Yerevan State Medical University after M.

Heratsi

Department of Medical Microbiology

M.S. Hovhannisyan

SPECIAL MICROBIOLOGY

Edited by V.A. Shekoyan

Handout on Special microbiology for foreign students

Yerevan 2016

1
UDC 579 (07)

Approved by YSMU Foreign students

Education Methodological and Medico-biological committees
and was guaranteed for publication by the academic council of the
YSMU after M. Heratsi by protocol N 3, 02.12.2015

Reviewers: Hasmik S. Hovhannisyan

Vigen A. Asoyan

Special Microbiology for foreign students

Edited by M.S. Hovhannisyan – Yerevan: M. Heratsi’s Yerevan
State Medical University publishing, 2015, 270 pages.

Language Editor: N. Nazaretyan

M. Bisharyan

Computer Design: Manyak Avetisyan

ISBN 978-9939-65-134-7 @ M. Heratsi’s

Yerevan State Medical University, 2016

2
 Brief Description of Special Microbiology

Medical microbiology, virology, immunology being basic sciences, studies microbes’

structure, physilogy, lows of deveolopment and belong to such sciences, which is nessesary
to each doctor and medworker, because it insures general biological knowleges and at the
same time as a propedeutics direction, is used in the practical sphera of medicine. Medical
microbiology consists of two parts: General and Special. The studying subject of Special
microbiology are pathogenic microorganisms, which are the source of infectious diseases
and different pathological processes, their morphology, physiology, biological and antigenic
properties and the role of distinct species in different infectious diseases etiology and
pathogenesis, as well as elaboration of methods of laboratory diagnosis, prophylaxis and
treatment of infectious diseases.

3
 MICROBIOLOGICAL LABORATORY DIAGNOSTIC METHODS

 Microscopic-preparation of smears from investigating material, staining by Gram method and

microscopy. Description of microorganisms by morphology and staining properties.
 Bacteriological method-isolation of pure culture from investigating material and identification
of them by morphology, staining properties (tinctorial), cultural properties, bio-chemical
activities, antigenic structure, toxigenicity, etc.
 Serological: revealing of specific antibodies or antigens in blood serum by immunological
reactions (agglutination, precipitation, complement fixation, lysis, immune enzyme, RIA).
 Skin-allergic tests-revealing of development of infectious allergy (DTH) in some infectious
diseases (tuberculosis, brucellosis, anthrax).
 Biological: infection of pathogenic material into susceptible animal organism and isolation of
pure culture from these organisms.
Choosing of pathological material (feces, urine, blood, mucous, sputum, etc.) depends on clinical
diagnosis, pathogenesis of infections, stages of infections, and choused method of examination.

ANTIGEN-ANTIBODY REACTIONS IN THE LABORATORY

Reactions of antigens and antibodies are highly specific. An antigen will react only with
antibodies elicited by itself or by a closely related antigen. Because of the great specificity, reactions
between antigens and antibodies are suitable for identifying one by using the other. This is the basis of
serologic reactions.
Types of diagnostic tests: Many properties of diagnostic tests are performed in the
immunological laboratory. Most of these tests can be designed to determine the presence of either
antigen or antibody. To do this, one of the components, either antigen or antibody, is known and the
other is unknown.
Immunoelectrophoresis- mmunoelectrophoresis combines electrophoresis immunodiffusion
(immune precipitation in gel). This method can be used for analyzing complex antigens in biological
fluids. A glass slide is covered with molten agar or agarose. A well for antigen and a trough for
antiserum is cut on it. Antigen well is filled with antigen mixture (human serum). The slid is then
placed in an electric field for about an hour to allow for the electrophoretic migration of various
antigens. Different antigens will migrate at different rates or even in different directions, depending
upon their size and charge and the conditions of electrophoresis. After the completion of
electrophoresis, antiserum trough is filled with appropriate antiserum (antiserum to whole human
serum). Antigens and antibodies diffuse towards each other, resulting in the formation of precipitin
bands, for individual antigens and antibodies, whenever they are both in zones of optimal proportions,
in 18-24hours. Because immunoelectrophoresis uses electric charge in addition to diffusion, it is more
likely to separate antigen than is simple diffusion alone. By this method, over 30 different antigens can
be identified in human serum. This technique is useful for detection of normal and abnormal serum
proteins.
Radioimmine assay (RIA)-This method is used for the quantitation of antigens or haptens that
can be radioactively labeled. It is based on the competition for specific antibody between the labeled
(known) and unlabeled (unknown) concentration of material. The complexes that form between the
antigen and antibody can be separated and amount of radioactivity measured. The more unlabeled
antigen is present, the less radioactivity there is in the complex. The concentration of the unknown
(unlabeled) antigen or hapten is determined by comparison with the effect of standards. RIA is a
highly sensitive method and is commonly used to assay hormones or drugs in serum. The
radioallergosobent test (RAST) is a specialized RIA that is used to measure the amount of serum IgE
antibody which reacts with a known allergen (antigen).
Enzyme-Linked Immunosorbent Assay (ELISA) – the method can be used for the
quantitation of either antigens or antibodies in patient specimens. It is based on covalently linking an
enzyme to a known antigen or antibody, reacting the enzyme -linked material with the patient’s
specimen, and the assaying for enzyme activity by adding the substrate of the enzyme. The method is
nearly as sensitive as RIA yet requires no special equipment or radioactive labels.
4
 For measurement of antibody, known antigens are fixed to a surface (eg, the bottom of small wells on
a plastic plate), incubated with dilutions of the patient’s serum, washed, and then reincubated with
antibody to human IgG labeled with an enzyme, eg, horseradish peroxidase. Enzyme activity is
measured by adding the substrate for the enzyme and estimating the color reaction in a
spectrophotometer. The amount of antibody bounds in proportional to the enzyme activity. The titer of
antibody in the patient’s serum is the highest dilution of serum that gives a positive color reaction.
Immunofluorescence (Fluorescent antibody)- Fluorescent dyes eg, fluorescein and
rhoamine, can be covalently attached to antibody molecules and made visible by ultraviolet (UV) light
in the fluorescence microscope. Such “labeled” antibody can be used to identify antigens, eg, on the
surface of bacteria(such as streptococci and treponemas), in cells in histologic section, or in other
specimens. The immunofluorescence reaction is direct when known labeled antibody interacts
directly with unknown antigen and indirect when a two-stage process is used. For example, known
antigen is attached to a slide, the patient’s serum (unlabeled) is added, and the preparation is washed. If
the patient’s serum contains antibody against the antigen, it will remain fixed to it on the slide and can
be detected on addition of a fluorescent dye-labeled antibody to human IgG and examination by UV
microscopy. The indirect test is often more sensitive than direct immunofluorescence, because more
labeled antibody adheres per antigenic site. Furthermore, the labeled antiglobulin becomes a “universal
reagent”, ie, it is independent of the nature of the antigen used because the antibody to IgG is reactive
with all human IgG.
Immunoblot (Western Blot)- is a technique that combines electrophoresis with ELISA to
separate and identify protein antigens in a sample. It has many research applications, but its main
clinical use is to confirm positive ELISA screening tests for antibodies against HIV. The ELISA tests
are simpler and cheaper, but they give a small percentage of false positive results; therefore, positive
results need to be confirmed by different separated by electrophoresis in a polyacrylamide gel. Smaller
proteins migrate faster than larger ones. The resulting bands of separated antigens can react with
specific antibodies, but antibodies do not diffuse well into these gels. Therefore, it is necessary to
transfer the antigens present in the bands from the gel by blotting them onto a filter. To test for
antibodies against HIV, commercial kits are available in which antigens of the HIV virus have been
electrophoresed and blotted onto the filter, which is then cut into strips for each test. Each sample to be
tested for antibodies to HIV is applied to a strip and incubated to allow specific antibodies to react with
the viral antigens. To detect the antigen – antibody combinations formed, a label is used, usually an
enzyme – labeled anti-HGG, as in ELISA, or sometimes a radioactive label. The Western blot is a
more laborious and expensive technique than the ELISA; however, it offers greater specificity, since
antigens are identified by two criteria: their size and their reactivity with antibodies.

PATHOGENIC COCCI
STAPHYLOCOCCI
The Staphylococcus, S. aureus, was discovered by R. Koch (1878), and later isolated
from furuncle pus by L. Pasteur (1880). It was described as the causative agent of numerous
suppurative processes and studied in detail by F. Rosenbach (1884).
Staphylococci are included in the class Bacteria, family Micrococcaceae, genus
Staphylococcus. According to the contemporary classification Staphylococci are classified
into 20 species and 15 subspecies, which we classify into 2 groups: coagulase positive,
coagulase negative. Three of them ecologically are connected with human organism:
S. aureus produces golden pigment (S. aureus)
S. epidermidis produces white pigment (S. albus)
S. saprophyticus produces yellow pigment (S. citreus)
Other species are parasitic on animals.
Some are members of the normal flora of the skin and mucous membranes of humans;
others cause suppuration, abscess, a variety of pyogenic infections, and even a fatal
septicaemia, they can cause 120 nosologic forms.
5
 Morphology: Staphylococci are spherical in shape, 0.6-1.5µm in diameter. They are
arranged characteristically in grape-like clusters (in smears from pure culture). Cluster
formation is due to cell division occurring in more than one plane with daughter cells
remaining close together. In smears from liquid media and pus, the cocci appear singly or in
pairs, short chains, but not clusters. Staphylococci are Gram-positive organisms which are
nonmotile, non-sporing and, with the exception of rare strains, non-capsulated.

Cultivation: Staphylococci are facultative anaerobes. They grow well on ordinary

nutrient media with a pH of 7.2-7.4 at a temperature of 37º C. At room temperature with
adequate aeration and subdued light the organisms produce golden, white, lemon-yellow,
and other pigments known as lipochromes. These pigments do not dissolve in water but are
soluble in ether, alcohol. They are most readily formed on milk agar and potatoes at tempera-
ture of 20-25º C. The selective medias for Staphylococcus are salt agars: yolk-salt agar
and milk- salt agar: Salt (6-15%) agars are good selective plating media and are useful for
staphylococci isolation from food, dust, faeces and pus where mixed bacterial flora is
expected. On yolk-salt agar they form smooth (S-form), convex, shiny, opaque colonies.
Round the colonies they form pearl zone (lecithinase activity). On milk-salt agar pigment
production is enhanced. Pigment is not formed in liquid media.
On blood agar colonies are similar to those on salt agar, but may be surrounded by a
zone of ß-hemolysis (haemolytic activity).
Sugar broth (as an enrichment media) is used for examination of blood (sepsis).
Fermentative properties: Staphylococci produce enzymes which cause lysis of
proteins and sugars. They hydrolyze proteins and form H2S, liquefy gelatin (as a funnel),
don't form indole; fermentation of sugars (glucose, maltose, lactose, saccharose) with acid
formation, without gas. Fermentation of glucose and mannite in anaerobic condition has
differential diagnostic meaning. There is no indole production in cultures. S. aureus is
catalase-positive and oxidase –negative.
Staphylococcus aureus strains usually exhibit the following characteristics: 1. coagulase
positive; 2.greater biochemical activity, (differential diagnostic is fermentation of mannite and
glucose in anaerobic condition); 3.produce clear hemolysis on blood agar; 4. produce a
golden yellow pigment; 5.liquefy gelatine; 6.produce phosphatase; 7.in a medium containing
potassium tellurite, reduce tellurite to form black colonies; 8.produce pearl zone on yolk-salt
agar; 9.produce thermostable nucleases which can be demonstrated by the ability of boiled
cultures to degrade DNA in an agar diffusion test 10.Urease activity is positive for S. epider-
midis and S. saprophyticus.
Staphylococci are variably sensitive to many antimicrobial drugs. They are resistant to many
types of penicillin (β-lactamates) because they produce beta-lactamase enzyme.
Antigenic structure: Antigenic structure of S. aureus is very complex. Staphylococci
contain antigenic polysaccharides and proteins as well as other substances important in cell
wall structure. These include:
1.Capsular polysaccharide: A few strains of S. aureus are capsulated and these tend to
be more virulent than non-capsulated strains. The capsule is composed of antigenic
polysaccharide. It prevents ingestion of the organism by polymorphonuclear leucocytes. The
capsule may promote adherence of the organism to host cells and to prosthetic devices.
2.Teichoic acid (which are polymers, are linked to the peptidoglycan and can be
antigenic). Teichoic acid is a major antigenic determinant of all strains of S. aureus. Teichoic
6
acids function in the specific adherence of gram-positive bacteria to mucosal surface.
Teichoic acid participates in staining of bacteria also.
3.Peptidoglycan: (is a polysaccharide polymer containing linked subunits, provides
rigid exoskeleton of the cell wall). It stimulates both humoral and cellular immune responses
in the host. The antibodies against peptidoglycan possess opsonizing activity; however,
increased levels of antibodies may predispose some patients to immune complex disorders.
In addition to their role in providing rigidity and resilience to the staphylococcal cell wall,
peptidoglycan and teichoic acid also have several biologic activities that are thought to
contribute to virulence. These properties include the ability to activate complement, to inhibit
chemotaxis of inflammatory cells, and to stimulate antibody production.
4.Protein A: Protein A is a group - specific antigen and is a cell wall component of
many S. aureus strains that binds IgG molecules, non-specifically, through Fc region leaving
specific Fab sites free to combine with specific antigen. When suspension of such sensitized
cells is treated with homologous (test) antigen, the antigen combines with free Fab sites of
IgG attached to staphylococci cells. This is known as co-agglutination.
5.Cross reacting antigens.
Pathogenicity: Pathogenicity of Staphylococcus aureus depends on invasive
properties, capsule formation, which has antiphagocytic ability; protein A, which inactivate
complement. The pathogenic factors are combined with toxin production and enzymes too.
They produce several toxins:
Leukocidin: Leukocidin act to damage polymorphonuclear leucocytes and especially
macrophages (antiphagocytic action), and can produce dermonecrosis.
Hemolysins (ά, β, γ and δ): These toxins have haemolytic activity. Alpha toxin is the
most important in pathogenicity. This toxin lyses erythrocytes. It is also leucocidal (is toxic for
human macrophages and platelets and causes degranulation of polymorphonuclear
leucocytes through disruption of their lysosomes), dermonecrotic, cardiotoxic (alfa toxin has a
powerful action on the vascular smooth muscle) and neurotoxic. This is lethal toxin.
Enterotoxins: An important cause of food poisoning. Enterotoxins (A to F) are
soluble, heat-stable (they resist boiling for 30 minutes) and resistant to the action of gut
enzymes. Enterotoxins are produced when S. aureus grows in protein food (e.g., creams,
creamery products, dairy products). By mechanism of action it is cytotoxin. The usual
incubation period ranges from 2-6 hours after the ingestion of food. Patient develops nausea,
vomiting and diarrhoea. The duration of acute symptoms is usually less than 24 hours. In
addition to the ability to cause nausea and vomiting, enterotoxins are pyrogenic, mitogenic
and are capable of producing thrombocytopenia and hypotension. Enterotoxin F is reported
to be responsible for the Toxic shock syndrome (TSS): In humans, the toxin is associated
with fever, shock, and multi-system involvement, including a desquamative skin rash.
Toxic shock syndrome toxin (TSST): TSST is the same as the enterotoxin F.
TSST and the enterotoxins are recognized as superantigens, i.e. they are potent activators
of T- lymphocytes without relation to their epitope specificity resulting in the liberation of
cytokines such as tumour necrosis factor, interleukins 1, 2; interferon gamma and they bind
with high affinity to mononuclear cells. These characteristics partly explain the florid and
multisystem nature of the clinical conditions associated with these toxins.
Exfoliative toxin: This is epidermolytic toxin. This toxin is responsible for scalded skin
syndrome in young children, causes impetigo of the new-born (characterized by isolated
pustules that become crusted and rupture).
Staphylococci produce enzymes:
Hyaluronidase, which breaks down hyaluronic acid, a component of connective tissue,
which facilitates the spread of bacteria to adjacent areas(known as spreading factor).
Coagulase accelerates the formation of a fibrin clot from its precursor, fibrinogen (this
clot may protect the bacteria from phagocytosis by walling off the infected area and by
coating the organisms with a layer of fibrin).
Fibrinolysin (staphylokinase): This enzyme dissolves coagulated plasma and
probably aids in the rapid spread of bacteria through tissues.

7
 Lecithinase, which is hydrolyses lecithin in the cell membrane resulting in destruction of
the membrane and widespread cell death.
Lipase ,which participates in adhesion and invasion.
DNA-ase –plays a role in pathogenesis of diseases.
Resistance: Staphylococci are among the more resistant of non-sporing bacteria. They
are relatively resistant to drying, heat (they withstand 50º C for 30 minutes), a 3 per cent
phenol solution kills the organisms in 15-20 minutes. 1% chloramine kills the staphylococci in
2-5 minutes. Staphylococci are very sensitive to certain aniline dyes, particularly to brilliant
green which is used for treating pyogenic skin diseases caused by these organisms.
Staphylococci are variably sensitive to many antibacterial drugs, they can develop drug
resistance. They produce Beta-lactamase which causes resistance to penicillin and similar
drugs (beta-lactamates).
Pathogenesis and diseases in man: Staphylococci, particularly S. epidermidis, are
members of the normal flora of the human skin and respiratory and gastrointestinal tracts.
Nasal carriage of S. aureus occurs in 40-50% of humans. Staphylococci are also found
regularly on clothing, bed linen, and other fomites in human environment.
Staphylococci enter the body through the skin and mucous membranes. When they
overcome the lymphatic barrier and penetrate into the blood, staphylococcal septicaemia sets
in. Both the exotoxins and the bacterial cells play an important role in pathogenesis of
diseases caused by these organisms.
Staphylococci are responsible for a number of local lesions in humans: abscess,
furuncle, carbuncle, osteomyelitis, dermatitis eczema, peritonitis, meningitis, etc, 120
nosologic forms.
In some cases Staphylococci may give rise to a secondary infection in individuals
suffering from smallpox, influenza, and wounds, as well as postoperative suppurations.
Staphylococcal sepsis and staphylococcal pneumonia in children are particularly severe
diseases.
Staphylococci play an essential part in mixed infections, and are found together with
streptococci in cases of wound infections, diphtheria, and tuberculosis.
S. aureus infection can also result from direct contamination of a wound, e.g.,
postoperative staphylococcal wound infection or infection trauma (chronic osteomyelitis
subsequent to an open fracture, meningitis following skull fracture).
If S. aureus disseminates and bacteremia ensues, endocarditis, acute hematogenous
osteomyelitis, meningitis, or pulmonary infection can develop (table 1).
Food poisoning due to staphylococcal enterotoxin is characterized by a short
incubation period (1-8 hours); violent nausea, vomiting, and diarrhoea; and rapid
convalescence. There is no fever

8
Table 1 Staphylococcal diseases
Skin and soft tissue Folliculitis, furuncle (boil), abscess(particularly breast abscess)
wound infection, carbuncle, impetigo, paronychia, less often
cellulitis
Musculoskeletal Osteomyelitis, arthritis, bursitis, pyomyositis
Respiratory Tonsillitis, pharyngitis, sinusitis, otitis, bronchopneumonia, lung
abscess, empyema, rarely pneumonia
Central nervous Abscess, meningitis, intracranial thrombophlebitis
system
Endovascular Bacteremia, septicaemia, pyemia, endocarditis
Urinary Staphylococci are uncommon in routine urinary tract infections,
though they do cause infection in association with local
instrumentation, implants or diabetes

Immunity: Immunity acquired after staphylococcal diseases is due to phagocytosis and

the presence of antibodies (antitoxins, precipitins, agglutinins). The phagocytic and humoral
factors act together and supplement each other. Post-infectious immunity following
staphylococcal diseases is of low grade and short duration.
Treatment: diseases are treated with antibiotics, sulphonamides, and anti-
staphylococcal gamma-globulin. During Staphylococcal chronic infections anatoxin,
autovaccine are used.
Prophylaxis: The general precautionary measures include: hygiene in working and
everyday-life conditions, treatment of vitamin deficiency, prevention of traumatism and
excess perspiration, observance of rules of hygiene in maternity hospitals, surgical
departments, and children’s institutions.
Routine disinfection of hospital premises (surgical departments, maternity wards) and
bacteriological examination of the personnel for carriers is used. Examination of pathogenic
staphylococci resistant to antibiotics is important.
For prophylaxis is recommended staphylococcal anatoxin and bacteriophage. For the
formation of passive response antistaphylococcal immunoglobulin and donor's
antistaphylococcal hyperimmune sera is recommended.
Laboratory diagnosis: Diagnostic methods are (picture -1):
1.Microscopic
2.Bacteriological

9
 STREPTOCOCCI
The streptococcus was discovered by T. Billroth (1874) in tissues of patients with
erysipelas and wound infections and by Pasteur in patients with sepsis. Streptococci are
placed in the family Streptococaceae. They are part of the normal flora of humans and
animals. Some of them are human pathogens. The most important of them is Streptococcus
pyogenes causing pyogenic infections.

Streptococcus pyogenes
Morphology: The streptococci are gram-positive spherical
cells measuring 0.5 to 1.0μm in diameter and form chains (they
divide in only one plane and tendency of cells to remain united
results in the development of the characteristic chains). They are
non-motile, do not form spores. Some strains are capsulated. In
smears from cultures grown on solid media the streptococci are
usually present in pairs or in short chains, while in smears from
broth cultures they form long chains or clusters.

Cultivation: The majority of streptococci are aerobes and facultative anaerobes. The
optimal temperature for growth is 37º C.
The organisms show poor growth on ordinary meat-peptone agar, and grow well on
selective medias - sugar, blood, serum, ascitic agars. On solid media they produce small,
translucent colonies. In sugar broth medium growth is in the form of fine-granular
precipitates on the walls and at the bottom of the tube.
Some streptococcal strains cause haemolysis on blood agar. Based upon their
haemolytic properties, streptococci can be classified into three groups:
1.ß-haemolytic streptococci: these organisms produce a wide (2-4mm in diameter)
clear zone of complete haemolysis in which no red blood cell is visible on microscopic
examination.
2.α-haemolytic streptococci: The colonies are surrounded by a narrow zone (1-2 mm)
of haemolysis; they produce a green zone round the colony, as a result of conversion of
haemoglobin into methaemoglobin. The streptococci producing α-haemolysis are also known
as streptococci viridans.
3.γ-haemolytic streptococci: These organisms do not produce any haemolysis on blood
agar. Enterococcus faecalis is an important organism of this group.

10
 Fermentative properties: Streptococci are non-proteolytic, do not liquefy gelatine, and
do not reduce nitrates to nitrites. They coagulate milk; dissolve fibrin, ferment glucose,
maltose, lactose, saccharose with acid formation. S. pyogenes is soluble in bile (40%) and
are not soluble in 10per cent bile, unlike pneumococci. Streptococci are catalase negative
(table 1).
Antigenic structure: By the group-specific polysaccharide (C substance) the
streptococci are classified into 20 serological groups which are designated by capital letters:
A, B, C, D….(Lancefield classification). S. pyogenes is in A group.
Depending on the type-specific M, T and R protein antigens the serological group A is
subdivided into serotypes (about 100 serotypes). The M protein is the most important of
these. It acts as a virulence factor by inhibiting phagocytosis (it binds IgG molecules, non-
specifically, through Fc region leaving specific Fab sites free to combine with specific
antigen). The T and R proteins have no relation to virulence.
Various structural components of S. pyogenes exhibit antigenic cross reaction with
different tissues of the human body. Antigenic relationships have been demonstrated
between capsular hyaluronic acid and human synovial fluid, cell wall protein and
myocardium, group A carbohydrate and cardiac valves, cytoplasmic membrane antigens and
vascular intima and peptidoglycan and skin.
Pathogenicity: The pathogenetic factors are: adherence, colonization, invasion,
secretion of toxins and enzymes.
Toxin production: Streptococci produce exotoxins with various activities:
Haemolysins:
a)Streptolysin-S is nucleoprotein with haemolytic and cytotoxic activities, non-antigenic,
oxygen stable and sensitive to heat and acid. It is responsible for hemolysis around the
surface colonies. In addition to causing ß-hemolysis, it is able to inhibit chemotaxis and
phagocytosis. It can cause destruction of cell’s lysosomal membranes. Therefore, this
haemolysin appears to be an important factor of group A streptococcal infections.
b)Streptolysin-O (oxygen labile) is protein and is strongly antigenic. It is heat labile. SLO
induces brisk response, usually within 10-14 days. Anti streptolysin O(ASO) appears in sera
following streptococcal infection. Estimation of this antibody (the titre of ASO antibodies can
be important in the diagnosis of rheumatic fever) is a standard serological procedure for the
retrospective diagnosis of infection with S. pyogenes.
It can cause lysis of erythrocytes, destruction of cell’s lysosomal membranes leading to
necrosis of tissues. Streptolysin-O is toxic for leucocytes, produces haemolytic,
cytotoxic, leukotoxic, cardiotoxic activities.
Leukocidin, which destructive to leucocytes (inhibition of phagocytosis); occurs in
highly virulent strains.
Erythrogenic (Dick, scarlatinal) toxin: It is a protein, antigenic, relatively heat stable.
This is superantigen. This toxin is responsible for characteristic skin rash with pharyngitis and
tonsillitis in scarlet fever. It is produced only by certain strains of S. pyogenes lysogenized by
a bacteriophage carrying the gene for the toxin. This toxin is neutralized by antibodies found
in the convalescent sera. This property has been used for developing susceptibility and
diagnostic tests for scarlet fever (Dick test gives positive result in persons lacking antitoxin,
i.e., susceptible person). This test is known to be only of historical importance as scarlet fever
is no longer a common or serious disease.
Cytotoxins: Peptides, which destroy kidney cells and cause glomerulonephritis.
Cardiohepatic toxin, which causes myocardia’s affection and forms granules in liver.
Enzymes:
Fibrinolysin (streptokinase) activates plasminogen to form plasmin, which dissolves
fibrin clots. It can be used to lyse thrombi in the coronary arteries of heart attack patients.
Fibrinolysin plays a biological role in streptococcal infections by breaking down the fibrin
barrier around the lesions and facilitating the spread of infection.
Desoxyribonucleases – D Nase (streptodornase) depolymerizes DNA in exudates or
necrotic tissue. Pyogenic exudates contain large amounts of DNA, derived from the nuclei of

11
necrotic cells. Sterptodornase helps to liquefy the thick pus and may be responsible for the
thin serous character of streptococcal exudates.
Hyaluronidase degrades hyaluronic acid, which is the ground substance of
subcutaneous tissue. Hyaluronidase is known as a spreading factor because it facilitates the
rapid spread of S. pyogenes in skin infections.
Pathogenesis: Streptococci cause a wide variety of infections. Streptococci are mainly
spread by the air droplet route. S. pyogenes causes three types of diseases: 1. Pyogenic
diseases such as local pyogenic inflammation, abscesses, phlegmona, lymphadenitis,
pyelitis, otitis, sinusitis, meningitis, peritonitis, and pharyngitis, tonsillitis (inflammation of the
pharyngeal and tonsillar mucosa). S. pyogenes is the most common bacterial cause of sore
throat. Invading the blood, streptococci produce a serious septic condition. 2. Toxigenic
diseases such as scarlet fever and toxic shock syndrome. 3. Immunologic diseases such
as rheumatic fever and acute glomerulonephritis (certain strains of group A streptococci
contain cell membrane antigens that cross-react with human heart tissue antigens-
autoimmune processes develop).
Epidemiology and resistance: Many streptococci are members of the normal flora of
the human body. The source of infection is carriers and sick persons. The main ways of
spreading are droplet; and endogenous which occur during immunodeficiency.
S. pyogenes is a delicate organism. It can be killed by heating at 54º C for 30minutes. It
is also killed by usual strengths of disinfectants, but is more resistant to crystal violet than
many other bacteria. Streptococci live for a long time at low temperature, are resistant to
desiccation, and survive for many months in pus and sputum. When exposed to a
temperature of 70ºC, they are destroyed within one hour. A 3-5 per cent phenol solution kills
the organisms within 15 minutes.
Immunity: Resistance against streptococcal diseases is type-specific. Thus, a host who
has recovered from infection by one group A streptococcal is relatively insusceptible to
reinfection by the same type but fully susceptible to infection by another type.
Immunity against erythrogenic toxin is due to antitoxin in blood. Immunity after scarlet
fever is stable, life-long. Antibody to streptolysin O (antistreptolysin O) develops following
infection; it blocks hemolysis by streptolysin O but does not indicate immunity.
Immunity after streptococcal infections is of a low grade and short duration.
Treatment: Antibiotics.
Prophylaxis: There is no specific prophylaxis (vaccines). Streptococcal infections are
prevented by the practice of general hygienic measures in everyday life.

12
 STREPTOCOCCUS PNEUMONIAE

Morphology: Streptococcus pneumoniae is gram-positive,

about 1μm in diameter, lancet (flame) shape, and arranged in
pairs (diplococci), capsule forming, non-motile and non-sporing
organisms. It is inhabitant of upper respiratory tract of human and
some animals, causes infection primarily of the respiratory tract,
conjunctivitis, otitis, meningitis. They differ from streptococci
chiefly in their morphology, optochin sensitivity and possession of
a specific polysaccharide capsule.

Cultural characteristics: Pneumococcus is aerobe and

facultative anaerobe. Optimum temperature is 37ºC, pH 7.8 and grows
only in enriched media (blood, serum, ascitic agar). Pneumococci form
a small round colony, primarily dome-shaped and later developing a
central plateau with an elevated rim. In broth they produce diffuse
turbidity. Pneumococci are ά-hemolytic on blood agar. For growth
they need 5-10℅ CO2 for primary isolation.
Biochemical reactions: Pneumococci ferment glucose, saccharose, lactose and inulin
with the production of acid but no gas. Fermentation of inulin by pneumococci is a useful
test for differentiating them from streptococci which do not ferment it. Pneumococci are bile
soluble (40%). Bile solubility is a constant property of pneumococci and hence is of
diagnostic importance. They are highly sensitive to optochin (ethyl hydrocuprein
hydrochloride). Optochin sensitivity test is used for identification of pneumococci and
distinguishing them from viridans streptococci, both of which produce α –haemolysis on blood
agar. Pneumococci are catalase and oxidase negative.
Antigenic structure: The most important antigen of the pneumococci is the capsular
polysaccharide which is type specific. By the capsular antigen pneumococci are
subdivided into 85 serotypes. The antigenic structure depends on M protein. The somatic
portion of the pneumococcus contains an M protein that is characteristic for each type and a
group-specific carbohydrate that is common to all pneumococci. The carbohydrate can be
precipitated by C-reactive protein, a substance found in the serum of certain patients.
Production of disease: Pneumococci produce disease through their ability to multiply
in the tissues. The virulence of the organism is a function of its capsule, which prevents or
delays ingestion of encapsulated cells by phagocytes. The virulence is connected with C-
polysaccharide, M-protein, which is antiphagocytic, hemolysins (S-streptolysin), leukocidin,
and enzymes: peptidase, which destroys SIgA, hyaluronidase, which is a spreading factor
(degrades hyaluronic acid).
Immunity: Immunity to infection with pneumococci is type-specific and depends on both
antibodies to capsular polysaccharide and intact phagocytic function.
Treatment: Streptococci are sensitive to many antimicrobial drugs.
Some species are interesting for medical microbiology:
Group A streptococci are among the most important human pathogens. The great
majority of haemolytic streptococci that produce human infections belong to group A.
Haemolytic streptococcus group A is known as S. pyogenes.
13
 Group B (S. agalactica) streptococci colonize the genital tract of some women and
cause neonatal meningitis and sepsis.
Group C streptococci colonize the respiratory and urinary tracts.
Group D streptococci (Enterococcus faecalis), which are pathogenic for human and
animals occur as a part of the normal flora in the gut, are noted for their ability to cause
urinary, biliary, and cardiovascular infections.
Group H and K is noted during endocarditis.
Oral streptococci (S. mutans, S. salivarius) occur in dental caries.
Diagnostic laboratory tests for Streptococcal infections (picture 2):
1.Microscopic: Test material is obtained from the pus of wounds, inflammatory
exudate, tonsillar swabs, blood, urine, and foodstuffs. Tests include microscopy of pus
smears.
2.Bacteriological: Isolation of the pure culture and its identification (Table1).

14
 3.Biological: White mice are sensitive to pneumococcus.

GRAM–NEGATIVE COCCI
The genus Neisseria consists of gram negative, aerobic, non-sporulating, nonmotile,
oxidase-positive cocci, typically arranged in pairs (diplococcus). N. meningitidis and N.
gonorrhoeae are the primary human pathogens of the genus. Besides the two important
pathogens, N. meningitidis and N. gonorrhoeae, the family contains many other genuses:
Moraxella, Acinetobacter, Kingella, which occur as commensals (conditionally pathogenic)
and saprophytes.
N. meningitidis and N. gonorrhoeae are pathogenic for human and typically are found
associated with or inside polymorphonuclear cells. Most importantly, the two species are
differentiated by the usual clinical presentations of the diseases they cause: meningococci
typically are found in the upper respiratory tract and cause meningitis, while gonococci cause
genital infections.
GONORRHOEAE
Neisseria gonorrhoeae (Gonococcus)
Morphology: N. gonorrhoeae is Gram-negative, non-motile diplococcus, approximately
0.8 µm in diameter, non-capsulated. They are kidney (coffee-bean) shaped. Gonococci
possess pili on their surface. Pili facilitate adhesion of the cocci to mucosal surfaces and
promote virulence by inhibiting phagocytosis. Under the action of chemotherapeutic
preparations they can transfer into L-forms.
Cultural characteristics: Gonococci grow best on media containing complex organic
substances such as heated blood, hemin, and animal proteins and in an atmosphere con-
taining 5-10% CO2. They are aerobic. Optimal temperature for growth is 37ºC; pH-7.2-7.4.
On ascites agar they form small, round, transparent, convex colonies (S-type).
Biochemical reactions: Gonococci ferment glucose with producing acid only. They
are catalase and cytochrome oxidase positive. They lack protein lysing activity.
Antigenic structure: Gonococci are antigenically heterogenous. They are capable of
changing their surface structures and antigenic structure. The antigenicity is connected with:
1. Pili: which are hairlike structures, act as virulence factors by promoting attachment to
host cells. Pili undergo antigenic variations (16serotypes).
2. Cell membrane proteins: Outer membrane proteins show antigenic diversity, which
helps in typing gonococcal strains. These proteins act as ligands attaching the coccus to the
host cells. They also form transmembrane channels (proteins) which play a role in the
exchange of molecules across the outer membrane.
3. Lipopolysaccharides of the cell wall.
4. Superficial polysaccharide K antigen.
Toxicity in gonococcal infections is largely due to the endotoxic effect of LPS.
Virulence factors are: fimbria, capsule, LPS of the cell wall which possesses endotoxic
and antiphagocytic activities, SIgA- protease, β-lactamase enzyme, production of which
depends on R plasmids.
Resistance: The gonococcus is a very delicate organism, readily killed by heat, drying
and antiseptics.
Pathogenicity: Gonorrhoeae is a venereal disease which has been known since ancient
times. The name gonorrhoeae (meaning flow of seed) was first employed by Galen in150AD.
The disease is acquired by sexual contact. Gonococci require cylindric epithelium. They
attack mucous membrane of the genitourinary tract, eye, rectum, and throat producing acute
suppuration that may lead to tissue invasion; this is followed by chronic inflammation and
fibrosis. The incubation period is 2-8days. In males the disease starts as an acute urethritis
with yellow, creamery pus containing gonococci in a large number, and painful urination. The
process may extend to the epididymis. In females, the primary infection (acute
gonorrhoeae) is in the endocervix and extends to the urethra and vagina, giving rise to
mucopurulent discharge. It may then progress to the uterine tubes, causing salpingitis,

15
fibrosis and obliteration of the tubes. Infertility occurs in 20℅ of women with gonococcal
salpingitis. Chronic gonococcal cervicitis or proctitis are often asymptomatic.
Blennorrhea, gonococcal ophthalmia neonatorum, an infection of eye of the newborn, is
acquired during passage through an infected birth canal. Initial conjunctivitis rapidly
progresses and, if untreated, results in blindness. For prevention of gonococcal ophthalmia
neonatorum, instillation of tetracycline, erythromycin or silver nitrate into the conjunctival sac
of the newborn is used.
Immunity: Repeated gonococcal infections are common. Protective immunity to
reinfection does not appear to develop as part of the disease process, because of the
antigenic variety of gonococci.
Treatment: Antibacterial drugs (-lactamates-penicillin, cephalosporins and other
antibiotics).
Epidemiology, prevention, control: It is transmitted by sexual contact, often by
women and men with asymptomatic infections. The infection rate can be reduced by avoiding
multiple sexual partners, rapidly eradicating gonococci from infected individuals, by means of
early diagnosis and treatment, and finding cases and contacts through education and
screening of populations at high risk. Vaccination has no place in prophylaxis.
Diagnostic laboratory tests (picture 3):
1.Acute gonorrheae: Microscopic method- direct examination of gram-stained smear of pus.
This can be used to demonstrate the characteristic intracellular gram negative diplococci in
symptomatic urethritis (phenomenon of incomplete phagocytosis).
2.Chronic gonorrheae:
a)bacteriological method
b)serological method: Bordet-Gengou CFR

MENINGITIS
(Neisseria meningitidis)
Morphology: Meningococci (N. meningitidis) are gram-negative cocci that resemble
paired coffee bean, non-motile diplococcus, and 0.6-1m in diameter. They form prominent
polysaccharide capsule. They are non-sporing and non motile.
Cultural characteristics: Meningococci do not grow on ordinary media. The organisms
grow best on blood agar (chocolate agar), serum agar incubated at 37C in an atmosphere of
5-8 CO2. They are strict aerobes. On solid media, after incubation for 24hours, the colonies
are small (about 1mm in diameter), translucent, round, convex, bluish grey, with a smooth
surface(S-type). Growth is poor in liquid media, producing a granular turbidity with little or no
surface growth.
The biochemical activity of meningococci is feebly marked: they ferment glucose and
maltose with acid formation. Indole and hydrogen sulphide are not produced and nitrates are
not reduced. They are catalase and oxidase positive.
Antigenic structure: N. meningitidis possesses a polysaccharide capsule and on the
basis of this it has been subdivided into 13 serogroups. The most important serogroups
associated with disease in humans are A, B, C, D, X, Y, Z, 29E, W-135, H, I, K, L.
Serogroups H, I, K and L have been isolated from carriers and have not been associated with
disease. Meningococcal antigens are found in blood and cerebrospinal fluid of patients with
active disease. By the cell wall proteins they are subdivided into serotypes, which are
designated by Arabic numerals (1,2,3, ….).
Ecology: Meningococci are very delicate organisms, being highly susceptible to heat,
desiccation, alterations in pH and disinfectants.
Epidemiology: The human nasopharynx is the only reservoir of the meningococcus.
Asymptomatic nasopharyngeal carriers rarely contract the illness but serve to infect their
contacts. Transmission is essentially by airborne droplets or less often by fomites. During
interepidemic periods, the carrier rate is about 5-10 per cent. An increase in carrier rate
heralds the onset of an epidemic. During epidemics the carrier rates in closed communities
may go up to 90 per cent. Meningitis is common in children between 3 months and 5 years of
16
 age. Epidemics usually occurs in semi-closed communities living in crowded conditions, as in
jails and ships formerly, and in army camps in recent times.
Meningococci colonize the membranes of the nasopharynx and become part of the transient
flora of the upper respiratory tract without producing symptoms. The organisms attach to
epithelial cells with the aid of pili. From nasopharynx meningococci can enter the
bloodstream producing bacteremia and spread to specific sites, such as the meninges or
joints, or be disseminated throughout the body. The most important manifestations of disease
are nasopharyngitis, meningococcemia and meningitis.
Fulminant meningococcemia (Waterhouse-Friderchsen syndrome) is more severe,
with high fever, shock and hemorrhagic rash; there may be disseminated intravascular
coagulation, and adrenal insufficiency.
Cerebrospinal meningitis is the most common complication of meningococcemia. It
usually begins suddenly with fever, headache, vomiting, stiff neck and progresses to coma
within a few hours.
The important virulence factors of meningococci are:
1. A polysaccharide capsule that enables the organism to resist phagocytosis by
polymorphonuclear leukocytes.
2. Endotoxin-LPS of the cell wall, which causes fever, shock and other pathophysiologic
changes (in purified form, endotoxin can reproduce many of the clinical manifestations of
meningococcemia).
3. An immunoglobulin A protease, which by cleaving secretory Ig A helps the bacteria to
attach to the membranes of the upper respiratory tract.
4. Pili: Meningococci possess pili on their surface which allow intimate contact with host cell
and organisms release endotoxin.
5. Outer membrane proteins.
6. They produce enzymes hyaluronidase, neuraminidase, fibrinolysin, which promote
their invasion in tissues, SIgA- protease and plasmocoagulase.
Immunity: Immunity to meningococcal infection is associated with the presence of
specific, complement-dependent, bactericidal antibodies in the serum. Immunity is tense,
lifelong and relapse occurs rarely.
Prophylaxis: Monovalent and polyvalent vaccins containing the capsular
polysaccharides of groups A, C, W-135 and Y are available. Meningococcal chemical vaccine
is used.
Laboratory diagnosis:
Methods (picture 3):
1. Microscopic (RIF)
2. Bacteriological -isolation of pure culture (in ristomycin containing media (ristomycin inhibits gram
positive microbes in investigated material) and identification (table 1).
3.Serological(PHAR).
Table 1 Differences between pathogenic and non-pathogenic meningococci

Non-pathogenic Pathogenic
1.Growth on ordinary + -
nutrient media
2.Growth temperature the borders are high 22o-39 o C
3.Biochemical activity high low (only fermentation of
glucose and maltose)

4. Pigment formation +
-

17
 Sample tests
Staphylococcus
1. Which of the following enzymes are typical of Staphylococcus aureus:
1. lecithinase
2. hyaluronidase
3.coagulase
4. mucinase
a)1.2.3. b)1.2.4 c)1.3. d)3.4.
2. Which toxins are produced by Staphylococcus aureus:
1. hemolysin
2. enterotoxin
3. erythrogenic toxin
4. leukocidin
a)1.2.3. b)1.2.4. c)1.3. d)2.3..
3. Which medias are used for Staphylococci cultivation:
1. milk-salt agar
2. egg- yolk agar
3. peptone water
4. sugar broth
a)1.2.3. b)1.2.4. c)1.3. d)1.4.

