By Ms. Kostiuk from Microbiology department

1. Lesson N12 LABORATORY DIAGNOSIS OF ZOONOSIS (PLAGUE, TULARAEMIA, BRUCELLOSIS
AND ANTHRAX)
1.Scientifically methodical ground of theme
Thise group of diverse organisms is presented togerther as the mode of transmission is
zoonotic. The resorvoir of infection is animals. Tramsmission may occur directly by
contact or bites of animals or through an insect vector where the insect feeds on an
animal 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 and
specific prophylaxis of plague ,tularemia, brucellosis and anthrax
3.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 in
antiplague protective clothing. A strict work regimen must be observed. Depending on
the clinical form of the disease and the location of the causative agent, test specimens are
collected from bubo content (in bubonic plague), ulcer secretions (in cutaneous plague),
mucus from the pharynx and sputum (in pneumonic plague), and blood (in septicaemic
plague). Test matter is also recovered from necropsy material (organs, blood, lungs,
contents of lymph nodes), rodent cadavers, fleas, foodstuff's, water, air, etc. Examination
is performed in the following stages.
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2. 1. Microscopy of smears, fixed in Nikiforov's mixture and stained by the Gram
method or with methylene blue by Loeffler's method.
2. Inoculation of the test material into nutrient media, isolation of a pure culture and
its identification. To inhibit the growth of the accompanying microflora, 1 ml of a 2.5 per
cent sodium sulphite solution and 1 ml of a concentrated alcohol solution of gentian
violet, diluted in distilled water in a ratio of 1 :100, are added to 100 ml of meat-peptone
agar. Prior to inoculation 0.1 ml of antiphage serum is added to the culture to render the
plague bacteriophage harmless.
3. Biological tests of the isolated pure culture and of material from which isolation
of the organism is difficult are conducted on guinea pigs. In the latter case a thick
emulsion prepared from the test material is rubbed into a shaven area of skin on the
abdomen. If plague bacilli are present the animals die on the fifth-seventh day. To hasten
diagnosis the infected guinea pigs are killed on the second-third day and the plague
bacillus 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 is
differentiated from the causative agent of rodent pseudotuberculosis (see Table 1). The
biological 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 accelerated
diagnostic 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 have
common symptoms. For differentiation of tularaemia from other diseases laboratory tests
are the most effective. Those peculiarities of the disease which can be revealed easily and
quickly 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. In
tularaemia patients the test gives a positive reaction 6-12 hours after inoculation of
tularine. In distinguishing tularaemia from other infections one must bear in mind that
allergic 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 and
volume methods. In some cases this test may give a positive reaction with material
containing brucella organisms, since they possess antigens common to tularaemia
bacteria.
3. The tularaemia culture is isolated by the biological method as it is impossible to
recover the pathogen directly from a tularaemia patient. For this purpose white mice or
guinea pigs are infected by material obtained from people suffering from the disease
(bubo punctate, scrapings from ulcers, conjunctiva! discharge, throat films, sputum, and
blood). Biological tests are conducted in special laboratories where a standard regimen is
observed. The laboratory animals die in 4-12 days if tularaemia bacteria are present in the
test material. Autopsy is performed, smears from organs are made and organ specimens
are inoculated onto coagulated egg medium for culture isolation. Microscopic,
microbiological, and biological studies of the cultured organisms are made. If no culture
can be isolated from the first infected guinea pig, an emulsion, obtained from the latter's
organs, is inoculated into a second guinea pig, etc.
