Lesson № 21

Summary of the lecture on the topic

Main characteristics of special danger infections: definition of the term.

To be an infection of special danger the infection should possess at least one of the next character-
istics: the tendency to pandemic distribution or the infectious agent has to be extremely virulent.

Main characteristics of special danger infections: classification.

The bacterial special danger infections are cholera, plague, tularaemia, brucellosis, anthrax and
glanders.

Main characteristics of special danger infections: the significance for humans.

To be infection of special danger the infection should be characterized as disease creating the pos-
sibility of fatal consequences for all human population or for individual human because of high
probability of an individual to get infected and/or high probability to die from this disease.
Nowadays only single cases of special danger infections are registered. Sometimes the cases of in-
fection in bigger groups of humans (but not epidemics) are revealed. The cases of using the patho-
gens producing special danger infections by bioterrorists are also known.

Main characteristics of special danger infections: specificity of microbiological diagnostics.

All manipulations with infectious agents and pathological material containing live bacteria should
be performed only in specialised laboratories called laboratories with special safety regime. If
pathogen producing special danger infection was occasionally isolated in normal bacteriological
laboratory the culture of this pathogen should be immediately transported to the special safety re-
gime laboratory or killed.

Main characteristics of special danger infections: special safety regime for laboratories.
The regime is very strict: strict control of following the safety rules is comprehensive for ever y-
body, strict control of permission of the personal to the laboratory and its sanitary decontamina-
tions after the termination of the working day before leaving of the laboratory.

Vibrios: classification and the role in human disease.

Vibrios are the members of the family Vibrionaceae. Two biological variants of V. cholerae are of
medical importance – var. asiatica (cholera) and var. eltor which cause cholera. Another one spe-
cies of Vibrio is V. parahaemolyticus, it produces diarrhoeas.

Vibrio cholerae: morphology.

Gram-negative curved rod (comma-shaped), highly motile (monotrichous) and it is readily stained
with aqueous fuchsine.

Vibrio cholerae: metabolic characteristics and cultivation.

Vibrio cholerae grows well on alkaline media at usual temperature. The alkaline 1% peptone liq-
uid medium is selective for it. When the medium is inoculated with Vibrio cholera the bacteria
produce delicate pellicle on the surface after 6-8 hrs of incubation. Thiosulfate-citrate-bile sucrose
(TCBS) agar is differential-diagnostic medium. The pathogen causing cholera when growing on
the medium produces yellow S-shaped colonies – the evidence of sucrose fermentation.

Vibrio cholerae: biochemical characteristics.

The basic biochemical characteristics are positive nitroso-indole reaction (“cholera red test”) and
high amylase activity.

Vibrio cholerae: antigenic composition.

According to the О-antigen all vibrios are classified into serological groups. The vibrios belonging
to the group О1 are main pathogens causing cholera. They are classified into three serological var-
iants: Ogawa, Inaba and Hikojima. As the result of the phenomenon of dissociation of S-shaped
vibrios into R-shaped the RO-antigen is revealing in the bacteria. Quite recently it was discovered
that vibrios from the serological group О139 are also able to cause cholera.

Vibrio cholerae: the diagnostic sera used for complete serological identification.
Five diagnostic sera are necessary for complete serological diagnostics of vibrios producing chol-
era: О1-serum, RO-serum, type-specific serum Ogawa, type-specific serum Inaba and О139-
serum.
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Vibrio cholerae: the algorithm of serological identification.