Gonorrhea:
1. Name diseases caused by gonococci:
1. enterities
2. blenorea
3. rheumatic fever
4. gonorrhea
a)1.2.3. b)2.4. c)1.2. d)1.4.
2. Which of the following are typical of Gonorrhea:
1. gram (+)
2. motile
3. gram(-)
4. non-motile
a)3.4. b)1.2.4. c)1.3. d)2.3.4.
3. Which are the characteristic features of N. gonorrhea:
1.arranged in pairs
2.arranged in chains
3.resemble coffee bean
4.like grape clusters
a)1.2.3. b)1.2.4. c)1.3. d)3.4.

Meningococcus:
1. Which of the following cultural properties are typical of N. meningitis:
1. don’t require nutrient media
2. form fine, colorless colonies on serum agar surface
3. biochemical activity is weak
4. growth in anaerobic conditions
a)2.3. b)1.2.4. c)1.3. d)2.4.
2.The clinical forms of meningococcal infections are:
1. Nasopharyngitis
2. Epidemic cerebrospinal meningitis
3. Meningococcemia 1,2,3
4. Endocarditis
a)1.2.3. b)1.2.4. c)1.3. d)3.4.

18
3. Specific prophylaxis of meningococcal infections is carried out by:
a)meningococcal chemical vaccine
b)antimicrobial serum
c)antitoxic serum
d)auto-vaccine

Streptococcus:
1. Which of the following are typical of S. pyogenes:
1. growth on ordinary nutrient media
2. formation of small colonies on agar
3. formation of precipitate on the wall and at the bottom of the tube
4. formation of turbidity in liquid media
a)2.3. b)1.2.4. c)1.3. d)1.4.
2. Virulence of S. pneumonia is determined by:
1. peptidase
2. O-streptolysin
3. hyaluronidase
4. capsule
a)1.2.3. b)1.3.4. c)1.3. d)3.4.

19
20
21
22
 ENTEROBACTERIACEAE FAMILY
The Enterobacteriaceae form a large family of gram-negative rods found primarily in the colon
of humans and other animals, many as part of the normal flora.
The family Enterobacteriaceae includes 30 genera: of all of these the most pathogenic for human
organism are: Escherichia, Salmonella, Shigella, Proteus, Klebsiella, Yersinia, etc.
Morphology: they are short gram-negative rods with rounded ends and measure 1.5-5.0μm in
length, 0.3-0.8μm in breadth. They do not form spores, they are facultative anaerobes. They differ in
fermentative properties (they ferment a wide range of carbohydrates) and antigenic structure.
Antigenic structure: They possess a complex antigenic structure. There are 3 main types of
antigens:
1. The cell wall somatic O antigen is the outer polysaccharide portion of the cell wall. Somatic O
antigen is the basis for the serologic typing of many enteric rods.
2. The H antigen is the flagellar protein. Only flagellated organisms have H antigen (Escherichia,
Salmonella).
3. The capsular or polysaccharide K antigen, which contain the thermolabile L and B antigens and
thermostable A antigen.

ESCHERICHIA COLI
This genus is named after T. Escherich who was the first to describe the colon bacillus in 1885.
Morphology: E. coli are rods 0.4-0.7 in breadth and 1-3 in length. The ends are rounded. They
are motile-peritrichous. Non-sporing, do not form capsule. Capsule and fimbriae are found in some
strains.
Cultural characteristics: E. coli is a facultative anaerobe. The optimum temperature for
growth is 37 C and the optimum pH 7.2-7.5. Good growth occurs on ordinary media (meat-peptone
agar), on which colonies are large, slightly convex semitransparent, greyish (S-type colonies). They
can give S-R dissociation. The S-R variation occurs as a result of repeated subcultures and is
associated with the loss of surface antigens and usually of virulence. Some strains of E. coli can
produce polysaccharide capsule. The colonies of these strains are “mucoid”. Many strains, especially
those isolated from pathogenic conditions, are haemolytic on blood agar.
On differential diagnostic media - Endo’s medium (MacConkey’s medium) the organism produce
red colonies with metallic hue due to lactose fermentation. On Levin media they produce blue
colonies. On Ploskirev media they form red colonies.
Fermentative properties: E. coli possesses sugar lysing and protein lysing activities. Glucose,
levulose, lactose, maltose and many other sugars are fermented with the production of acid and gas.
Typical strains do not ferment saccharose. The fermentation of lactose with acid and gas formation is
differential diagnostic property for E. coli. It produces indole (indole is produced by bacteria that have
tryptophanase-enzyme. They metabolised tryptophane from meat-peptone broth into indole). Gelatine
is not liquefied, hydrogen sulphide is not formed. They haven’t urease activity (urea is not splited). E.
coli reduces nitrates to nitrites; Voges-Proskauer (VP) reaction is negative. Methyl red (MR) test is
positive.
Antigenic structure: It has three antigens that are used to identify the organism in
epidemiological investigations:
Somatic “O” antigen or cell wall antigen. This is thermostable, lipopolysaccharide antigen of
the cell wall. By O antigen they are subdivided into 173 serological groups. The normal colon strains
of E. coli belong to early O groups (1,2,3,4,5, etc.) and enteropathogenic strains belong to the latter O
groups (26,55,111, 112, etc.).
“H” or flagellar antigen –H antigen is type specific. There are 75 H antigens. This antigen is
thermolabile protein.
Capsular “K” antigen: there are 100 K antigens. (K antigen consists of three components A
thermostable; B and L thermolabile). K antigen masks O antigen (for revealing O it is necessary to boil
the culture and destroy the K antigen). K antigen is type specific too.
On the basis of O antigens, E. coli is subdivided into a number of O groups. Each O group is then
divided into subgroups on the basis of K antigen. Each of these subgroups includes strains with
different H antigens. Thus, on the basis of antigenic structure an antigenic formula is derived which
fully reflects the antigenic properties of the strain. For example, E. coli O26:K60:H12. Specific
23
serotypes are associated with certain diseases; eg, O55 and O111 cause outbreaks of neonatal
diarrhoea.
Virulence factors: Virulence factors depend on:
1. Adhesive factors (fimbriae-which are chromosomally determined) and colonisation
2. Invasive factors which depend on surface proteins by which microbes penetrate into intestinal
epithelial cells, multiply and destroy them.
3. Exotoxins: a)enterotoxins: Enterotoxins: heat labile protein toxin, it activates adenylate
cyclase in the enterocysts to form cyclic AMP. The accumulation of cAMP in the intestinal
mucosa initiates the hypersecretion of electrolytes and fluids into the lumen, resulting in
watery diarrhoea. Heat stable enterotoxin is a low molecular weight polypeptide and poorly
immunogenic toxin. It activates guanylate cyclase causing the increased production of cyclic
guanosine monophosphate (cGMP) and subsequent hypersecretion of electrolytes resulting in
diarrhoea. b)cytotoxin: is phage encoded cytotoxin identical to Shiga toxin produced by
Shigella dysentery, which can cause destroying of vessel’s endothelium and intestinal wall.
4. Hemolysins: many strains of E. coli produce hemolysin. A larger proportion of E. coli strains
recovered from extra-intestinal lesions of a man are haemolytic than are those isolated from
human faeces.
5. Endotoxin - the somatic lipopolysaccharide surface O antigen, besides exerting endotoxin
activity, also protects the bacillus from phagocytosis and the bactericidal effects of
complement. Diseases caused by E. coli subdivided into:
Endogenous: Causes diseases outside the intestinal tract. It causes urinary tract infection-cystitis,
pyelonephritis. It can cause meningitis in association with the B streptococci; can cause sepsis (by the
immunodeficiency). These infections are caused by conditionally pathogenic E. coli (the member of
normal microflora of our organism) and named coli-bacteriosis.
Exogenous: is an acute infection of the intestinal tract and is named esherichiosis (diarrhoea). E.
coli causing diarrheal diseases is subdivided into 5 groups. They produce diarrhoea with different
pathogenic mechanisms:
1.Enteropathogenic E. coli (EPEC): They cause coli-enteritis in infants and children
(serogroups O26, O55, O111) usually occurring as institutional outbreaks but they can also cause
sporadic diarrhoea in children and less often in adults. The pathogenesis of EPEC diarrhoea is not fully
understood. EPEC do not ordinarily produce enterotoxins, nor they are invasive. They are seen to be
adherent to the mucosa of the upper small intestine by superficial proteins, intimately attached to cup-
like projections of the enterocyte membrane, causing disruption of the brush border microvilli (they
produce inflammatory process and erosive surfaces). The pathogenesis is limited by endotoxin which
causes inflammatory reaction.
2.Enterotoxigenic E. coli (ETEC) causative agent of cholera-like diarrhoea infection. The first
step in pathogenesis is adherence of the bacteria to the cells of the jejunum and ileum by pili that
protrude from the bacterial surface. ETEC is not invasive but produce enterotoxin, heat-labile toxin
(functional blockater), which acts by stimulating adenylate cyclase, the resultant increase in
intracellular cyclic AMP (cAMP) concentration stimulates cAMP-dependent protein kinase, causing
an outpouring of fluid, potassium, and chloride from the enterocytes. Watery diarrhoea occurs
(resembles a mild form of cholera). Its severity varies from mild watery diarrhoea to fatal diseases
indistinguishable from cholera. Persons from developed countries visiting endemic areas often suffer
from ETEC diarrhoea, a condition known as “travellers diarrhoea”. Though plasmids with enterotoxin
genes may be present in any strain of E. coli, in practice only a small number of serotypes become
enterotoxigenic (e.g. O6, O8, O15, O25, O27, O167).
3. Enteroinvasive E. coli (EIEC): Causative agent of dysentery-like disease. These resemble
shigella in many respects. Many of these strains are non motile, do not ferment lactose or ferment it
late with acid, but without producing gas. Many of these show O antigen cross reaction with shigella.
Clinically EIEC infection resembles shigellosis, ranging from mild diarrhoea to frank dysentery:
bloody diarrhoea accompanied with inflammation. EIEC usually belong to serogroups O25, O114,
O124, O144, O152, O154. They penetrate into enterocytes and produce heat-stable toxin (Shigella-like
toxin) which inhibits protein synthesis by removing adenine from rRNA of human ribosomes.
4.Enterohemorhagic E. coli (EHEC): EHEC strains have become well known as the cause of
several outbreaks of the disease associated with ingestion on undercooked hamburger or raw milk.
24
Cattle are suspected as the reservoir. These organisms produce verotoxin or shigella-like toxin, which
is cytotoxin. This toxin is responsible for hemorrhagic colitis (an inflammation of the colon with
bleeding). It penetrates into the blood and can defeat kidney. Many patients, especially children,
infected by this organism produce stools combined with copious amounts of blood, but without fever.
Sometimes it can have fatal result. The typical EHEC is serotype O157:H7
5. Enteroaggregative E. coli (EAEC): These strains are so named because they appear
aggregated in a “stacked brick” formation on Hep-2cells or glass. They have been associated with
persistent diarrhoea, especially in developing countries. They produce a low molecular weight heat
stable enterotoxin called EAST1(enteroaggregative heat stable enterotoxin-1). In animal experiments
they cause shortening of villi, hemorrhagic necrosis and mild oedema with mononuclear infiltration of
the submucosa. Most of them are O-untypable, but many are H-typable.
Resistance: E. coli is more resistant to physical and chemical factors of the external environment
than the other members of Enterobacteriaceae family. At 55 C the organism perishes in 1 hour, and at
60 C in 15 minutes. E. coli is sensitive to brilliant green and other disinfectants. E. coli is a common
inhabitant of the large intestine of humans and mammals. The bacteria are excreted in great numbers
with the faeces and are always present in the external environment (soil, water, foodstuffs, and other
objects). Detection of E. coli in drink water is used as the indicator of faecal contamination (coli titer,
coli index) Coli-titer is the minimal quantity of the water which contains one E. coli. It equals 300.
Coli-index is the quantity of E. coli in one litre water. It equals 3.
As a member of normal flora E. coli (with bifidum bacteria, lactobacilli) participates in
nutritional function by producing several vitamins B, K, D.
E. coli produce colicins (antibiotic like substances) which suppress exogenous and endogenous
toxic products, and suppress the pathogenic flora (fungi, strepto-staphylococci, other intestinal
pathogens). Antibiotic therapy inhibits the predominant normal flora and some diseases can occur
(e.g., mycoses can occur and during antibiotic therapy we use anti fungal drugs). E. coli takes parts in
stimulation of formation of immune system (due to presence of muramil peptide in their cell wall).
Immunity: During endogenous infection there is humoral immune response, but in this case
immunoglobulins haven’t protective property. Immunodeficiency assists depressing phagocytosis.
In the time of exogenous infection humoral immune response takes place too. IgG cross the
placenta and from the blood it penetrates into the intestine. Here participate secretory IgA too.
Treatment and prophylaxis: Antibiotics-penicillin, cephalosporins. Eubiotics: bifidobacterin;
lactobacterin.
There is no specific prophylaxis. These infections are prevented by the practice of general
hygienic measures in everyday life.

25
The role and functions of normal microflora are numerous:
1. The normal microflora is non- specific defense factor of the organism.
2. The normal microflora has antagonistic role against pathogenic and putrefactive
(saprogenous) microflora due to production of lactic acid, acetic acid, antibiotics,
bacteriocines; at powerful biological potential’s expense can compete with foreign
microflora.
3. The normal microflora participates in water - salt metabolism, regulation of gas composition
of intestine, in metabolism of proteins, carbohydrates, fatty acids, cholesterin, nucleic acids,
and also in production of biologically active compounds: antibiotics, vitamins (k, B group
and others), toxins, etc.
4. The normal microflora participates in digestion and detoxication of exogenous substrates and
metabolites, which is close to liver’s function.
5. The normal microflora participates in regulation of steroid hormones and bile salts as a result
of excretion of metabolites from liver into intestine and following restitution in it.
6. The normal microflora plays morphokynetic role in development of different organs and
systems of the organism, participates in physiological inflammation of mucous membrane
and in replacing the epithelium.
7. The normal microflora plays anti-mutagenous function due to the destruction of cancerogenic
substances in the intestine. At the same time some bacteria can produce powerful mutagens.
So, enzymes of intestinal bacteria transform artificial cyclomate into active cancerogene
(cyclohexamine) for urinary bladder.
8. Exopolysaccharides (glycocalyx) of microorganisms, which is the part of biological
membrane, prevent microbial cells from different physical-chemical influence. Intestinal
mucous membrane is also under the protection of biological membrane.
9. The normal microflora possesses a significant influence on formation and support of immune
system. There are approximately 1,5kg of microorganisms in intestine, which antigens
stimulate immune system. Natural non-specific stimulator of immunogeneses is
muramyldipeptide, which is formed from bacterial peptidoglycane under the influence of
intestinal lysozyme and other lytic enzymes. It results in abundant saturation of intestinal
tissue with lymphocytes and macrophages, so, in norm the intestine is in chronic
inflammation -`like conditions
10. The important function of normal microflora is participation in colonizative resistance.
Laboratory diagnosis:
1. Bacteriological method-isolation of pure culture and identification (picture1)
2. Serological method: slide agglutination, tube agglutination.

SALMONELLA
Genus Salmonella has been named after American microbiologist, D.E Salmon.
The genus Salmonella consists of bacilli that parasitise the intestine of large number of vertebrate
species and which infect man, they cause enterocolitis, enteric fevers such as typhoid fever,
septicaemia and the carrier state.
The morphology of salmonella corresponds with general characteristics of the
Enterobacteriaceae family: gram-negative, middle-sized rods (2-5μm in size), they are motile
(peritrichous), do not form capsule and spore.
Antigenic structure: Salmonella possess three main types of antigens on the basis of which they
are serologically typed. These are: cell wall O, flagellar H, capsular Vi (virulence) which are
important for taxonomic and epidemiologic purpose. F. Kauffman and P. White classified the
Salmonella into a number of groups according to antigenic structure (table 1). By the somatic O
antigen, which is the outer polysaccharide of the cell wall Salmonella are subdivided into 65
serological groups (which are designated by capital letters A, B, C, D, ….) based on the presence of
distinctive O antigen, in which O antigen factors were designated by Arabic numerals (1, 2, 3, etc.).
H antigen: This antigen present on the flagella is a heat labile protein. It is composed of two
phases: phase I is specific, and the phase II is non-specific. Within each group this bacteria is

26
identified by the specific phase of H antigen (S. paratyphi A constitutes group A, paratyphi B belongs
to group B (table).
Vi antigen -surface antigen envelops the O antigen. It is heat –labile acidic polysaccharide. It is
destroyed by heating the bacteria at 100ºC for one hour. When fully expressed, it renders the bacterium
inagglutinable by O antiserum but agglutinable by Vi antiserum

SALMONELLA TYPHI
S. PARATYPHI A and S. PARATYPHY B
The morphology of the typhoid salmonella corresponds with the general characteristic of the
Enterobacteriaceae family.
Cultivation: Salmonella are facultative anaerobes. The optimum temperature for growth is 37º
C. They grow on ordinary media at pH 6.8-7.2. On meat-peptone agar S. typhi forms semitransparent,
large colonies (2-4 mm in diameter). Colonies of S. schottmulleri (S. paratyphi B) have a rougher
appearance and mucus swelling round the colonies. This is a characteristic differential cultural
property. On Ploskirev’s and Endo’s media S. typhi and S. paratyphi form colourless colonies due to
the absence of lactose fermentation. On Wilson and Blair’s brilliant-green bismuth sulphite medium
black colonies with metallic sheen are formed due to production of H2S. Salmonella paratyphi A and
other species that do not form H2S produce green colonies. Selenite broth and bile broth are employed
as enrichment medias. In Rappaport media they grow and produce turbidity.
Fermentative properties: S. typhi does not liquefy gelatine; they ferment proteins and liberate
hydrogen sulphide (H2S); does not produce indole and reduces nitrates to nitrites. They ferment
glucose, mannite, and maltose with acid formation. S. paratyphi ferments carbohydrates with acid and
gas formation. Lactose, saccharose are not fermented. They are MR positive, VP negative, urea is not

27
hydrolysed. S. paratyphi A and paratyphi B differentiated by the hydrogen sulphide production. S.
paratyphi B formed H2S, paratyphi A not (table 2).

By antigenic structure S. typhi in the D group, S. paratyphi A in the A group, S. paratyphi B in

the B group and S. paratyphi B can cause infection in animal organism too (zooanthroponose).
Pathogenicity: The main virulent factors are endotoxin, Vi antigen.
Epidemiology and Pathogenesis: The source of infection is a patient, or far more frequently, a
carrier. Man acquires infection by ingestion of contaminated water or food. Typhoid fever occurs in
two epidemiological types. The first is endemic or residual typhoid that occurs throughout the year
though seasonal variations may sometimes be apparent. The second is epidemic typhoid, which may
occur in endemic or non-endemic areas. Typhoid epidemics are water, milk or foodborne. On reaching
the gut, the bacilli attach themselves to microvilli of the ileal mucosa and penetrate to the lamina
propria and submucosa. They are phagocytosed there by polymorphs and macrophages. The ability to
resist intracellular killing and to multiply in these cells is a measure of their virulence.
For laboratory diagnosis of this infection it is necessary the pathogenesis of disease, which
depends on different stages:
1.Digestive stage –penetration of microorganism into stomach.
2.Mesenterial lymphadenitis stage: On reaching the gut, the bacilli attach themselves to the
epithelial cells of the intestinal villi and penetrate to the lamina propria and submucosa (invasive
stage). After it they enter into the mesenteric lymph nodes where they multiply. This is incubation
period which is usually 10-14 days. After it they enter the bloodstream and the next stage is developed
(bacteremeia) .
3.Bacteremia period(prodrome period) during which the bacilli are seeded in all the organs and
tissues (liver, gall bladder, spleen, bone marrow, lymph nodes, lungs, kidney where further
multiplication takes place). Prodromal period is usually 2-3 days during which non-specific symptoms
such as fever, malaise and loss of appetite occur (The first week of the diseases).
4.Parenchymatouse dissemination period (specific-illness period) As bile is a good culture
medium for the Salmonella, it multiplies abundantly in the gall bladder and specific-illness period
occurs during which the overt characteristic signs and symptoms of diseases occur: headache, malaise,
anorexia, a coated tongue and abdominal discomfort with either constipation or diarrhoea. Red rash
(rose spots) appears on the skin during the second and or third week. Some develop psychoses,
deafness or meningitis. Cholecystitis, arthritis, abscesses, periosteitis, nephritis, haemolytic anaemia,
venous thromboses and peripheral neuritis are other complications found
5.Allergic-secreted period when the bacteria are discharged into the intestine where involves the
Peyer’s patches and lymphoid follicles of the ileum which had been previously sensitized by the
Salmonellae in the initial stage. These become inflamed; undergo necrosis and slough off, leaving
behind the characteristic typhoid ulcers and may be followed by perforation of the intestine and

28
peritonitis and circulatory collapse. Ulceration of the bowel leads to the two major complications of
the disease-intestinal perforation and hemorrhage.
6.Convalescence (recovery period) is slow. The typhoid-paratyphoid salmonellae together with
products of their metabolism induce antibody production and promote phagocytosis. These processes
reach their peak on the fifth-sixth week of the disease and eventually lead to recovery from the disease.
In about 5-10% cases, relapse occurs during convalescence. The relapse rate is higher in patients
treated early with chloramphenicol.
Clinical recovery does not coincide with the elimination of the pathogenic bacteria from the
body. The majority of convalescents become carriers during the first weeks following recovery.
Patients who continue to shed typhoid bacilli in faeces for three weeks to three months after clinical
cure are called convalescent carriers. Those who shed the bacilli for more than three months but less
than a year are called “temporary carriers”(acute carriers), and 3-5 per cent of the cases continue to
excrete the organisms for many months and years after the attack and, for life (chronic carriers).
Inflammatory processes in the gall bladder (cholecystitis) and liver are the main causes of a carrier
state since these organs serve as favourable media for the bacteria, where the latter multiply and live
for long periods. Besides, this typhoid-paratyphoid salmonella may affect the kidneys and urinary
bladder, giving rise to pyelitis and cystitis. In such lesions the organisms are excreted in the urine.
Immunity: Post infections immunity is tense, stable, lifelong, which depends on humoral and
cellular immune response.
Prophylaxis: General measures amount to rendering harmless the sources of infection. This is
achieved by timely diagnosis, hospitalization of patients, disinfection of sources, and identification and
treatment of carriers. Of great importance is prevention of typhoid fever and paratyphoids are such
measures as disinfection of water, safeguarding water supplies from pollution, systematic and thorough
cleaning of inhabited areas, fly control, and protection of foodstuffs and water from flies. Regular
examination of personnel in food-processes factories for identification of carriers is also extremely
important.
In the presence of epidemiological indications specific prophylaxis of typhoid infections is
accomplished by vaccination. The TABte vaccine was used which contains O and Vi-antigens of
typhoid, paratyphoid A; B, and a concentrated purified and sorbed tetanus anatoxin. A new
areactogenic vaccine (chemical vaccine) consisting of the Vi-antigen of Salmonella typhi has been
produced. It is marked by high efficacy and used in immunization of adults and children under seven
years of age. Specific bacteriophage can be used too.
Treatment: Patients with typhoid fever and paratyphoids are prescribed chloraminphenicol and
the other antibiotics which act on gram-negative bacteria.
Laboratory diagnosis:
1.Bacteriological: Isolation of haemo-culture (bacteremic phase). For this examination
Rappaport media is used (MPB, 10% bile, glucose, indicator, float for indication of gase formation), in
which differentiation of Salomella typhi and paratyphi is possible. If S. typhi is present fermentation of
glucose with acid formation is occurs; for Paratyphi –acid and gas formation. A pure culture is isolated
from faeces and urine copro-culture or urino-culture (recovery period). The test material is inoculated
into bile broth; Ploskirev’s, Endo’s medias or bismuth sulphite agar (pict.2).
2.Serological method –Widal tube agglutination reaction. This is a test for the measurement
of O and H agglutinins for typhoid and paratyphoid bacilli in the patient’s sera (sufficient number of
agglutinins accumulate in blood on the second week of the disease -specific illness period).
3.Passive hemagglutination test.
4.Skin-allergic test.
5.Diagnosis of carriers: The detection of carriers is important for epidemiological and public
health purpose. The demonstration of Vi agglutinins has been claimed to indicate the carrier state.

SALMONELLA-GASTROENTERITIS
(causative agents of food toxinfection)
The genus salmonella comprises many species and types of bacteria which cause food
toxinfection (S. typhimurium; S. enteritidis; S. anatum; S.heidelberg, S. derby, S. haifa, S. infants). It
may be caused by any salmonella except S. typhi. Salmonella gastroenteritis or food poisoning as

29
distinct from typhoid fever and paratyphoids A and B is generally is anthropo-zoonotic disease, the
source of infection being animal products.
Morphology: Morphologically Salmonella organisms possess the general characteristics of the
family Enterobacteriaceae.
Fermentative properties: They do not liquefy gelatine and do not produce indole. The majority
of species produce hydrogen sulphide and ferment glucose, maltose with acid and gas formation.
Resistance: Salmonella are relatively stable to high temperatures (60-75ºC), high salt
concentrations, and to acids. They withstand 8-10 per cent solution of acetic acid for 18 hours, and
survive for 75-80 days at room temperature.
A characteristic feature of foodstuffs contaminated by salmonella is that they show no changes
which can be detected organoleptically.
Virulence factors are:
1.adhesion and colonization.
2.enterotoxin which acts by adenylate cyclase mechanism It is necessary to note that salmonella's
protein toxins have intracellular location and pass internal medium of macroorganism in the case of
microbe's structure destruction.
3.cytotoxin, which is similar to shigella exotoxin
4.different enzymes-protease, mucinase, decarboxylase, etc.
5.endotoxin
Pathogenesis: Human infection result from the ingestion of contaminated food. The most
frequent sources of salmonella food poisoning are poultry, meat, milk, and milk products. Of great
concern are eggs and egg products. Meat may be infected while the animal is alive or after its death.
Depend on virulence, infectious dose of bacteria and immune state of macroorganism, there are
following clinical forms of infection:
1.food toxinfection
2.salmonellas’ diarrhoea
3.generalized (typhoid)
Intoxication develops in a few hours following infection. Masses of microbes ingested with the
food are destroyed in the gastrointestinal tract and in the blood (bacteremia is infrequent). This results
in the production of large amount of endotoxin which, together with the endotoxin entering the body
with the ingested food, gives rise to intoxication. Salmonella produce exotoxin-enterotoxin too, which
is similar to E. coli thermolabile toxin. The mechanism of enterotoxin is disturbances of water-salt
metabolism, which cause diarrhoea.
Clinically, the disease develops after short incubation period (18-48 hours or less), with diarrhoea
(with or without blood), which can vary from mild to severe, vomiting, abdominal pain and fever. It is
self-limited, causes non-bloody diarrhoea, and does not require medical care except in the very young
and very old. By the generalization of the processes the diseases are of long duration or become
chronic.
 It may vary in severity from the passage of one or two loose stools to an acute cholera-
like disease
 It usually subsides in 3-5 days, but in some cases more prolonged enteritis develops,
with passage of mucus and pus in faeces resembling dysentery.
 In a few, typhoidal or septicemic type of fever may develop.

Immunity: Immunity acquired after salmonellosis is tense and type specific.

Treatment: Treatment of uncomplicated, non-invasive salmonellosis is symptomatic.
Antibiotics should not be used. But for the serious invasive cases antibiotic treatment is needed.
Prophylaxis: Veterinary-sanitary and other anti-epidemiological measures.
Control of salmonella food poisoning requires the prevention of food contamination. Food may
become contaminated at various levels, from natural infection in the animal or bird, to contamination
of the prepared food. Proper cooking of food destroys salmonellae.
Laboratory diagnosis: Bacteriological method: Isolation of pure culture from the faeces of
patients and food. In outbreaks of food poisoning, the causative article of food can often be identified
by taking a proper history.

30
 KLEBSIELLAE
They are in family Enterobacteriaceae, genus Klebsiellae (the discoverer was Klebs) which
contain two species: Klebsiella pneumoniae and Enterobacter. For human organism the most important
is Klebsiella pneumoniae, which includes three subtypes: K. pneumoniae, K. ozaenae, K.
rhinoscleromatis.
Morphology and physiology: The Klebsiella are gram-negative thick short bacilli 0.6-6.0μm in
length and 0.3-1.5 µm in breadth. They have rounded ends, are nonmotile. They occur mainly in pairs
but may be seen frequently as single organisms, and are normally surrounded by polysaccharide
capsule, non spore forming.
Cultivation: The Klebsiella are facultative anaerobes, which grow readily on common nutrient
media at pH 7.2-7.4 and at temperature of 35-37º C. They form turbid mucilaginous colonies on agar
and produce intense turbidity in broth. On differential diagnostic media (Endo, Ploskirev) they form
red colonies due to fermentation of lactose (table 3).
Antigenic structure: Klebsiella contain O and K antigens. There are 11 “O” antigens and 80 and
more “K” antigens.
Ecology: Klebsiella are widely distributed in nature. They are contained in normal intestinal
biocenosis. They may be found on the skin and mucous membranes. They are as commensals in the
intestine and as saprophytes in soil and water. The source of infection is patient. K. pneumonia is
excreted from human throat (pharynx), gastroenteric (gastrointestinal) tract in 5% healthy people.
Pathogenesis: The virulence factors are: adhesion (fimbria, superficial proteins), capsular
polysaccharide (antiphagocytic factor), colonization, endotoxin, enterotoxin and cytotoxin. K.
pneumonia produces enterotoxin (which is very similar to the heat-stable toxin of E. coli) which
stimulates secretion of liquids and development of diarrhoea.
Three species of bacteria play most important role in human pathology: the causative agents of
pneumonia, ozaenae and rhinoscleromatis.
K. pneumonia (Friedlander rod): is responsible for pneumonia and hospital infections
(bronchitis and bronchopneumonia), which involves one or several lung lobes, sometimes producing
fused foci and lung abscesses. The death rate is quite high. In some cases the organisms may be
responsible for meningitis, appendicitis, cystitis. They may also cause inflammation in cases of mixed
infections. As they produce enterotoxin they are causative agents of enteric infection in new-borns.
K. ozaenae: morphological characteristics are given above. They are responsible for rhinitis
which is characterized by an offensive nasal discharge. K. ozaenae affects the mucous membranes of
the nose, nasal sinuses, and conchae. This results in production of a viscid discharge which dries up
and forms thick scabs with an offensive odour. These scabs make breathing difficult. Ozaenae is
transmitted by the air-droplet route. It is possible that other factors (trophic and endocrine
disturbances, etc.) also contribute to its development.
K. rhinoscleromatis: These organisms are responsible for chronic granulomatosis of skin and
mucous membranes of the nose, pharynx, larynx, trachea, and bronchi, with the formation of
granulomas. Rhinoscleroma is mildly contagious disease. Treatment is a matter of great difficulty and
involves complex therapeutic measures which must be carried out over a long period of time.
Immunity: Diseases caused humoral and cell immune response. The formed antibodies don’t
have protective properties. Immunity is low and in short duration.
Treatment: Antibiotics; cephalosporins.
Prophylaxis: is ensured by recognition of the early stages of ozaenae and rhinoscleroma, active
antibiotic therapy, and prevention of healthy individuals from being infected by the sick. There is not
specific vaccino-prophylaxis.
Laboratory diagnosis: Includes the following methods:
1. Microscopic: examination of smears made from sputum (from patients with pneumonia) nasal
mucus discharge (from patients with pneumonia), and tissue specimens (from patients with
rhinoscleroma).
2. Bacteriological method: Isolation of the pure culture and identification by cultural, biochemical, and
serological properties (table1).

31
3. Serological reaction: Complement fixation reaction.

PROTEUS
Proteus was discovered in 1885 by G. Hauser. Genus Proteus contains the following species P.
vulgaris, P. mirabilis, P. morgani, P. rettgeri, P. inconstans. P. vulgaris and P. mirabilis cause
toxinfections and pyo-inflammatory processes in human organism frequently. The species are
differentiated by studying their fermentative properties.
Morphology: They are polymorphous (in young cultures most of them are long curved and
filamentous), gram–negative rods (1-3x0.6μm), motile (peritrichous). Non-flagellar, non-motile
variants are also encountered. The organism does not form either spores or capsules.
Cultural properties: Facultative anaerobes, and grows readily on common media. They are
characterized by creeping growth (H-forms – hauch in German means breathing. These are motile
forms). Some strains are non motile and form large, S-form colonies (O-forms – one hauch non-
breathing). Cultures of Proteus have a characteristic putrid odour described as “fishy”. Proteus
liquefies gelatine and coagulates serum. They produce indole, ammonia, hydrogen sulphide,
ornithindecarboxilase, reduce nitrates to nitrites and ferment levulose, glucose, galactose, saccharose,
maltose with acid and gas formation(table 1).
Antigenic structure: Proteus contains O, H and K antigens. “O” antigen is cell wall LPS by
which they are subdivided into 49 serogroups; by “H” antigen – into 19 serovars. Weil and Felix
observed that certain nonmotile strains of P. vulgaris , called “X” strains, were agglutinated by sera
from typhus fever patients. This can be used in agglutination reaction for revealing Rickettsias (Weil
and Felix agglutination reaction-three nonmotile Proteus strains OX2, OX19, and OXK are used in this
test)
Pathogeneses and pathogenicity: Proteus is conditionally pathogenic bacteria (common
members of the indigenous microflora of the colon; opportunistic pathogens; a fairy common cause of
cystitis). The virulence factors are the following:
 Fimbriae
 Endotoxin-LPS of the cell wall
 Protease, increases permeability of the vessels and affects immunoglobulins
 Urease
 Hemolysin
 Hemagglutinins
In gastroenteric tract they can cause toxinfections and acute gastroenteritis. P. mirabilis is the
most frequently associated bacterium with urinary tract infection (especially dangerous

32
 infection in new-borns: Proteus transferred from umbilicus which often leads to bacteraemia
and meningitis).
In association with other conditionally pathogenic microorganisms they can cause local pyo-
inflammatory processes and septic infections: cystitis, pyelitis, pleuritis, abscesses,
pneumonia, meningitis, sepsis.