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3. 4. Laboratory diagnosis of rodent tularaemia is made by microscopy of smears from
organs, 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 bc
B. 1 – – –++ ++ –+ – + – – –
melitensi
s
2 – – –++ ++ +– – + – – –
3 – – –++ ++ ++ – + – – –
B. 1 +(–) + ––– ++ +– + + – + –
abortus
2 +(–) + ––– –– +– + + – + –
3 +(–) + + + + ++ +– + + – + –
4 +(–) + ––– ++ –+ + + – + –
5 – – –++ ++ +– + + – + –
6 – –+ –++ ++ +– + + – + –
7 – +– –++ +–+ ++ + + – + –
8 + – –++ ++ –+ + + – + –
9 – + –++ ++ –+ + + – + +
B. suis 1 – + +++ –– +– – – + + –
2 – – –++ –– +– – + + + +
3 – – +++ ++ +– – + + + +
4 – – +++ ++ ++ – + + +
B. 1 – + ––+ –– +– – + – ± –
neotoma
e
B. ovis + – +++ –– – + – – –
B. canis – – – ++ – – – – + + +
Laboratory diagnosis. The patient's blood and urine (for isolation of the pathogen),
serum (for detection of agglutinins), milk and dairy products (for detection of brucellae
or agglutinins in milk) are examined. The microbe is isolated in special laboratories.
1. Culture isolation. Since brucellosis is often accompanied by bacteraemia, blood is
examined during the first days of the disease (preferably when the patient has a high
temperature). For this purpose, 5-10 ml of blood is collected and transferred into two or
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4. three flasks (2-5 ml per flask) containing 100 ml of liver-extract or ascitic-fluid broth (pH
6.8). The cultures are grown for 3-4 weeks or more. Five to ten per cent of carbon
dioxide is introduced into one of the flasks (for growth of the 23 bovine species of the
bacteria). Inoculations on agar slants are made every 4-5 days for isolation and
identification 'of the pure culture.
An antiphage serum is introduced into the cultures for neutralization of the phage
which inhibits the growth of brucellae. The best results are obtained when the blood is
inoculated 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 into
each egg. The infected eggs are placed in an incubation chamber for 5 days, after which
0.3-0.5 ml of their contents is inoculated into the liquid nutrient media. Growth is
examined every 2-3 days.
If the blood culture produces a negative result bone marrow obtained by sternum
puncture 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 of
the precipitate is seeded onto agar plates containing 1 :200000 gentian violet. In some
cases faeces, cow's and human milk, and amniotic fluid of sick humans and animals are
examined for the presence of Brucella organisms.
Brucella cultures may be isolated by the biological method. For this purpose healthy
guinea pigs or white mice are injected with 0.5 or 3 ml of the test material. A month later
the guinea pigs' blood is tested for agglutinins, the allergic test is carried out, and the pure
culture is isolated. White mice are tested bacteriologically every three weeks.
2. Serological test. From the tenth-twelfth day of the disease onwards, the
agglutinins accumulate in the blood in an amount sufficient for their detection by the
agglutination tests. The Wright (in test tubes) and Huddleson (on glass) reactions are
carried 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 a
titre 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 positive
results with sera of healthy individuals who have normal antibodies in their blood.
3. Skin allergic test. To determine allergy, Burne's test is made beginning from the
fifteenth-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 is
considered 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. The
index of healthy individuals averages 0-1 and occasionally 3-5. In sick persons the
reaction is considered high if the index is 50-70, mild, if it is 25-49, and low, if it is
10-24.
For detecting brucellae in the external environment the reaction for demonstrating a
rise 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
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5. LF Causes pulmonary edema and death in rats;
cytolytic for macrophages
PA Required for the binding of both EF and LF
to host cell
EF, edema factor, LF, lethal factor, PA, protective antigen
Laboratory diagnosis. In cases of cutaneous anthrax the malignant pustular
exudate is examined; it is obtained from the deep layers of the oedematous area where it
borders 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 morphologically
characteristic capsulated bacilli, arranged in chains, allows a preliminary diagnosis.
2. For isolation of the pure culture the specimens are inoculated into meat-peptone
agar and meat-peptone broth. The isolated culture is differentiated from other
morphologically similar bacteria by its morphological and biochemical properties.