Serological identification of vibrios producing cholera includes the next steps: the isolated bacteri-
al culture is used for the agglutination reaction with О1 and RO diagnostic sera. If the result of the
reaction is positive the vibrios are classified as O1 serological group and the next step is revealing
of serotype of the pathogen by applying agglutination reaction with use of type-specific sera Oga-
wa and Inaba. When the titre of agglutination reaction with Ogawa serum is higher than the titre of
agglutination with Inaba serum the pathogen is classified as serotype Ogawa. If the titre of agglu-
tination reaction with Inaba serum is higher than the titre of agglutination with Ogawa serum the
pathogen is classified as serotype Inaba. In the case when agglutination reactions applying the both
diagnostic sera is resulted in low titres the vibrios are classified as serotype Hikojima. The vibrios
which are not agglutinated by О1 and RO sera are repeatedly inoculated several times into peptone
water and analysed by applying the agglutination reactions with the same diagnostic sera again. In
the case if negative results of the agglutination by O1, RO and also by O139 sera are secondary
obtained the vibrios are classified as non – agglutinating. Such pathogens can produce acute intes-
tinal infections but not cholera and are belonging to other serological groups (not to O1 or O139
groups). If the result of the agglutination reaction with O139 diagnostic serum is positive the vib-
rios are classified as O139 serological group.

Vibrio cholerae: main characteristics used for differentiation of biological variants.

To detect biological variants (biotypes) of vibrios producing cholera type-specific bacteriophage is
used for phage typing and also the biological variants differ from each other by their biochemical
characteristics.

Vibrio cholerae: pathogenicity factors.

The pathogenicity factors of Vibrio cholerae are the bacterial enzymes such as mucinase, which
dissolves the protective mucus coating over the intestinal cells and helps vibrios to penetrate to the
surface of the cells, and neuraminidase involved into binding of the bacteria to the microvilli of the
brush border of the gut. Vibrio cholerae like other Gram- negative bacteria possesses endotoxin
and also produces protein toxin – choleragen. The effects of the choleragen are diarrhoeas and wa-
ter-electrolyte imbalance.

Vibrio cholerae: resistance to unfavourable conditions of the environment.

Resistance of Vibrio cholerae to unfavourable conditions of the environment is low. It is killed
immediately by boiling and is highly sensitive to disinfectants. The vibrios survive for a long time
in stagnant water, выгребных ямах and on the surface of the objects contaminated with the faeces
of ill individuals.

Vibrio cholerae: main approaches to revealing of non-cultivated forms.

When Vibrio cholera is living in the environment for a long time the bacteria can lose the ability to
be cultivated at the laboratory on the culture media. Such forms of Vibrio cholera are called non-
cultivating and could be revealed only by applying PCR.

Parahaemolytic vibrio: distribution in the natural environment.

V. parahaemolyticus is a marine organism.

Parahaemolytic vibrio: epidemiology.

It is transmitted with water penetrate into the digestive tract when swimming or by ingestion of
raw or undercooked seafood,

Parahaemolytic vibrio: pathogenicity factors.

Main pathogenicity factor of V. parahaemolyticus is haemolysin which is similar to enterotoxin.

Parahaemolytic vibrio: the role in human disease.

Parahaemolytic vibrio causes food poisoning and dysentery-like infections.

Cholera: the source of the infections and the mechanism (ways) of its transmission.
The source of the infections in the case of cholera is ill individual. If the disease is caused by V.
cholerae, var. eltor the carriers can also be the sources of the infection. The mechanism of its
transmission is faecal-oral and the main way is the water-born.
Cholera: specific features of the 7th pandemics.
This pandemics was the last one listed in the history of this disease. The 7th pandemics has started
in the 60-s of the ХХ century and it was not in India (like it was before) but in Indonesia. This
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pandemics is still continuing.

Cholera: pathogenesis of the disease.

If vibrios manage to come through acidic barrier of stomach they colonize the epithelial cells of
small intestine, liberate cholera toxin which stimulates marked hypersecretion of water and chlo-
ride into the lumen and impaired absorption of sodium resulting in water-electrolyte imbalance.

Cholera: immunity.
Naturally acquired (post-infectious) immunity is strong and prolonged but the possibility of new
infection is not completely excluded.

Cholera: laboratory diagnostics.

The diagnostics is based on the microscopic investigations of the specimens of stool in the smears
stained with aqueous fuchsine and on isolation of pure culture of the pathogen by growing in 1%
Peptone water and on the TCBS agar. Identification includes study of morphological, biochemical
characteristics and antigenic composition. The procedure of identification should include revealing
of the serological variant (serotype) and biological variant (biotype) of the pathogen.