Specific prophylaxis is absent; treatment by antibiotics and Proteus phage can be used also.

YERSINIA
Yersinia are short, pleomorphic, gram-negative rod that can exhibit bipolar staining. They do not
form spores and are catalase-positive, oxidase-negative, and microaerophilic or facultative anaerobes.
The natural hosts for them are animals but they can produce a serious disease in humans.
Genus Yersinia contains 7 species. The causative agents of human infections are:
Y. pestis- causative agent of plague.
Y. pseudotuberculosis-causes fatal typhoid-like illness with hepatoslenomegaly.
Y. enterocolitica- important causes of human diarrheal diseases.

YERSINIA ENTEROCOLITICA
These are non-lactose-fermenting, gram-negative ovoid coccobacillus showing polymorphism in
older cultures, motile (peritrichous) rods. In 37ºC they loose motility, they are active motile in 18-
22ºC. 1-3x0.5-0,8μm, non-capsulated (some virulent strains can form capsule), non-spore forming
organisms. They are urease-positive and oxidase-negative.
Cultivation: These organisms grow readily on common nutrient media at temperature 28-30 C.
In liquid media they produce diffuse turbidity, on solid media they form small (0.1-0.2mm), shinny,
light blue, S-type colonies.
They are found in the intestinal tract of a variety of animals, in which they may cause a disease,
and are transmissible to humans, in whom they can produce a variety of clinical syndromes.
Antigenic structure: Y. enterocolitica possesses “H”-flagellar and somatic “O” antigens.
According to somatic “O” antigen they are subdivided into 34 serovariants. Causative agents of
infections in human organism belong to O3, O5 - O9 serovariants.
Ecology: Y. enterocolitica has been isolated from rodents and domestic animals (eg, sheep,
cattle, swine, dogs, and cats) and waters contaminated by them. Transmission to humans probably
occurs by contamination of food, drink, or fomites.
Person to person transmission with either of these organisms is probably rare.
Pathogenicity and pathogenesis: Y. enterocolitica is facultative intracellular parasite.
Pathogenicity depends on adheisins (fimbria, microcapsule), cytotoxins and invasive properties
(invasion depends on superficial proteins, which can inhibit activities of bactericidal factors-
phagocytosis, complement), enzymes (phosphatase, proteinkinase), endotoxin, thermostable
enterotoxin (guanilate cyclase mechanism of action).
33
 An inoculum of 108-109 Yersinia must enter the alimentary tract to produce infection. During the
incubation period of 5-10 days, Yersinia multiplies in the gut mucosa (enterocytes, Peyer’s patches),
particularly in the ileum. This leads to inflammation and ulceration, and leukocytes appear in faeces.
The process may extend to mesenteric lymph nodes and rarely, to bacteremia.
Early symptoms include fever, abdominal pain, and diarrhoea. Diarrhoea may be due to an
enterotoxin or to the invasion of the mucosa, and it ranges from watery to bloody. At times, the
abdominal pain is severe and located in the right lower quadrant, suggesting appendicitis. One to 2
weeks after the onset some patients develop arthralgia, arthritis. Very rarely, Yersinia infection pro-
duces pneumonia, meningitis, or sepsis. The clinical forms are gastroenteritis, lymphadenopathy, and
acute appendicitis.
Treatment: Y. enterocolitica are generally susceptible to aminoglycosides, chloramphenicol,
tetracycline, third-generation cephalosporins.
Prevention and control: Contact with farm and domestic animals, their faeces, or materials
contaminated by them probably accounts for most human infections. Meat and dairy products have
occasionally been indicated as sources of infection, and group outbreaks have been traced to
contaminated food or drink. Conventional sanitary precautions are probably helpful. There are no
specific preventive measures.
Laboratory diagnosis:
Bacteriological method: Isolation of pure culture and identification by biochemical activity (table
1).
Serological method: agglutination reaction.

SHIGELLAE
Shigellas are the causative agents of bacterial dysentery. In 1898 this organism was studied in
detail by K. Shiga in Japan and according to the current International Nomenclature, all dysentery
bacilli are grouped together in one genus known as Shigella.
Morphology: Morphologically dysentery bacilli correspond to the organism of the family
Enterobacteriaceae. They are slender gram-negative rods, non-sporing, non-capsulated, nonmotile, the
difference from other members of Enterobacteriaceae is dysentery bacteria have no flagella. They
possess microvilli, fimbriae, pili like other members of enterobacteriaceae.
Cultural characteristics: Shigellas are facultative anaerobes and grow readily on common
media (but less readily than other enterobacteria) at pH 6.7-7.2, optimal temperature for growth - 37º
C. On solid media they form small (1-1.5 mm), fragile, semitransparent colonies. In meat broth
dysentery bacilli produce a diffuse turbidity. Colonies on Endo (MacConkey agar), Levin Ploskirev
medias are colorless due to the absence of lactose fermentation. An exception is S. sonnei which
ferments lactose late (after 48-72 hours) with acid formation (without gas) and forms pale pink
colonies.
Antigenic structure: The antigenic structure of Shigella is associated with somatic O-antigen
and surface K-antigens. By somatic O antigen Shigella are subdivided into four groups (A, B, C, D)
and 40serotyps (table 1).
1. Group A - S. dysenteriae which subdivided into 1-10 serovars;
34
 2. Group B - S. flexneri ( 1-6 serovars);
3. Group C – S. boydii (1-15 serovars)
4. Group D – S. sonnei (-).

Dysentery bacilli are differentiated on the basis of the whole complex of antigenic structure and
biochemical properties.
Dysentery bacilli ferment glucose with acid formation, don’t ferment lactose except S. sonnei,
which ferment lactose during 48-72 hours (in this case they differ from E. coli-fermentation of lactose
during 18-24 hours). None of species of dysentery bacilli liquefy gelatine nor produce hydrogen
sulphide. Fermentation of mannite is of importance in classification and Shigella has traditionally been
divided into mannite positive, mannite negative. S. dysenteria is mannite negative, does not ferment
mannite, others are mannite positive, they ferment mannite. Shigella are MR positive and reduce
nitrates to nitrites (table 2).

Ecology and spreading:. They are killed at 56ºC in one hour and 1% phenol in 30 minutes. In
water and ice they remain viable for 1-6 months, boiling or chlorination of water and pasteurisation of
milk destroy the bacilli. In faeces they die within a few hours due to the acidity produced by the
growth of coliforms. S. sonnei is, in general, more resistant than other species.
Dysentery is anthroponose infection; the only source of infection is man-cases or carriers. The
modes of transmission are:
35
 1. direct, through contaminated fingers and hand (dirty hands infection) 2.through fomites such
as door handles, water taps, lavatory seats. 3.through water 4.through contaminated food and drink.
5.through flies which may transmit the infection as mechanical vectors.
Virulent factors are the following:
 adhesion, colonization which depends on fimbriae, superficial proteins of external
membrane, mucinase
 invasion which depends on superficial proteins and enzymes(neuraminidase,
hyaluronidase)
 endotoxin: all shigella release an endotoxin after autolysis. It is thermostable
lipopolysaccharide of the cell wall. It has irritating action on the intestinal wall which
causes diarrhea and subsequent ulcers.
 Exotoxins:
 (Shiga toxins): Shigella dysentery in addition to endotoxin, produces a powerful
exotoxin. It is heat-labile protein and acts as enterotoxin and neurotoxin. As
enterotoxin, it acts on the intestinal mucosa causing transudation of fluid in the
lumen and as neurotoxin, it damages endothelial cells of small blood vessels of
the central nervous system which results in neurological complications like
polyneuritis, coma and meningism.
 Cytotoxins: its synthesis is controlled by plasmid genes. It acts enterocytes,
neurons and myocard. It means they have three types of activities-enterotoxic,
neurotoxic and cytotoxic.
Pathogenicity: Shigella cause bacillary dysentery. This is anthroponose infection, human beings are
the only natural hosts for shigella. Infection occurs by ingestion of contaminated food or water.
Shigellas are highly communicable; the infective dose is 103 organisms (whereas it usually is 105-1011
for salmonella and vibrio). The bacilli infect the epithelial cells of the villi in the large intestine and
multiply inside them. It leads to necrosis of the mucous membrane, superficial ulceration, bleeding.
Shigella produces endotoxin lipopolysaccharide of cell wall which enters into the blood and actes on
neurons and vascular system. S. dysentery produces thermolabile exotoxin which displays marked
tropism to the nervous system and intestinal mucous membrane. They produce cytotoxin which defeats
enterocysts, neurons, and myocardia cells. These testify that this toxin has three kinds of action:
enterotoxic, neurotoxic, cytotoxic.
After a short incubation period (1-2 days) there is a sudden onset of abdominal pain, and watery
diarrhoea. Diarrhoea has been attributed to an exotoxin action in the large intestine. A day or so later,
as the infection involves the ileum and colon, the number of stools increase; they are less liquid but
often contain mucus and blood. Each bowel movement is accompanied by straining and tenesmus
(rectal spasms), which resulting lower abdominal pain. However, loss of water and electrolytes may
lead to dehydration, acidosis, and even death. Upon recovery from the infection, most people develop
circulating antibodies to Shigella, but do not protect against reinfection.
Bacillary dysentery has a short incubation period(1-7days, usually 48 hours)
 The onset and clinical course are variable and are largely determined by the virulence of
the infecting strain
 The main clinical features are frequent passage of loose, scanty faeces containing blood
and mucus, along with abdominal cramps and tenesmus.
 Fever and vomiting may present.
 Complications are most often seen in infection with S. dysentery and include arthritis,
toxic neuritis, conjunctivitis, parotitis and, in children, intussusception.
 Haemolytic uremic syndrome may occur as a complication in severe cases.
 The severity of the disease may vary from acute fulminating dysentery to mild diarrhoea.
 As the term bacillary dysentery, refers only to the more severe cases, the term
“shigellosis” has been employed to include the whole spectrum of the disease caused by
shigella.
Immunity: Immunity acquired after dysentery is specific but relatively weak and of a short
duration. For these reason the disease may recur many times and in some cases, may become chronic.

36
 Treatment: Patients are given antibiotics (tetracyclines) and sulphonamides (phthalazole, etc.).
Dysentery accompanied by vitamin deficiency; for this reason, a patient with dysentery should be
given a high-caloric diet, rich in vitamins.
Prophylaxis: Dysentery control is ensured by a complex of general and specific measures:
Early and a completely effective clinical, epidemiological, and laboratory diagnosis;
 Hospitalization of patients or their isolation at home with observance of the required regimen;
 Thorough disinfection of sources of the diseases;
 Adequate treatment of patients with highly effective antibiotics and use of chemotherapy;
 Control of disease centres with employment of prophylactic measures;
 Surveillance over foci and the application of prophylactic measured there;
 Treatment with a phage of all persons who were in contact with the sick individuals;
 Observance of sanitary and hygienic regiments in children’s institutions, at home and places of
work, in food industry establishments, at catering establishments, in food stores.
Laboratory diagnosis:
Bacteriological method(picture3) . Diagnosis is made by isolating the bacillus from faeces and
identification by biochemical activity Reliable results of laboratory examination depend, to a large
extent, on correct sampling of stool specimens and its immediate inoculation onto a selective
differential medium. The procedure should be carried out at the patient’s bedside, and the plate sent to
the laboratory (because E. coli is the antagonist of Shigella and attack and kill them).
Serological method: Antigenic identification by slide agglutination with polyvalent and
monovalent sera.

CAMPYLOBACTER
Campylobacter were known mainly as pathogens for various animals, in which they caused
sepsis, abortion, or enteritis. Some of them cause diarrhoea in humans. The medically important
Campylobacters are: C. jejuni, C fetus and Helicobacters.
Morphology: The genus Campylobacter (Greek, meaning curved
rod) contains slender spirally curved Gram-negative rods, 0.2-0.3x0.5-
5μm in size. They are typically comma shaped but may occur as “S” or
multispiral chains.They are non-spore-forming, motile (amphitrichous or
lophotrichous).
Cultural characteristics: Growth occurs under microaerophilic
condition, 5% oxygen concentration being optimal. They are
capnophilic, (they need 10-15% CO2). C. jejuni is thermophilic, growing well at 42C. They require
nutrient media, cannot grow on ordinary medias. Selective medias are Butzler’s, Skirrow’s, Preston’s
medias (H2, formiate, fumorate containing medias). Colonies appear usually by 42hours. They are
nonhemolytic, grey or colourless, moist, and flat or convex.
Biochemical activities: Catalase, oxidase positive; urease negative, nitrate reduction test is
positive.
Campylobacters are important veterinary pathogens. Campylobacters of medical importance are
the following:
Causing diarrheal disease: C. jejuni, C. coli, C. lari
Causing extraintestinal infection: C. fetus
Causing abscesses: C. sputorum, C. conciscus.
Epidemiology and pathogenesis: Domestic animals such as cattle, chickens, and dogs are the
source of these organisms. Transmission is usually fecal-oral. Human-to-human transmission occurs
but is less frequent than animal-to-human transmission. Food and water contaminated with animal
feces is the major source of human infection. The organisms multiply in the small intestine, invade the
epithelium, and produce inflammation that results in the appearance of red and white blood cells in the
stools. Occasionally, the bloodstream is invaded and a clinical picture of enteric fever develops. The
clinical symptoms are: headache, malaise, fever, crampy abdominal pain, profuse diarrhoea that may
be grossly bloody. Pathogenesis of infection depens on adhesion, which occurs by superficial
microvilli; invasion by mucinase enzyme and toxins. They produce LPS endotoxin and thermolabile
enterotoxin. Some strains can produce cytotoxin too.

37
 Treatment: Erythromycin or ciprofloxacin is used and aminoglycosides are used.
Prevention: there is no vaccine. The measure is personal hygiene measures.
Laboratory diagnosis: If the patient has diarrhoea, a stool specimen is cultured on a blood agar
plate containing antibiotics that inhibit most other fecal flora. The plate is incubated at 42C in a
microaerophilic atmosphere containing 5 oxygen and 10 carbon dioxide.

HELICOBACTER
Helicobacter pylori causes gastritis and peptic ulcers.
Morphology: H. pylori is a gram-negative spiral bacillus with many
characteristics in common with campylobacter species. H. pylori grows in
3-6 days when incubated at 37ºC in a microaerophilic environment. H.
pylori attaches to the mucus-secreting cells of the gastric mucosa. The
natural habitat of H. pylori is the human stomach, and it is probably
acquired by ingestion. However, it has not been isolated from stool, food,
water, or animals. Person-to person transmission probably occurs, because
there is clustering of infection within families.
Clinical findings: Gastritis and peptic ulcer are characterized by recurrent pain in the upper
abdomen, frequently accompanied by bleeding into the gastrointestinal tract. No bacteremia or
disseminated disease occurs.
Treatment and prevention: Antibiotics such as amoxicillin, metronidazole are used. There is no
vaccine or other specific preventive measure.
Laboratory diagnosis: Gram staining of the smears of biopsy specimens of the gastric mucosa.
The presence of IgG antibodies can also be used as an evidence of the infection.

38
 Sample tests
1.The virulence factors of E. coli are:
1.adheision
2.endotoxin
3.enterotoxin
4.protein A
a)1.2.3. b) 1.2.4. c)1.3. d)1.4..

2.The role of conditionally pathogenic E. coli in microorganism:

1.is an antagonist against decaying bacteria
2.produces colicine
3.is a synergist of candida genus
4.inhances digestion
a)1.2.3. b) 1.2.4. c)1.3. d)1.2.4.

3.Typical properties of E. coli are:

1.Gram (+)
2.Gram (-)
3.forms spore in environment
4.is motile
a)1.2.3. b) 2.4. c)1.3. 4. d)1.4.

4.Which of the following are virulence factors of Shigella:

1.invasion
2.musinase production
3.toxins
4.cord factor
a)1.2.3. b) 1.4. c)1.3. d)1.2.4.

5.Cytotoxin, produced by Shigella dysentery has the following actions:

a)inhibits transmission of impulses
b)affects leukocytes
c)neurotoxic
d)hemolytic

6.Which of the following are diseases which caused by Klebsiella:

1.pneumonia
2.rhiniscleromatis
3.ozenae
4.reumatic fever
a)1.2.3. b) 1.2.4. c)1.3. d)1.2.

7.Mark the transmission routs of Klebsiella:

1.cutaneous
2.respiratory
3.sexual
4.alimentary
a)1.2.3. b)2.4. c)1.3. d)1.2

8.Which of the following properties are typical of Klebsiella:

1.non-capsulated
2.mucous colonies
3.urease positive
4.capsulated
a)1.2.3. b)2.3.4. c)1.3. d)1.4.
39
9.Which of the following are typical of P. vulgaris:
1.require to nutrient media
2.cause food toxinfection
3.virulence is connected with histotoxin
4.virulence is connected with lipopolysaccharide layer
a)1.2.3. b)2.4. c)1.3. d)1.2.

10.Proteus vulgaris are:

1.Gram(+) cocci, arranged in pairs
2.Gram(-), middle sized rods with irregular arrangement
3.virulence is determined by histotoxin
4. virulence is determined by lipopolysaccharide
a)1.2.4. b)2.4. c)1.3. d)1.3.4.

11.Which of the following is typical of S. typhi, Except:

a)protein degrading with H2S
b)glucose fermentation
c)cytotoxin production
d)growing on Ploskirev media

12.How would you diagnose carriers of Salmonella:

a)complement fixation reaction
b)passive hemagglutination with Vi antigen
c)bile culture
d)urine culture

40
41
42
43
 CHOLERA
(V. cholerae)

Vibrio cholerae belongs to family Vibrionaceae, genus Vibrio. Genus vibrio has different well
described species. The most important pathogenes of man are V. cholerae (the classical cholera
vibrio); V. cholerae el-Tor(the el-Tor vibrio); serotype O-139(Bengal- identified in 1992 causes
epidemics of cholera).
The causative agents of cholera (V. cholerae) was discovered by R. Koch in 1883, V. cholerae
biovar el Tor was discovered by Gotshlich in 1906.
Morphology: Cholera vibrios are short, curved (comma-shaped-hence the old name is V.
comma), polymorphic(polymorphism is frequent in old culture), gram-negative microorganisms
measuring 1.5 µm in length and 0.2-0.4 µm in breadth. In stained films of mucus flakes from acute
cholera cases, the vibrios are seen arranged in parallel rows, described by R. Koch as the “fish in
stream” appearance. They are motile, monotrichous, non-spore forming, non-capsulated.
Cultivation: Cholera vibrios are facultative anaerobes. The optimum growth temperature is
37ºC. The organisms grow readily on alkaline media at pH 8.5-9.0. On solid media the colonies are
transparent with a light-blue hue, forming domes with smooth edges. In alkaline 1% peptone water
growth occurs in about six hours as a fine surface pellicle. Vibrio cholerae grows well on TCBS
medium which contains thiosulfate-citrate-bile salts and sucrose, on which it produces yellow, convex
colonies. On Endo media (MacCon-key’s agar) the colonies are colourless at first, but become reddish
on prolonged incubation due to the late fermentation of lactose. On Mansur’s gelatine taurucholate
trypticase tellurite agar (GTTA-this medium is useful for the isolation of cholera and other vibrios
from faeces. pH and tellurite are inhibitory to most enterobacteria and gram positive bacteria) Cholera
vibrios produce small, translucent colonies with a greyish black centre and turbid halo.
Holding or transport media are: Venkatraman-Ramakrishman (VR) medium, Cary-Blair medium.
Fermentative properties: they possess proteolytic activity: coagulated serum and liquefies
gelatine (funnel shape). Indole is formed and nitrates are reduced to nitrites. These two properties
contribute to the “cholera red” reaction or “nitroso-indole” reaction, which is tested by adding a
few drops of concentrated sulphuric acid to a 24-hours peptone water culture. With cholera vibrios, a
reddish pink colour develops due to the formation of nitroso-indole. They hydrolyse casein, coagulate
milk. Cholera vibrios ferment glucose, galactose, maltose, saccharose, mannite, mannose and lactose
(late fermentation) with acid formation. Heiberg classified vibrios into eight groups based on the
fermentation of mannose, saccharose and arabinose. V. cholerae and vibrio el Tor biovars belong to
biochemical variant I. They ferment mannose and saccharose but not arabinose. The cholera
vibrio possesses lysin and ornithine decarboxylases and oxidase activity. The ability of forming
acetylmethylcarbinol from glucose is used in differentiating microbes of the genus Vibrio (VP
reaction). Catalase and oxidase positive, MR and urease negative. They ferment starch (Kodam
reaction-diastase activity) and glycogen.
Antigenic structure: The cholera vibrio has two antigens-thermostable somatic O antigen and
thermolabile flagellar H antigen. The H-antigen is common for the genus Vibrio. According to the O
antigen there are more than 100 serotypes (139 serotype). The cholera vibrios and El-Tor biovars
belong to O1 serotype. In O1 serotype there are three antigens A, B, C. According to the combination
of which three serological variants Ogawa (AB); Inaba (AC) and Hikojima (ABC) serovars are
distinguished. Therefore in the diagnostic laboratory group O-1 antiserum came to be used for
identifying pathogenic cholera vibrios (which are referred to as “agglutinable vibrios”. Other vibrio
isolates which were not agglutinated by the O-1 antiserum came to be called non-agglutinable or
NAG vibrios. They are considered non-pathogenic.
Toxin production: Vibrio cholerae produces a heat-labile enterotoxin called cholerogene (cholera
toxin) which plays an important role in the pathogenesis of cholera. Cholerogene is a functional
blockator, thermolabile enterotoxin which acts by activation of adenylate cyclase and accumulation of
cAMP, leading to outpouring into the small intestinal lumen, of large quantities of water and
electrolytes and the consequent watery diarrhoea. CT production is determined by a filamentous phage
integrated with the bacterial chromosome. It can also replicate as plasmid which can be transmitted to
non-toxigenic strains, rendering them toxigenic.
44
This exotoxin can reproduce the symptoms of cholera even in the absence of the vibrio organisms.
Cholera toxin consists of two polypeptides: A (active) and B (binding). The B component binds to
plasma membranes of epithelial cells lining the small intestine, and the A component induces the
stimulation of adenylate cyclase. The resulting overproduction of cyclic AMP stimulates secretion of
chloride ions and water, leading to massive watery diarrhoea without inflammatory cells that may be
accompanied by vomiting resulting in dehydration, shock, acidosis, and death. The mortality rate
without treatment is 40%.
Vibrio cholerae also possesses lipopolysaccharide endotoxin (like gram-negative rods), which
protect the bacteria from phagocytosis.
The cholera vibrios produce enzymes: fibrinolysin, hyaluronidase, collagenase, lecithinase,
mucinase and neuraminidase (receptor destroying enzyme), plasmocoagulase. They possess lysin and
ornithine decarboxylases, cytochromoxidase activities.
Pathogenesis: Cholera is anthroponose infection, under natural conditions V. cholerae is
pathogenic only for humans. The infective dose is high because they are alkaline bacteria and they are
killed in a few minutes in gastric juice. The source of infection is sick people and carriers. The vibrios
are transmitted by food, water, contaminated hands. If they survive the barrier of gastric acidity they
reach the epithelial cells by chemotaxis, motility mucinase and other proteolytic enzymes. V. cholerae
organisms attach to the microvilli of epithelial cells. They multiply in the alkaline contents of the
small intestine and liberate cholera toxins (CT):
Cholera is characterized by a short incubation period of several hours up to 6 days. After the
incubation period there is sudden onset of vomiting and profuse diarrhoea with abdominal cramps.
Stools, which resemble “rice water”, contain mucus, epithelial cells, and large numbers of vibrios.
There is rapid loss of fluid and electrolytes, which leads to profound dehydration, collapse and anuria.
Three phases can be distinguished in the development of the disease:
1.Cholera enteritis which lasts 1-2 days (choleric diarrhoea).
2.Cholera gastroenteritis: Profuse diarrhoea and continuous vomiting lead to dehydration of the
patients body and this results in lowering of body temperature, drastic decrease in the number of
mineral and protein substances, and the appearance of convulsions.
3.Cholera algid which is characterized by severe symptoms. The skin becomes wrinkled due to the loss
of water, cyanosis appears and the voice becomes husky and is sometimes lost completely. The
temperature falls to 34-35º C. As a result of blood concentration cardiac activity is drastically
weakened and urination is suppressed. In severe cases the algid period is followed by asphyxia which
is characterized by cyanosis, dyspnoea, uraemia, amoniaemia (uraemia), and unconsciousness (cholera
coma), which leads to prostration and death. The mortality rate is high.
Fulminate form of cholera and dry cholera (or cholera sicca) may occur in a number of cases.
In fulminate form sudden beginning of symptoms and stormy development of symptoms are
characteristic. Cholera sicca is characterized by the absence of diarrhoea and vomiting and results in
death due to severe intoxication.
Resistance: The cholerae vibrio survives for a long time at low temperatures. It lives in faeces
for up to a month, in water for several days, on foodstuffs from 1 to 10 days. They survive for 1-5 days
at room temperature and for a week in the refrigerator. The el Tor vibrio is marked by high resistance.
El Tor is formes development of carrier state.
The vibrios show a low resistance to sunlight, X-rays, desiccation, and high temperatures. It is
destroyed instantly at 100ºC, and in 5 minutes at 80ºC. Cholera vibrios are highly sensitive to
disinfectants; particularly to acids (e.g. a 1:10.000 solution of hydrochloric acid kills them within one
minute). The organism is also very sensitive to the action of gastric juice.
Infection takes place from water and foodstuff and it is possible the contact way. Cholera is one
of the ancient infections, which cause pandemia. The epidemiological centre is considered to the
Ganga and Brahmaputra rivers in India. Till the 19th century, cholera was confined to its endemic
areas. Then the disease spread far and wide in pandemics (International health organisation described 7
pandemia which were caused by Vibrio cholera-6 and el Tor-1). There are several distinctive
differences between the epidemiology of el Tor and classical cholera vibrio: Infection with el Tor
vibrio leads to a larger proportion of mild cases, higher incidence of carriers and greater chances for
endemicity as compared to classical cholera. The el Tor is able to survive longer in the environment.

45
 Immunity: Immunity acquired after cholera is high-grade, stable and is of an antibacterial and
antitoxic character. Essential role has the secretor IgA which prevents the adherence of vibrios (local
immunity).

Treatment: Treatment consists of prompt, adequate replacement of water and electrolytes, either
orally or intravenously. Antibiotics such as tetracycline are not necessary, but they do shorten the
duration of symptoms and reduce the time of excretion of the organism.
Prophylaxis: Prevention is achieved mainly by public health measures that ensure a clean water
and food supply. The vaccine, composed of killed organisms, has limited usefulness; it is only 50%
effective in preventing disease for 3-6 months and does not interrupt transmission.
Alive vaccine and cholerogene anatoxin are available in certain countries (in endemic foci).
Neither the killed nor the alive vaccine is recommended for routine use in travellers. Prompt detection
of carriers is important in limiting outbreaks. For prophylaxis tetracyclines are used.
Cholera is an acute diarrheal disease caused by V. cholera. In its most severe form, cholera is
a dramatic, pandemic and terrifying illness in which profuse painless watery diarrhoea and copious
effortless vomiting may lead to hypovolemic shock and death in less than 24 hours. In treated cases,
the disease may last 4-6 days, during which the patient may pass a total volume of liquid stool equal to
twice his body weight.
 all the clinical features of severe cholera result from this massive loss of fluid and electrolytes
 the cholera stool is typically a colourless watery fluid with flecks of mucus, said to resemble
water in which rice has been washed (hence called “rice water stool”).It has characteristic
inoffensive sweetish odour. In composition it is a bicarbonate-rich isotonic electrolyte solution,
with little protein. Its outpouring leads to diminution of extracellular fluid volume,
hemoconcentration, hypokalemia, base-deficit acidosis and shock.
 the common complications are muscular cramps, renal failure, pulmonary oedema, cardiac
arrhythmias and paralytic ileus.
 the clinical severity of cholera varies widely, from the rapidly fatal disease to a transient
asymptomatic colonization of the intestine by the vibrios.
 the incidence of mild and asymptomatic infections is more with El-Tor vibrios than with the
classical cholera vibrios.
Laboratory diagnosis(picture 1):
1.Microscopic
2.Bacteriological(identification of pathogenic and non-pathogenic vibrios and differentiation
between classical, el Tor vibrios and O139 is necessary (tables 1,2).
3.Serological
4.Express diagnosis.

46
 Sample tests:

1. Name the typical properties of Vibrio El-Tor:

1. is sensitive to polymixin
2. is sensitive to C II group of phages
3. sensitivity to C IV group of phages
4. diastase activity
a)1.2 b)2.4. c)2.3. d)3.4.

2. Which laboratory methods are used in cholera:

1. bacteriological
2. express diagnosis
3. microscopic
4. skin-allergic
a)1.2.3. b)2.3.4. c)2.3. d)3.4.

3.Which of the following is typical of V. cholerae:

a)ovoid form
b)monotrichous
c)peritrichous
d)spore formation

4.V. cholerae:
1.is comma-shaped
2.is monotrichous
3.is peritrichous
4.is polypeptide capsule forming organism
a)2.3. b)2.4. c)1.2. d)1.3.

5.Which nutrient media is used for V. cholerae cultivation:

a)alkaline agar
b)Klauberg's media
c)blood agar
d)yolk broth

47
48
 DIPHTHERIA
(C. diphtheriae)

Diphtheria is an acute infection with fibrinous inflammation in the portal of entry, toxic affecting
of cardiovascular and nervous systems. Diphtheria caused by Corynebacterium diphtheriae (the
discoverer was Klebs, 1883).
Morphology: C. diphtheriae is a straight or slightly curved rod
(Latin coryna-club), 1-8mμ in length, and 0.3-0.8m in breadth.
Characteristically, they possess irregular swellings at one end that give
them a club-shaped appearance. It depends on the granules distributed
near the poles (volutin or Babesh-Ernst granules) stained deeply with
aniline dyes that gives the rod a beaded appearance. The organisms in
stained smears are arranged at various angles to each other, resembling
Latin letters L, X, W, Y (this has been called the Chinese letters or
cuneiform arrangement). This is due to the incomplete separation of the daughter cells after binary
fission. They are Gram-positive and produce no spores, capsules, non-motile. They are pleomorphous.
C. diphtheriae may change into cone-shaped, thread-like, fungi-like, and coccal forms. Normal flora of
human organism contains conditionally pathogenic Corynebacteria: C. pseudodiphthericum, C. xerosis
which are normal inhabitants of the mucous membranes. They are commonly called diphtheroids and
can cause pyo-inflammatory diseases. They are on the skin, conjunctiva and in the throat.
Morphologically they are different. Conditionally pathogenic Corynebacteria don’t arrange at an angle,
volutin granules are spread over the whole surface of the rod.
Cultivation: C. diphtheria is facultative anaerobe. The optimal temperature for growth is 37º C
and the pH of medium is 7.2-7.6. The organism grows readily on the media which contain protein
(coagulated serum, blood), for growth they require amino acids, mineral elements (magnesium, zinc,
cooper, iron), for growth they require carbohydrates too. The selective media for C. diphtheria are:
Roux’s and Loeffler’s media; Klauberg’s media (contains potassium tellurite, blood, glycerine).
Tellurite inhibits the growth of most other bacteria, acting as a selective agent. On Roux’s (coagulated
horse serum) and Loeffler’s (three parts of serum and one part of sugar broth) media the organisms
grow very rapidly; and colonies can be seen in 6-8hours, resembling shagreen leather (dry, crumbling
colonies). On Klauberg media (McLeod’s, Hoyle’s) they form three types of colonies: gravis, mitis
and intermedius. The names were originally proposed to relate to the clinical severity of the disease
produced by the three types: gravis causing the most serious, and mitis the mildest variety, with
intermedius being responsible for the disease of intermediate severity.
These three types differ in a number of properties. Corynebacterium of the gravis biovar
produce large, rough (R-forms), daisy-like (camomile) grey colonies. These biovars ferment dextrin,
starch, and glycogen; they produce a pellicle and granular deposit in meat broth. They are highly toxic
with very marked invasive properties. The colonies produced by the mitis biovar on tellurite agar are
dark, smooth (S-forms), and shining. Starch and glycogen are not fermented, and dextrin fermentation
is not constant property. They cause hemolysis of erythrocytes and produce diffuse turbidity in meat
broth. Organisms of the intermedius biovar are intermediate strains. They produce small (RS-forms),
black colonies on tellurite agar. Starch and glycogen are not fermented. Growth on meat broth
produces turbidity and granular deposit.
Fermentative properties: They all reduce nitrates to nitrites, catalase positive. They possess
cystinase activity they ferment cystine with H2S formation (Pizzu reaction). They don’t have urease
activity (this property have conditionally pathogenic Corynebacteria). They ferment glucose with acid
formation. They don’t ferment saccharose.
Antigenic structure: Corynebacteria forms superficial protein microcapsule, which contains K-
antigen. By K antigen they are subdivided into 10 serotypes. They contain group-specific O antigen,
which cross reacts with Mycobacteria and Nocardia. Serologic tests are not usually employed in
identification, because they all produce the same exotoxin. The cell wall of these organisms contains
specific lipids (corynemicolic and corynemicolenic acids), glycolipid (trehalose dimicolate or cord
factor), mannose, and inositol phosphates.
Resistance and Ecology: Humans are the only natural host of C. diphtheriae. The sources of
infection are patients and carriers. Corynebacteria reside in the upper respiratory tract and are
49
transmitted by airborne droplets. Transmission by contact route by various objects (toys, bed-cloths,
dishes, books, etc.) and foodstuffs (milk, cold dishes, etc.) contaminated with C. diphtheriae is also
possible.
C. diphtheria is relatively resistant to harmful environmental factors. They survive for two
months at room temperature and for several days on children’s toys. They survive in dust about 5
months. They are sensitive to disinfectants. The organisms are killed by a 5% carbolic acid in
1minutes, by 1% phenol solution in 10minutes.
Pathogenicity: The pathogenic factors are:
1.Adhesion, which occurs by microvilli and microcapsule
2.Colonisation
3.Invasion- the invasive factors are hyaluronidase, neuraminidase, fibrinolysin and protease.
4. Glycolipids, which is the part of cell wall and they destroy the cells in which they multiply.
5.Cord factor (trehalose dimicolate), which destroys mitochondria disturbing the processes of
respiratory phosphorylation
6.Toxins (exotoxins): the organisms produce several toxins but in pathogenesis the principle role
has a)Histotoxin, which is heat-labile polypeptide. This toxin is cytotoxin, which inhibits protein
synthesis in the cells and inactivates transferase, the enzyme responsible for the formation of the
polypeptide chain (anti-elongated factor). It contains two subunits: A and B. By B toxin the bacteria
are fixed on the mucous membrane. Toxin A penetrated into the cell and intoxication occurs. The
adrenal gland, kidney, liver, heart muscles are mainly infected The toxin affects all eukaryotic cells
regardless of tissue type but has no effect on the analogous factor in prokaryotic cells. C. diphtheriae
produces the Histotoxin (diphtheria toxin) only when it is infected by a lysogenic phage carrying the
tox gene. C. diphtheriae cells that are not lysogenized by this phage do not produce exotoxin and are
non-pathogenic. b)Dermonecrotoxin, which causes destruction of tissues. c)Haemolysins (posses
haemolytic activity).
Pathogenesis: The source of infection is patient or carrier. Children (1-4 years old) are most
susceptible to diphtheria. Entering into organism the bacteria are attached on the portal entry,
colonized and exotoxin is produced. Diphtheria toxin is absorbed into the mucous membrane and
causes destruction of epithelium and a superficial inflammatory response. The necrotic epithelium
becomes embedded in exuding fibrin and red and white cells, so that a greyish ‘pseudomembrane is
formed-commonly over the tonsils, pharynx, or larynx. Any attempt to remove the pseudomembrane
exposes and tears the capillaries and thus results in bleeding. The regional lymph nodes in the neck
enlarge, and there may be marked oedema of the entire neck. The diphtheria bacilli within the
membrane continue to produce toxin activity. This is absorbed and results in distant toxic damage,
particularly parenchymatous degeneration, fatty infiltration, and necrosis in heart muscle, liver,
kidneys, and adrenals, sometimes accompanied by gross haemorrhage. The toxin also produces nerve
damage often resulting in paralysis of the soft palate, eye muscles, or extremities.
Incubation period is 2-10 days. When diphtheritic inflammation begins in the respiratory tract,
sore throat and fever usually develop. Prostration and dyspnoea soon follow because of the obstruction
caused by the membrane. This obstruction may even cause suffocation if not promptly relieved by
intubation or tracheostomy. Irregularities of the cardiac rhythm indicate damage to the heart. Later,
there may be difficulties with vision, speech, swallowing, or movement of the arms or legs. All of
these manifestations tend to subside spontaneously.
Immunity: Immunity following Diphtheria is antitoxic in character, and the grade of the
immunity is depends on the content of antitoxins in blood. Immunity depends on antimicrobial
immunoglobulins too. Reinfection in 6-7 cases follows.
Prophylaxis and treatment: Specific prevention: DPT vaccine. The carriers treated by
erythromycin. For treatment antitoxic serum is used which is effective in early period of diseases (till
the toxin is not fixed on the tissue) and antibiotics (penicillin, erythromycin).
Nowdays pentavaccine is recommended- DPT+HBV+HIb(hemophilus influenzae). Vaccination
starts in new born infants at 1.5-2months.
Laboratory diagnosis (picture 1):
1. Microscopic: Specimens: Swabs from the nose, throat. Smears stained by Methylene blue or
Neisser’s method.