3. Laboratory animals (white mice, guinea pigs and rabbits) are inoculated with the
pathological material and with the pure culture de-rived from it. B. anthracis causes the
death 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 anthrax
bacilli which are surrounded by a capsule.
A rapid biological test is also employed. The culture obtained which has to be
identified is introduced intraperitoneally into white mice. Several hours after inoculation
smears are prepared from the peritoneal contents. Detection of typical capsulated bacilli
gives a basis for con-firming the final result of the biological test.
The allergic test with anthracin (a purified anthrax allergen) is employed when a
retrospective diagnosis is required in cases which have yielded negative results with
microscopical and bacteriological examination.
Postmortem material as well as leather and fur used as raw materials are examined
serologically by the thermoprecipitin reaction (Ascoli's test) since isolation of the bacilli
is a matter of difficulty in such cases.
The result in the first test tube (containing the test material) may be either positive
or 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 the
possibility of the presence of bacteria identical with B. anthracis in their biological
properties (see Table 1). These sporing aerobes are widely distributed in nature and are
normally sporeforming saprophytes. They include B. cereus, B. subtilis, B. megaterium,
etc.
The anthrax bacilli may be differentiated from anthracoids (false anthrax
organisms) and other similar sporing aerobes by phagodiagnosis. The specific
bacteriophage only causes lysis of the B. anthracis culture.
4. Student’s independent study program
1. Structure, staining properties of causative agent of plague, tularemia, brucellosis
and anthrax . Cultivation.
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6. 2. Biochemical and other differential diagnistic properties of causative agent of
plague, 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 of
transmission, 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 in
museum preparations. Drow your observation.
Y.pestis stains with ordinary aniline dyes and given bipolar appearance, its ends
staining more intensively. It is Gram negative. Y.pestis is characterized by marked
individual variability (pleomorphism). In smears from organs and in young cultures it has
an 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 cocci
measuring 0.2-0.7 mcm. In old 'cultures the organisms retain the coccal form. They are
non-motile, and Gram-negative. In the animal body they are sometimes surrounded by a
fine 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 electron
microscope Brucella organisms of cattle, sheep and goats appear as coccal and
coccobacilary 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 sometimes
present).
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 before
the cells have separated after the previous division, which leads to the formation of
streptobacilli. Outside the host's body they produce oval-shaped central spores which are
smaller in diameter than the bacillus. In the bodies of man and animals the bacilli produce
capsules 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 of
brucellosis. Make up conclusion and record your observation.
To carry out a standard agglutination test, on the second week of the disease (the
10th–15th day) withdraw 2-3 ml of blood from the patient's cubital vein. Dilute
successively the obtained serum (Table 2) with isotonic saline in titres from 1:25 to
1:400.
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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 violet
and containing 1 mird of brucellae (per ml) killed with formalin or phenol is used as a
diagnosticum. Currently a uniform colour brucellar diagnosticum for Wright's and
Huddleson's tests is commercially available.