The brucellae: classification.

The bacteria belonging to the genus Brucella cause brucellosis in humans and animals. The most
of the cases of the disease in humans are produced by B. melitensis. The species of Brucella
B.abortus and B.suis rarely cause the disease in humans.

The brucellae: morphology.

Brucellae are Gram-negative coccobacilli.

The brucellae: cultivation.

Brucellae grow on enriched media (best on the liver- containing ones) at normal temperature. They
grow slowly: usually for 2 two weeks in the first generations. B. abortus is capnophilic bacterium
so requires increased concentrations of СО2 in the atmosphere of cultivation.

The brucellae: biochemical characteristics.

Biochemical characteristics of brucellae are very diverse but not used for their identification.

The brucellae: antigenic composition.

The antigenic composition of Brucellae includes the surface antigens and R-forms of the bacteria
contain R-antigen which can be revealed with use of diagnostic R- antisera.

The brucellae: the characteristics used for differentiation of the species.

The species of brucellae are differentiated in agglutination reactions with mono-receptor sera
which react with the surface antigens of the bacteria specifically. The species are also differentiat-
ed by their biochemical characteristics and by their sensitivity to bacteriostatic effect of microbio-
logical dyes. B. abortus is also differentiated by its lysability by species-specific bacteriophage.

The brucellae: pathogenicity factors.

The virulence of brucellae is mainly based on their ability to inhibit phagosome-lysosome fusion
in the cells of phagocytes.

The brucellae: resistance to unfavourable conditions of the environment.

Brucellae are quite resistant to unfavourable conditions of the environment especially to low tem-
peratures. But they are sensitive to disinfectants and to high temperature: they immediately dye
when they are boiled.

Brucellosis: the source of the infections and the mechanism (ways) of its transmission.
The sources of the brucellosis are domestic animals and the mechanism of its transmission is fae-
cal-oral.

Brucellosis: main characteristics of the disease.

The main symptoms of brucellosis are fever and infection of various organs and systems of human
organism. More frequently the musculoskeletal and nervous systems are involved. The prolong
course of the disease and diversity of the clinical forms are characteristic for brucellosis. The in-
fection is classified as special danger one because of high virulence of the infectious agent.
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Pathogenesis of brucellosis.
Brucellae disseminate from the portals with lymph getting via lymphatic channels into regional
lymph nodes, multiply in macrophages and cause appearance of granulomas composed of large ep-
ithelioid cells. Later they are getting into the bloodstream and disseminate into internal organs and
bones where they produce appearance of necrotic foci surrounded with infiltrations.

The brucellae: immunity.

Post-infectious immunity is developed in brucellosis which is cross-reactive and mainly based on
appearance of T lymphocytes, specific antibodies and on development of DTH. The antibodies
which appear in the beginning of the disease are agglutinins. In chronic brucellosis the appearance
of incomplete antibodies and opsonins (which stimulate phagocyte to complete phagocytosis) is
registered. Despite of development of immunity the possibility of new infection is not completely
excluded.

Laboratory diagnostics of brucellosis.

Laboratory diagnostics is based on immunological tests, such as: Burne skin test to reveal sensiti-
sation to the pathogen and on revealing of antibodies in blood serum. In acute brucellosis the
glass-agglutination test called Heddleson reaction and agglutination reaction in tubes – Wright re-
action are applied. In diagnostics of chronic forms of the disease Coombs test (to reveal incom-
plete antibodies) and opsono-phagocytic probe are usually performed. The procedure of isolation
of pure culture of this pathogen is very difficult to perform and it is dangerous because of high vir-
ulence of brucellae.

Revealing of the humans infected by brucellae.

To reveal fresh cases of brucellosis the agglutination reaction is performed and the titre of specific
antibodies 1:160 is showing the infection in the patient. For retrospective analysis revealing of op-
sonins and incomplete antibodies confirms the disease in the past.

Specific prophylaxis of brucellosis.