50
 2. Bacteriological: Isolation of pure culture and identification of diphtheroids by morphology,
biochemical activity: fermentation of carbohydrates, cystinase and urease activity(table 1) and
determination of toxigenicity in gel: Auckterloni test (Elek’s test).
3. Schick test (the susceptibility test).

Sample tests

1.Which of the following are typical of C. diphtheriae:

1.fermentation of glucose
2.cystinase activity
3.urease activity
4.fermentation of saccharose
a)1.2.4. b)1.2. c)1.5. d)1.2.

2.The causative agent of diphtheriae:

a)is a slightly curved gram negative rod
b)is spiral shaped
c)forms spores
d) possesses volutin granules

3.The following is typical of diphtheria toxin, Except:

a)it is coded by the «tox» gene
b)diphtheria toxin is histotoxin which is functional blockator
c)it is an exotoxin mediated by the temperate phage
d)diphtheria toxin has local and generalized action

4.Which of the following is typical of C. diphtheria:

a)gram negative
b)presence of Babesh-Ernst's granules
c)growth on Loewenstein-Jensen’s media
d)prevented by antitoxic serum only

5.In laboratory diagnosis differentiation of C. diphtheriae is carried out by:

1.lactose fermentation
2.cystinase activity
3.plasmacoagulase activity
4.urease activity
51
a)1.3. b)2.4. c)1.2. d)2.3.4.
WHOOPING COUGH
(B. pertussis)
Whooping cough is an anthroponose infection with an acute trachobronchitis, paroxysmal cough,
is transmitted by airborne droplets. The causative agents are Bordetellas. To this family belongs
Bordetella pertussis - the causative agent of Whooping cough, B. parapertussis - the causative agent
of parapertussis (produce mild infection), B. bronchiseptica - the causative agent of bronchitis and
pertussis-like illness. They differ by morphology, biochemical activity
and by antigenic structure.
Morphology: Bordetella are minute gram-negative coccobacilli,
0.2-0,5 x 1.0-1.2 mμ in size. B. parapertussis is larger (0.6-2mμ). They
are arranged single or in pairs. They don’t form spore. B. pertussis can
form tender capsule, and they are non-motile, except B. bronchiseptica,
which is peritrichous. Bipolar metachromatic granules may be
demonstrated on staining.
Culture: They are strict aerobes. Bordetella requires enriched media. Bordet-Gengou medium
(potato-blood-glycerol agar) that contains penicillin can be used. Blood is required apparently not to be
providing additional nutritive factors, but rather to neutralize inhibitory agents like toxic fatty acids.
Casein - charcoal media; charcoal or ion-exchange resins incorporated in culture media may serve
the same purpose. Growth is slow. They grow during 3-7 days. On Bordet-Gengou agar they form
small, dome shaped, smooth, opaque, viscid colonies resembling bisected pearls or mercury drops. On
blood agar they form a hazy zone of heamolysis. The colonies of fresh culture is S-type, during re-
inoculation they form R-type colonies.
Biochemical activity: It is biochemically inactive. It does not ferment sugars, proteins, doesn’t
reduce nitrates. They possess catalase and oxidase activity.
Antigenic structure: The antigenic structure is compound. They possess 14 antigenic
components agglutinogenes. For genus is common agglutinogene 7. Factors 1-6 are specific for B.
pertussis. Factor 12 is specific for B. bronchiseptica, and factor 14 for B. parapertussis.
Virulence factors are: 1.adhesion, which depends on capsule, superficial fimbriae,
filamentous hemagglutinin. 2.Colonization 3.Penetration-they penetrate into the epithelium of the
respiratory tract and internal medium of macrophages. They produce a number of factors that are
involved in the pathogenesis of the disease. Protein on the pilli, which is called filamentous hemagg-
lutinin (FHA) plays role in adherence of the bacteria to the ciliated epithelial cells of the upper
respiratory tract. Antibody against the FHA inhibits attachment and protects against the disease.
4.They produce protein toxins: a)Tracheal toxin –cytotoxin (non-secreted toxin with cytotoxic
action) b)Pertussis toxin (whooping cough toxin) promotes lymphocytosis, sensitization to histamine
and enhanced insulin secretion. It causes spasm of bronchial vessels and lead to paroxysmal cough.
This toxin is functional blockator and activates ATP system which leads to disturbances in water-salt
metabolism. It inhibits phagocytosis. c)Dermonecrotoxin is cytotoxin. It acts on myocardic cells by
activation of ATP-ase. It promotes lymphocytosis, sensitization to histamine. This toxin causes
necrosis of upper respiratory tract’s mucous membrane. d)LPS endotoxin is heat stable toxin, which
damages the epithelial cells of the upper respiratory tract and causes intoxication.
5.They produce enzymes: hyaluronidase, lecithinase, coagulase.
6.They form capsule, which an antiphagocytic factor (phagocytosis during this infection is
incomplete).
Pathogenesis: B. pertussis is a pathogen only for human, is transmitted by airborne droplets.
Bordetellas are attached to the ciliated epithelium of the upper respiratory tract but do not invade the
underlying tissue. The organism multiplies rapidly on the epithelial surface of the trachea and bronchi
and interferes with ciliary action. Decreased cilia activity and epithelial cell death occur. The infection
is limited to the respiratory tract and bacilli do not invade the bloodstream. The bacteria liberate the
toxin and substances that irritates surface cells, causing coughing and marked lymphocytosis. Later
there may be necrosis of the epithelium parts and polymorphonuclear infiltration with peribronchial
inflammation and interstitial pneumonia.
Incubation period is about 3-14 days. After incubation period the disease takes a protracted
course comprising three stages: catarrhal, paroxysmal, convalescence stages.
52
 Catarrhal stage develops, with mild coughing and sneezing, malaise, headache. Loss of
appetite, nervous irritation occurs. Laryngitis, pharyngitis develops. The temperature is high 39-40 C.
This stage continues 11-14 days. During this stage, a large number of organisms are sprayed in
droplets, and the patient is highly infectious but not very ill. The next is paroxysmal stage (spasmatic
cough) stage; the cough develops its explosive character and characteristic “whoop” upon inhalation.
This leads to rapid exhaustion and may be associated with vomiting, cyanosis, and convulsions. The
“whoop” and major complications occur predominantly in infants; paroxysmal coughing predominates
in older children and adults. This stage is prolonged 2-8 weeks. The next is convalescence stage which
is slow (2-6 months). The disease usually lasts 6-8weeks though in some it may be very protracted.
Complications may be:
1.due to pressure effects during the violent bouts of coughing (subconjunctival hemorrhage,
subcutaneous emphysema).
2.respiratory (bronchopneumonia, lung collapse)
3.neurological (convulsions, coma). Respiratory complications are self-limited, the atelectasis
resolving spontaneously but the neurological complications may result in permanent squeal such as
epilepsy, paralysis, retardation, blindness or deafness.
Immunity: High grade humoral immunity acquired after whooping cough is antimicrobial,
which is stable and tense in character.
Epidemiology and control: Whooping cough is endemic in most densely populated areas
worldwide and also occurs intermittently in epidemics. The source of infection is usually a patient in
the early catarrhal stage of the disease. Communicability is high, ranging from 30 to 90. Most cases
occur in children under age 5 years; most deaths occur in the first year of life. Control of whooping
cough rests mainly on adequate active immunization of all infants (DPT vaccine).
Prophylaxis: Specific prophylaxis - DPT vaccine.
Laboratory diagnosis (picture 2):
1. Bacteriological: Isolation of pure culture and differentiation of different species(table 1) a)The
cough plate method-A culture plate is held about 10-15cm in front of the patient’s mouth during a bout
of spontaneous or induced coughing so that droplets of respiratory exudates impinge directly on the
medium. This has the advantage that specimens are directly inoculated at the bedside. b)The post nasal
(peroral)swab: secretions from the posterior pharyngeal wall are collected with a cotton swab on a bent
wire passed through the mouth. c)The per nasal swab: here a swab on flexible nichrome wire is passed
along the floor of the nasal cavity and material collected from the pharyngeal wall.
2. Serological: Complement fixation reaction (Bordet-Gengou CFR).
3.Direct fluorescent antibody test.
Bordetella parapertussis: This organism may produce a disease similar to whooping cough;
this infection is often subclinical.
Bordetella bronchiseptica: is a small gram-negative bacillus that inhibits the respiratory tracts
of canines, in which it may cause ‘kennel cough” and pneumonitis. It grows on blood agar medium. B.
bronchiseptica has a silent copy of the pertussis toxin gene.

53
 Sample tests

1.Bordetella pertussis is characterized by the following:

1.is a coccobacteria
2.produces hemolytic zone on blood agar
3.grows on Bordet-Gengou media
4.urease activity is negative
a)1.2.3. b)1.4. c)1.3.4. d)2.3.

2. Bordetella pertussis is characterized by the following, Except:

a)is an aerobe
b)grows on Bordet-Gengou media
c)forms capsule
d)grows in bile broth

3.Specific prophylaxis in pertussis is carried out by:

a)DPT vaccine
b)TAB-te vaccine
c)STI vaccine
d)Vi antigen

4.How would you proceed for the bacteriological diagnosis of Bordetella pertussis infection:
1.microscopic examination
2.culture on cough plate
3.culture on Endo media
4.culture on Bordet-Gengou's media
a)1.2.4. b)2.4. c)1.3.4. d)1.3.

5.Causative agent of Whooping cough:

1.has catalase
2.releases tracheal toxin
3.has protective antigen
4.forms S and R types of colonies
a)2.3. b)1.2.4. c)1.2.3. d)2.4.5.

6.Bordetella pertussis:
a)is gram positive, large rod
b)forms capsule
c)is arranged in chains
d)forms spores

54
 TUBERCULOSIS
(M. tuberculosis)
Tuberculosis is chronic infectious diseases which caused by pathogenic Mycobacteria which can
defeat all the organ systems and tissues especially the lungs. Mycobacteria belong to order
Actinomycetales, family Mycobacteriaceae, species Mycobacterium (discovered by R. Koch in 1882).
Morphology: There are several types of pathogenic Mycobacteria
for human organism: 1.M. tuberculosis (90%), 2.M. bovis (5%). 3.M.
africanus (3%) 4. M.avium. These types are different by their morphology,
cultural properties and pathogenicity.
The organisms are slender, straight or slightly curved rod, 1-4 mμ in
length and 0.3-0.6 mμ in breadth. M. tuberculosis is long and slender
organism. M. bovis is a plump and short rod. The organisms are non-
motile, gram-positive, pleomorphous (rod-like, thread-like, branching,
granular, coccoid) and do not form spores and capsule. They are aerobic, acid-fast bacilli. Mycobacte-
ria contain high concentration of lipids (40-60%): fatty acids, phosphatide, sulfatides, glycolipids,
trehalose dimycolate, tuberculostearic acid, mycolic acids, wax D, which contribute to the organism’s
acid-fastness. They are stained poorly by the dyes used in Gram’s stain (they have been described as
Gram positive). They are stained by the Ziehl-Neelsen method. Spheroplasts are formed when grown
in presence of lysozymes. L-forms are also seen. In cytoplasm metaphosphate inclusions can be
obtained (Much granules), which have differential diagnostic meaning.
Cultivation: M. tuberculosis is an obligate aerobe. The organisms grow on selective media,
which contains egg, potato, milk, coagulated serum (Soton’s synthetic media, Petrangnini’s media,
Dorset media). More often Loewenstein-Jensen’s media (egg, potato, salt, glycerine, aspargine,
malachite green which acts as a selective agent for inhibition of other bacteria) is used. This media is
without starch. The optimal temperature for growth is 37º C, pH is 6.4-7.0. Mycobacteria grow slowly,
the generation time in vivo being 14-15hours. Colonies appear in about 2-8 weeks. Growing on a solid
media they produce R-form dry, brittle, with a wrinkled surface, yellow colonies. On broth they
produce a thin delicate film in 10-15 days, which later becomes thick brittle, wrinkled and yellow,
which sinks to the bottom of the tube; the broth remains clear. Scarcely visible growth appears in 8-10
days after inoculation. During micro-cultivation (cultivation in citrated rabbit or sheep blood) growth
becomes visible in 3-6 days, they form microcolonies and in the smears prepared from these colonies
the mycobacteria are arranged in parallel chains. This phenomenon is named “cord” factor, which is
trehalose dimycolate.
Biochemical activity: They possess urease, nicotinamidase activity. M. tuberculosis form niacin
(they produce nicotinic acid) when grown on an egg medium; reduces nitrates to nitrites. Tubercle
bacilli are catalase negative (weakly catalase positive).
Antigenic structure: Antigenic structure of M. tuberculosis depends on proteins, lipids, and
particularly large amounts of phosphatides and polysaccharides, which are in the cell wall, tuberculin.
The tuberculin is a protein and possesses high antigenic property. It is revealed in complement fixation
and agglutination reactions.
Ecology: Tuberculosis is an ancient disease. Evidence of spinal tuberculosis has been found in
some Egyptian mummies. Tuberculosis has been for many centuries the most important of human
infections, in its global prevalence, devastating morbidity and massive mortality. It has been called the
“white plague”. In nature M. tuberculosis causes tuberculosis in human and other primates
(experimentally, it is highly infectious for guinea pigs, non-pathogenic for rabbits). M. bovis cause
tuberculosis generally in cattle. They cause tuberculosis in man rarely (experimentally, it is highly
pathogenic for rabbits, guinea pigs). M. avium causes natural tuberculosis in birds and rarely in
humans. It is non-pathogenic for laboratory animals (guinea pigs, rabbits). M. africanum spread in
African countries (They show properties intermediate between M. tuberculosis and M. bovis).
Infection with tuberculosis takes place through the respiratory tract by the droplets and dust and
sometimes alimentary through contaminated food, and by contact through skin and mucous memb-
ranes. Tubercule bacilli are more resistant to external effects compared with other non-spore forming
bacteria because of their high lipid content (25-40%). The organisms survive in dried sputum for 2
months, on the pages of books, on bed - clothes for 3 months, in water - 1year, in soil - 6 months, in
butter and milk - 200-250 days. They are easily rendered harmless at temperatures from 100 to 120º C.
55
Mycobacteria are sensitive to exposure to sunlight. They aren’t sensitive to disinfectants. It is
necessary high concentrations and prolonged exposure of disinfectants for killing these bacteria.
Pathogenicity: Mycobacteria don’t produce toxins: exotoxins and endotoxins. Pathogenic
factors depend on the components of microbial cell:
1. Lipids (wax D, muramin dipeptide, phtionic acids, sulfatides), which causes specific granuloma
and destruction of tissues.
2.Cord factor (virulent strains of tubercle bacilli form microscopic “serpentine cords”, in which
bacilli are arranged in parallel chains), which possesses the following actions:
a)Cord factor destroys the mitochondria of the cells of the infected body and causes disorders in
respiration and phosphorylation.
b) It inhibits migration of leukocytes, causes chronic granulomas, and can serve as an
“immunologic adjuvant”.
c) Has antiphagocytic action.
3. Tuberculin, it plays the main role in pathogenesis of the infection. Tuberculin. R. Koch is the
first scientist, who excretes a substance known as tuberculin from the tubercle bacillus (a 6-8 week
culture filtrate of the bacillus grown in 5% glycerol broth) and this tuberculin named “Alt tuberculin
Koch” (old tuberculin - OT). Koch employed OT in the treatment of tuberculosis but it also caused a
serious illness because it contained heterologous (waste) substances. In 1937 Seibert excreted a clear
preparation new tuberculin “PPD-Purified Protein Derivative” (tubercle culture grown in synthetic
medium). This protein is used for skin-allergic reactions (Mantoux and Pirquoe).
Pathogenesis: M. tuberculosis causes natural infection in humans, other primates, dogs, and some
other animals which have close contact with humans. The source of infection is usually an open case
of pulmonary tuberculosis. Other forms of tuberculosis are of much less importance in public health.
In 90% cases the infection is caused by M. tuberculosis, in 10-18 % -M. bovis. The other mycobacteria
rarely cause infection. The risk of infection and disease is the highest among socioeconomically
disadvantages people, who have poor housing and poor nutrition. Tuberculosis is social infection.
The duration of incubation period in tuberculosis comparatively is long, from several weeks to
several years. The M. tuberculosis is transmitted by respiratory aerosol, and its initial site of infection
is the lung. On the site of entering Mycobacteria cause caseous necrosis of the tissues, sensitization,
and rising of specific inflammatory processes lymphadenitis and lymphangitis. Inflammatory focus
occurs: primary affect or infectious granuloma. Primary affect is included: infectious granuloma,
lymphangitis and lymphadenitis. The parenchymal exudative lesion and the draining lymph nodes
together are called a Ghon complex. (During alimentary infection the same processes occur in the
intestine). Primary lesions usually occur in the lower lobes. The primary complex can get either acute
or chronic form. When body resistance is low and conditions of work and life are unfavourable, the
organisms may leave the site of primary localization and spread throughout the body, causing a
generalized infection involving urogenital organs, bones, joints, skin, and eyes (the tuberculosis
bacterium can infect all organs and systems except muscles). Since the tubercle bacillus can involve
every organ system, its clinical manifestations are protean; fever, fatigue, night sweats, and weight loss
are common. Pulmonary involvement gives rise to chronic cough and hemoptysis. Scrofula is myco-
bacterial cervical adenitis that presents as swollen non-tender lymph nodes, usually unilaterally. M.
tuberculosis causes scrofula. Miliary tuberculosis is characterized by multiple disseminated lesions,
which are resembled to millet seeds. Tuberculous meningitis and tuberculous osteomyelitis, especially
vertebral osteomyelitis are important disseminated forms.
The development of the primary tuberculous foci takes a benign course if the conditions of the
life are favourable and there are no aggravating factors present. This stage usually terminates with
resorption and healing of the caseous foci, which become calcified and enclosed in a dense connective-
tissue capsule (Ghon complex). The organisms survive in the lymph nodes and other tissues and
organs of the primary focus for many years and sometimes even for life. People infected in such a way
acquire, on the one hand, relative immunity and, on the other hand, a potentially latent form of
tuberculosis which may become active under the influence of a number of infectious diseases and
psychic and physical traumas.
Immunity: Man is naturally resistant to tuberculosis, this property being hereditary. On the basis
of the allergic reaction, X-ray examination, and patho-anatomical changes it has been shown that in a
great number of cases infection does not result in the disease. There are approximately 80 per cent of
56
adults over 20 years of age among infected persons and no more than 10 per cent of them become ill,
and only 5 per cent immediately after infection.
After recovery from the primary infection, resistance to the organism is mediated by cellular
immunity but not phagocytosis, because phagocytosis in this case incomplete. Primary role belongs to
T lymphocytes. The changes in the T-lymphocytes indices (significant) are adequate to the illness.
Experimentally suppressing T- lymphocytes causes the development of infection processes.
Circulating antibodies are also formed, but they play no role in resistance and are not used for
diagnostic purposes.
In the course of primary infection, the host also acquires hypersensitivity to the tubercle bacilli.
This is made evident by the development of a positive tuberculin reaction. Tuberculin sensitivity can
be induced by whole tubercle bacilli or by tuberculo-protein in combination with the chloroform-
soluble wax D of the tubercle bacillus, but not by tuberculo-protein alone. Hypersensitivity and
resistance appear to be distinct aspects of related cell-mediated reactions.
Treatment: The primary treatment for mycobacterial infection is specific chemotherapy. The
most widely used antituberculosis drugs: isoniazid, rifampin, ethambutol, and pyrazinamide,
streptomycin. These are the first line antituberculosis drugs. The second-line antituberculosis drugs
include streptomycin, kanamycin, ethionamide, cycloserine and ciprofloxacin.
Prevention and control:
1.Prompt and effective treatment of patients with active tuberculousis and careful follow-up of
their contacts with tuberculin tests, X-rays, and appropriate treatment are the mainstays of public
health tuberculosis control.
2.Drug treatment of asymptomatic tuberculin-positive persons in the age groups most prone to
develop complications (eg, children) and in tuberculin – positive persons who must receive
immunosuppressive drugs greatly reduces reactivation of infection.
3. Individual host resistance: Non-specific factors may reduce host resistance, thus favouring
the conversion of asymptomatic infection into disease. Such factors include starvation, gastrectomy,
and suppression of cellular immunity by drugs (eg, corticosteroids) or infection. HIV infection is a
major risk factor for tuberculosis.
4. The eradication of tuberculosis in cattle and the pasteurization of milk have greatly reduced
M. bovis infections.
Immunization: Living avirulent (attenuated) tubercle bacilli, particularly BCG (bacillus
Calmette-Guerin, attenuated bovine organisms) vaccine, have been used for specific prophylaxis. It is
given in a single injection to newborn infants. Revaccination is carried out at the age of 7, 12, 17, 23,
and 27-30 years. Postvaccinal immunity is produced within 3 or 4 weeks and remains for 1-1.5 to 15
years. Revaccination is given to persons, whose Skin-allergic test (PPD) is negative.
Laboratory diagnosis (picture 3):
1. Microscopic: Microscopy of smears from sputum, pus, spinal or pleural fluid, urine, faeces,
lymph nodes, etc., stained by the Ziehl-Neelsen method. For concentration of the organism, the sputum
is subjected to enrichment methods: homogenization and flotation.
2. Bacteriological: Isolation of the pure culture and differentiation of mycobacteria(table 1):
Pryce’s micro-culture method is the most effective.
3. Biological method.
4. Tuberculin (allergic) tests are used for detecting the infection in children with tuberculosis
and for tuberculosis diagnosis.
5. Complement fixation reaction (positive in 80 per cent of cases with chronic pulmonary
tuberculosis)
6. Indirect hem-agglutination reaction: Sheep erythrocytes, on which polysaccharides of M.
tuberculosis or tubeculin are adsorbed, are agglutinated in serum of tuberculosis patients.

57
 LEPROSY
(M. leprae)
The organisms responsible for leprosy are M. leprae, were discovered by Hansen in 1901.
Anthroponose, chronic infection disease defeating skin, mucous membranes, peripheral nerve system
and generalization of the processes can occur. Leprosy is an antiquity infection and described in India
and Egypt in 1000-1500 A.C. (ante Christum – in Latin). Leprosy has always been held in
superstitious dread and the person suffering from leprosy considered “unclean” and social outcaste.
These patients are isolates in lepra colonies. The first Lepra colonies were built in Antique Armenia in
260-270. Nowadays there are Lepra colonies in Italy, Greece, Siberia and the USA. In the USA the
Lepra colony is great scientific centre.
Morphology: M. leprae have many properties in common with the
tubercle bacilli. They are straight or slightly curved bacilli, and club
shaped swellings and granular forms sometimes occur. The organisms
are 1-8m μ in length and 0.3-0.5m μ in breadth. They usually occur in
groups resembling packets of cigars or clusters. M. leprae is non-motile,
produces neither spores nor capsules, and is gram-positive. The
organisms are pleomorphous. They can transfer into L-form too. M.
leprae are similar to M. tuberculosis in chemical composition. Their
lipid content ranges from 9.5-18.5 per cent. Besides mycolic acid, they contain leprosinic oxy acid,
free fatty acids, wax (leprosine), alcohols, and polysaccharides.
Cultivation: Cultication on artificial media is unsuccessful. Cultivation is possible in cell
cultures, on Wasserman media (serum, potatoes, blood, egg, glycerine containing media).The growth
is prolonged (6-8weeks) due to the generation time (12-30days); they form wrinkled pellicle on the
surface of the medium. When bacilli from human leprosy (ground tissue nasal scrapings) are
inoculated into footpads of mice, local granulomatous lesions develop with limited multiplication of
bacilli. Inoculated armadillos develop extensive lepromatous leprosy.
Fermentative properties, antigenic structure and classification have not been worked out enough.
Pathogenesis and disease in man: M. leprae are extremely resistant, and survive in human
corpses for several years. Humans are the natural hosts.
The source of infection is sick person. The causative agent is transmitted by the air – droplet
route through the nasopharynx and injured skin. Intimate and prolonged contact between healthy
individuals and leprosy patients is the main mode of infection. The organism replicates intracellulaly,
58
typically within skin histiocytes, endothelial cells, and the Schwann cells of nerves. Incubation is 4-6
to 15-35 years. There are three clinical forms of leprosy: 1. lepromatous; 2. tuberculoid; 3.
intermediate (undifferentiated).
Lepromatous form is a severe infection. In lepromatous leprosy, multiple nodular skin lesions
occur, resulting in typical leonine (lion like) facies. In the lepromatous type the course is progressive
and malignant.; slow symmetric nerve involvement; abundant acid-fast bacilli in the skin lesions;
continuous bacteremia; and negative lepramin skin test (the cell mediated response to organism is
poor, the skin and mucous membrane lesions contain large number of organisms, foamy histiocytes
rather than granulomas are found, and the lepramin skin test result in negative). After the onset of
therapy, patients with lepromatous leprosy often develop erythema nodosum leprosum (ENL), which is
interpreted as a sign that cell-mediated immunity is being restored. ENL is characterized by painful
nodules, especially along the extensor surfaces of the tibia, and ulna, neuritis, and uveitis. The
disfiguring appearance of the disease results from several factors 1.The skin anaesthesia results in
burns and other traumas, which often become infected; 2. Resorption of bone; leads to loss of features
such as the nose and fingertips; 3. Infiltration of the skin and nerves leads to thickening and folding of
the skin.
In tuberculoid leprosy, the cell-mediated immune response to the organism limits its growth;
very few acid-fast bacilli are seen, granulomas containing giant cells form, and the lepromin skin test
result in positive. The lepromin skin test is similar to the tuberculin test. In tuberculoid leprosy,
hypopigmented macular skin lesions, thickened superficial nerves, and significant anaesthesia of the
skin lesions occur.
Intermediate leprosy is intermediated between tuberculoid and lepromatous forms.
Treatment: Several specialized sulphones (dapsone-diaminophenylsulfone,) and rifampin
suppress the growth of M. leprae and the clinical manifestations of leprosy if given for many month.
Thalidomide is the treatment of choice for severe ENL reactions.
Prevention: Isolation of all lepromatous patients, coupled with chemoprophylaxis with dapsone
for exposed children, is required. There is no vaccine.
Immunity: Antibodies are formed, but they don’t have high protective function. During
infection changes in immunocompetent cells are detected, especially changes of T lymphocytes. The
activation and number of T cells decrease. The activation of immunity can be detected by Lepromin
test (Mitsuda test). As an allergen lepramin is used, this injected intracutaneous. The first is the early
reaction, which consists of erythema and induration developing in 24-48 hours and usually remaining
for 3-5 days. This is analogous to the tuberculin reaction. Histologically the lesion consists of serous
exudate with lymphocytic infiltration. The second is the late reaction, appearing in three weeks
reaching a peak in four weeks and gradually subsiding in the next few weeks. The reaction consists of
an indurated skin nodule, which may ulcerate. Histologically, there is infiltration with lymphocytes,
epithelioid cells and giant cells. Both the early and late reactions are usually correlated in the same
individual, but most leprologists use late reaction as an evidence of lepramin positivity.
The lepromin test is not used to diagnose leprosy. It isn’t indicated prior contact with the lepra
bacillus either. Healthy persons in non-endemic areas with no chance of contact with the bacillus may
sometimes give a positive lepromin test, probably due to sensitisation by other mycobacterial antigens.
The test is employed for the following purposes:
1. To classify the lesions of leprosy patients. The lepromin test is positive in tuberculoid,
negative in lepromatous and variable in intermediate type of the disease.
2. To assess the prognosis and response to treatment. A positive reaction indicates good
prognosis and a negative one bad prognosis. Conversion to lepromin positivity during treatment is an
evidence of improvement.
3. To assess the resistance of individuals to leprosy. It is desirable to recruit only lepromin
positive persons for work in leprosoria as lepromin negative persons are more prone to develop the
disease.
4. To verify the identity of candidate lepra bacilli. Cultivable acid fast bacilli claimed to be lepra
bacilli should give matching results when tested in parallel with standard lepromin.
Laboratory diagnosis: Microscopic: Acid-fast staining.

59
 Sample tests

1.Which of the following are specific for M. tuberculosis:

1.growth on simple media
2.forms of S type colonies on solid nutrient media
3.grows on Loewenstein-Jensen’s media
4.reduction of nitrates into nitrites
a)1.4. b)3.4. c)1.3. d)2.3.4.

2.Mark the user of tuberculin test:

1.diagnosis of active Tb infection
2.indication of hypersensitivity(Tb)
3.diagnosis of diphtheria
4.for formation of immunity
a)1.2. b)1.4. c)1.2.4. d)2.3.4.

3.Hypersensitivity in tuberculosis:
1.is provided by lymphocytes
2.develops in 24-72hours
3.develops in several minutes
4.is revealed by Pirque test
a)1.2. b)1.4. c)1.2.4. d)2.3.4.

4.Which of the following statements concerning M. tuberculosis is Wrong:

a)forms S type of colonies on solid media
b)possesses urease activity
c)forms R type of colonies
d)colonies are formed in 3-4weeks

5.The causative agent of leprosy belongs to the following family:

a)Enterobacteriaceae
b)Spirochetaceae
c)Micrococcaceae
d)Mycobacteriaceae

6.Clinical forms of leprosy are:

1.lepromatous
2.ocular
3.tuberculoid
4.septic
a)1.3. b)1.4. c)1.2.4. d)2.3.4.

60
 LEGIONELLA
(L. pneumophilia)
Legionellosis is an acute infection with intoxication, fever, respiratory syndrome defeating
kidney and CNS. The genus is named after famous outbreak of pneumonia among people attending the
American Legion convention in Philadelphia in 1976 (Legionnaires’ disease).
These microorganisms are in the family Legionelaceae, genus Legionella. The genus contains 9
species. For human organism the most important is L. pneumophilia.
Morphology: Legionella are gram negative, polymorphic rods 2-3 μm
tin length, 0.3-04 μm in breadth (middle sized). They are motile,
lophotrichous in some cases amphitrichous, non-capsulated, non-sporing.
They often poorly are stained by Gram’s method and are not seen in stains of
clinical specimens. Gram-stained smears should be made to suspect
Legionella growth on agar media. Presence of lipidic acids in the cell wall
ensures staining of Legionellas by Romanovsky – Giemza staining method.
Culture: They are facultative anaerobes with aerobic prevalence. They don’t grow on ordinary
media. Growth occurs on media enriched with amino acids (cysteine, tyrosine), iron, hemoglobins, 5%
CO2. Macrophagal cellular cultures can be used too for cultivation. The temperature for growth is 25-
42C (optimum 35C, pH- 6.9- they are very sensitive to changes in pH). Legionella grows slowly;
visible colonies are usually present after 3 -5days of incubation. Colonies are round or flat with entire
edges, with grey sheen (S-type colonies). They vary in colour from colourless to iridescent pink or
blue and are translucent or speckled.
Biochemical activity: L. pneumophilia is oxidase and catalase positive, they ferment starch,
hydrolyse gelatine. They don’t have urease activity; they don’t reduce nitrates to nitrites. Sugar lysing
activity is absent.
Antigenic structure is not known well. Antigenic specificity of L. pneumophilia is thought to be
due to complex antigenic structures. There are more than ten serogroups of L. pneumophilia.
Serogroup I is the most common serogroup for humans. Legionella species cannot be identified by
serogrouping alone, because there is cross reactive antigenicity among different species. Occasionally,
Bacteroides, Bordetella, and some Pseudomonades also cross-react with L. pneumophilia.
Pathogenicity: Pathogenicity depends on capsular substances, surface proteins, protein
exotoxin, LPS endotoxin, aggression enzymes.
By these pathogenic factors L. pneumophilia enters and grows within human alveolar
macrophages and monocytes and is not effectively killed by polymorphonuclear leukocytes. Peptide
toxins - inhibit phagolysosome formation (they reduce oxidative metabolic burst); binds with C3b of
complement system and inactivate it. These toxins inhibit expression of MHC antigens on the surface
of macrophages and by this mechanism they inhibit presentation of antigen to CD4+ cells. Bacterial
catalase inhibits activation of oxygen’s toxic metabolites, which formed due to activation of
macrophages. Thermolabile exotoxin (cytotoxins and haemolysins) causes defeating lungs and
intoxication. Thermostable endotoxin is the cause of the action which is characteristic for gram
negative microorganisms. In pathogenicity participates exoenzymes: protease and hemolysins.
Ecology: The Legionellae are ubiquitous in the environment and worldwide in distribution. They
are in soil and in freshwater lakes and streams and have been found in high numbers in air –
conditioning systems and washing facilities, eg, shower stalls. The later sources have been responsible
for outbreaks of human disease, especially in hospitals. Water’s chlorination, heating and cleaning can
help to control the multiplication of Legionella in water and air – conditioning systems. Legionella is
not communicable from infected patients to others. Despite airborne transmission of the organism,
person to person spread does not occur.
Pathogenesis and clinical findings: The portal of entry is the respiratory tract, and pathogenic
changes occur primarily in the lungs. In severe cases bacteremia occurs accompanied by damage to the
vascular endothelium in multiple organs, especially the brain and kidneys.
Incubation period is 2-7 days. The incidence of clinically significant disease is highest in men
over the age of 55 years. Factors associated with high risk include smoking, chronic bronchitis and
emphysema, steroid and other immunosuppressive treatment, cancer chemotherapy, and diabetes
mellitus.

61
 The clinical picture can vary from a mild influenza-like illness to a severe pneumonia
accompanied by mental confusion, non-bloody diarrhoea, proteinuria, sand microscopic hematuria.
Although cough is a prominent symptom, sputum is frequently scanty and non-purulent. Most cases
resolve spontaneously in 7-10 days, but in older and immunocompromised patients, the infection can
be fatal.
The clinical forms of the infection are:
1.Disease of legionnaires’ (severe pneumonia).
2.Pontiac fever: The syndrome is characterized by fever and chills, myalgia, malaise, ness and
headache that develop over 6-12 hours. Dizziness. Photophobia, neck stiffness, and confusion,
mild cough and sore throat also occur. In this form pneumonia does not occur.
3.Fort – Bregg fever is characterized with fever and exanthema.
4.Intrahospital infection the fatal rate is high and the causative agent is L. micdadei, which cause
pneumonia of both lungs and kidney insufficiency.
Immunity: Infected patients make antibodies against Legionella, but the peak antibody response
may not occur until 4-8 weeks after the infection. The roles of antibodies and cell-mediated responses
in protective immunity in humans have not been defined. Both humoral and cell-mediated immune res-
ponses occur. The cell-mediated response is important in protective immunity because of the
intracellular infection and growth of Legionella.
Prophylaxis and treatment: Legionella are susceptible to erythromycin and some other drugs
(Rifampin). There aren’t specific prophylaxis. The prophylaxis is non-specific: disinfection of
reservoirs and air-conditioning systems.
Laboratory diagnosis:
1.Microscopic (direct immunofluorescence)
2.Bacteriological method
3.Complement fixation reaction
4.Immune enzyme assay.

Sample tests

1.By which of the following procedures Legionella disease is most rapidly diagnosed:
a)immunofluroscence
b)tube agglutination test
c)toxin neutralization test
d)cultivation on Klauberg agar

2.Virulence factors of Legionella are:

1.capsular substances
2.outer membrane proteins
3.endotoxin
4.leukocidin
a)1.3.4. b)1.2.3. c)2.3. d)2.3.4.

