis can be made onthe basis of this data? A. Lung plague. B. Streptococcal pneumonia. C. Staphylococcal pneumonia. D. Pneumonia caused by Klebsiella. E. Diphtheria. 39. Cattle raw material (a leather, a wool) should be checked up for the presence ofthe anthrax agent. For making this procedure we have to prepare soluble thermoresistant 11
  • 12. antigen in water-salt extract from raw material. What reaction may be used for thispurpose? A. Neutralizations. B. Precipitation in gel. C. Agglutinations. D. Indirect hemagglutination. E. Precipitation in liquid. 40. In one of mountain settlements the mass destruction of rodents was observed.Simultaneously inhabitants of this district have been ill. Illness was accompanied by fastrise of body temperature up to 400 C, the expressed intoxication, increase groin lymphnodes. In preparations (swabs from a cadaveric material) gram-negative bipolar stainedoval rods were revealed. What microorganisms are agents of this infectious disease? A. Staphylococci. B. The agent of plague. C. The agent of tularemia. D. The activator anthrax. E. Clostridia. 41Each of following statements concerning plague is correct EXCEPT: A. The causative organism, Y. pestis, is gram-negative rod with a very low ID50 B. Thereis an animal resorvoir for this diseas, including rodent C. The diagnosis must be serological because the organism does not grow in culture D. People should not handle dead wild animals since fleas associated with the animals can transmit the disease Real–life situation to be solved: 1.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? 2.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-nagative bacilli with delicate capsule. On the MPA R-form colonies like crumpled lace handkerchief twere 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. 3.The group of doctors have to go to Uganda. It is known there are unforable epidemic situation, becouse 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? 12
  • 13. 4. After examination of the patient К., which complained of a high temperature,headache, pain in muscles, oedematous lids, a hyperaemia of conjunctiva, bubo on leftparaauricularis 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 subcutaneouslyto white mice. The smear was prepares too. There were small gram-negative bacteria inthe smear, there were not any signs of growth on nutrient medium, and the mouse died in5 day after inoculation of material tested. A. Whether do these results of bacteriological examination confirm the clinicaldiagnosis? B. What tests are necessary to carry out? 5. In the patient with high temperature enlarged painless lymph node (bubo) wasfound. The physician carried out allergic test with a tularin and took a blood foragglutination test. In 48 hours he has made the diagnosis of tularemia. А. On what data has he made this conclusion? 6. Burnet’s intracutaneous allergic test was carried out for milkmaids. In five of 30assays the tests were was positive. All persons were vaccinated against brucellosisbefore. А. Is it possible according to positive Burnet’s test make conclusion, that thesepeople 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? 7. The patient К., has complaints of increased sweating, wavy fever, headache andmuscle pain, joint pain. The initial diagnosis of brucellosis was made. Laboratoryexamination for confirmation of the diagnosis was assigned. А. Enumerate laboratory methods, which allow to confirm the diagnosis ofbrucellosis; B. From what day of disease is it possible to perform Wright’s reaction? C. What criteria do testify about positive Wright’s reaction? D. How is it possible to differentiate the sick persons from vaccinated onesaccording Wright’s reaction? 8. A patients with initial diagnosis of anthrax was admitted to the infectiousdiseases 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 rightforearm. In a few day anthracic carbuncle was formed. It is known that three weeks agothe patients bought a new sheepskin coat in the market. A. What disease may be in these patients? 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? 9. The tested material from the carbuncle was delivered to the bacteriologicallaboratory. There were revealed gram-positive rods with capsules in paires or in shotchains resembling bamboo canes with elbow-shaped articulation. A. Can we make diagnosis according these signs? B. What methods of examinations can confirm your diagnosis? 10. The patient complaints of high temperature (39 °C), headache, generalweakness. On his forearm there is carbuncle of purple colour outside and black spot in 13
  • 14. the centre. The doctor made allergic test with anthracin. In 48 hrs hyperemia andinfiltrate (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?7. List of literature:1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology,Lugansk, 2004, chapter19, p. 72-100. 14