For active specific prophylaxis the immunisation by avirulent live strain of brucellae is applied.
This vaccine is highly allergic. So nowadays the chemical vaccine is applied which is also highly
immunogenic but less allergic.

The pathogen causing tularaemia: classification.

The pathogen causing tularaemia is Francisella tularensis.

The pathogen causing tularaemia: morphology.

Francisella is Gram-negative encapsulated coccobacilli.

The pathogen causing tularaemia: cultivation.

Francisella grows on complex solid media at normal temperature and during 3-5 days produces
small whitish colonies. It practically never is cultivated in liquid media because these bacteria
practically don’t grow in these media.

The pathogen causing tularaemia: biochemical and metabolic characteristics.

It is difficult to detect biochemical activity of Francisella because of its complex growth require-
ments.

The pathogen causing tularaemia: antigenic composition.

The pathogen causing tularaemia contains somatic О-antigen and also Vi-antigen localised in its
cell wall.

The pathogen causing tularaemia: pathogenicity factors.

Francisella is resistant to phagocytosis because it is able to leave phagolysosome and to migrate to
the cytoplasm of the phagocytic cell.

The pathogen causing tularaemia: resistance to unfavourable conditions of environment.

The pathogen causing tularaemia can survive in the environment but it is highly sensitive to boil-
ing and to the effect of disinfectants.

Tularaemia: the source of the infections and the mechanism (ways) of its transmission.
The sources of tularaemia are small rodents. The main the way (the route) of its transmission in
nature in the populations of animals is vector-born (transmissive) one. The humans are getting in-
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fected by contact, alimentary, air born routes and very rarely the vector-born route is involved.

Tularaemia: main characteristics of the disease.

It is characterised by fever, intoxication, infection of lymph nodes leading to formation of buboes,
infection of lungs and also skin and mucous membranes.

Tularaemia: pathogenesis of the disease.

The pathogen penetrates into human organism through the skin and mucous membranes (even if
they are intact). The inflammation centres, the ulcers, are forming at the sites of inoculation of
Francisella. Then the lymphogenous spread of the pathogens to the regional lymph nodes occurs
leading to appearance of primary buboes in the lymph nodes. The buboes break open and drain and
Francisella bacteria are getting into the blood-stream. Haematogenous dissemination of the bacte-
ria is accompanied by sensitization of human organism and resulted in getting of the pathogens in-
to organs and tissues (liver, spleen, etc.) where they multiply and cause appearance of lesions and
necrotic ulcers in the effected organs.

Tularaemia: immunity.
Post-infectious immunity is developed in tularaemia based on appearance of T lymphocytes and
phagocytes. The phagocytosis in the individuals having specific immunity to tularaemia is com-
pleted and the immunity is strong and prolonged. Specific allergic reaction as a response to the
antigens of the pathogen producing tularaemia continues for whole life of the individual and can
be revealed by the skin test.

Tularaemia: laboratory diagnostics.

Laboratory diagnostics of tularaemia is based on immunological test, such as skin test (intradermal
injection of tularin) and on the revealing of specific antibodies in the serum of patients. The specif-
ic antigen of the pathogen can be detected in the corpses of rodents by applying Ascoli test. The
pure culture of the pathogen can be isolated by infecting laboratory animals (biological method).

Tularaemia: specific prophylaxis (vaccine).

For the specific prophylaxis of tularaemia the live Gajski-Elbert vaccine is applied.

The pathogen causing plague: classification.

The pathogen causing plague is Yersinia pestis, it is member of the family Enterobacteriaceae.

The pathogen causing plague: morphology.

The yersiniae causing plague are small Gram-negative ovoid rods (coccobacilli) which when are
growing at 370С produce capsule and show bipolar staining when stained with methylene blue –
the phenomenon called metachromatic way of staining and resulted in appearance of intensive blue
staining only at the edges of bacterial cell.

The pathogen causing plague: cultivation.