3.Which of the following is typical of L. pneumophilia, Except:

a)requires enriched nutrient media
b)multiplies in macrophages and monocytes and is transported within them
c)in pathogenicity participates protein toxin which is exfoliatin toxin
d)exotoxin causes defeating of lungs

4.Which of the following is typical of Legionella pneumophilia:

a)is acquires by inhalation of contaminated droplets from air conditioners
b)transmission is by alimentary route
c)it requires simple media for growth
d)is a motile, gram-positive rod

62
 PSEUDOMONADS
Pseudomona
ds are in the family Pseudomonadaceae and contain more than 20 types. They are widely distributed in soil and
water. Some of them are pathogens for humans. The pathogens are: 1. P. mallei the causative agent of malleus
(glanders), 2. P. pseudomallei, the causative agent of melioidosis 3. P.
aeruginosa, the causative agent of blue-green pus infection.
Pseudomonas aeruginosa:
The first person, who described this microorganism in 1862 was Lukke.
P. aeruginosa is widely distributed in nature and is commonly present in
moist environments, in hospitals. It can colonise normal humans, in whom it is a
saprophyte. It causes disease in humans with abnormal host defences. Approxi-
mately 10 of people carry it in the normal flora of the colon
Morphology: P. aeruginosa is motile (lophotrichous or monotrichous), rod-shaped microorganism,
measuring about 0.6 x 2 μm. It is gram-negative and occurs as single bacteria, in pairs, and occasionally in short
chains. They are non-spore-forming and non-capsule-forming. They produce mucous which is capsule-like
substance and it covers the microbe.
Cultivation: They are obligate aerobes. They grow readily on many types of culture media, sometimes
producing a sweet or grape-like or corn taco-like odour. On broth they form pellicle. P. aeruginosa forms large
(2-3mm), smooth, translucent, round colonies with a fluorescent greenish color. P. aeruginosa produces 4
distinct pigments: 1.It is nonfluorescent, bluish pigment pyacyanin, which is soluble in chloroform and water. It
diffuses into the surrounding medium and water. This pigment is not produced by other species of this genus,
therefore, its detection is diagnostic of P. aeruginosa.
2 Pyoverdin: Fluorescent pigment, which is insoluble in chloroform but soluble in water. It gives a
greenish color to the agar. 3. Pyorubin: It is bright red, water soluble and insoluble in chloroform. 4.
Pyomelanin: It is brown to black pigment and its production is uncommon. It is chemically unrelated to animal
melanin pigment or the black pigment pyomelanin.
They grow well at 37-42 C; its growth at 42C helps differentiate it from other Pseudomonas species. It
is oxidase-positive. It does not ferment carbohydrates, but many strains oxidase
glucose. Identification is usually based on colonial morphology, oxidase
positivity, the presence of characteristic pigments, and growth at 42 C. Strains
of P. aeruginosa isolated from cystic fibrosis patients have a prominent slime
layer (glycocalyx), which gives their colonies a very mucoid appearance. The
slime layer mediates adherence of the organism to mucous membranes of the
respiratory tract and prevents antibody from binding to the organism.
Biochemical activity: They are active. Pseudomonas aeruginosa possess
carbohydrate-lysing activity: they ferment glucose with acid formation without gas. Lactose and maltose are not
utilized. They liquefy gelatine, serum, hydrolyse casein. They have haemolytic activity: on blood agar they form
hemolysis zone around the colony. These microorganisms reduce nitrates to nitrites. They don’t form indole and
H2S.
Antigenic structure: They have O and H antigens but these antigens don’t have practical significanse.
Antigenic properties possess mucous, toxins, adhesins, fimbria.
Pathogenic factors: The main factor is Exotoxin A, which is thermolabile protein. This toxin causes
tissue necrosis. The toxin blocks protein synthesis. Although the mechanism of action of exotoxin A is identical
to that of diphtheria histotoxin, it suppresses immunogens. This is the severe toxin of P. aeruginosa and is
named lethal toxin and causes oedema, necrosis, metabolic acidosis.
Exotoxin S is thermolabile protein. It causes pathogenic action on lungs.
Cytotoxin causes cytotoxic action on neutrophils, which ensure changes in cell ultrastructure,
disturbances in NA, K, Ca. This toxin causes rising of membrane permeability and the cell loses large molecules
(e.g., proteins).
Hemolysins: Hemolysin of I type - thermolabile protein with lecithinase activity which promotes
formation of necrotic foci. Hemolysin of II type - thermostabile protein, which stimulates the action of the I
type toxin. It has hemolytic activity, too.
Leukocidin causes lysing of leukocytes.
Endotoxin: It is a lipopolysaccharide exhibiting many biological properties of enterobacterial LPS
including pyrogenic action.
Some strains can produce enterotoxin.
The pathogenic factors are enzymes: neuraminidase, protease, which inhibits activity of immune system
proteins. Collagenase, which lyses the connective tissue. It is spreading factor. They produce lecithinase like
toxin, elastase, hyaluronidase.

63
 Epidemiology: P. aeruginosa is found chiefly in soil and water, although approximately 10 of people
carry it in the normal flora of the colon. Its ability to persist and multiply in moist environment of the hospital
wards, bath rooms, kitchens, equipments and antiseptic or disinfectant solutions is of particular importance in
cross-infection. It is found on the skin in moist areas and can colonize the upper respiratory tract of hospitalized
patients. Its ability to grow in simple aqueous solutions has resulted in contamination of respiratory therapy and
anaesthesia equipment, intravenous fluids, and even distilled water. They are resistant to disinfectants and
antibiotics and can contaminate them.
P. aeruginosa is primarily an opportunistic pathogen that causes infections in hospitalized patients, eg,
those with extensive burns, in whom the skin host defences are destroyed; in those with chronic respiratory
disease (eg, cystic fibrosis), in whom the normal clearance mechanisms are impaired; in those who are
immunosuppresed; in whom neutrophil counts less than 500/L; and in those with indwelling catheters. It
causes 10-20 per cent of hospital – acquired infections.
Pathogenesis and clinical findings: P. aeruginosa can cause infections virtually anywhere in the body,
but urinary tract infections, pneumonia and wound infections (especially burns) predominate. They participate
in mixed infections. They cause pyo-inflammatory infection in association with Staphylococci, Proteus, and
Escherichia. The bacteria can spread to the skin, where they cause black, necrotic lesion called ecthyma
gangrenosum. The organism can enter the blood and cause sepsis. Patients with P. aeruginosa sepsis have
mortality rate greater than 50. P. aeruginosa can cause meningitis, osteomyelitis, arthritis, otitis, pneumonia,
pleuritis, abscesses of liver, brain, inflammation of the genital-urinary tract. They play a role in surgery, and
burn wounds and lethal rate in these infections is very high. It accounts for 5-15% of nosocomial infections.
The commonest infections caused by it are:
1. Urinary tract infection when it is mechanically placed into urinary tract during catheterization.
2. It is able to multiply on respiratory ventilators and deliver large numbers of organisms directly into the lungs
of an already debilitated person leading to respiratory infections like necrotizing pneumonia.
3. Septicaemia may develop in persons with leukaemia or persons receiving immunosuppressive drugs, in
newborn babies and old debilitated persons.
4. In drug addicts, it may cause endocarditis and septic arthritis.
5. In addition, it may cause wound and burn infection, chronic otitis media and otitis externa, eye infection and
acute necrotizing vasculitis which leads to haemorrhagic infarction of skin and internal organs.
The infections, caused by this bacterium, are very severe and treatment is difficult, because the resistance
of P. aeruginosa to antibiotics is very high. This resistance depends on plasmids (R-plasmids).
Treatment: Because P. aeruginosa is resistant to many antibiotics, treatment must be tailored to the
sensitivity of each isolated and monitored frequently; resistant strains can emerge during therapy. The treatment
of choice is antipseudomonal penicillin, eg, ticarcillin or piperacillin, plus an aminoglycoside, eg, gentamicin or
amikacin.
Prevention: Prevention of P. aeruginosa infections involves keeping neutrophil counts above 500/L,
removing indwelling catheters promptly, taking special care of burned skin, and taking other similar measures to
limit infections in patients with reducing host defences.
Laboratory diagnosis:
Bacteriological method: P. aeruginosa grows as non-lactose fermenting (colourless) colonies on Endo’s,
MacConkey’s agar (oxidase-positivity). On ordinary nutrient media they form colonies with blue-green pigment
and with fruity aroma.

Sample tests
1.Pseudomonas aeruginosa:
1.are straight, little curved, gram (-) rods
2.require special nutrient media
3.do not require additional components
4.are motile, non-spore forming, spherical bacteria
a)2.4. b)1.2. c)3.4. d)1.3.
2.Pseudomonas aeruginosa:
1.is middle sized, little curved, gram (-) rod
2.grows only in complex media with pigment formation
3.forms blue-green pigment in simple nutrient media
4.possesses proteolytic activity
a)1.3.4. b)2.4. c)1.4. d)1.2.
3.Pseudomonas aeruginosa:
1.are straight, little curved, gram (-) rods
2.require special nutrient media

64
3.do not require additional components
4.are motile, non-spore forming, spherical bacteria
a)2.4. b)1.2. c)3.4. d)1.3.

65
66
67
68
 `ZOONOSES
 Yersinia pestis
 Bacillus anthracis
 Brucellas
 Francisella tularensis

PLAGUE
(Yersinia pestis)
Plague is an acute, pandemic infection with intoxication, high fever, which defeats lymph nodes
and lungs. It is a quarantine infection. Plague is an ancient scourge of mankind. The disease was
familiar to the ancient civilizations of Asia. Serious infection often results, which in previous centuries
produced pandemics of “black death” with millions of fatalities.
The history of epidemic diseases has no more terrifying chapter than the plague. Historians of
plague identify 41 epidemics before the birth of Christ and 109 epidemics in the next 15 centuries. The
first documented pandemic occurred during the region of Emperor Justinian in 542A.D. It probably
began in the Middle East; spread widely, and lasted 60 year. Approximately 100million people died of
the infection, and towns were completely decimated. The second pandemic known the «black death»
(the name black death may have been derived from the extensive cutaneous hemorrhages and gangrene
often seen in fatal cases of plague), which swept over Europe during the fourteenth century killed
about 25 million people, which accounted for about one quarter inhabitants. The third pandemic began
in 1894 and obversed in 1939 killed about 12-15 million people.
Plague is an infection of wild rodents, transmitted from one rodent to another and occasionally
from rodents to humans by the bites of fleas. Y. pestis was discovered by the French microbiologist A.
Yersin in Hong-Kong in 1894. Russian scientists D. Samoilovich, D. Zabolotny, N. Gamaleya and
others contributed greatly to the study of the mechanisms of its transmission.
R. Karamchamdani and K. Rao from India introduced the extremely effective antibiotic strepto-
mycin into practice for the treatment of all forms of plague.
Morphology: Y. pestis is a plump, short, ovoid, gram-negative rod,
about 1.5 µx 0.7 µm in size that exhibits striking bipolar staining with
special stains (methylene blue), occurring singly or in pairs or, when in fluid
culture, in chains. They are non-motile, non-sporing. The organisms are
surrounded by a slim layer glycoprotein capsule. Y. pestis is characterized
by marked individual variability (pleomorphism): in old cultures they may
be coccoid, club shaped, filamentous, etc.
Cultural characteristics: Y. pestis is aerobic or facultative anaerobe. It can grow on ordinary
media with pH 5-9; the optimum temperature is 25-30º C. Growth is more rapid in media containing
blood or tissue fluids. In cultures on blood agar colonies may be very small at 24 hours. On agar slants
the culture forms a viscid translucent mucilaginous mass. On agar plates after 10-12hours it forms
colonies which resemble broken glass; after 18-24hours colonies are with turbid white centre, and
scalloped borders resembling lace or crumpled lace handkerchiefs (R-colonies). In meat-broth the
cultures form a pellicle on the surface with thread-like growth resembling stalactites and a flocculent
precipitate.
This organism has little biochemical activity, and this is somewhat variable. Y. pestis does not
liquefy gelatine. Indole and H2S is not produced. It reduces nitrates, ferments glucose, maltose,
galactose, mannite but not lactose with acid formation, but no gas. They are citrate negative catalase
positive, oxidase and urase negative. MR reaction is positive, VP-negative.
Antigenic structure: Antigenic structure is complex. 20 antigens have been detected by gel
diffusion and biochemical analysis. Many of them are virulence factors. They include the following:
F-1 antigen is a heat labile surface protein, which inhibits phagocytosis and is generally present
only in virulent strains. V and W antigens are produced together. They are cell wall products. Antigen
V is protein, W antigen is lipoprotein. These antigens are virulence factors and they inhibit
phagocytosis and intracellular killing of the bacillus. V-W antigens are encoded by genes on plasmids.
Somatic «O» antigen: is similar to O antigens of Enterobacteriaceae family.

69
 Virulent strains produce a bacteriocin (pesticin I), coagulase, fibrinolysin, superficial proteins,
toxins, pH6 protein. Y. pestis hasn't serovars.
Resistance: The plague bacillus can withstand low temperature. At 0ºC it lives for 6 months. It
survives in water for 30 days, in vegetables and fruits for 6-11 days, in sputum for 10 days. Y. pestis is
very sensitive to drying and high temperatures. Boiling kills the organisms within 1 minute. In a 5%
phenol solution it is killed in 5-10 minutes and in 5% solution of lysol in 2-10 minutes.
Pathogenicity and pathogenesis: Virulence factors are: Adhesion on epithelial cells of different
tissues of portal of entry. Adhesive factors are fimbriae, neuraminidase participates in adhesion too,
because it releases (sets free) receptors for Y. pestis. Colonization, invasion, aggression (suppression
of phagocytosis). In pathogenicity takes part various enzymes and toxins. Virulent strains produce
bacteriocin –Pesticin I, coagulase, fibrinolysin, which are located on the surface membrane of the
cell wall. They inhibit activation of complement system. Toxin production plays an essential role in
pathogenicity. Y. pestis produces Endotoxin, a LPS similar to the endotoxin of enteric bacilli, which
produces allergization of the organism. Exotoxin is protein in nature (by mechanism of action is
functional blockader-toxicoblockador), possessing some properties of both exotoxins and endotoxins
called «mice toxin» (non-secreted) which blockaded the function of cell mitochondria of heart and
liver. On injection into experimental animals Plague toxins produce local edema and necrosis with
systemic effects on the peripheral vascular system and liver. These organisms contain protein toxins,
which consist of two subunits: A - toxic and B for adhesion. Toxic and allergic properties depend on
these toxins too. Virulence depends on unidentified surface components, which absorbs hemin and
basic aromatic dyes in culture media to form colored colonies. Virulence has also been associated
with the ability for purine synthesis.
Epidemiology: Plague is a zoonotic disease. The plague bacillus is naturally parasitic for rodents
(field mice, skunks and other animals) that occur in many parts of the world. Infection is transmitted
among them by rat fleas.
Contagiousness is very high and the rate of death is also very high. This is an ancient scourge of
mankind. The chief enzootic area is Central Asia, but it caused epidemics and pandemics. In the 14th
century Plague spread in Europe, it was named Big death or Black death and 25% of population were
died.
Pathogenesis and clinical findings depend on portal of entry and by this case human plague may
be of the following forms: Cutaneous, bubonic, cutaneous-bubonic, primary septicaemic, secon-
dary septicemic, primary pneumonic, secondary pneumonic and intestinal. In human beings,
plague occurs in three major forms: bubonic, pneumonic and septicemic.
Y. pestis enters the human organism through the skin and sometimes through mucous
membranes. These are cutaneous forms of infection. In the pneumonic form of the disease the plague
bacilli are spreaded in the air with sputum expelled by the patient when he coughs or talks.
In bubonic plague, after an incubation period (2-5 days) the lymph nodes draining the site of
entry of the bacillus become infected. The temperature is high 39-40º C. The face and conjunctiva are
hyperaemic. Lips are dry, coated tongue (white as whitening, chalk) and the tongue becomes large. As
the plague bacillus usually enters through flea bites on the legs, the inguinal nodes are involved and
hence the name bubonic (bubon, meaning groin). The gland becomes enlarged and suppurates. The
bacilli enter the bloodstream and produce septicaemia. Pneumonic plague may be seen sometimes
during epidemics of bubonic plague. Pneumonic plague is spreaded by droplet infection. The bacilli
spread through the lungs producing haemorrhagic pneumonia. The main symptoms: cough, bloody
mucoid sputum, which contains bacilli in enormous numbers. Cyanosis is very prominent. Pneumonic
plague is highly infectious and the death rate is very high. Septicemic plague is usually the terminal
event in the bubonic or pneumonic plague but may sometimes occur primarily. Meningitic
involvement may occur rarely. Human carriers have not been recorded but asymptomatic
oropharyngeal infection has been observed in some contacts.
Treatment: Streptomycin is used for treatment of plague especially, this drug is effective in
pneumonic form of diseases. Good results have been obtained from combination of streptomycin with
Chloromycetin or tetracycline with anti-plague serum. Anti-plague gamma-globulin and specific
bacteriophage are also used for treatment of plague patients.
Prophylaxis: General prophylaxis comprises the following measures:
1. Early diagnosis of plague
70
2. Immediate isolation and hospitalization of patients and enforcement of quarantine; individuals who
have been in contact with patients are placed under quarantine for six days and prescribed prophylactic
streptomycin treatment;
3. Thorough disinfection and extermination of rats in disease foci;
4. Individual protection of medical personnel and prophylactic treatment with streptomycin and
vaccination;
5. Prophylactic measures and systematic observation carried out by plague control laboratories,
stations, and institutes in endemic areas.
Specific prophylaxis is accomplished with alive EV vaccine. It is produced in dry form and
administered subcutaneously, intracutaneously or percutaneously once or twice. Immunity lasts for no
longer than one year. Depending on the epidemiological condition, revaccination is conducted in 6 or
12 months.
Laboratory diagnosis(picture 1):
1.Microscopic.
2.Bacteriological (isolation of pure culture and differentiation-table 1)
3.Biological
4.Serological (passive hemagglutination)
5.Skin allergic test with pesticin.
6.Express diagnosis (immunofluorescence and diagnosis with specific phage).

71
 SPORE-FORMING
GRAM-POSITIVE BACILLI
The gram-positive spore-forming bacilli are the Bacillus and Clostridium species. These bacilli
are ubiquitous, and because they form spores, they can survive in the environment for many years.
Bacillus species are aerobes, Clostridia species are obligate anaerobes. Of the many species of both
Bacillus and Clostridium genera, most do not cause important disease, several species, however, cause
important disease in humans.
BACILLUS SPECIES
The genus bacillus includes large aerobic, gram-positive rods occurring in chains. Most members
of this genus are saprophytes prevalent in soil, water, and air and on vegetation, such as Bacillus
cereus and Bacillus subtilis. The causative agent of infection disease in human organism is Bacillus
anthracis.
ANTHRAX (Ba c i l l u s a n t h r a c i s)
Morphology: The Anthrax bacillus is one of the largest of pathogenic bacteria measuring 3-12
m in length and 3-5 m in breath. Gram-positive, have square ends (truncated) resembling bamboo
canes and are arranged in long chains (in smears from pure culture). In pathogenic material
arrangement is in pairs or in short chains. The bacilli are non-motile. They produce oval shaped central
spores, which are smaller in diameter than the bacillus. In the human and animal body the bacilli
produce polypeptide capsules, which surround single organism or are continuous over the whole chain.
Capsules are also produced on nutrient media, which contain blood, serum, egg yolk.

Cultivation: The bacillus is aerobic and facultative anaerobic. The optimum growth temperature
is 37º C. It grows well on all ordinary media at pH 7.2-7.6. They do not require nutrient media: on
meat-peptone agar the bacilli form rough colonies (R-form), which have uneven edges and resemble
the head of a medusa (jelly-fish) or locks of matted hair (named lion’s mane). Broth cultures of the
anthrax bacillus produce flocculent growth resembling cotton wool which sinks to the bottom of the
tube or flask, leaving the broth clears (with little or no turbidity). On blood agar the colonies are non-
haemolytic.
When Bacillus anthracis is grown on the surface of a solid medium containing 0.005-0.5 units of
penicillin/ml, in 3-6 hours the cells become large spherical (protoplasts) and occur in chains on the
surface of the agar, resembling a string of pearls. This “string of pearls reaction” differentiates clearly
B. anthracis from anthracopids and other aerobic spore bearers.

72
 Fermentative properties: The anthrax bacilli possess great biochemical activity. They ferment
carbohydrates: glucose, levulose, saccharose, maltose with acid formation.
They contain enzymes: dehydrogenase, lipase, diastase, lecithinase and catalase. In gelatine stab-
cultures growth resembles an inverted fir tree, the gelatine being liquefied in layers. The organisms
cause late liquefaction of coagulated serum and produce ammonia and hydrogen sulphide. They
peptonize milk.
Antigenic structure: B. anthracis contains: Somatic group-specific polysaccharide antigen.
Antibodies to these antigens are not protective. Revealing of this antigen is by Askoli thermo-ring-
precipitation reaction. Capsular type-specific polypeptide antigen and antibodies to this antigen are
not protective also. Protective antigen, which is thermolabile protein and possesses marked
immunogenic properties, gives rise to the formation of protective antibodies, which neutralize the
aggressive enzyme of the anthrax bacillus.
Resistance: The vegetative bacilli are not resistant and are killed in 40 min at 55º C, in 15-30
min at 60º C, and in 1-2 min at 100º C. The spores are thermo-resistant, and withstand boiling for 15-
20 min and autoclaving at 110º C for 5-10 min. They are destroyed by 1 per cent formalin and 10 per
cent sodium hydrate solutions in 2 hours. Destruction of the spores in animal products is important and
2 per cent formaldehyde solution is used at 39-40º C for 20 min for disinfection of wool.
Pathogenicity and Pathogenesis: B. Anthracis produces exotoxin (by the mechanism of action
is functional blockator, toxicoblockator), which contains three components: 1. oedema factor, caused
accumulation of cAMP and development of oedema in tissues; 2. lethal factor which has cytotoxic
action and causes oedema of the lungs and hypoxia. 3. protective antigen which is immunogenic and
antibodies which synthesized against this antigen are high protective.
Pathogenicity depends on the polypeptide capsule, which is virulence factor. It possesses
antiphagocytic activity.
Anthrax is zoonosis. Sheep, cows, horses, camels, pigs are susceptible to anthrax infection. The
animals are usually infected alimentary, ingesting the spores in the fodder. The Bacilli localize in the
intestine. The disease is characterized by lassitude, cyanosis, and sanguineous discharge from the
intestine, nostrils, and mouth. Septicaemia sets in preceding death, which occurs in 2-3 days.
Humans acquire the disease from sick animals or articles and clothes manufactured from
contaminated raw materials: sheepskin coats, fur mittens, collars, hats. The portals of entry are skin,
mucous membranes and respiratory tract. Anthrax occurs in three main clinical forms: cutaneous,
respiratory, and intestinal.
In the cutaneous form the causative agent enters the body most frequently through injured
integuments, mainly those parts of the body, which are not covered with clothes (face, neck, hands,
and forearms). An anthrax carbuncle (malignant pustule) develops at the site of bacilli localization.
The disease prevails in individuals who come in contact with sick animals or raw materials con-
taminated with anthrax bacilli. This has a professional meaning (milkmaids, stock-breeders, furriers,
etc.).
The respiratory form is acquired through the air when working with material contaminated with
bacillary spores. The disease assumes the form of a severe bronchopneumonia. The bacilli are dischar-
ged in the sputum.
The intestinal form is due to ingestion of meat of sick animals. It is characterized by grave
lesions in the intestinal mucosa with haemorrhages and necrotic foci. The bacilli are excreted in the
faeces.

73
 The respiratory and intestinal forms are very serious (hard) and the death rate is very high (about
100).
Immunity: Immunity is antimicrobial and depends on the presence of protective antigens.
Immunity is high grade and stable. During infection specific sensitization develops, which is revealed
by skin-allergic reaction (with allergen anthraxin).
Prophylaxis and treatment: General measures of anthrax control are carried out in joint action
with veterinary workers. These measures are aimed at timely recognition, isolation, and treatment of
sick animals. Animals which have died of anthrax are burnt or buried on specially assigned territory;
not less than 2 meters deep, and covered with lime chloride.
Persons at high risk can be immunized with cell-free vaccine containing purified protective
antigen. (In our country we use STI alive vaccine
Treatment comprises timely intramuscular injection of anti-anthrax globulin and the use of
antibiotics (penicillin, tetracycline, and streptomycin).
Laboratory diagnosis (picture 2):
1.Microscopic.
2.Bacteriological: isolation of pure culture and differentiation (table 1).

3.Serological (Askoli thermo-ring-precipitation for revealing of thermostable polysaccharide antigen

of raw material).
4.Skin-allergic test (with anthraxin)
5.Biological.

BRUCELLOSIS
Brucella is the causative agent of brucellosis. The synonyms: undulant fever, Malta fever, Bruce
septicaemia, etc.
Brucellosis is a zoonotic allergic infection which leads to chronic state. Brucellosis is
characterized by fever, effecting motive and suppurating apparatus, cardiovascular, nervous (neural),
genitourinary systems of human organism. The discoverer was David Bruce in (1887). He isolated B.
melitensis from the spleen of British soldier stationed at Malta, who had died of Malta fever.
The brucellae are obligate parasites of animals and humans and are characteristically located
intracellularly. Brucella are subdivided into several species:
 B. melitensis (brucella of goats and sheep)
 B. abortus (brucella of cattle)
 B. suis (brucella of pigs)
 B. neotomae (brucella of forest rats)
 B. canis (brucella of dogs)
74
Causative agents of human organism are the first three species. Differentiation is based on CO 2
requirements, H2S production, sensitivity to dyes (basic fuchsin and thionine), and agglutination by
mono-specific sera, phage lysis, etc.
Morphology: Brucella are small, coccal, ovoid-shaped micro-organisms (coccobacilli) 0.5-0.7
µm in size. They are gram-negative, non-motile, and do not form spores or capsules.

Cultivation: The organisms are aerobic. The optimum temperature for growth is 37º C, the pH
6.8-7.2. Brucella organisms may be cultivated on ordinary media, but they grow best on liver-extract
agar and liver-extract broth. B. abortus requires 5-10 per cent CO2 for growth, whereas the other
species grow in air. On liver-extract agar the organisms form round, smooth (S-form) colonies with a
white or pearly hue. Dissociation of the organisms from S-form to the R-form has been demonstrated.
They grow slowly over a period of 2-3 weeks (the generation time -13 hours). In liver-extract
broth they produce turbidity and subsequently a mucilaginous precipitate settles at the bottom of the
tubes. Brucella organisms grow well on unfertilized eggs and on the yolk sac of a 10-12 day-old chick
embryo. Under the action of antibiotics they become L-forms.
Fermentative properties: Brucella does not liquefy gelatine, does not produce indole. Some
strains produce hydrogen sulphide, break down urea and asparagine, reduce nitrates to nitrites, and
hydrolyse proteins, peptones and amino acids, with release of ammonia and hydrogen sulphide. No
carbohydrates are fermented, although a small number of strains ferment glucose and arabinose.
Antigenic structure: Brucella contains surface Vi-antigen and somatic type-specific A and M
antigens, which are present in different amounts in the three major species. Brucella abortus and suis
contain much A as M; B. melitensis contains high quantity of M antigen. For identification of Bru-
cellae by antigenic structure absorption of agglutinins reactions (Kastellani reaction) is used.
Epidemiology and Resistance: Brucellosis is zoonotic infection. Brucellae are animal pathogens
transmitted to humans by accidental contact with infected animal faeces, urine, milk, and tissues. The
common sources of infection for humans are non-pasteurized milk, milk products and cheese and
occupational contact (eg, farmers, veterinarians, slaughterhouse workers) with infected animals.
Occasionally the airborne route may be important.
Brucellae are characterized by high resistance and viability. They survive for a long time at low
temperatures. The organisms live for 18 weeks in soil, urine, animal faeces, manure, hay dust and bran,
up to 16 weeks in ice, snow, butter and sheep’s milk cheese, from 12 to 16 weeks in sheep’s wool, for
30 days in dust, for 20 days in meat, and for 7 days in milk. Brucellae are sensitive to high
temperatures and disinfectants. At 60º C they are destroyed in 30 min., at 70º C in 10 minutes, and at
boiling point in a few seconds. They are very sensitive to phenol, formalin, chloramine and other
disinfectants.
Pathogenicity and Pathogenesis: The common routes of infection in humans are alimentary
(intestinal tract-ingestion of infected milk), droplet (mucous membranes), and contact route and the
organisms can enter through not damaged surfacing.
More pathogenic for human organism is B. melitensis.
The organisms progress from the portal of entry, via lymphatic channels and regional lymph
nodes, to the thoracic duct and the bloodstream, which distributes them to the parenchymatous organs
(liver, spleen, bone marrow, and other parts of the reticuloendothelial system and give rise to orchitis,
ostitis, arthritis, etc.). They localize intracellularly and can survive prolonged. Pathogenicity
determines endotoxin. They produce hyaluronidase, which promotes invasion and spreading of
bacteria. In pathogenicity takes place ability of these bacteria to reproduce in lymphoid-macrophagal

75
system. Human infection may be of following types: 1.Prymary latent infection with only serological,
but not clinical evidence
2.Acute septic with fever 39-40ºC, weakness, generalized lymphadenopathy, enlargement of
liver and spleen. Duration of fever is 3-4 days.
3.Prymary chronic metastatic-subfebrile fever, intoxication enlargement of liver and spleen.
Affection of nerves, genito-urinary systems and motor system(polyarthritis)
4.Secondary chronic metastatic
5.Secondary latent
After an incubation period of 1-3 weeks, non-specific symptoms resembling influenza occur. The
undulating (rising-and-falling) fever pattern. Weakness and fatigue are marked. Acute brucellosis
known as undulant fever is mostly due to B. melitensis. It is associated with prolonged bacteraemia
and with atypical and polymorphous symptoms. The structural and motor systems, haemopoetic,
hepatolienal, nervous and genital systems are often involved. Pregnant woman may have miscarriage.
Often brucellosis recurs, continuing for months and years. The death rate is not high, about 1-3 per
cent. Brucellosis in human has many clinical symptoms common to other diseases (tuberculosis,
rheumatic fever, enteric fever, typhoid fever). For this reason, the differential diagnosis of brucellosis
is of great importance.
Immunity: A characteristic immunity is acquired following brucellosis, the patient becoming
insusceptible to repeated infection. The activity of T-lymphocytes forms basis for infectious and post-
infectious immunity in brucellosis; phagocytosis plays a particularly important role. They produce
cross immunity in the body. The humoral factors; i.e. the production of opsonins, agglutinins take
place in rendering of brucellae. Cellular immunity causes delayed hypersensitivity, which is revealed
by skin-allergic tests.
Treatment: Antibiotics – tetracycline, erythromycin, streptomycin, levomycetin.
Prophylaxis: Comprises a complex of general and specific measures carried out in conjunction
with veterinary services. This includes:
1. Early recognition of brucellosis, hospitalization of sick individuals, exposure of the sources of the
disease.
2. Sanitary-veterinary measures.
3. Immunization by alive vaccine in an additional measure in districts, where there are cases of goat-
sheep brucellosis.
Laboratory diagnosis: The patient’s blood and urine (for isolation of the pathogen), serum (for
detection of agglutinins), milk and dairy products are examined. The methods are (picture 3):
1. Serological - The Wright (tube agglutination) and Huddleson (slide agglutination) reactions
are used. In some cases the complement-fixation test is used. Opsono-phagocyte test
2. Skin allergic test - Burne’s test is used. 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.
3. Bacteriological - Isolation of pure culture and identification of brucella species (table 1).

Table 1 DIFFERENTIAL CHARACTERISTICS OF BRUCELLA SPECIES

B.ovis,
Properties B.melitensis B.abortus B.suis B.canis,
B.neotomae
CO2 requirement - + - +
H2S production - + - +
Growth on dye media Thionin - - - +
fuchsin + + - +
Lysis by T6 phage RTD - + - -
RTDx104 - + + +
Agglutin. by mono- A - + + +
sp.ser. M + - - -
Catalase + + - +

76
Oxidase + + - +
Hyaluronidase + + + +
Reducing of nitrates + + + +

77
 TULARAEMIA
(F. tularensis)

The specific cause of tularaemia, Francisella tularensis, was discovered in 1912 by G. McCou
and C. Chapin in Tulare (California) and studied in detail by E. Francis.
Morphology: The tularaemia bacteria are pleomorphic short coccal-shaped (coccobacilli) or rod-
like bacteria measuring 0.2-0.7m. They are non-motile. In the animal body they sometimes are
surrounded by a fine capsule. They don’t form spores.
Cultivation: The tularaemia organism- facultative anaerobes, which does not grow on ordinary
media, but grows well at 37C in enriched media (yolk medium, blood agar with glucose and cystine).
Milk-white colonies are formed. The bacteria can be cultivated on media containing brain, spleen, and
liver tissues, heart extracts. They grow slowly. Culture collections on solid media may be well
preserved for 2-6 months.
Antigenic structure: The tularaemia bacteria contain somatic cell wall O antigen, and Vi
antigen, which induce antibody formation (agglutinins, precipitins). The common character of the
antigens of tularaemia and brucellosis agents in the agglutination reaction has been ascertained. This
fact must be taken into account in serological diagnosis of these diseases.
Ecology and resistance: These bacteria survive for long periods (over 3 months) at low
temperatures, in water 90, in soil 10 days. It is killed in several minutes by 3 per cent solutions of
lysol, formalin and alcohol. When heated to 60C it is destroyed in 10-15 minutes.
Tularaemia is a zoonotic infection. The nature hosts of this causative agent are water rats, field
voles, grey rats, field and house mice and other animals. Among the domestic animals: camels, sheep,
cats, dogs, pigs. Humans are infected with air borne dust, alimentary (by infected water and food). The
pathogen may also gain entrance into the body by the bites of arthropods and insects (ticks, mos-
quitoes, etc.).
Transmission from patients to healthy people is not noted.
Pathogenesis: The routes of entry are skin, mucous membranes of eye, mouth, nose, respiratory
tract and gastro-intestinal tract. The invasive properties are very high and the organisms can enter
through not damaged surfacing.
Pathogenesis and clinical findings depend on portal entry and by this case tularaemia may be of
the following forms: bubonic, ocular, anginose bubonic, abdominal or intestinal, pneumonic, and
generalized or primary septicaemia.
Incubation period is 2-7 days. After incubation the bacteria enter the lymph nodes where they
multiply and enter the blood and bacteraemia occur. The infectious properties depend on endotoxin.
Tularaemia is accompanied by specific allergy and remains for years and sometimes for life. In
recent years the death rate has been low and most patients recover owing to the wide use of antibiotics.
Immunity: Immunity is acquired humoral and cellular. Cellular immunity leads to delayed
hypersensitivity. The humoral immunity is strained (tension).
Prophylaxis and treatment: Specific prophylaxis: alive vaccine (Haisk’s-Elbert alive vaccine)
is used. For treatment antibiotics of large spectrum is used.
Laboratory diagnosis:
1.Biological
2.Serological: Indirect hemagglutination. Thermo- ring-precipitation.
3.Skin-allergic test:

78
 Sample tests

1.Which of the following factors are specific for Anthrax bacillus:

1.protective antigen
2.spores
3.endotoxin
4.exotoxin
a)1.2.4. b)1.2. c)2.4. d)2.3.

2.Which of the following vaccines used in Anthrax prevention:

a)EV vaccine
b)alive-corpuscular of Elbert-Haisky vaccine
c)DPT vaccine
d)STI alive attenuated vaccine

3.The virulence of B. anthracis is connected with:

1.capsule
2.exfoliatine
3.erythrogenic
4.oedaemic factor
a)1.2.4. b)1.2.3. c)1.4. d)2.4.

4.Which of the following are typical of Tularemia:

1.immediate hypersensitivity
2.delayed hypersensitivity
3.endotoxin
4.production of neuotoxins
5.superinfection
a).2.4. b)1.2. c)1.3. d)2.3.

5.The causative agent of Tularemia:

1.is gram negative
2.is coccobacterium
3.forms spore
4.is motile
5.
a).2.4. b)3.2. c)1.4. d)1.2.

6.Which of the following are typical of Brucellas:

1.aggression
2.cord-factor
3.invasion
4.W-antigen
a).2.4. b)1.2. c)1.3. d)2.3.

7.Which of the following are typical of Brucellas:

1.protein toxin
2.lipopolysaccharide toxin
3.mice toxin
4.hyaluronidase
a)2.3. b)1.2. c)1.3. d)2.4.

79
8.Which of the following are typical of the bacteriological method of Brucellosis diagnosis:
1.colonies appear after 18-24hours
2.colonies appear after 2-3weeks
3.meat-pepton agar is used
4.liver media is use
a)1.2.4. b)2.4. c)1.3. d)2.3.

9.The pathogenicity of causative agents of Plague depends on:

1.mice toxin
2.pesticine
3.F-antigen
4.M and A antigens
a)1.2.3. b)2.4. c)1.3. d)2.3.

10. Each of the following statements concerning Plague is correct, Except:

a)Plague is caused by a gram negative rod
b)Plague is transmitted to human by flea bite
c)the main reservoirs in nature are small rodents
d)Plague is caused by gram positive cocci

80
81
82
83
 CLOSTRIDIA – RESPONSIBLE FOR ANAEROBIC INFECTIONS:

1.Gas gangrene (polymicrobial anaerobic infection)

2.C. tetani – causative agent of tetanus
3.C. botulinum- causative agent of botulism

Anaerobes are classified into two groups:

1.Spore-forming gram positive bacteria which belong to family Bacillaceae and genus
Clostridium.
2.Non-spore-forming gram negative bacteria, which belong to family Lactobacillaceae, genus
Bacteroides and Fusobacterium. Important role in human organism plays family Bacillaceae,
genus Clostridium.
Medically important Clostridium species are: C. teteni (which causes tetanus); C. botulinum
(which causes botulism), Clostridiums which are causative agents of gas gangrene
(polymicrobial anaerobic wound infection).
The members of these species possess common properties: they are anaerobes, Gram positive,
large rods 3-10μm in size. They form spores. The spores are larger than the bacillary bodies, giving the
bacillus a swollen appearance, resembling a spindle (kloster-means a spindle). They are generally
motile (peritrichous), except C. perfringens, which is non-motile. They all produce exotoxin. They are
the members of normal flora in humans and animals, and are excreted into environment by the faeces
and form spores in soil.

GAS GANGRENE
Wound infection is characterized by toxaemia, intensive local oedema, intensive necrosis of
tissues and gas formation.
Gas gangrene is polymicrobial infection. This infection is caused by several species of
Clostridia in association with various aerobic and facultative anaerobic organisms (pathogenic
staphylococci and streptococci, decaying bacteria, etc.).
Gas gangrene is caused by 1. C. perfringens (was discovered by Welch in 1892 and named C.
welchii). This is the most important of the clostridia causing gas gangrene. The organism from
anaerobic infections is isolated in 90-100%. 2. C. novyi, isolated from wounds in 20-35℅ of cases. 3.
C. septicum (was discovered by Pasteur and Joubert in 1878) – 4-12 per cent. 4. C. histolyticum –
0.2-0.4%. Except these clostridia gas gangrene can be caused by C. sordelli (Sordell, 1912); C. fallax
(Sechen and Veinberg, 1915), which are met rarely in wounds.
Morphology: They all are gram-positive rods 4-22x1.5 µm in size.
C. perfringens is plump and short bacilli with truncated ends, about 4-6 µm. C. novyi is the largest rod
about 4-22 µm in size. The ends are rounded, arrangement is in chains. C. septicum is a pleomorphic
bacillus with rounded ends, about 3-8x 0.6 µ in size. C. histolyticum - small rods 1.5-3 µm in size, the
ends are rounded. They are aerotolerant and some growth may occur even in aerobic cultures.
All are motile except C. perfringens and only C. perfringens forms capsule.