The procedure of performing the Wright test is similar to that used in other
agglutination tests, with the exception of the fact that the serum is diluted in a multiple
order with isotonic NaCI solution containing 0.5 per cent of phenol. The test tubes are
placed in the incubator for 18–20 hrs and then left at room temperature for 2 hrs. The
results of agglutination are denoted with pluses. The reaction is considered positive if the
titre is 1:200 and doubtful if it is 1:50. Wright's reaction may be positive in vaccinated
subjects and patients with brucellosis. So, it is to be repeated in the course of the disease
to 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 frequently
used in brucellosis foci (in field conditions) since it is simple to perform. Using a wax
pencil, divide a glass plate into six squares, then pipette on them undiluted serum to be
studied in 0.08, 0.04, 0.02, 0.01, and 0.02-ml portions, and add 0.03 ml of the same
diagnosticum that is used in Wright's reaction to each dose of the serum but the last (the
fifth square) into which 0.03 ml of isotonic sodium chloride solution (control of the
serum) is placed. In the sixth square, place 0.03 ml of the antigen and 0.03 ml of isotonic
saline (control of the diagnosticum). Mix drops of the serum and antigen with a glass rod
and after slight rocking of the plate read the results of the reaction which is manifested
already in the first minutes by the appearance of microgranular stained agglutinate. The
absence of agglutination in all doses of the serum is assessed as a negative reaction, the
presence of agglutination in the first dose (0.08 ml of the serum) as a doubtful one, in the
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8. second dose (0.04 ml), as a weakly positive one, in the third or fourth doses (0.02–0.01
ml), as a positive one; the agglutination reaction expressed by 4 pluses in all doses is
evaluated as a drastically positive one. The value of Huddleson's agglutination test is
diminished by the fact that not infrequently it produces positive results with healthy
people's sera due to the presence of normal antibodies. Hence, sera showing positive
Huddleson's reaction should be subjected to the tube agglutination test.
Table 3
Schematic Representation of Huddleson's Agglutination Test
Number of square Control
Ingredient, ml of of
1 2 3 4 serum antigen
5 6
Isotonic saline – – – – 0.03 0.03
Patient's 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 laboratory
diagnosis and specific prophylaxis of plague ,tularemia, brucellosis and anthrax
6. 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 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.
9.F. tularensis has such properties:
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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 of
material tested which was taken from the man; e – grows onto media with a blood and
cystine.
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 – sensitive
to 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 – 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.
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 % 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.
18. 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.
19. For bacteriological diagnosis of brucellosis are used: a – blood; b – punctate of
a 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-shaped
articulation.
23. 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.
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:
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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 at
120 °С 90 min; d – boiling in 1 % solution of soda for 2 hrs; e – burning or immersion in
concentrated 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 cause
brucellosis 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 of
viable spores of the causative agent of especially dangerous disease. Name
microorganisms, 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.
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11. 34. A 34 year-old- patient complained of carbuncle on his face. His examination
revealed subcutaneous painful edema with black eschar in the center, vesicular eruption
on circumference. Microbiologic examination revealed nonmotile, capsule-, forming
streptobacillli. 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 (Askoly
test). Result: in some minutes after adding immune serum to skinr extract albescent ring
has 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 of
high body temperature, weakness, absence of appetite, pain in joints. For 10 days she was
treated for flu. But infectionist has suspected brucellosis. Using what reaction is it
possible 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 an
animal streptobacilli surrounded with a capsule were revealed. It gives the basis to
diagnose:
A. Tularemia.
B. Anthrax.
C. Plague.
D. Brucellosis.
E. Streptococci pneumonia
38. There is the patient with the presumptive diagnosis of acute pneumonia. In the
smear of sputum there are revealed chaotically located microorganisms of oval forms in
length up to 2 microns bipolar stained. What the most likely diagnosis can be made on
the 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 of
the anthrax agent. For making this procedure we have to prepare soluble thermoresistant
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12. antigen in water-salt extract from raw material. What reaction may be used for this
purpose?
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 fast
rise of body temperature up to 400 C, the expressed intoxication, increase groin lymph
nodes. In preparations (swabs from a cadaveric material) gram-negative bipolar stained
oval 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?
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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 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 on nutrient 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?
5. 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 for
agglutination 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 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?
7. 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’s reaction?
C. What criteria do testify about positive Wright’s reaction?
D. How is it possible to differentiate the sick persons from vaccinated ones
according Wright’s reaction?
8. A patients 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 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 bacteriological
laboratory. There were revealed gram-positive rods with capsules in paires 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?
10. 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
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14. the centre. The doctor made allergic test with anthracin. In 48 hrs hyperemia 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?
7. List of literature:
1.I. S. Gaidash, V.V. Flegontova, Microbiology, virology and immunology,
Lugansk, 2004, chapter19, p. 72-100.
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