The pathogen causing plague grows on ordinary nutritive media. The optimal temperature for
growth is 25-280С although all yersiniae can grow at the temperatures varying in wide range.
When growing in liquid media the pathogen produces pellicle on the surface with thread-like
growth resembling stalactites and a flocculent precipitate. On the solid agar media it produces R-
shaped colonies with a dense center scalloped edges called “lace handkerchiefs “. Selective media
for isolation of pure culture of Y. pestis contain haemolysed blood and sodium sulphate to accel-
erate the growth of bacteria and gentian violet to inhibit the growth of concomitant microflora.

The pathogen causing plague: biochemical characteristics.

Biochemical activity of the pathogen causing plague is high. The ability of these bacteria to fer-
ment glycerol is used to differ between continental (plague of natural foci) and oceanic (imported
plague) strains of plague.

The pathogen causing plague: antigenic composition.

Y. pestis cells contain О-antigen (endotoxin) similar to the O-antigen of other enterobacteriae cap-
sular antigens V, W and F1.

The pathogen causing plague: pathogenicity factors.

The pathogenicity factors of the pathogen causing plague are numerous, such as: glycoprotein of
its capsule F1, Vi-antigen composed of the protein V and lipoprotein W fractions, etc. In many
cases the virulence of the pathogen is dependent on the presence of special plasmid genes.
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The pathogen causing plague: resistance to unfavourable conditions of environment.

Y. pestis pathogens can survive for a long time in the environment especially to low temperatures.
But they dye during 1 minute when are boiled.

Plague: the source of the infections and the mechanism (ways) of its transmission.
Plague is zoonotic infection but in the case of pneumonic plague humans can be the sources of the
infection. The pathogen is transmitted from the rodents to humans by the bites of fleas (the vector-
born or transmissive route). In the case of pneumonic plague the air born route is involved into
transmission.

Plague: main characteristics of the disease.

he disease is characterized by severe course with severe intoxication, fever, appearance of the skin
and lymphatic nodes lesions (formation of buboes), involvement of lungs and other organs result-
ed in high mortality.

Plague: pathogenesis of the disease.

The skin bubonic plague is developed after the bites of humans by the infected fleas. When the
buboes are drained and the pathogens are getting into the bloodstream the septic (or hemorrhagic)
form of plague is developed. When Y. pestis penetrate the lungs they cause secondary pneumonic
plague when the ill individual is becoming the source of the infection: the patient becomes to ex-
crete the pathogen with respiratory discharges and to infect other humans with involvement of air
born route of spreading of the infection. The individuals who were infected by such a way develop
the primary pneumonic plague.

Plague: immunity.
Post-infectious immunity developed in plague is strong and prolonged. The immune defense is
based on the phagocytosis which is completed in the individuals which possess specific immunity
to plague.

Plague: specific prophylaxis (vaccine).

Life vaccine EV is applied fro specific active prophylaxis (prevention) of plague.

Methods of laboratory diagnostics of plague.

Five approaches of laboratory diagnostics are applied in the case of plague: microscopic investiga-
tions of the smears prepared using the content of the buboes or phlegm; isolation of pure culture
by growing the bacteria on blood agar containing haemolysed blood and treated on the surface
with the anti-phage serum (Tumansky medium) followed by the agglutination reaction on the glass
slide and identification of the species of the pathogen with use of species-specific phages; use of
IFR for revealing of specific antigens of Y.pestis in specimens or applying of IHAR or ELISA
techniques for the express-diagnosis; in the case if the pure culture can’t be isolated for identifica-
tion (because of high contamination of the specimens with spoilage microflora) the termostable an-
tigen of the pathogen can be revealed by applying Ascoli’s precipitation test; and biological meth-
od of diagnostics can be used (the by infecting guinea pigs).

The pathogen causing anthrax: classification.

The pathogen causing anthrax is Bacillus anthracis. It belongs to the family Bacillaceae.

The pathogen causing anthrax: morphology.

It is large sporogenous Gram-positive rod arranged in the smears in chains and producing capsule.

The pathogen causing anthrax: cultivation.