Cultural properties: They all are anaerobes, but the level of anaerobic peculiarities is not the
same. C. novyi is strictly anaerobic. C. perfringens is less anaerobic than the other causative agents of
anaerobic infections. They grow on nutrient media, which are used for cultivation of anaerobes. The
optimum temperature for growth is 37 C, and optimal pH of medium is 6.0-8.0. Sediment and a uni-
84
form turbidity and large volumes of gas are produced in cultures grown on Kitt-Tarozzi medium. C.
histolyticum don’t produce gas but they rapidly dissolve liver tissue, which is in media. On blood agar
containing glucose (Ceisler media) C. perfringens forms smooth disk-like grey colonies with smooth
edges and raised centre. C. novyi form R-form colonies with haemolytic zone. C. septicum form
uniform pellicle on the background of haemolysis. C. histolyticum form small, greyish colonies. The
colonies of Clostridiums resemble discs or lentil deep in agar stab cultures.
Biochemical activity: The biochemical activity is high. The organisms lyse proteins and
carbohydrates. C. perfringens possess high carbohydrate and protein lysing activities. They lyse
glucose, lactose and mannite, saccharose with acid and gas formation; intensively coagulate milk about
1-3 hours, which is differential-diagnostic property. In litmus milk medium, it ferments lactose and
produce acid and gas. The acid clots the milk and the gas breaks up the clot resulting in stormy clot
reaction (stormy fermentation). Production of acid is also indicated by change in the colour of the
litmus from blue to red. They liquefy gelatine (during 2-3hours), possess lecithinase activity, reduce
nitrates to nitrites. C. histolyticum possess high protein lysing properties. They form H2S. This
organism peptonizes milk, liquefies gelatine and coagulates serum. C. septicum possess high
carbohydrate activity. They liquefy gelatine, don’t form indole. These bacteria reduce nitrates to
nitrites. The biochemical activity of C. novyi is low. They ferment some carbohydrates.
Toxins: They produce toxin with complex (compound) antigenic and chemical structure (about
12 distinct toxins), designated by Greek letters. Alpha () toxin: this is the most important toxin
biologically and responsible for the profound toxaemia of the gas gangrene. It is lethal, dermonecrotic
and haemolytic. It is thermostable. Beta (), Epsilon (€), Iota () toxins are lethal and necrotising, and
non-hemolytic, Etta (ή), Gamma (γ) toxins are minor lethal toxins. Delta (δ) toxin has a lethal effect
and is haemolytic. Theta (θ) toxin is an oxygen labile haemolysin antigenically related to streptolysin
O. It is also lethal and a general cytotoxic toxin. The kappa(κ) toxin is a collagenase. The lambda (λ)
toxin is a proteinase and gelatinase. The mu toxin (μ) is a hyaluronidase.
The toxins possess: 1. necrotic; 2. lethal; 3. neurotoxic; 4. leukotoxic 5. haemolytic activities
Gas gangrene Clostridia produce enzymes: fibrinolysins, hyaluronidase, collagenase, urease and
deoxyribonuclease.
Antigenic structure: C. perfringens strains are classified into 6 serologic types: A-F. It doesn’t
have practical importance. It is necessary the antigenic structure of toxins, which are the basic factors
of pathogenicity.
Pathogenicity: C. perfringens cause Gas gangrene (myonecrosis) and food poisoning. Habitat is
soil, where they form spores and are preserved for a long time. Myonecrosis results from
contamination of wound with soil or faeces. Food poisoning is transmitted by ingestion of
contaminated food.
Gas gangrene in wounds is caused by germination of spores under anaerobic conditions and the
production of toxins and enzymes, which damage cell membranes, vascular system resulting in
hemolysis. Degradative enzymes produce gas in tissues. Pain, oedema, and cellulitis occur in the
wound area. Crepitation indicates the presence of gas in tissues. Hemolysis and jaundice are common,
as are blood–tinged exudates. Shock and death can ensue. Mortality rate is high. Clinically gas
gangrene possesses several forms, which depend on associations of anaerobes and other flora: 1.
Emphysematous. In this form are associated C. perfringens and C. septicum and gas formation,
intoxication is indicated. 2. Toxoid form is caused by C. perfringens and C. novyi association. In this
form the high amount of toxins is excreted. The quantity of gas is low. This is the critical form and the
rate of death is high. 3. Mixed form is the association of anaerobes and aerobes. During this form
oedema and emphysema are described. 4. Phlegmonous form – anaerobes and cocci. 5. Suppurative
form – anaerobes and decaying bacteria.
Food poisoning: Spores are located in soil and can contaminate food. Spores are heat-resistant
and can survive cooking and germinate. The organisms grow to large numbers in reheated foods,
especially meat dishes. Entering the gastrointestinal tract, heat labile enterotoxin is produced. The
mechanism of action is the same as that of enterotoxin of S. aureus (it acts as a super antigen). After a
short incubation period is 8-16 hours; watery diarrhoea with cramps and vomiting occurs. It resolves in
24 hours.
Clostridium difficile –it is a long, slender, Gram-positive bacterium. Spore-forming, spores are
large, oval and subterminal. It is non-hemolytic, saccharolytic and weakly proteolytic. C. difficile is
85
the most common cause of nosocomial diarrhea. The disease usually follows from the use of broad-
spectrum antibiotics to which the organism is resistant.
C. difficile causes acute colitis with bloody diarrhea and pseudomembranous colitis. C. difficile
is an opportunistic organism that rarely causes disease and does so only when normal flora is lost.
There appears to be little protective immunity following infection. The disease is prevented by
restricting the use of antibiotics associated with C. difficile outbreaks, for treatment, the antibiotic
causing the disease should be discontinued and the disease treated with vancomycin and
metronidazole. Two high molecular weight exotoxins, A and B, are involved in the pathogenesis of the
condition.
Diagnosis can made by demonstrating the toxins in the faeces of the patients by its characteristic
effect on Hep-2 and human diploid cell cultures or by ELISA. The toxin is specifically neutralised by
the C. sordelli antitoxin. C. difficile can also be grown from the faeces of patients.
C. difficile strains are usually resistant to most antibiotics. Treatment is with metronidazole.
Vancomycin and bacitracin are also useful.
Imunity: Immunity acquired after gas gangrene is not high and in short duration it is of an
antitoxic and anti-enzymatic character. Antitoxic immunoglobulins cannot produce tense, high
immunity because toxic dose of the toxins is lower than immunogenic dose. Antimicrobial
immunoglobulins haven’t protective function.
Treatment: Surgery is the most important prophylactic and therapeutic measure in gas gangrene.
Wounds should be cleans and debrided. For treatment antibiotics are used, penicillin is choice.
Penicillin may be given for prophylaxis too. Polyvalent antitoxin (anti-gas gangrene serum which
included C. perfringens, C. septicum, C. novyi antitoxins 10.000 units) is injected intramuscularly or,
in emergencies, intravenously as a prophylactic measure. If the antitoxic serum gives for treatment
the dose which recommended is 50.000 units. Treatment by antibiotics with antitoxic serum gives
good effect. For specific prophylaxis associated, sorption, clean polyanatoxin is used, which included
Tetanus anatoxin, C. perfringens and C. novyi anatoxins and Botulinum anatoxin.
Laboratory diagnosis(picture 1):
1.Microscopic.
2. Bacteriological(isolation of pure culture and identification-table 1).
3.Biological-toxin neutralization test As an investigated material are used: infected, necrotized
tissues, discharge from the wound, and dressing (bandaging) materials.

TETANUS
Tetanus is an acute wound infection with contraction of the muscles, asphyxia, and affection of
organs and systems. The causative agent is C. tetani (was discovered by Monastirsky, and Kitasato
isolated the pure culture).
Morphology: Gram positive, slender rods about 4-8 x 0.5-1.0μm.
They form spherical, terminal spores, which give the bacilli drumstick
appearance. They are non-capsulated and motile (peritrichate).
Cultural properties: Obligate anaerobes. They grow only in the
absence of oxygen. The optimum temperature is 37º C and pH 7.4. The
86
growth is improved by blood and serum. On solid media they form translucent, greyish colonies and a
zone of haemolysis is produced around colonies. Sediment and a uniform turbidity are produced in
broth (Kitt-Tarozzi broth) with libration of gas and a peculiar odour (as a result of proteolysis).
Biochemical activity: No carbohydrates are usually fermented. The protein lytic activity is low
too. They coagulate milk slowly. C. tetani causes slow gelatine liquefaction.
Antigenic structure: C. tetani is not serologically homogeneous. They contain somatic O
antigen and flagellar H antigen by which they are subdivided in 10 serotypes. It hasn’t got diagnostic
meaning, because all 10 types produce the same exotoxin.
Resistance: Vegetative cells of the tetanus organism withstand a temperature of 60-70º C for 30
minutes, and are destroyed quite rapidly by all commonly used disinfectants. The spores are very
resistant, and survive in soil and on various objects over a long period of time and withstand boiling 1-
3 hours. For destruction of spores autoclaving at 121º C for 20 minutes is recommended. The spores
are resistant to disinfectants and are killed by exposure to 5 per cent phenol solution for 8-10 hours.
Pathogenesis and pathogenicity: Spores are widely spread in soil. The portal of entry is usually
a wound site. C. tetani spores implanted in a wound can germinate and multiply only if the conditions
are favourable (necrotic tissue, and poor blood supply in the wound). In a wound when the organisms
multiply they produce exotoxins:
1. Tetanospasmin (functional blockator, neurotoxin).
2. Tetanolysin is a membrane toxin, heat labile haemolysin.
Tetanolysin is heat labile, oxygen labile haemolysin.
Tetanospasmin is responsible for the clinical manifestation of tetanus, powerful toxin and yields
only botulinum toxin. The gastro-intestinal enzymes can’t destroy this toxin but given orally is not
dangerous because by the gastrointestinal mucous membrane this toxin can’t be absorbed.
Tetanospasmin is oxygen stable, heat labile. It is good antigen and is specifically neutralized by the
anatoxin. The tetanospasmin reaches the motor centres of the spinal cord via the peripheral nerves (it
moves along the axial nerve cylinders). The toxin is specifically and avidly fixed by gangliosides of
the grey matter of the nervous tissue. The tetanus toxin specifically blocks synaptic inhibition in the
spinal cord. The abolition of spinal inhibition causes uncontrolled spread of impulses initiated
anywhere in the central nervous system. This results in muscle rigidity and spasms due to the
simultaneous contraction of muscles (agonists and antagonists) in the absence of reciprocal inhibition.
Incubation period is 6-14 days (but sometimes it is shorter and in this case the disease is severe). The
onset of the disease is characterized by persistent tonic muscular spasms at the site of penetration of
the causative agent. This is followed by tonic spasms of the jaw muscles (trismus), face muscles (risus
sardonicus), and extremities are affected. This is the development of the clinical picture of descending
tetanus (the disease in experimental animals-white mice is ascending). The patient lies in bed, resting
on his head and hips with body bent forward like an arch (opistotonus).

The death rate is 35-70%. Death usually is the result of the spasms of the respiratory tract.
Healthy people and animals, which discharge the organism in their faeces into the soil, are sources of
the infection. Spores of C. tetani can be demonstrated in 50-80℅ of examined soil specimens. The
majority of tetanus cases in adults occur among farm workers, and more than 30-35 per cent of total
incidence of the disease is associated with children from 1-15 years old. In more than 50 per cent of
cases tetanus is acquired as the result of wounds of the lower extremities inflicted by spades, nails, and
stubbles during work in the field. C. tetani may gain entrance into the body of a newborn infant
through the umbilical cord and into a woman during childbirth, through the injured uterine mucosa.
87
 Immunity: Immunity following tetanus is mainly antitoxic in character, and of low grade.
Reinfections may occur.
Treatment: Tetanus immune globulin is used. Penicillin or metronidazole is useful. An adequate
airway must be maintained and respiratory support given. Benzodiazepines should be given to prevent
spasm.
Prevention: Prevention includes: 1. An active immunisation (vaccination). 2. Special
(extremely) immunization: immune prevention during trauma.
1. Active immunisation: Polyvalent vaccine is (DPT) usually given to children. This vaccine
contains diphtheriae anatoxin, pertussis vaccine (killed Bordetella) and tetanus anatoxin. Vaccination
starts in newborn infants at 3-5 months. Vaccine is given three times once 45 days. First revaccination
is injected at the age of 1-2. II revaccination is after 5 years at the age of 6-7 years. III revaccination is
at the age of 11-12 years (DT). IV revaccination is at the age of 16-17 years (DT).
Nowdays pentavaccine is recommended-DPT+HBVvaccine+hemophilus influenzae vaccine.
Vaccination starts in new born infants at 1.5months.
Special immunization: When trauma occurs, the wound should be cleaned, debrided and
depending on conditions passive or active-passive immunisation is given. 1. Passive immunisation:
antitoxic serum is injected. 2. Active–passive immunisation: First tetanus anatoxin is injected, after 30
minutes antitoxic serum or extremely revaccination is administered.
Laboratory diagnosis:
1.Microscopic
2.Bacteriological
3.Biological: toxin neutralization.
BOTULISM
Clostridium botulinum is the causative agent of severe food intoxication botulism, which is
characterized by damaging the central nervous system especially medulla oblongata and medulla
spinalis (spinal cord) with bulbar and ophthalmoplegic syndromes.
C. botulinum was first isolated by van Ermenghem (1896) from a piece of ham that caused an
outbreak of botulism (the name botulism is derived from sausage- in Latin botulus means sausage).
Morphology: It is a gram-positive large rod 4-9 x 0.6-1μm in size, the arrangement is irregular,
and sometimes they form short chains. They are motile, peritrichous, non--
capsulated. They form subterminal, oval, bulging spores (it gives bacilli
tennis racket appearance).
Cultural properties: They are strict anaerobes. They grow only under
anaerobic conditions. The optimal temperature for growth is 30-40º C. They
grow on ordinary media. The colonies are large, irregular and semitranspa-
rent. On glucose-blood agar they form S-type colonies and zone of
hemolysis around the colonies. In broth they form sediment and diffuse turbidity. In deep agar they
grow and form colonies like a lentil.
Biochemical activity: This activity is variable. All the species of C. botulinum produce
gelatinase, lecithinase and H2S. They possess saccharose-lysing activity too. They ferment glucose,
levulose, and fructose with acid and gas formation.
Antigenic structure: There are seven antigenic groups A, B, C, D, E, F, G. Group C is
subdivided into Cα and C groups. They are similar by morphology and by cultural properties. For
identification the antigenic specificity of toxins which they produce is necessary. The toxins produced
by the different types are identical in their pharmacological activity, but are neutralised only by the
homologous antiserum (they cannot produce cross-reacting immunity). For human organism the most
important species are A, B, E, the others cause infections in humans’ rarely.
Pathogenesis: Botulism is severe toxinfection, which develops during the use of food, which
contains toxins and microbial cells. The most common offenders are spiced, smoked, vacuum-packed,
or canned alkaline foods that are eaten without cooking. In such foods, spores of C. botulinum
germinate; under anaerobic conditions, vegetative forms grow and produce toxin. The main pathogenic
factor is neurotoxin, and the main clinical symptoms depend on this toxin. This is a powerful toxin and
0,001 mg (1-2 μg) is fatal for humans. It is produced intracellularly and appears in the medium only on
the death and autolysis of the cell. This toxin is stable to digestive enzymes and is not destroyed by the
action of gastric juice. C. botulinum E species produces pro-toxin which toxicity is weak; trypsin and
88
other proteolytic enzymes activate this progenitor toxin to active toxin, which toxicity rises by 10-100
times. The absorption of botulinum toxin from gastrointestinal mucosa into bloodstream is very fast.
By bloodstream toxin spreads through all the organism and selectively acts on medulla oblongata and
cardiac system. The toxins are neurotoxic proteins, which are relatively stable, being inactivated only
after 30-40 minutes at 80º C and 10 minutes at 100º C. It is made up of heavy and light chains linked
by a disulfide bond. The heavy chain is thought to specifically and avidly bind the toxin to the motor
nerve end plates and with the internalization of the toxin. The light chain blocks the calcium-mediated
release of acetylcholine and blockades functional activity of neurons. It suppresses the parasympathica
nervous system. Dissociation between sympathica and parasympathica nervous systems is detected.
The toxin is polypeptide encoded by lysogenic phages. Incubation period is 6-24 hours, symptoms
begin with vomiting, thirst, constipation, visual disturbances (incoordination of eye muscles, double
vision), inability to swallow, and speech difficulty; signs of bulbar paralysis are progressive, and death
occurs due to respiratory paralysis or cardiac arrest. Gastrointestinal symptoms are not regularly
prominent. There is no fever. The patient remains fully conscious until shortly before death. The
mortality rate is high (60). Patients who recover do not develop antitoxin in blood. Postinfectious
immunity is absent. It depends on weak immunogenic properties of botulinum toxin.
Ecology: Their natural habitat is the soil or the intestinal tract of animals and humans, where
they live as saprophytes. In the environment they form spores. Since spores of C. botulinum are widely
distributed in soil, they often contaminate vegetables, fruits, and other materials. In the soil and animal
organisms C. botulinum form spore, which is stable to temperature (by boiling and in minus 253º C -
6-7 hours). In 5 phenol solution they survive 1 day. The toxin is relatively heat-labile; boiling for
several minutes inactivates it. Thus, sufficient cooking can prevent disease.
When foods are canned or otherwise preserved, they either must be sufficiently heated to ensure
destruction of spores or must be boiled for 20 minute before consumption. Strict regulation of
commercial canning has largely overcome the danger of widespread outbreaks, but commercially
canned mushrooms and vichyssoise cause death. At present, the chief danger lies in home-canned
foods, particularly string beans, corn, peppers, olives, peas, and smoked fish or vacuum-packed fresh
fish in plastic bags. Toxic foods may be spoiled and rancid, and cans may swell; or the appearance
may be innocuous. The risk from home-canned foods can be reduced if the food is boiled for more
than 20 minutes before consumption.
Treatment: Polyvalent (A, B, E) antitoxin must be promptly administered intravenously with
customary precautions. Adequate ventilation must be maintained by mechanical respirator, if
necessary. These measures reduce the mortality rate from 60 to below 25.
Prevention: Proper sterilization of all canned and vacuum-packed foods is essential. Food must
be adequately cooked to inactivate the toxin. Swollen cans must be discarded (clostridial proteolytic
enzymes form gas, which swell cans).
1. Laboratory diagnosis (picture2):
2. Microscopic.
3. Bacteriological(isolation of pure culture) Serological: passive hemagglutination
4. Biological (toxin neutralization test).

89
 Sample tests
1.Botulotoxin is:
a)cytotoxin
b)membrane toxin
c)erythrogenic toxin
d)functional blockator

2.C. tetani is:

1.coccobacteria
2.large gram (+) rod
3.forms terminal spore
4.non-motile
a)2.3. b)3.4. c)2.4. d) 1.3.

3.C. tetani is:

1.strict anaerobe
2.has W-V-antigens
3.grows on Kitt-Tarozzi media
4.has flagellar antigen
a)2.3. b)3.4. c)2.4. d) 1.3.4.

4.The tetanospasmin is:

1.cytotoxin
2.neurotoxin
3.erythrogenic toxin
4.functional blockator
a)2.3. b)3.4. c)1.2.4. d)2.4.

5.The following statements concerning C. perfringens are correct, Except:

a)is an important cause of gas gangrene
b)is an important cause of food poisoning
c)produces an exotoxin that degrades lecithin and causes necrosis and hemolysis
d)it produces a toxin that inhibits the release of acetylcholine at the synapse

6.Gas gangrene is an:

1.air-droplet infection
2.clostridial infection
3.polymicrobial infection
4.anaerobic infection
a)1.3. b)3.4. c)1.2.4. d)2.3.4.

90
91
92
 CHLAMYDIA

They are in the genus Chlamydia, family Chlamydiaceae, order Chlamydiales. This family
contains a single genus Chlamydia and three species: C. trachomatis; C. psittaci; C. pneumonia.
Chlamydiae are obligate intracellular parasites, because they lack mechanisms for the production
of metabolic energy and cannot synthesize ATP. This defect restricts them to an intracellular existence,
where the host cell furnishes energy-rich intermediates. The organisms are characterized by low
metabolic activity and are cultivated at 33-41º C in yolk sac of a chicken embryo. They are small in
size and measured 0.2-1.5μm in diameter. Examination of Chlamydiae indicates that the outer cell wall
resembles the cell wall of gram-negative bacteria. It has relatively high lipid content. It is rigid but
does not contain a typical bacterial peptidoglycan. Chlamydia contains both RNA and DNA to that in
bacteria. Chlamydiae have a replicative cycle different from that of all other bacteria(picture1). The
extracellular forms of Chlamydiae are named elementary bodies -EB(metabolically less active),
which are about 0.3μm in diameter attached to the susceptible host cell. The type and range of
susceptible host cell vary with the species and biovar. The host cell phagocytizes the elementary body,
housing it in a vacuole. Chlamydia dependent modification of the endocytic membrane prevents
lysosomal fusion and thus escapes degradation. This elementary body is reorganized into initial bodies
(vegetative forms), after transformation of them to the large, metabolically active reticulate body-
RB). The reticulate body grows in size and divides repeatedly by binary fission, producing multiple
reticulate bodies. After it the reticulate bodies begin to convert back into elementary bodies. The new-
formed elementary bodies may be librated from the host cell (by rupture) to infect new cells. Within
cells, the site of replication appears as an inclusion body, which can be stained (by Romanovsky-
Giemsa method) and visualized microscopically. These inclusions are useful for the identification of
these organisms in the clinical laboratory. Growth, reproduction and maturation of Chlamydia
organisms are completed in 40-72 hours.
Antigenic structure: They contain 1.Thermo-stable, lipopolysaccharide antigen, which is genus
specific and common to all Chlamydia. This antigen is present in all stages of the development cycle
and can be identified by the CFR. 2. Species- specific protein, thermo-labile antigen present at the
envelope surface. These are present in all strains of a Chlamydial species. They help in classifying
Chlamydia into the species (trachomatis, psittaci, etc.). 3. Outer membrane proteins by which
Chlamydiae have been classified into many serological variants.
General characters:
1.They are small, obligate intracellular, gram negative bacteria
2.They possess both RNA and DNA, ribosomes and cell wall similar to that of gram negative
bacteria. However, they differ from most true bacteria in that they do not have peptidoglycan.
3. they lack the ability to produce their own ATP, therefore, they use host’s ATP.
4.They multiply by binary fission
5.They are non-motile and stain poorly with Gram, but readily with Romanovsky – Giemsa.
They are gram negative. Giemsa staining is preferable for staining inclusions in cell culture. Inclusion
bodies of Chlamydia are basophilic in nature and look like beef eye.
6.They can also be demonstrated by direct immunofluorescence staining
7.They multiply in the cytoplasm of the host cell forming microcolonies or inclusion bodies
which drape around the nucleus like a cloak or mantle (chlamys means mantle)
8.Like gram negative bacteria, the outer membrane of various Chlamydia possess several
proteins of which major outer membrane protein has species-specific epitopes
9.They possess a genus–specific lipopolysaccharide-protein complex antigen
10.They infect a wide spectrum of vertebrate hosts including birds, mammals and humans
11.They are susceptible to a wide range of antibiotics such as tetracyclines, erythromycin,
macrolides and rifampicin.
Pathogenic factors: The pathogenic properties depend on surface antigens, which suppress the
response mechanisms of human organism. In pathogenicity Endotoxins and Exotoxins participate.
The Endotoxin is lipopolysaccharide of the cell wall, which is similar to gram-negative
bacteria’s LPS toxins.
Exotoxin is thermolabile protein substance.

93
 Chlamydia can blockade formation of phagolysosoms (development of incomplete
phagocytosis).
On the basis of antigenic structure Chlamydiae are subdivided into: Chlamydia trachomatis
causative agent of trachoma, conjunctivitis and lymphogranuloma venereum, urethritis. Chlamydia
psittaci is the causative agent of ornithosis, pneumonia, meningoencephalitis and gastroenteritis.
Chlamydia psittaci (the discoverer was K. Meyer, 1933) is the causative agent of ornithosis,
which is an acute infection with intoxication and defeated of lungs, CNS and with hepatolienal
syndrome.
Ornithosis (GK. ornis bird) is disease of many birds.
Chlamydia psittaci infects birds (parrots, pigeons, chickens and many other species of birds) and
many mammals. Humans are infected primarily by
inhaling organisms in dry bird faeces (they may also
contract the disease cutting poultry, cleaning cages,
and taking care of birds).
The agent enters the human body through the
respiratory tract. The adhesion is on the epithelium of
bronchus, bronchiolus where the reproduction occurs.
Then, the aetiological agent invades the blood and
produces bacteraemia, which lasts for a week or even
longer. As a result of various cycles, which undergo
within the tissues and organs, disturbances of
metabolism appear and intoxication and allergy
develop.
Incubation period is 6-17 days. The onset is sudden, with fever 39-40º C, severe headache,
myalgia, nausea, vomiting, cough, anorexia and sore throat. The clinical picture often resembles that of
influenza, pneumonia, or typhoid fever (clinically there are 3 forms: pneumonia, influenza, typhoid).
Immunity: During infection immunoglobulins are produced but they don’t have high protective
properties and immunity following the disease is relative and of a short duration. Repeated infections
occur.
Prophylaxis and treatment: For treatment antibiotics are used, especially tetracyclines are the
drugs of choice. The prophylaxis comprises sanitary-veterinary measures: early diagnosis, isolation
and hospitalization of patients. Sick birds are killed and their nesting places are disinfected. In view of
a high ornithosis incidence among pigeons, it is necessary to double veterinary control measures and
restrict or ban pigeon breeding in towns or near poultry farms.

CAUSATIVE AGENT OF TRACHOMA

By biological properties they are similar to Chlamydia genus. They grow in embryonated eggs at
35 C and in some cell cultures. Chlamydia trachomatis is subdivided
into 15 serotypes: L1; L2; L3 are causative agents of venereal
lymphogranulomatosis. A; Ba; B and C are the causative agents of
trachoma. D-K serotypes cause urethritis and eye infections.
Trachoma is an anthroponose infection. The reservoir of the
causative agent is human. Trachoma spreads by contact, by using
common towels, washing in communal basin, and by dirty hands and
flies. This is a family infection.
Trachoma (trachys-GK, means rough) is chronic infection with
chronic inflammation of the conjunctiva and cornea with hyperplasia
of adenoid tissue and hypertrophy of the follicles, which resemble
transparent granules. Clinically there are four phases: 1. The
inflammation of eye conjunctiva, and mucopurulent secretion. 2.
Development of inflammatory processes and cicatrix formation 3. The cicatrix formation process is
high. 4. Ending of cicatrix formation. It may lead to blindness. Cytoplasm of the affected cells contains
microcolonies of large primary and transitory bodies of Chlamydia organisms surrounded by a
vesicular membrane. These bodies resemble inclusions, which L. Halberstaedter and S. Prowazek
described in 1907, and they have diagnostic meaning.
94
 Treatment: Antibiotics such as Tetracyclines and Sulphonamides are used.
Immunity: Acquired immunity is of a short duration.
Prophylaxis: Prophylaxis comprises timely recognition and proper treatment of patients, a
dispensary service in disease foci, observance of hygiene in working and living conditions, and
improvement of the welfare and cultural level of the population.
Chlamydia trachomatis is the causative agent of conjunctivitis in newborns, or inclusion
blennorrhoea, which is marked by infiltration of the conjunctiva, particularly that of the lower eyelid.
The sources of infection are mothers in whose genitourinary system the causative agent is preserved, it
is transmitted to the infant during delivery. Adults are infected when they go swimming in small ponds
and non-chlorinated swimming pools. The disease in them follows the course of acute follicular
conjunctivitis. The incubation period is 10-12 days.
Treatment: with sulphanilamides and antibiotics (the administration of silver nitrate for the
prophylaxis of gonorrhoeal blennorrhoea does not prevent the development of inclusion blennorrhoea).

CAUSATIVE AGENT OF VENEREAL

LYMPHOGRANULOMATOSIS
Chlamydia trachomatis organism is responsible for venereal lymphogranulomatosis in human.
Genital routes transmit the infection. Chlamydia trachomatis causes non-gonococcal urethritis and
occasionally, epididymitis, prostatitis, or proctitis in men. In women, C. trachomatis causes urethritis,
cervicitis, and pelvic inflammatory disease, which can lead to sterility and predispose to ectopic
pregnancy. Patients with genital tract infections caused by C. trachomatis have a high incidence of
Reiter’s syndrome, which is characterized by urethritis, arthritis, and uveitis. Reiter’s syndrome is
an autoimmune disease caused by antibodies formed against C. trachomatis cross-reacting with
antigens on the cells of the urethra, joints, and uveal tract.
C. trachomatis L1-L3 immune types cause lymphogranuloma venereum, a sexually transmitted
disease with lesions on genital and in lymph nodes.
Infection by C. trachomatis leads to formation of antibodies and cell-mediated reactions but not
to resistance to reinfection or elimination of organisms.
Treatment –treatment is by the new macrolide antibiotics(clarithromycin, azithromycin).
Laboratory diagnosis – CFR, IF, ELISA

Sample tests

1.The following are typical of Chlamydia, Except:

a)formation of elementary body during the proliferation
b)formation of reticular body during the proliferation
c)presence of gram negative cell wall
d)spore formation
2.Which of the following are typical of Chlamydia:
1. are obligate intracellular parasites
2.have cellular structure
3.appearance in the form of elementary bodies
4.growth in artificial nutrient media
a)3.4. b)1.2.3. c)1.3.4. d)2.4.
3.Which of the following are virulence factors of Chlamydia:
1.surface antigens
2.endotoxin
3.exotoxin
4.M-protein
a)3.4. b)1.2.3. c)1.3.4. d)2.4.
4.Trachoma is characterized by:
1.is chronic infectious disease
2.defeating of urethra
3.defeating of conjunctiva
4.transmission by sexual rout a)1.3 b)1.2.3. c)3.4. d)2.4.
95
 Picture 1

96
 RICKETTSIAE
RICKETTSIAL DISEASES
Rickettsiae belong to the order Rickettsiales, family Rickettsiaceae.
Rickettsias are obligate intracellular parasites. They cannot grow and multiply on artificial
nutrient media. They can grow and multiply only within host cell. They grow readily in the yolk sac of
the embryonated egg, and in cell culture. In this respect, they are similar to viruses. However, in
morphological and limited biochemical aspects, they resemble bacteria (Prokaryotes) and are therefore
classified as such. Purified Rickettsiae contain both RNA and DNA in a ratio similar to that in
bacteria. Rickettsiae have cell wall that are made up of peptidoglycan containing muramic acid and
diaminopimelic acid and thus resemble the cell wall of gram-negative bacteria. They divide like
bacteria (binary fission). Rickettsias are pleomorphic appearing either as rods, or as cocci, and they
occur singly, in pairs, in short chains or in filaments (picture1). Rickettsiae grow in different parts of
the cell. Those of the typhus group are usually found in the cytoplasm; those of the spotted fever
group, in the nucleus. Rickettsiae are subdivided into the following groups:

Typhus fever group- this group of diseases consists of epidemic typhus, recrudescent typhus (Brill-
Zinsser disease) and endemic typhus.

EPIDEMIC TYPHUS DISEASE

(R. prowazekii)
BRILL-ZINSSER DISEASE
Epidemic typhus (louseborne typhus) is an acute, cyclic infectious disease with fever and skin
rash, known as status typhousus (the name typhus comes from the cloudy state of consciousness in the
disease) and by damaging nervous and cardiovascular systems. The causative agent is R.
prowazekii(named after von Prowazek, who died of typhus fever while investigating the disease).
Morphology: Rickettsias are pleomorphic Prokaryotes. It may be spherical, thread-like and rod-
like. The organisms are 0.8-4.0 μm in length and 0.3-0.6 μm in breadth. The thread-like forms may
reach 40 μm in length. Rickettsiae are gram-negative, and readily are stained red according to

97
Zdradovsky. According to Romanovsky-Giemza they are stained bluish purple. They are non-spore
forming, non-capsulated. They contain powerful mucous microcapsular coat (layer)-capsule like
structure. They are non-motile.
Culture: They aren’t reproduced on artificial nutrient media.Growth generally occurs in the
cytoplasm of the infected cells. Rickettsia are multiplied in chicken embryo (they cause death of
embryon after 6-13 days of inoculation) and in cell culture (fibroblast, HeLa, Hep-2 with patches
formation. Laboratory animals such as guinea pigs and mice are useful for the Rickettsiae from
patients. The optimum temperature for growth is 32-35ºC.
Antigenic structure: They contain two antigens: 1. Surface, group-specific, soluble antigen,
which is soluble in ether. This is thermo-stable lipopolysaccharide complex. This antigen protective
antigen, and possess immunogenic properties. This kind of antigens has other Rickettsias too (e.g., R.
rickettsii the causative agent of Rocky Mountain spotted fever). The polysaccharide of this antigen is
similar to polysaccharide of Proteus vulgaris strains and patients with rickettsial infections develop
antibodies that variably agglutinate Proteus vulgaris strains. OX19, OX2 antigens of Proteus vulgaris
are used in serological reactions to diagnose rickettsial diseases (Weil-Felix reaction). 2. Internal, type
specific, thermolabile, non-soluble antigen. It is protein-polysaccharide complex.
Pathogenicity and pathogenesis: The pathogenicity depends on adherence on cholesterol
containing cell receptors. Entering into the cell occurs by endocytosis and they are multiplicated in
macrophages, fibroblasts and in endothelial cells of small blood vessels causing death of these cells.
After destroying the cells, Rickettsia enters into the blood causing defeating of endothelial cells.
Damage to the vessels of the skin results in the characteristic rash and oedema and hemorrhage caused
by increased capillary permeability Rickettsias as other gram-negative bacteria contain endotoxin,
which is lipopolysaccharide of the cell wall which causes fever and petechiae, but its role has not been
confirmed. The organisms contain thermolabile protein in the capsule like structure, which possess
toxic properties (injection of this protein into the white mice caused the death of mice). The source of
infection is human. The patient is the source of infection, and the body lice are the vector. Rickettsias
are transmitted to humans by the bite of the arthropods.
R. prowazekii has a life cycle limited to humans and to the human louse (Pediculus humanus corporis
and Pediculus humanus capitis). After sucking blood of a typhus patient, the body louse becomes
infective in 3-10 days. Rickettsias are multiplied in the guts of lice. Infection with typhus takes place
not through the bite of a louse but by rubbing the organisms, which are discharged during defecation
into the skin, or when the lice are crushed and organisms penetrate abrasions and scratches on the skin
and mucosa. Rickettsiae are not transmitted from one generation of lice to another.
Rickettsiae multiply in endothelial cells of small blood vessels and produce vasculitis. The cells
become swollen and necrotic; there is thrombosis of the vessel, leading to rupture and necrosis.
Vascular lesions are prominent in the skin but vasculitis occurs in many organs and appears to be the
basis of hemostatic disturbances. Disseminated intravascular coagulation and vascular occlusion may
develop. In the brain, aggregations of lymphocytes, polymerphonuclear leukocytes, and macrophages
are associated with the blood vessels of the grey matter; these are called typhus nodules. The heart
shows similar lesions of the small blood vessels. Other organs may be also involved.
The incubation period is 5-15 days. After a short prodrome period the infection begins with high
fever (39-40C), chills and fever lasts for about 2 weeks, headache (status typhousus). The
characteristic rash appears on the 4th and 6th day starting on the trunk and spreading over the limbs but
sparing the face, palms and soles. Towards the second week, the patient becomes stuporous and
delirious. The recovery is slow, because the pathogenic effect is very high in the nervous and cardio-
vascular systems. The mortality rate is about 1-2.
Immunity: The post infectious immunity is stable. The incidence of reinfections has been
described. This is recurrences of the first disease and is known as Brill-Zinsser’s disease. In some
individuals, who recover from Epidemic typhus, Rickettsia can persist for many years in the lymph
nodes of an individual without any symptoms being manifest. In some conditions infection can recur.
The signs and symptoms of Brill-Zinsser’s disease are similar to those of epidemic typhus but are less
severe (milder), the duration of the disease is shorter (less than two weeks), skin rash is rare, fever is
erratic and case fatality rate is lower. Patient develops severe headache, malaise and myalgia.
Treatment: The treatment of choice for all rickettsial diseases is tetracycline, with
chloramphenicol as the second choice.
98
 Prophylaxis: 1.Early recognition, isolation, and hospitalisation of patients. 2. Sanitation
measures in the disease foci (fumigation). 3. Registration and observation of all individuals who have
been in contact with patients; systematic application of delousing measures among the population and
improvement of their education in hygiene. 4. For specific vaccination several types of vaccins are
used (alive combined- from Rickettsia and their antigens and chemical-from antigens).

MURINE TYPHUS
(ENDEMIC TYPHUS DISEASE)
The causative agent is Rickettsia typhi (R. mooseri) was discovered in 1928 by H. Mooser.
Morphologically, antigenic properties cultivation is the same with R. Prowazekii. The organism
is more pleomorphic than R. Prowazekii. R. Mooseri measure of 0.3-1.3 μm in size.
Pathogenicity depends on endotoxin and protein toxin.
Endemic typhus is a milder disease than epidemic typhus. Rats are reservoir and the rickettsia are
passed from rat to rat by ectoparasites
(rat flea - Xenopsylla cheopis and rat louse (polypax spinulosa). Rat flea acquires infection by feeding
upon a rickettsiaemic rat. The rickettsia is multiplied in the gut of the flea. It may infect other
susceptible rats and thus a natural cycle of flea-rat-flea infection may become established. Infection in
flea is not transmitted transovarially.
Rats and mice are main reservoirs of the causative agent in nature. Humans contract endemic
typhus from rodents. Human beings may also be infected by the bite of the rat tick. When infected flea
takes a blood meal, it defecates on the host. The latter rubs the infected faeces into the minute
abrasions (produced by scratching) that function as portal of entry for rickettsiae in the infected faeces.
Infection also may be conveyed with foodstuffs contaminated with the urine of infected animals, by
contact (by rubbing in faecal matter of infected mice when scratching the skin) and by inhalation of
dried flea faeces. This infection is usually endemic or sporadic in character. Depending on epizootic
state, local outbreaks of the disease may occur in some cases. The disease is seasonal, being more
prevalent in August-November (the period of increased density and activity of rodents). Clinically,
human endemic typhus is identical to epidemic typhus, but it is usually a mild illness of shorter
duration, has fewer complications and fatality rate is less. The diseases is characterised by fever and
rash on the face, chest, abdomen, back, palms and soles, headache, malaise and myalgia.
Immunity: The postinfectious acquired immunity is stabile and cross reacted to epidemic
typhus.
Murine typhus control is ensured by extermination of rats and mice, prevention of rats
penetrating into dockyards from incoming ships, protection of foodstuffs from rats, and extermination
of rat fleas, lice, and ticks. The specific prophylactic measure is immunization of individuals living in
endemic areas by killed vaccine.
Laboratory diagnosis: R. typhi and R. prowazekii are similar by different properties but may be
differentiated by immunological tests- agglutination and CFR with R. prowazekii and R. typhi and
biological male guinea pigs are sensitive to endemic typhus fever and they develop fever and a
characteristic scrotal inflammation (scrotal phenomenon).
1.Serological: Agglutination reaction, passive hemagglutination, CFR
2.Biological: (scrotal phenomenon).

Q–FEVER
The causative agent of Q-fever (Q-query) is Coxiella burnetti (discoverer was F. Bernett).
Coxiella burnetii is an obligate intracellular pathogen. It is polymorphic bacterium 1.0X0.3μm in
size (very small size and can pass through bacterial filter). It has gram negative cell wall. It grows
within the phagolysosome of macrophages of the vertebrate host.
Culture: They aren’t reproducd on artificial nutrient media; they grow well in chicken embryos.
Antigenic structure: Coxiella burnetti contains two antigens: Surface polysaccharide antigen of
I phase, which is immunogenic activates synthesing of immunoglobulins; and internal antigen of the
II phase (chemical structure is unknown), which plays role of adjuvants. Q-fever sera does not cross-
react with rickettsial or Proteus bacillus antigens.

99
 Pathogenesis: Q fever is the only rickettsial disease that is not transmitted to humans by the bite
of an anthropod. The important reservoirs for human infection are cattle, sheep and goats. Human
infections have been traced to transmission of infection by inhalation of aerosols of these materials:

1. consumption of infected milk 2. handling of infected wool or hides, 3.soil contaminated with
faeces of infected animals 4.infected straw 5.contaminated clothing. The transmission of infection by
inhalation of aerosols of these materials. Man to man transmission of Q fever can also occur. After
incubation period of 2-4 weeks patient develops fever, chills, headache, malaise, myalgia, pneumonia,
hepatitis, endocarditis and meningoencephalitis. Unlike most other rickettsial disease, no skin rash
occurs in Q fever. The rickettsia may remain latent in the tissues of patients for months or years and
latent infection may become activated naturally or by use of X-ray treatment or multiple cortisone
injections.
 The human disease is an acute systemic infection characterized by an interstitial pneumonia.
The clinical picture is very variable and asymptomatic infections is very common
 In chronic Q-fever, The Coxiella spreads through almost all organs and many cause hepatitis,
meningoencephalitis or endocarditis. Spontaneous recovery is usual.
 The Coxiella may remain latent in the tissue of patients for 2-3 years.
Treatment: tetracycline, doxycycline are used for many months. In endocarditis prolonged
treatment with combinations of tetracycline, cotrimoxazole or rifampicin may be required.
Prevention: Prophylactic measures are:
1. Separate facilities for animal parturition 2.Destruction of suspected placentas. 3. Heat
treatment of milk at 74º for 15 seconds. 4. Abattoir workers should take care like wearing of gloves,
glasses, mask, etc.; while handling carcasses and animal hides.
The Coxiella vaccine is used also. Vaccins have been developed from formalin killed whole
cells, attenuated C burnetii and trichloroacetic acid extracted antigens.
Laboratory diagnosis:
1.Serological: Agglutination, CFR
2.Skin-allergic
3.Biological
Sample tests
1.Which of the following are typical of Rickettsia:
1.multiply only within living cells
2.polymorphism
3.gram positive bacteria
4.gram negative bacteria
a)3.4. b)1.2.4. c)1.4. d)3.4.
2.Antigenic structure of R. prowazekii is:
a)surface group-specific antigens
b)superficial V antigen
c)protective antigen
d)Vi antigen
3.Cultivation of Rickettsia takes place on:
1.the yolk-sac of embryonated egg
2.tissue cultures
3.yolk salt agar
4.blood agar a)2.4 b)1.2. c)1.3. d)3.5.

100
 SPIROCHAETES

Spirochaetes (speira-coil and chaite-hair) belong to the order Spirochaetales, family

Spirochaetacae. Spirochaetes are elongated, motile, flexible
bacteria. The body of spirochaete consists of the hollow coil,
which is enclosed by the cytoplasmic membrane. The outer
membrane is a thin cell wall. All spirochaetes are actively
motile and achieve their motility by means of two or more
axial filaments. The axial filaments are inclosed in the space
between the outer sheath and the cell body. One end of each
axial filament is attached near the pole of the cell
(blepharoplasts). By rotating its axial filament, the cell
rotates in the opposite directions to move through liquids like
a corkscrew.
Generally spirochaetes are free-living saprophytes.
They are found in contaminated water, in sewage, soil, and decaying organic matter, and within the
bodies of humans and animals (conditionally pathogenic organisms are: T. orale, T. denticola, T.
refringens).
Three pathogenic genera belong to this family:
Treponema genus causes the following diseases in humans:
 T. pallidum, the causative agent of syphilis
 T. pertenue, the causative agent of Frambesia (Yaw)
 T. carateum, the causative agent of Pinta
 T. bejel, the causative agent of Beijel
Borrelia genus the causative agent of Relapsing fever and Lyme disease,
Leptospira genus the causative agent of leptospirosis.
These three genera are differentiated by the staining properties and the number of twists.

SYPHILIS
(Genus Treponema)
Syphilis is a venereal infection, cyclic infection and infects various organs and systems.
Treponema pallidum is causative agent of Syphilis, was discovered by Schaudinn and Hoffman in
1905.
Morphology: this organism is thin, delicate about 0.1- 0.2μm in width and 5-15μm in length
(trepin-turn, nema thread). It has 8-12 regular spirals at a distance of 1μm from one another. These
organisms are actively motile, rotating steadily around their axis even after attaching to cells by
tapered ends. They exhibit backward and forward movements, flexion of the whole body. During
motion, secondary curves appear and disappear in succession but the primary spirals are unchanged.
They do not stain well with aniline dyes. They are stained well by Romanovsky-Giemsa stain and are
stained pale-pink (the name pallidum refers to its pale staining). Besides the typical form, Treponema
may be seen as granules, L-forms and other structures especially under the action of antibiotics. Under
the unfavourable conditions they form cyst and can survive in this case for a long time.

Cultivation: They are strictly anaerobic organisms. Pathogenic Treponemes do not grow on
ordinary nutrient media. They require special nutrient media (they need 11 aminoacids, salts, vitamins,
serum albumin) and reproduce on special artificial media. Pure culture isolation of the Treponema
101
organisms is extremely difficult. On prolonged (3-5days) cultivation (generation time is 30hours) the
organisms lose their virulence. T. pallidum can be maintained by serial passage in rabbit testes. They
haven’t oxidase and catalase activity.
Biochemical activity is not clear. Some strains can ferment sugars; proteins with indole and H2S
formation; can liquefy gelatine.
Antigenic structure: The antigenic structure of T. pallidum is complex, and contains
polysaccharides, lipids and proteins of the cell wall. A group of lipoproteins are abundant and have
unknown functions, though they appear to be important in the immune response. Cardiolipin is an
important component of the treponemal antigens. The serotypes and serogroups are not defined.
Ecology: T. pallidum is very delicate. It can be inactivated by drying or by heating (in one hour
at 45-48 C). The organisms are sensitive to acids and other disinfectants (soap, arsenicals, bismuth)
and to desiccation. Hence fomites are of little importance in transmission of infection. It is killed in 1-3
days at 0-4 C, so that transfusion syphilis can be prevented by storing blood for at least four days in
the refrigerator before transfusion.
Pathogenicity: Syphilis is an anthroponose infection. Naturally infection with T. pallidum occurs
only in humans. Experimentally, monkeys may be infected and primary syphiloma develops. The
source of infection is the patient. The disease is transmitted by sexual contact, in the low per cent cases
it may be transmitted by fomites. Syphilis may also be transmitted through the placenta (congenital
syphilis).
The Spirochaetes enter the body through minute abrasions on the skin or mucosa. Infectivity of
patients to the sexual partner is maximum during the first two years of the disease- the primary,
secondary and early latent stages. After five years, the risk is considered minimal. The infective dose is
small, as few as 60 treponemes being capable of infecting 50 per cent of human volunteers. The
causative agent localizes primarily in the mucous membranes of the genital organs and mouth and in
the skin. At the site of penetration it multiplies and produces proliferative and destructive changes.
Clinical disease sets in after an incubation period.
Incubation period is about 3-4 weeks. During the incubation period Spirochaetes multiply
locally at the site of entry, and some spread to nearby lymph nodes. In this system the spirochaetes
multiply intensively as the concentration of the oxygen in lymph nodes is low, which ensures for them
normal conditions for growth. Then they rich the bloodstream and spread through the organs and
systems. During the incubation period the Treponema can be distinguished in perineural lymph area. It
explains the entering of Spirochaeta into the CNS. In spite of the spread of causative agent during the
incubation period into the internal organs there aren’t visible pathogenic changes in these organs. So at
the end of the incubation period the Spirochaetes are far from the site of entering although they aren’t
distinguished either clinically or in laboratory tests. After the incubation period three periods of
infection are distinguished:
Primary syphilis (ulcus durum, hard chancre), which lasts 6-7 weeks. It occurs at the site of
entering. In general the chancre is genital; other common sites are mouth and nipples. Hard chancre is
a hard infiltrate with an erosion or ulceration on its surface, at the point where the Treponema enters
the body. The floor and edges of the ulcer are of a cartilaginous consistency (for this reason the lesion
is known as ulcus durum, primary sclerosis, or hard chancre). The chancre is painless. The size of the
ulcer is 10-20 mm. Primary syphiloma is accompanied by the development of regional adenitis
manifested by enlarged and hard lymph nodes. During this phase the quantity of immunoglobulins
increases and after 2-4 weeks the serologic reactions such as Wasserman complement fixation,
precipitation reactions become positive. And this period is subdivided into sero-negative (first 3weeks)
and sero–positive (4-7weeks). During this period there are high amounts of Treponema in the tissue
liquid of the hard chancre as well as in the lymph nodes and during all the period microscopic method
of investigation can be used for diagnosis The chancre invariably heals in about 10-40days, even
without treatment leaving a thin scar.
Secondary syphilis (Syphilis secundaria) sets in 2-2,5 months after the primary lesion heals.
The secondary lesions are due to widespread multiplication of the spirochetes and their dissemination
through blood. Secondary syphilis is manifested by eruptions (roseolar or papular skin rashes) on the
skin and mucous membranes (anywhere on the body, including the hands and feet), and moist, pale
papules (condylomas) in the anogenital region, axillas and mouth and development of specific lesions
in the internal organs, bones, and peripheral and central nervous systems. There may also be syphilitic
102
meningitis, hepatitis, nephritis (immune complex type) or periostitis. This period may vary from 2-3 to
several years. Usually in this period regional adenitis and polyadenitis are investigated. In this period
the transmission from active form to the latent is described. The relapse rash is rare, not bright, and
during each relapse the quantity of rash becomes less. During this period usually no symptoms occur.
Both primary and secondary lesions are rich in spirochetes, and the patients are highly infectious.
Syphilis tertiary (Third period) is characterized by the development of granulomatous lesions
(gummas) in skin, bones, and liver; degenerative changes in the central nervous system
(meningovascular syphilis, paresis, tabes); or cardiovascular lesions (aortitis, aortic aneurysm, aortic
valve insufficiency). In all tertiary lesions, treponemas are very rare, and the exaggerated tissue
response must be attributed to hypersensitivity to the organisms. However, treponemas can
occasionally be found in the eye or central nervous system in late syphilis.
A pregnant syphilitic woman can transmit T. pallidum to the foetus through the placenta
beginning in the 10th to15th weeks of gestation. Some of the infected foetuses die, and miscarriages
result; others are born live but develop the signs of congenital syphilis in childhood: interstitial
keratitis, Hutchinson’s teeth, saddle-nose, periostitis, and variety of central nervous system anomalies.
Adequate treatment of the mother during the first month of pregnancy prevents congenital syphilis.
Immunity: The diseases produce no insusceptibility. Individuals who have recovered from the
disease may be re-infected. Immunity in syphilis is infectious (non-sterile), and characterized by
cellular defence reactions (the lymphocytes produce lipolytic enzymes which cause lysis of
treponemas). The presence of antibodies does not indicate of body resistance.
A state of infectious allergy, a peculiar manifestation of body reactivity, is a characteristic feature
of syphilis. During primary syphilis (hard chancre), the reactivity is lower. An increase in reactivity
occurs most frequently during the later periods and is accompanied by deep changes in the tissues and
organs.
The state of allergy may be revealed by the intracutaneous luetin reaction. Luetin is prepared
from T. pallidum cultures or ill tissues.
Treatment: Penicillin has treponemicidal activity (penicillin G, benzathine penicillin G).
Prophylaxis: prompt and adequate treatment of all discovered cases; follow up on sources of
infection and contacts so that they can be treated; safe sex with condoms is highly recommended.
Several sexually transmitted diseases can be transmitted simultaneously. Therefore, it is important to
consider the possibility of syphilis when any one sexually transmitted disease has been found.
Diagnostic laboratory tests: Specimens: Tissue fluid - expressed from early surface lesions for
demonstration of spirochetes; blood serum for serologic tests.
Methods (picture 1):
1.Microscopic: Dark-field examination. Immunofluorescence (fixed slide stained with a
fluorescein-labelled anti-treponemal serum, and examined by means of immunofluorescence
microscopy for typical fluorescent spirochetes). Staining by Romanovsky-Giemsa and Morozov’s
methods.
2.Serological tests: Wasserman CF reaction. During this reaction is used non-specific antigen:
the purified cardiolipin from beef heart. Precipitin reactions are widely used in syphilis diagnosis, i.e.
the Sachs-Witebsky test (the citochol reaction) and the Kahn test.
3.The T. pallidum immobilization test has a great value.
Diseases related to Syphilis
These diseases are all caused by Treponemes closely related to T. pallidum. None are sexually
transmitted diseases; all are commonly transmitted by direct contact. None of the causative organisms
has been cultured on artificial media.
Bejel, caused by T. pallidum, occurs chiefly in Africa, Arabian countries (Endemic Arabian
syphilis), particularly among children, and produces highly infectious skin lesions but does not affect
the cardiovascular and nervous systems; late visceral complications are rare. Penicillin is the drug of
choice.
Frambesia (Yaws)-causative agent is T. pertenue. It occurs in hot tropical countries (Africa,
Ceylon, South America, Central America, India, Indonesia China). Yaws is endemic, particularly
among children. The primary lesion, an ulcerating papule, occurs usually on the arms or legs.
Transmission is by person-to person contact in children under 15; transplacental, congenital infection
does not occur. Scar formation of skin lesions and bone destruction are common, but visceral or
103
nervous system complications are very rare. It has been debated whether yaws represented by
nonsexual means is not climates. There appears to be cross-immunity between yaws and syphilis.
Diagnostic procedures and therapy are similar to those for syphilis.
Pinta is a disease caused by T. carateum and occurs epidemically in all age groups in Mexico,
Central and South America, the Philippines. The disease appears to be restricted to dark – skinned
races. The primary lesion, a non – ulcerating papule, occurs on exposed areas. Some months later, flat,
hyper – pigmented lesions appear on the skin; depigmentation and hyperkeratosis take place years
afterward. Late cardiovascular and nervous system involvement occurs very rarely. Pinta is transmitted
by non-sexual means, either by direct contact or through the agency of flies or gnats. Diagnosis and
treatment are the same as for syphilis.

RELAPSING FEVER
(Genus Borrelia)
According to its vectors, relapsing fever is subdivided into two types: epidemic- transmitted by
louse, and endemic, transmitted by ticks.

EPIDEMIC RELAPSING FEVER

The causative agent of epidemic relapsing fever (louseborne) is B. recurrentis (was discovered
in 1868 by O. Obermeier). Relapsing fever is an acute infection for which is characterised by acute
fever, intoxication, enlargement of liver and spleen.
Morphology: B. recurrentis is an irregular spiral 10-30m in long and 0.3m in wide, gram
negative. They possess from 3-9 spirals with acute ends. The organisms are motile, gram-negative, and
readily stain blue violet by the Romanowsky-Giemsa method.
Cultivation: Strict anaerobes. B. recurrentis can be cultured in fluid media containing blood,
serum, or tissue under anaerobic conditions; but it rapidly loses its pathogenicity for animals when
transferred repeatedly in vitro. Multiplication is rapid in chick embryos when blood from patients is
inoculated onto the chorioallantoic membrane. Optimal temperature for growth is 30-37ºC; pH-7.2-7.4.
Antigenic structure and classification: B. recurrentis are non-fermentative organisms. They
have no serological variants.
Ecology and Pathogenesis: Relapsing fever is an anthroponose infection, which transmitted by
cloth’s lice. The lice are infected by sucking the patient’s blood and, 5-12 days later it becomes
capable of infecting human beings. The organisms gain entrance into the body when haemolymph of
crushed lice is rubbed into the skin. Lice remain capable of conveying infection throughout their life
25-40 days. Transovarial transmission of the organisms does not occur in lice. B. recurrentis multiplies
in the tissues of the reticuloendothelial system. At the end of incubation period, a great number of the
organisms invade the blood where some of them are destroyed by bactericidal substances.
The elaborated endotoxin affects the central nervous system and causes toxicos, fever,
functional
disturbances, and dystrophy in organs and tissues. The endotoxin affects the blood-vascular system
with resulting spleen infarction and necroses in the spleen and liver. These organisms are destroyed in
blood by lysins and phagocytes. The organisms, which are reserved in tissues, began to multiply, and
then enter into blood and new attack of disease occurs. These attacks vary from 3 to 5 in number and
last until the host body renders harmless all the new varieties present. For disease characteristic are
high temperature 39-40 C, nausea, vomiting, and spleen enlargement. Fever lasts for 6-7 days during
the first attack, then the temperature falls and apyrexia or remission of 5-7 day’s duration follows.
Each new attack (5-6attacks) is of less duration than the previous one, while the period of apyrexia
becomes longer.
Immunity: Immunity in relapsing fever is characterized by the presence of antibodies
(agglutinins, lysins), but it relatively weak and of a short duration.
Treatment: Antibiotics are used: penicillin, chlortetracycline and streptomycin.
Prophylaxis: Such disasters as wars, famine may give rise to epidemics of relapsing fever.
Improvement of living, the practice of anti-epidemic precautions, timely recognition of the disease,
hospitalization of patients, medical control of sources of disease (delousing-cleanliness, insecticides) is
the main prophylactic measures. No vaccins are available.

104
 ENDEMIC RELAPSING FEVER
Endemic relapsing fever (tick-borne relapsing fever): the causative agents are: B. persica, B.
caucasica, B. hispanica, B. latyschewi and etc.
Borrelia organisms responsible for tick-borne relapsing fever morphologically, by cultivation and
antigenic structure resemble the causative agents of epidemic relapsing fever.
Pathogenicity: In nature, the causative agents of endemic relapsing fever are parasitic within the
body of wild rodents and insectivores from which they gain entrance into the body of ticks
(Ornithodorus). After entering the tick’s gut, the organisms penetrate into the mouth cavity in 10-12
days and than spread throughout the body. The transovarial transmission of the causative agent is
possible. The tick remains infected for life, which may be over 10 years. Human beings acquire the
infection through tick bites, when a papula (primary affect) forms at the site of the bite.
Pathogenesis and clinical picture are similar to those of epidemic relapsing fever. The disease is
characterized by relapses of 1-2 days’ duration. The relapses may be 5-7-9 and more in number. The
duration of remissions varies from several hours to 6-8 days.
Treatment: Antibiotics.
Prophylaxis: Prophylaxis is ensured by measures aimed at extermination of ticks and rodents,
early recognition of the disease, hospitalization of patients, and observance of individual prophylaxis
(prevention of ticks attacking people).
Laboratory diagnosis:
1.Microscopic: Examination of thick smears of blood obtained during the febrile period.
Specimens are stained by the Romanowsky-Giemsa stains; the organisms are examined for motility in
the thick films by use of dark-field illumination.
2.Serological: Serologic test may be carried out during the period of apyrexia. A drop of serum
taken from a patient who has recovered from an attack of relapsing fever is mixed on a slide with a
drop of patient’s blood containing the causative agent. The slide is covered with cover slip and placed
in an incubation room. The Borrelia organisms lose their motility and are destroyed in 30-60 minutes.
3.Biological method: This method is employed for differentiating epidemic and endemic
relapsing fever. A guinea pig is sensitive to endemic relapsing fever.

LEPTOSPIRA
Leptospiras are the causative agent of leptospirosis, which is a zoonotic infection. To the genus
Leptospira belong more than 10 species and L. interrogans is pathogenic for human.
Morphology: Leptospirae are thin, tightly coiled (12-18 regular twists), flexible spirochetes, 5-
15 μm in length and 0.1-0.2 μm in breadth. The poles are thickening as a button. One end of the
organism is often bent, forming hook. They do not stain well with aniline dyes. They stain well with
Romanovsky-Giemsa stain and they stain pink. They are actively motile (there is active rotation
motion).
Culture: The organisms are aerobic and micro- aerophils. They grow best under aerobic
conditions at 28-30º C in protein rich (5-10% serum) and 0,05% of NaCl. They grow slowly in 5-7
days and produce round colonies 1-3mm in diameter. In broth they form turbidity.
Antigenic structure: L. interrogans contains 19 serogroups. By the surface protein antigen they
subdivided into 180 serovars.
Ecology: In nature the reservoirs of leptospirae are rodents and mammals. In rodents they cause
chronic infection and carrier state. They excrete leptospirae in urine and faeces both during active
illness and during the asymptomatic carrier state. Leptospira survive in water and soil 2-3 weeks; and
drinking, swimming, bathing, or food contamination may lead to human infection. Persons most likely
to come in contact with water contaminated by rats (eg, miners, sewer workers, farmers, fishermen)
run the greatest risk of infection. Children acquire the infection from dogs more frequently than do
adults. Control consists of preventing exposure to potentially contaminate by rodents control.
Doxicycline orally once weekly during heavy exposure is effective prophylaxis. Dogs can receive
distemper-hepatitis-leptospirosis vaccinations. This infection termed water fever.
Pathogenesis: Human infection results usually from ingestion of water or food contaminated
with leptospirae. Human infection results when leptospiras are ingested or pass through mucous
membranes or skin. They enter into the blood and by bloodstream circulate and multiply in various
organs especially parenchymatous organs particularly liver and kidneys (they don’t produce exotoxin
105
but produce toxic substances which defeated parenchymatous organs) causing hemorrhage and
necrosis of tissue and resulting in dysfunction of those organs (jaundice, hemorrhage, nitrogen
retention). They cause dysfunction of central nervous system and aseptic meningitis is occurs.
The incubation period is 4-14 days. The illness is typically biphasic. After an incubation period
the illness begins with chill, high temperature 39-40º C, intense headache, insomnia(sleeplessness),
anorexia, severe myalgia (the patient cannot move), the palpation of muscles is painful. Than a short
period of resolution of these symptoms occur as the organisms are cleared from the blood. The second,
immune phase is most often characterized by the findings of aseptic meningitis and, in severe cases,
liver damage (jaundice) and impaired kidney function and the IgM antibody titre is rises.

Immunity: Post infectious immunity is type specific, life-long.

Treatment and prevention: In very early infection, antibiotics (penicillin, tetracyclines) have
some therapeutic effect but do not eradicate the infection. Prevention primarily involves avoiding
contact with the contaminated environment. Doxycycline is effective in preventing the disease in
exposed persons.
In unfavourable epidemiological conditions killed polyvaccin can be used.
Diagnosis (picture1): Diagnosis is based on history of possible exposure, suggestive clinical
signs. Investigated materials are: blood, urine, spinal cord. The diagnostic methods are (picture 1):
1.Microscopic: dark field examination; “wet mount” and “hanging drop” microscopy; thick smears
stained by Giemsa’s technique.
2.Bacteriological- isolation of pure culture.
3.Biological - animal inoculation.
4.Serological – agglutination reaction and CFR.

106
 Sample tests
1. Treponema pallidum:
1. is a spiral shaped organism
2. forms cyst
3. forms 6-12 regular spirals
4. forms 6-9 irregular spirals
a)1.2. b)1.3.4. c)1.2.3. d)3.4

2.Epidemiology of Syphilis:
1. acquired by sexual contact
2. high risk groups are homosexuals, bisexuals
3. acquired by alimentary route
4. is transmissive infection
a)1.2. b)1.3.4. c)1.2.3. d)3.4.

3.Laboratory diagnosis of syphilis:

1. dark field microscopy
2. microscopy of smears stained by Romanovsky-Giemsa
3. isolation of pure culture in ordinary meda
4. Wasserman’s complement fixation reaction
a)1.2. 4. b)1.3.4. c)1.2.3. d)3.4.

4. Leptospira characterized by:

a) large irregular spirals
b)primary and secondary spirals
c)by Romanovsky-Giemza they stain blue violet
d)obligate intracellular parasitism

5. For cultivation of Borrelia it is important, Except:

a)simple media
b)serum and ascit containing media
c)anaerobic condition
d)embryonated egg

6. Enumerate Borrelia’s cultural properties:

1.are strict anaerobes
2.growth in serum containing media
3.growth in embryonated egg
4.are strict aerobes
a)2. 4. b)1.3.4. c)1.2.3. d)3.4.

107
108
 RNA ENVELOPED VIRUSES
(Orthomyxoviuses, Paramyxoviruses)

Influenza virus
Family Orthomyxoviridae (GK. myxa mucus): Three immunologic types are known, influenza
A, B and C viruses. Influenza A virus causes influenza in human and influenza in animals (pigs,
horse) and birds. Influenza B and C viruses are pathogenic only for human. The viral aetiology of
type A influenza was ascertained in 1933 by W. Smith, C. Andrewes, and P. Laidlaw.
Influenza is an acute viral infection, for which intoxication and defeated respiratory tract are
typical. This is pandemic infection.
Morphology: The influenza virus is spherical or oval (polymorphs: can be filamentous, is
frequent in freshly isolated strains; rod shape) middle sized virus with the diameter of 80-120 nm. The
nucleocapsid is formed of a RNA helix (connected
with endonuclease) enclosed in an outer lipid-
carbohydrate-protein membrane (enveloped virus),
which is connected with core by M matrix protein.
The virus is composed of single-stranded RNA of
negative polarity. The negative sense single -
stranded RNA is segmented and exists as eight
pieces. Viral RNA-dependent RNA polymerase is
present, which is essential for transcription of the
viral RNA in infected host cell. The nucleocapsid is surrounded by an envelope, which has an inner
membrane protein layer and an outer lipid layer (lipoprotein layer).The membrane protein is known as
the matrix “M” protein. From the envelope two types spikes are projecting Hemagglutinin (HA) and
Neuraminidase (NA), which are the important antigens that determine antigenic variation of influenza
virus and host immunity.
Resistance: Influenza viruses are sensitive to high temperature. The virus loses infectivity more
rapidly at 50C. They are sensitive to ether, which destroys infectivity.
Antigenic structure: The influenza viruses have two antigens: S (solutio) internal soluble
antigen, which is connected with the RNA. Because it is found free in the infected tissues and occurs
in the supernatant when the virus containing fluid is centrifuged, it was also called soluble “S” antigen.
By S antigen the influenza virus is subdivided into 3 serotypes: A, B and C. They possess no-cross
reactivity among the three groups. Revealing of this antigen by CFR and immunoprecipitation tests.
The next is V (viral) antigen, which is on the surface of spike like membrane, by chemical structure is
glycoprotein HA and NA, they are used to subtype the viruses. 14 subtypes of HA (H1-H14) and nine
subtypes of NA (N1-N9), in many different combinations, have been revealed from birds, animals, or
humans. Three HA (H1-H3) and two NA (N1, N2) subtypes have been revealed covered from
humans.
Hemagglutinin is complex glycoprotein. It is responsible for hemagglutination and
hemadsorbtion. It binds viral particles to susceptible cells and is the major antigen against which
neutralizing (protective) antibodies are directed. HA provided entering of viruses into the cell.
Functions of hemagglutinin are:
 Adhesion
 Antigenicity
 Penetration
 Hemagglutination
 Protective
 HA is a strain-specific antigen and capable of great variation.
Neuraminidase:
The antigenicity of Neuraminidase is also important in determining the subtype of influenza
virus isolates. The NA functions at the end of the viral life cycle. It is a sialidase enzyme. It facilitates
release of viral particles from the infected cell surface during the budding process and helps prevent

109
self-aggregation of virions by removing sialic acid residues from viral glycoproteins. It is not effective
in protection as the antihemagglutinin antibody. It is a strain-specific antigen and exhibits variations.
Influenza viruses are remarkable because of the frequent antigenic changes that occur in HA and
NA. Antigenic variants of influenza virus have a selective advantage over the parental virus in the
presence of antibody directed against the original strains. This phenomenon is responsible for the
unique epidemiologic features on influenza. Incidence of antigenic variation is highest in Influenza
virus A, less in B and it has not been demonstrated in Influenza C. Depending on the degree of
antigenic change of H and N, two distinct forms of antigenic variation are known:
Antigenic drift (minor antigenic changes) is due to the accumulation of point mutations in the
gene, resulting in amino acid changes in the protein. Sequence changes can alter antigenic sites on the
molecule such that a virion can escape recognition by the host’s immune system. A variant must
sustain two or more mutations before a new, epidemiologically significant strain emerges.
Antigenic shift (major antigenic changes in HA and NA) reflects drastic changes in the sequence
of a viral surface protein, changes too extreme to be explained by mutation.
Influenza virus replication: The first stage is interaction of the virus with the host cell. The
virus attaches to the glycoprotein receptors of the host cell. Viral particles are internalized within
endosomes then. The next step involves fusion of the viral envelope and cell membrane. The next is
uncoating by action of proteolytic enzymes.
Biosynthesis: Transcription and translation. The influenza virus has single-stranded RNA of
negative polarity as genetic material. The influenza virus’ RNA hasn’t mRNA function. mRNA is
synthesized by action of virus specific RNA-dependent RNA polymerase. After that translation takes
place on host cell’s ribosomes for virus-specific proteins synthesis (virus-specific RNA polymerase,
HA, NA, the structural proteins). The next stage is maturation. The virus matures by budding from the
apical surface of the cell. Individual viral components arrive at the budding site by different routes (the
nucleocapsids are assembled in the nucleus and move out to the cell surface, the matrix protein
synthesized in the cytoplasm). The viral multiplication cycle proceeds rapidly. New progeny viruses
are produced within 8-10 hours.
Epidemiology: The three types on influenza vary markedly in their epidemiologic patterns.
Influenza C is least significant. It causes mild, sporadic respiratory disease but not epidemic influenza.
Influenza B sometimes causes epidemics, but for influenza type A virus is typical massive epidemics
called pandemics. Every 10-40 years, when a new subtype of influenza A appears, a pandemic results
(There are in 1918 (H1N1-Spanish); 1957 (H2N2); 1968 (H3N2-Hong-Kong); 1977 (H1N1 re-
emerged). The reason why the virus is able to cause epidemics and pandemics is its ability to undergo
antigenic variations.
The incidence of influenza peaks during the winter.
Pathogenesis: Influenza virus spreads from person to person by airborne droplets or by contact
with contaminated hands or surface. The influenza virus penetrates the cells of the surface epithelial
layer of the upper respiratory tract mucosa. The influenza virus is strictly pneumotropic and the first
reproduction on this membrane occurs. After, the virus enters into the blood (viremia occurs). As the
virus multiplies and the infection develops, the trachea, bronchi, bronchioli, and alveolal epithelial
cells gradually become involved in the process. Influenza infections cause cellular destruction and
desquamation of superficial mucosa of the respiratory tract. This is attributed to loss of ciliary
clearance, dysfunction of phagocytic cells. They enter into the lymph nodes and infect the lymphocytes
and acquired immunodeficiency occurs. It serves as a favourable medium for the development of
secondary bacterial infections (bronchitis, pneumonia, encephalitis, influenzal meningitis).
The incubation period is 12-48 hours. Symptoms of influenza usually appear abruptly and
include chills, headache, and dry cough, followed closely by high fever, generalized muscular aches,
malaise, and anorexia.
Clinical symptoms of influenza in children are similar to those in adults, although children may
have higher fever and a higher incidence of gastrointestinal manifestations. Influenza A viruses are
important cause of croup in children under 1 year of age.
Immunity: Antibodies against HA and NA are important in immunity to influenza. They have
virus neutralization property. They form stable immunity. The three types of influenza viruses are
antigenically unrelated and therefore induce no cross-protection. Cytotoxic T cells and macrophages

110
take place in immune response too, but the role of cell-mediated immune responses in influenza is
unclear. Protection correlates with secretory IgA antibodies too.
Treatment: Amantadine, rimantadine, antiviral immunoglobulin, Interferon are used. Antibiotics
are used for prevention of secondary infections.
Prevention: Influenza spread by the air-droplet route. The source of infection is a patient who
may infect healthy people when sneezing, coughing, and talking. Influenza is highly contagious.
Spread of infection is prevented by isolation of patients, regularly ventilating the rooms and cleaning
them a damp cloth (moistened in chloramine solution).
Viral vaccines are the primary means of prevention of influenza. Inactivated, live vaccines are
used. Now purified HA and NA is used as a vaccine which is few reactogene and toxic.
Treatment: Amantadine, Rimantadine, antiviral immunoglobulin, Interferon are used.
Antibiotics are used for prevention of secondary infections.
Laboratory diagnosis (picture1-2): Clinical characteristics of viral respiratory infections can be
produced by many different viruses. Consequently, diagnosis of influenza relies on isolation of the
virus, identification of viral antigens in the patient’s cells, or determination of a specific immunologic
response by the patient.
Virological method: Isolation and identification of virus. Classically, embryonated eggs and
primary monkey kidney cells have been the isolation methods of choice for influenza viruses.
Rinocytoscopic method.
Immunofluorescence.
Serological: This based on hemagglutination inhibition and complement fixation reactions.

PARAMYXOVIRUS
Family Paramyxovirus:
Morphology: They are large, polymorphic (filamentous) viruses150-500 nm in diameter.
Nucleocapsid is spiral (helical). Genome is linear, single-stranded RNA, negative polarity. Unlike the
orthomyxoviruses, in which the segmented nature of the genome facilitates genomic reassortments and
antigenic variation so typical of influenza viruses, the Paramyxoviruses with their unsegmented
genome do not undergo genetic recombinations or antigenic variation. Hence all Paramyxoviruses are
antigenically stable. The nucleocapsid is surrounded by a lipid envelope which has the matrix M
protein and glycoprotein spikes at the surface: hemagglutinin (HA), neuraminidase (NA) and F
(fusion) protein, responsible for fusion of the viral envelope with the plasma membrane of the host
cells which is the essential early step of infection. It also brings about cell-to-cell fusion, causing large
giant cells or syncytia, which are characteristic of paramyxovirus infections. The F protein also
mediates the haemolytic activity of Paramyxoviruses.
Antigenic structure: Paramyxoviruses contain two antigens. 1. Internal, type specific and
nucleoprotein S antigen. 2. Surface V antigen, which is glycoprotein NA and HA and look as spike-
like projections.
Resistance: They are sensitive to physical and chemical factors. They are sensitive to ether.
They are killed relatively quickly by heating and exposure to the action of disinfectants. The source of
infection is patient and the route of transmission: air-droplet and in some cases by contact.
The Paramyxoviridae family is divided into three genera: Paramyxovirus which includes the
causative agent of Parainfluenza and Epidemic parotitis, Morbilivirus - the causative agent of
measles, Pneumovirus-respiratory syncytial virus the causative agent of lower respiratory tract
illness (bronchiolitis, pneumonia).