Bacilli causing anthrax grow on ordinary nutritive media at normal temperature during one day.
When growing in the liquid media it forms appearance of cotton-flake like growth, on solid agar
media the pathogen produces rough R-shaped colonies having disheveled form of edges (called
Medusa-head or lion mane appearance of colonies).

The pathogen causing anthrax: biochemical characteristics.

The pathogen causing anthrax unlike other representatives of the family possesses phosphatase ac-
tivity.
The pathogen causing anthrax: antigenic composition.
Serological characteristics of B. anthracis include capsular antigen, toxin and somatic polysaccha-
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ride antigens of the cell wall.

The pathogen causing anthrax: pathogenicity factors.

Protein capsule and complex protein toxin causing pulmonary edema and death of the infected in-
dividuals are pathogenicity factors of the bacilli causing anthrax.

The pathogen causing anthrax: resistance to unfavourable conditions of environment.

The resistance of vegetative cells of B. anthracis is not high – the same as resistance of usual bac-
teria but the spores of the pathogen are highly resistant in the environment so can survive in the
natural environment for tens of years and also they are resistant to disinfectants and survive during
20 minutes when boiling.

Anthrax: the source of the infections and the mechanism (ways) of its transmission.
The sources of infection in the case of anthrax are animals and the main route of transmission of
the infection to humans is contact. The alimentary and air born routes are also could be involved.
The spores of this pathogen could be used by bioterrorists.

Anthrax: pathogenesis of the disease.

High pathogenicity of B. anthracis is based on the resistance of these bacteria to phagocytosis (the
protective role of capsule) and on the production of protein toxin.

Anthrax: immunity.
Post-infectious immunity developed in anthrax is strong and is based on the phagocytosis, the de-
fensive effect of antibodies and infectious allergy.

Anthrax: specific prophylaxis (vaccine and immunoglobulin).

The life spore vaccine STI prepared from non-capsulated avirulent strain of B. anthracis is applied
for active specific immune prophylaxis. Specific gamma-globulin is used for urgent prophylaxis.

Methods of laboratory diagnostics of anthrax.

Four approaches of laboratory diagnostics of anthrax are applied: direct detection of the bacteria of
B. anthracis in the smears using light microscopy or detection of bacterial specific antigens in IFR;
isolation of pure culture of the pathogen by growing on serum agar (to detect the capsule) follow-
ing by final identification of the bacteria using their morphological and growth characteristics as
well as applying species-specific bacteriophages for phage indication; biological method of diag-
nostics can be applied to reveal the pathogen in the corpses of died laboratory animals; Ascoli’s
precipitation test is usually applied to reveal the specific antigens of the pathogenic bacilli in hair,
skin and fur of animals or in autopsy material.

Bacillus cereus: the role in human disease.

These bacilli are main pathogens producing food poisoning toxic infections – poisoning which are
the results of eating the food products massively contaminated but not only exclusively with the
bacterial toxins (as it happens in food poisoning intoxications) but contaminated with live patho-
gens.

Bacillus cereus: pathogenesis.

Pathogenesis of this bacillus is connected with exotoxin secretion and also releasing of the en-
zymes affecting on the food product and causing accumulation of the toxic substances in the prod-
ucts. The effects of the toxic substances and of the exoenzymes on human organism are leading
to development of food poisoning toxic infections.

Bacillus cereus: the comparative characteristics in comparison to the pathogen causing anthrax.
Unlike the pathogen causing anthrax and like other saprophytic bacteria Bacillus cereus doesn’t
produce capsule and these bacteria are not pathogenic for the laboratory animals. The anthrax spe-
cific bacteriophage never causes lysis of Bacillus cereus.
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Training algorithm of practical skills to be mastered at the lesson

Inoculation the liquid medium

Students have to revise the practical skill of the les-
son N3 to consolidate their proficiency: the training
algorithm is similar to inoculation the Hiss medi-
um with the bacteria grown on an agar slant.

Identification of Bacillus by smear

Students have to revise the practical skill of the
lesson N3 to consolidate their proficiency.