EPIDEMIC PAROTITIS
Mumps is an acute viral infection with intoxication. It is characterized by non-suppurative
enlargement of one or both parotid glands. Other organs may be involved in processes (pancreas,
testes, ovaries, CNS).
The causative agent was discovered by C. Johnson and E.
Goodpasture in 1934. It belongs to genus Paramyxovirus.
Morphologically are those of a typical Paramyxovirus.
Cultivation: Cell cultures are better suited for isolation-primary
monkey kidney being the preferred cell. The cytopathic effect is slow
111
and consists of syncytium formation and presence of acidophilic cytoplasmic inclusions. Growth is
best identified by hemadsorption. The virus can be grown in chick embryos-in the amniotic cavity for
primary isolation and allantoic cavity after adaptation. Eggs are inoculated at 6-8 days and incubated at
33ºC for five days before harvesting. By passaging of mumps virus in eggs and growing in chick
embryo they lose their infectivity and this is used for preparation of alive attenuated vaccine.
Antigenic structure: They contain S and V antigens and the virus of epidemic parotitis has no
serovars the virus has a single serotype. The virus has hemagglutination, haemolysis and
neuraminidase activities.
Resistance: The virus is weakly resistant to exposure to physical and chemical factors. A
temperature of 55-60 C kills the virus in 20 minutes. It is inactivated quickly by ultraviolet radiation
and destroyed by 0.1 per cent formalin, 1 per cent Lysol, 50 per cent alcohol, or ether. The virus
survives at low temperatures (-25 C and -70 C) up to 10 months.
Epidemiology: Humans are the natural host. Mumps virus is transmitted via respiratory droplets
and by direct contact or fomites contaminated with saliva or urine, primarily in the winter months. The
period of communicability is from the end of incubation period and during the prodromal and specific-
illness periods. The disease is prevalent among children of age group1-15 years, especially in boys.
Primary replication occurs in nasal or upper respiratory tract epithelial cells. After the virus
enters into the blood and viremia occurs, it disseminates the virus to the salivary glands and other
major organ systems. The incubation period is 11-23 days. The clinical features of Mumps reflect the
pathogenesis of the infection. The prodromal period of malaise and anorexia is followed by rapid
enlargement of parotid glands as well as other salivary glands. Swelling may be confined to one
parotid gland, or one gland may enlarge several days before the other. Gland enlargement is associated
with pain, (especially when acid substances are consumed) and fever (38C-40 C). Complications of
mumps include meningitis, meningoencephalitis, pancreatitis, nephritis and unilateral nerve deafness
(hearing loss is complete and permanent).
Immunity: Immunity is high grade and practically generated for life. Complement-fixing and
hemagglutinin-inhibiting antibodies appear in the serum. Cell mediated immune response also
develops. Its role in recovery and protection is unknown.
Treatment: Patients with epidemic parotitis are prescribed gamma globulin. Mumps immune
globulin does not prevent infection when administered to an exposed susceptible person and is of no
value for decreasing the incidence of orchitis even when given immediately after parotitis is first
noted.
Prophylaxis: General measures comprise isolation
of patients until they recover. All child contacts younger
than 10 years of age are isolated for a period of 21 days
(the longest incubation period. Specific prophylaxis is
immunization with attenuated live Mumps virus vaccine
(this vaccine made by cultivation of viruses in chick
embryo where they lose their infectivity). The vaccine is
recommended for children over age 1year and for
adolescents and adults who have not had mumps
parotitis. The vaccine is given as a single subcutaneous
injection, either alone or in combination with the measles and rubella vaccines(MMR vaccine).
Laboratory diagnosis (picture1-2): 1.Serological examination: The complement-fixation
reaction and hemagglutination-inhibition test are used. Antibodies may be detected a week after the
onset of the disease, their titre rising intensively. 2.Virological method: The virus may be isolated from
the saliva, urine or CSF.

MEASLES
Measles is acute viral infection. Measles is anthroponose infection with severe intoxication,
catarrhal syndrome, rhinitis, and laryngitis and skin rash.
Measles virus is in the family Paramyxoviridae, genus Morbilivirus. The virus has the general
morphology of Paramyxoviruses. It is a spherical, 150-500 nm in diameter. It has spiral nucleocapsid
which is surrounded by the lipoprotein envelope carrying on its surface hemagglutinin(H) spikes. The
measles virus does not possess neuraminidase(N) activity. The envelope also has the F protein which
112
mediates cell fusion and haemolytic activities. The genome consists of single-stranded RNA, negative
polarity. It contains internal S antigen and surface V antigen and has single serotype (antigenically is
uniform).
Resistance: The causative agent is very susceptible to high temperatures and is destroyed
quickly at 58 C. Outside the body it survives for not longer than 30minutes. It is sensitive to exposure
to sunlight.
Cultivation: The virus cultivated on various tissues and on monkey kidney cells or on human
amnion cells. Measles virus grows slow and typical cytopathic effects are develops (syncytium
formation, multinucleate giant cells are also formed in lymphoid tissues of patients).
Pathogenesis: Human is the only source of infection. Infection prevails in children. Patients
become infective from the first day of the prodromal period and remain so until the fourth and fifth day
after the appearance of eruption. Measles is spread by the air-droplet route. The disease prevails in
winter. The route of entry is the mucous membrane of upper respiratory tract. After entering the upper
respiratory tract the virus replicates (primary reproduction) and invades the blood and viremia occurs.
Virus affects the capillary endothelium and maculopapular rash appears. Except it, the virus suppresses
the activity of T-lymphocytes and secondary immunodeficiency develops. The virus can enter CNS
and cause encephalomyelitis.
The incubation period is 9-11 days. Clinically there are 3 periods: prodromal, catarrhal,
convalescence. Infection starts as severe with intoxication, fever 39-40o C, sneezing, coughing,
running nose, redness of eyes, anorexia. This is the prodromal period which lasts 3-4 days. The main
symptom in this period is appearance of Koplik’s spots, which are small tiny red patches with central
white specks on the oral mucosa opposite the molars. Later, the illness becomes weak, but on the 5 th
day the symptoms of prodromal period recover and the rash appears (Koplik’s spots disappear). The
red maculopapules rash of measles typically appears on the forehead first and spreads downwards
progressively to the chest, the trunk, and the limbs, to disappear in the same sequence 3-6 days later
leaving behind a brownish discolouration and finely granular desquamation. Symptoms are most
marked when the rash is at its peak but subside rapidly thereafter. Most patients recover uneventfully
but quite a few develop complications which may be due to the virus (croup, bronchitis) or to the
secondary bacterial infections(pneumonia, otitis media). The most serious complication is involving
the central nervous system. Acute encephalitis, meningoencephalitis occurs; late complication is
subacute sclerosing panencephalitis (SSPE). The mortality rate in encephalitis associated with measles
is about 15 per cent.
Immunity: Infection confers lifelong immunity. The presence of humoral antibodies indicates
immunity. However, cellular immunity must also be relevant to protection.
Prevention and control: In specific prevention, attenuated live measles virus vaccine is used.
Each child should receive two doses of measles vaccine, the first at 15 months of age and second just
before entering school. The vaccine is given either by itself, or in combination, as the MMR vaccine.
Prevention includes isolation of patients. Sick children are normally not sent to the hospital but are
isolated at home. Rooms which have been occupied by measles patient must be ventilated and be
looked after in adequately hygienic conditions and immunisation of all individuals who have been in
contact with patients should be done with anti-measles immunoglobulins.
Treatment: there are no available antiviral drugs effective against measles or its complications.
Bacterial superinfections should be treated with antibiotics. Passive immunization indicates neonates,
susceptible pregnant woman, and immunosuppressed patients. Passive immunity persists for 30 days.
If a child was exposed to contact with measles patient for the second time, the gamma-globulin
injection is repeated. Usually gamma-globulin does not completely provide protection from the
disease, but delays its onset, significantly lessens its severity, and prevents fatal cases.
Laboratory diagnosis (picture1-2):
1.Serological-hemagglutination inhibition, complement fixation tests.
2.Virological.

PARAINFLUENZA
Parainfluenza is an acute viral infection with intoxication, which initiates infection via the
respiratory tract. The Parainfluenza virus was isolated in 1956 in the USA by R. Chanock.

113
 Morphology: The morphology of Paramixoviridae resembles that of influenza viruses, but
Paramyxoviruses are larger, 150-350 nm in size, enveloped virus. The viral genome is linear, single
stranded RNA of negative polarity. They contain two antigens: Internal S antigen and surface V
antigen, which is NA and HA. All members of Paramyxovirus are antigenitically stable. By antigenic
structure they are subdivided into five groups. For humans pathogenic forms are I, II, III serogroups.
Parainfluenza virus is cultivated on primary human and monkey kidney cells. They grow slowly and
produce very little cytopathic effect. They don’t grow in chick embryo. They cause hemolysis of
erythrocytes. To detect the presence of virus, hemabsorption using guinea pig erythrocytes is
performed.
Pathogenesis: Parainfluenza viruses are transmitted by direct person–to- person contact or by
large droplet aerosols. First reproduction occurs especially in upper respiratory tract. The infection
may involve only the nose and throat, resulting in a harmless “common cold” syndrome. Infection may
be more extensive, can involve the larynx and upper trachea, resulting in croup (laryngo-
tracheobronchitis). Croup is characterized by respiratory obstruction due to swelling of the larynx and
related structures. The infection may spread deeper to the lower trachea and bronchi, culminating in
pneumonia or bronchiolitis (or both). Viremia, if it occurs at all, is uncommon, and cannot develop
intoxications.
The incubation period is short 3-4 days. The clinical symptoms are: malaise, headache, myalgia,
fever, harsh cough and hoarseness. Bronchitis can develop. The severe illness is associated with croup
(laryngotracheobronchitis).
Immunity: Post infectious immunity is developed, but antibodies do not prevent reinfections.
Treatment: There is no antiviral therapy. The treatment mainly is symptomatic.

Sample tests
1.Koplik’s spots are typical of which of the following infection:
a)infectious mononucleosis
b)rabies
c)measles
d)small pox
2.Causative agent of measles:
1.belongs to Paramyxoviridae family
2.belongs to Orthomyxoviridae family
3.RNA-containing large virus
4.gains entrance into organism via alimentary rout
a)3.4. b)2.4. c)1.3. d)2.3.
3.Measles’ virus is characterized by:
a)ability to cause pandemics
b)cytopathic activity
c)ability to affect mostly parotid glands
d)all of the above
4.The causative agent of Mumps:
1.belongs to the family Orthomyxoviridae
2.belongs to the family Paramyxoviridae
3.is a complex virus presented by DNA
4.reproduction occurs in tissue cultures
a)2.4. b)3.4. c)2.3. d)1.4.
5.Which of the followings are typical of Mumps virus, Except:
a)belongs to the family Paramyxoviridae
b)is RNA containing complex virus
c)does not reproduce in tissue culture
d)contains S and V antigens
6.Which of the followings are typical of Epidemic parotitis:
1.is an acute infection with affection of parotid glands
2.is an acute infection with affection of gastro- intestinal tract
3.animals are the source of infection
114
4.human beings are source of infection
a)2.3. b)1.4. c)1.3. d)2.4.
7.Supercapsid of influenza virus:
1.is composed of lipids and glycoproteins
2.promotes adhession of virus on the surface of epithelial cells
3.contain lipids and polysaccharides which have cellular nature
4.contains hemagglutinin and neuraminidase
a)1.2.5. b)1.2.4. c)1.3.4. d)1.3.5.
8.Which is true for influenza virus:
1.B and C types are pathogenic only for human
2.growth of viruses in chick embryo is revealed by hemagglutination reaction
3.supercapsid is composed of neuraminidase and dismutase
4.hemagglutinin is a complex glycoprotein, promoting adhesion of viruses on the surface of epithelial
cells
a)1.2.4. b)1.2.3. c)1.4. d)2.3.
9.Influenza virus is characterized by the followings:
1.is composed of double stranded DNA and M protein
2.nucleocapsid is presented by ribonucleoprotein
3.core of virus is composed of single stranded RNA of negative polarity
4.affects only human
a)1.3. b)2.3. c)2.4. d)3.4.

115
116
117
 PICORNAVIRUS

There are 120 viruses, which cause acute intestinal infections. These viruses are: Enteroviruses;
Rotaviruses; Coronaviruses. The Picornaviridae family (pico-small; rna-RNAviruses) includes two
groups of medical importance: Enteroviruses and Rhinoviruses. Among the major Enteroviruses are
Poliovirus (types1-3), Coxsackievirus A(types1-24), Coxsackievirus B (types1-6); ECHOvirus (types
1-34) and Hepatitis A virus.
Enteroviruses:
There are common properties for Enteroviruses: the virion is spherical, about 28-30 nm in
diameter; non-enveloped viruse composed of an icosahedral nucleocapsid and single stranded RNA
genome, which has positive polarity, isn’t sensitive to ethers
In human organism they cause various illnesses ranging from neural infections to infections in
several organ-systems.
Poliomyelitis:
Poliomyelitis is an acute infectious disease which affects the central nervous system. The
destruction of motor neurons in the spinal cord results in flaccid paralysis (polios GK grey; myelitis-
inflammation of spinal cord).
Properties of the virus (In 1908-1909, K. Landstainer and E. Popper proved poliomyelitis to be of
viral aetiology) are common to Picornavirus.
Cultivation: The poliomyelitis virus is cultivated on kidney cells of monkeys and on diploid
human cells (HeLa). The cytopathogenic effect is attended by destruction and the formation of
granules in the infected cells.
Antigenic structure: By antigenic structure they are subdivided into three groups: I, II, III.
During epidemic outbreaks, type I is most frequently isolated (65-95 per cent of cases). Paralytic forms
of the disease are more frequently produced by the type I organism.
Resistance: The virus survive in sterile water at room temperature for a period of more than 100
days, in milk 90 days, in faeces in the cold for more than 6 months, and sewage for several months. It
withstands exposure to 0.5-1 per cent phenol solutions. The poliomyelitis virus is sensitive to calcium
chlorate lime, chloramine, formalin, hydrogen peroxide and potassium permanganate solutions,
Pathogenesis and pathology: Natural infection occurs only in humans (experimentally,
monkeys may be infected by intracerebral or intraspinal inoculation). The virus is transmitted by the
fecal-oral route through ingestion. Air droplet route is possible too. Primary multiplication of virus
takes place in the oropharynx or intestine. In incubation period the virus reproduced in the lymphoid
substances of the throat and intestine. Later they enter into the blood and viremia occurs. Poliovirus
can spread along axons of peripheral nerves to the central nervous system, where it continues to
progress along the fibres of the lower motor neurons to increasingly involve the spinal cord or the
brain. Poliovirus invades certain types of nerve cells, and in the process of its intracellular
multiplication it may damage and completely destroy these cells. The anterior horn cells of the spinal
cord are most prominently involved, but in severe cases the intermediate grey ganglia and even the
posterior horn and dorsal root ganglia are often involved. In the brain, the reticular formation,
vestibular nuclei, and deep cerebellar nuclei are most often affected. The cortex is virtually spared,
with the exception of the motor cortex along the precentral gyrus. In addition to pathologic changes in
the nervous system, there may be myocarditis, lymphatic hyperplasia, ulceration of Peyer’s patches,
prominence of follicles, and enlargement of lymph nodes.
The incubation period is usually 7-17days, but it can range from 3 to 35 days. The clinical forms
are: 1. inapparent asymptomatic infection; 2. abortive poliomyelitis; 3. nonparalytic poliomyelitis; 4.
paralytic poliomyelitis. Only about 1 per cent of infections are recognized clinically.
Abortive poliomyelitis: This is the most common form of the disease. The patient has only the
minor illness, characterized by fever, malaise, drowsiness, headache, nausea vomiting, constipation,
and sore throat in various combinations. The patient recovers in a few days spontaneously.
Non-paralytic poliomyelitis manifests as aseptic meningitis with fever, headache, and a stiff
neck. The disease lasts 2-10 days, and recovery is rapid and complete. In a small percentage of cases,
the disease advances to paralysis.
Paralytic poliomyelitis: The major illness may follow the minor illness described above. In
paralytic poliomyelitis flaccid paralysis is the predominant finding resulting from lower motor neuron
118
damage. Painful muscle spasms also occur. The motor nerve damage is permanent, but some recovery
of muscle function occurs as other nerve cells take over. Muscle atrophy and loss of neuromuscular
functions can occur.
Immunity: Stable immunity is produced. Virus-neutralizing antibody forms soon after exposure
to the virus, often before the onset of the illness, and apparently persists for life.
Treatment: There is no antiviral therapy. Treatment is limited to symptomatic relief and
respiratory support, if needed. Physiotherapy for the affected muscles is important.
Prevention: Poliomyelitis can be prevented by both the killed vaccine (Salk vaccine, inactivated
vaccine, IPV) and alive, attenuated vaccine (Sabin vaccine, oral vaccine, OPV). Both vaccines induce
humoral antibodies. A. A. Smorodinzev and M. P. Chumakov prepared alive vaccine, which is
currently preferred for two main reasons: It interrupts faecal-oral transmission by inducing secretory
IgA in the gastrointestinal tract. 2. It is given orally and so is more readily accepted than the killed
vaccine, which must be injected.
Passive immunization is accomplished by injecting human immunoglobulins. Passive immunity
lasts 3-4 weeks.
Laboratory diagnosis (picture1-2): 1.Virological method: isolation of the virus. Virus can be
recovered from the throat, stool, or spinal fluid by inoculation of the cell cultures. The virus causes
cytopathic effect and can be identified by neutralization of the CPE with specific antisera.
2.Serological method: CFR, virus neutralization reaction. Precipitation reaction. 3.Biological method.
4.Immunofluorescence.

COXSACKIEVIRUS

Coxsackievirus, a large subgroup of the enteroviruses, is divided into two groups, A (they have
tropism to muscular tissues) and B (neurotropic virus). They produce a variety of illnesses in humans.
Group A viruses cause herpangina (Herpangina-vesicular pharyngitis; is characterized by fever, sore
throat, and tender vesicles in the oropharynx) and hand-foot-and mouth disease (this disease
characterized by a vesicular rash on the hands and feet and ulcerations in the mouth, mainly in
children). Group B viruses cause pleurodynia (Bornholm disease, epidemic myalgia, is characterized
by fever, chest pain); myocarditis and pericarditis (are characterized by fever, chest pain and signs of
congestive failure). In addition to these, a number of group A and B serotypes can give rise to aseptic
meningitis, respiratory and undifferentiated febrile illnesses, hepatitis, and paralysis.
General properties are typical to enteroviruses.
Antigenic structure: There are 30 serotypes of Coxsackie virus. 24 are listed as group A, and 6
as group B.
Coxsackie viruses are transmitted by the faecal–oral route, but respiratory aerosols also play a
role. They replicate in the oropharynx and the intestinal tract. Humans are the only natural hosts.
The incubation period of Coxsackievirus infection ranges from 2-9 days.
Treatment and prevention: There is neither antiviral drug therapy nor a vaccine available
against these viruses. No passive immunization is recommended.
Postinfectious immunity is life long. Virus neutralizing antibodies appear early during the course
of infection.

ECHO VIRUSES

ECHO–enteric cytopathic human orphan virus. General properties are typical to enteroviruses.
By antigenic structure they are subdivided into 34 serotypes. They are called orphan because they were
not initially associated with any disease, they are known to cause a variety of diseases such as aseptic
meningitis, upper respiratory tract infection, febrile illness without rash infantile diarrhoea and
hemorrhagic conjunctivitis. They are transmitted by the faecal-oral route. Pathogenesis is similar to
that of the other enteroviruses. Epidemics may occur especially in summer. Vaccination has not been
attempted.

119
 Sample tests

1.The following belong to the family Enteroviridae:

1.Hepatitis C virus
2.ECHO virus
3.Hepatitis A virus
4.Coxsacki virus
a)2.3.4. b)1.4. c)1.2.4. d)2.3.

2.Primary reproduction of Enteroviruses takes place in the :

a)lymphoid tissue of small intestine
b)neurons of spinal cord
c)epithelial cells of large intestine
d)nucleus of hepatocytes

3.The following are typical of Picornaviridae family, Except:

a)small size
b)helical(spiral) symmetry
c)icosahedral symmetry
d)cytopathic action

4.Which of the following is typical of causative agent of Poliomyelitis:

a)primary reproduction is in lymphoid tissue of small intestine
b)possesses dermatrophic action
c)is cultivated on liver containing media
d)nucleocapsid is spiral symmetry

5. The following is typical of Hepatitis C virus, Except:

a)is a simple, small sized DNA containing virus
b)transmission by sexual, vertical routs
c)can be integrated in genome of the target cell
d)is provided by oncogenic activity

6.The following are typical of Hepatitis B virus, Except:

a)is spherical
b)is small, RNA simple virus
c)can be transmitted by sexual route
d)can be integrated in the genome of target cell

120
 Togaviridae family

Togaviridae family (toga-meaning the Roman mantle or cloak, and refers to the viral envelope)
has two genuses: Alpha virus and Rubivirus.
Rubella virus is in Rubivirus genus.
Rubella (German measles)
Virion is spherical, 50- 70nm in diameter. The genome is composed of single-stranded RNA,
an icos ahedral nucleocapsid and a
lipoprotein envelope. On the surface of virion there
are E1 and E2 glycoprotein spoke-like projections.
These surface spikes contain hemagglutinin. Rubella
virus possesses hemolytic and neuraminidase
activities. However, unlike the Paramyxoviruses,
such as measles and mumps viruses, it has a positive
strand RNA and therefore has no virion polymerase.
The virus has a single antigenic type. Antibodies,
against hemagglutinin, neutralizes infectivity.
Humans are the natural hosts.
Antigenic structure: Rubella virus possesses nucleoprotein (nucleocapsid), protein “C”
antigen, which is revealed by CFR and E2, E1 antigens; revealing of them is by neutralization
reaction. E1 possesses hemaglutination activity. Antibodies against E2 antigens have expressed
protective action.
This virus is sensitive to environmental factors. It is inactivated by ether, chloroform,
formaldehyde. It is destroyed by heating at 56ºC, but survives for several years at 60ºC.
Cultivation: The virus can been grown in many primary cell cultures and continuous cell lines
and they cause cytopathic changes. Experimental infection can be produced in animal organisms. A
suitable experimental model for the teratogenic effects of Rubella is the pregnant rabbit in which the
virus infects the fetus transplancentally, leading to congenital malformations.
Rubella is primarily a mild childhood fever. It may be acquired congenitally or postnatally.

A. Postnatal Rubella
Rubella virus is excreted in oropharyngeal secretions and infection is acquired by inhalation.
Virus multiplies locally in the upper respiratory tract and in the cervical lymph nodes, followed by
dissemination throughout the body by the way of the blood stream.
Incubation period is 2-3weeks. After an incubation period a brief prodromal period with fever
and malaise is followed by a generalized fine, pink, maculopapular rash development, which starts
on the face and progresses downward to involve the extremities. The rash is generally discrete and
ordinarily disappears by the third day. Fever is usually inconspicuous, but a characteristic feature is
that postauricular, suboccipital and posterior cervical lymph nodes are enlarged and tender from in
a very early stage of illness. The illness is of short duration and recovery usually complete, within
3-4 days after the appearance of the rash. Mild polyarthritis, usually involving hands, occurs in
about 60% adult women caused by immune complexes. Complications like thrombocytopenic
purpura, postinfectious encephalopathy and rubella progressive panencephalitis are sometimes
observed.

B. Congenital Rubella
The significance of rubella virus is not as a cause of mild childhood disease but as a teratogen.
Rubella virus can cross the placental barrier in early pregnancy, and infect the fetus where it
disseminates and grows in every fetal organ. It may result in a large variety of congenital
abnormalities or death of fetus. Congenital annormalities may include total or partial neurosensory
deafness, cataract, glaucoma, microphthalmia or retinopathy leading to blindness, congenital heart
disease especially patent ductus arteriosus, sometimes accompanied by septal defects and
pulmonary artery stenosis, microcephaly with mental retardation, thrombocytopenic pupura and
hepatosplenomegaly. This is known as congenital rubella syndrome(CRS).

121
 About 20% of all infants infected in utero during the first trimester of pregnancy are born with
severe and usually multiple congenital abnormalities and many of the remainder have milder
defects. Severe congenital abnormalities may lead to intrauterine death with abortion or stillbirth.
If rubella occurs in the fourth month of pregnancy, the risk reduces to approximately 5% and only
abnormally likely to be seen in neurosensory deafness. If infection occurs after the fourth month of
pregnancy, congenital abnormalities are very infrequent.
The rubella virus is present in all excretions of congenitally infected infants. About third of
them continue to shed the virus for six months, and a few for a year or more. The virus may persist
in tissues such as cataractous lenses for several years, infected babies constitute an important
source of infection to the staff in nurseries.
After postnatal Rubella acquired, stable, life long immunity is formed, which depends on
hemaglutinin and virus neutralization immunoglobulins. After congenital Rubella immunity is not
stable.
Prophylaxis: Alive attenuated MMR vaccine is recommended for all infants in the second
year life (15months). The vaccine is effective and long lasting (10years) and causes few side
effects. Because it is alive vaccine, it should not be given to immunocompromised patients or to
pregnant women.
Laboratory diagnosis: 1.Virological: the virus may be isolated from blood during the early
stages or more successfully from throat swabs in rabbit kidney or vero cells. 2.Serological: ELISA
for IgM and IgG antibodies gives valuable information. A finding of IgM alone, without IgG,
means current acute infection. IgG antibody alone, without IgM means past infection or
vaccination and denotes immunity.
In congenital rubella, the virus may be isolated from variety of sources such as urine, throat
swabs, leucocytes, bone marrow or cerebrospinal fluid. In a new born baby, demonstration of
Rubella IgM antibodies are diagnostic of congenital rubella as IgM antibodies do not cross the
placenta. However, many babies have rubella IgG antibodies, acquired transplacentally.
Radioimmune assay, heamagglutination inhibition test are used too.

Sample tests

1.Which of the following are typical of Rubella:

a)Rubella is bacterial infectious disease, which is accompanied by lymphadenopathy
b)Rubella is infectious disease, which is accompanied by lymphadenopathy, rash, Koplik’s spots
c)Rubella is infectious disease, which is accompanied by high temperature, catarrhal
symptoms, conjunctivitis
d)Rubella is infectious disease, which is accompanied by petechial rash, exanthema, enanthema

2.Which of the following are typical of Rubella:

1.Rubella virus contains(-) RNA (8fragments)
2.Rubella virus has complex structure, contains RNA, as a complication causes subacute
panencephalitis
3.has E1 and E2 glycoproteins
4.Rubella is typical childhood infection
a)1.4.5. b)4.5. c)3.4. d)2.5.

3.The following are typical of Rubella, Except:

a)prevention of Rubella is carried out by alive (MMR-mumps, measles, rubella) vaccine
b)acquired and congenital immunity is typical for Rubella
c)congenital Rubella causes formation of subacute panencephalitis
d)virus contains E1 and E2 glycoproteins

122
 RABIES

Rabies is an acute infection of the central nervous system that is almost always fatal.
Rabies virus is in the Rhabdoviridae family(rhabdos-rod), genus Lyssavirus (Greek-lyssa-
rabies). It causes mortal infection in animals and humans. The virus is transmitted by the bite of a rabid
animal that manifests aggressive, biting behaviour induced by the viral encephalitis.
Structure: The Rhabdoviruses are rod or bullet shaped 50-60x180 nm in size. Nucleocapsid is
surrounded by an envelope with protruding spikes. They
possess spiral nucleocapsid symmetry. The spikes are
composed of the viral glycoprotein G. Between
supercapsid and capsid M matrix protein is present.
Virions contain an RNA-dependent RNA polymerase.
Rabies virus has a single antigenic type. They possess
superficial glycoprotein antigen and cor, nucleoprotein,
complement fixing antigen. The antigenicity resides in the
envelope G glycoprotein spikes.
There are two types of Rabies viruses:
1. Freshly isolated virus from natural human or animal infection is termed street virus. It
produces fatal encephalitis in laboratory animals, inoculated by any route, after long and
variable incubation period of 1-12weeks. Intracytoplasmic inclusion bodies (Negri bodies)can
be demonstrated in the brain of animals dying of street virus infection.
2. After several serial intracerebral passages in rabbits, the virus undergoes certain changes and
becomes what is called fixed virus. It is used for vaccine production.
Cultivation of viruses is in cell cultures or in experimental animal organism(intradural injection). In
brain neurons of experimental animals appearance of eosinophilic intracytoplasmic Babes-Negri
inclusions is detected.
Rabies virus survives storage at 4C for weeks but it is inactivated by CO2. This virus is killed
rapidly by exposure to ultraviolet radiation or sunlight, by heat (one hour at 50C), by lipid solvents
(ether, 0,1 sodium deoxycholate), by trypsin, by detergents, and by extremes of pH.
Virus replication and pathogenesis: Rabies virus attaches to the acetylcholine receptor on the
cell surface. After entry into the cell, the virion RNA polymerase synthesizes mRNA that code for
viral proteins. After replication of the genome viral RNA by a virus-encoded RNA polymerase,
progeny RNA is assembled with virion proteins to form the nucleocapsid, and the envelope is acquired
as the virion buds through the cell membrane.
Rabies is a natural infection of dogs, foxes, wolves, skunks, cats and bats. Rabies virus is
excreted in the saliva of effected animals. Man acquires infection by the bite of rabid dog or other
animals. Rarely, infection can occur following licks on abraded skin and intact mucosa. Infection has
occurred through the inhalation of massive virus aerosols generated in bat caves and laboratory
accidents.
The virus multiplies locally at the bite site (primary reproduction), infects the sensory neurons,
and moves by axonal transport to the central nervous system. The virus multiplies in the central
nervous system and then travels down the peripheral nerves to the salivary glands and other organs.
From the salivary glands, it enters the saliva to be transmitted by the bite. There is no viremic stage.
Within the central nervous system, encephalitis develops, with the death of neurons, and
demyelination. Infected neurons contain an eosinophilic cytoplasmic inclusion called a Negri body,
which is important in laboratory diagnosis of rabies.
Clinical findings: The incubation period varies, according to the location of the bite. It lasts
from 2 weeks to 16 weeks or longer. It is shorter when bites are sustained on the head rather than on
the leg, because the virus has a shorter distance to travel to reach the central nervous system.
Clinically there are three phases.
1. Prodromal period lasting 2-10 days. This period is with non-specific symptoms such as
fever, anorexia, malaise, headache, photophobia, nausea, vomiting and sore throat. Usually there is an
abnormal sensation around the site of infection. 2. Acute neurologic phase. During this phase the
signs of nervous system dysfunction such as nervousness, apprehension, and hallucinations occur.
123
General sympathic overactivity is observed, including lacrimation, pupillary dilatation, and increased
salivation and perspiration. A large fraction of patients will exhibit hydrophobia (fear of water). Most
notable is painful spasm of the throat muscles on swallowing. This phase is followed by convulsive
seizures or coma and death usually in 2-7 days after onset (the third period – coma phase). The
major cause of death is respiratory paralysis. Progressive paralytic symptoms may develop before
death. The disease course is slower, with some patients surviving 30 days. Death is preceded by coma
and is due to respiratory arrest.
The usual incubation period in dogs ranges from 3 to 8 weeks, but it may be short as 10 days.
Clinically, the disease in dogs is divided into the same three phases as human rabies.
Immunity, treatment and prevention: Because so few individuals have survived rabies, there
is no information regarding immunity to disease upon being bitten again. There is no antiviral therapy
for a patient with rabies. Only supportive treatment is available.
There are two approaches to prevent rabies in human: pre-exposure and postexposure. Pre-
exposure immunization with rabies vaccine should be given to individuals in high risk groups, such as
veterinarians, zoo keepers, and travellers to areas of hyperendemic infection.
The rabies vaccine is the only vaccine that is routinely used postexposure. The long incubation
period of the disease allows the virus in the vaccine sufficient time to induce protective immunity. The
rabies vaccine contains inactivated virus grown in human diploid cells (Vaccine grown in monkey lung
cells or chick embryo cells is also available).
The duck embryo vaccine or various nerve tissue vaccine are available as well. Duck embryo
vaccine has low immunogenicity, and the nerve tissue vaccines can cause an allergic
encephalomyelitis as a result of a cross-reaction with human myelin; for these reasons, the human
diploid cell vaccine is preferred.
Postexposure immunization involves the use of both the vaccine and human rabies immune
globulin (RIG, obtained from hyperimmunized persons) plus immediate cleaning of wound. This is an
example of passive-active immunization.
The decision to give postexposure immunization depends on a variety of factors, such as: 1. Type of
animal (all wild animal attacks demand immunization); 2. Whether an attack by a domestic animal was
provoked, whether the animal was immunized adequately, and whether the animal is available to be
observed; 3. Whether, rabies is endemic in the area. The advice of local public health officials should
be sought. Hospital personnel exposed to a patient with rabies need not be immunized unless a
significant exposure has occurred, eg, a traumatic wound to the health care worker.

Sample tests
1.Which of the following infections is characterized by formation of Babes-Negri’s inclusions:
a)Influenza
b)Parainfluenza
c)Rabies
d)Poliomyelitis
2.Rabies agent:
1.belongs to Paramyxoviridae family
2.belongs to Rhabdoviridae family, Lissavirus genus
3.affects only human beings
4.produces lethal infection in human and animal organisms
a)2.4. b)1.3 c)3.4. d)1.4.
3.Which of the following are typical of rabies virus, Except:
a)has spiral nucleocapsid
b)is provided by matrix protein
c)is presented by single-stranded RNA
d)has icosahedral capsid
4.Laboratory diagnosis in rabies includes the following:
1.is carried out after death
2.Babes -Negri’s inclusions are revealed
3.bacteriological examination

124
4.immunofluorescence is used a)2.3.4. b)1.3 c)3.4. d)1.2.4.

125
 POLIOVIRUSES
METHODS

Virological
Serological
(BNR, CFR, HAIR,
PR)
with pair serums

Virological method
Isolation of enteroviruses
Investigated
material:
feces,
smear from
nasopharynges,
blood,
liquor,
sectional
material

INDICATION

Paralysis death,
pathomorphological
changes of organs
IDENTIFICATION (BNR, CFR, HAIR)

Biological
neutralization
test
(color test)

126
 HERPES
HERPESVIRIDAE FAMILY

Family Herpesviridae has been divided into three subfamilies. The virions of this family have
the capacity to establish lifelong latent infections from which virus may be reactivated. They are
frequently reactivated in AIDS and following immunosuppressive therapy for organ transplantation or
in cancer.
Morphology: Herpesviruses are large (120-200nm in diameter). They comprise of four distinct
structural elements: envelope, tegument, capsid and core. Envelope is the outermost; it is composed of
lipid which carries surface glycoprotein spikes. Tegument is the electron-dense material present
between envelope and capsid. It contains several proteins. Inner to the tegument is icosahedral capsid.
Core, inside the capsid, consists of double-stranded DNA.

With the exception of Epstein-Barr virus, members of the family Herpesviridae can be
cultivated in cell cultures and produce giant cells and intranuclear inclusion bodies in infected cells.
Herpesviruses replicate in the host cell nucleus, form intranuclear inclusions, and are the only viruses
that obtain their envelopes by budding from the nuclear membrane. They are susceptible to fat solvents
like alcohol, ether, chloroform and bile salts. They are heat labile and have to be stored at -700C.The
family Herpesviridae is divided into three subfamilies based on biological, physical and genetic
properties:

Herpes simplex virus (HSV)

The virus is of two types: type 1 (HSV-1) and type 2 (HSV-2) are distinguished by two main
criteria: antigenicity and location of lesions. Lesions caused by HSV-1 are genital, above the waist,
whereas those caused by HSV-2 are below the waist.
HSV type 1 is usually isolated from lesions in and round the mouth and is transmitted by direct contact
or droplet spread from cases or carriers. HSV type 2 is responsible for the majority of genital herpes

127
infections and is commonly transmitted venereally. The infections caused by herpes simplex viruses
can be divided into primary infection, latent infection, reactivation and recrudescence.
HSV-1causes:
1.acute gingivostomatitis
2.herpetic whilow
3.keratoconjunctivitis
4.eczema herpeticum
5.encephalitis
6.generalized infection
HSV-1causes:
1.genital herpes
2.aseptic meningitis
3.neonatal infection
It may rarely cause head and neck infections.
Transmission and epidemiology: HSV-1 is transmitted primarily in saliva; whereas HSV-2 is
transmitted by sexual contact. As a result, HSV-1 infections occur mainly on the face, whereas HSV-
2lesions occur in the genital area. However, oral-genital sexual practices can result in HSV-1
infections of the genitals and HSV -2 lesions in the oral cavity(this occurs in about 10-20% of cases).
Although transmission occurs most often when active lesions are present, asymptomatic shedding of
both HSV-1 and HSV-2 does occur and plays an important role in transmission.
Pathogenesis: The virus replicates in the skin or mucous membrane at the initial site of
infection, then migrates up the neuron and becomes latent in the sensory ganglion cells. In general,
HSV-1 becomes latent in the trigeminal ganglia, whereas HSV-2 becomes latent in the lumbar and
sacral ganglia. The virus can be reactivated from the latent state by a variety of inducers, eg, sunlight,
hormonal changes, trauma, stress, and fever, at which time it migrates down the neuron and replicates
in the skin, causing lesions.
The typical skin lesion is a vesicle that contains serous fluid filled with virus particles and cell debris.
When the vesicle ruptures, virus is
liberated and can be transmitted to other
individuals. Multinucleated giant cells
are typically found at the base of herpes
virus lesion.
Immunity is type specific, but some
cross-protection exists. However,
immunity is incomplete, and both reinfection and reactivation occur in presence of circulating IgG.
Cell mediated immunity is important in limiting herpesviruses, because its suppression often results in
reactivation, spread, and severe disease.
Varicella –Zoster Virus (VZV): causative agent of Varicella(chickenpox) is the primary
disease; Zoster(shingles) is the recurrent form.
VZV is structurally and morphologically identical to other herpesviruses but is antigenically different.
It has single serotype. The same virus causes both variclla and zoster. Humans are the natural hosts.
Transmission and epidemiology: The virus is transmitted by respiratory droplets and by direct
contact with the lesions. Varicella is a highly contagious disease in childhood.
Pathogenesis: VZV infects the mucosa of the upper respiratory tract, then spreads via the
blood to the skin, where the typical vesicular rash occurs. Multinucleated giant cells with intranuclear
inclusions are seen in the base of the lesions. After the host becomes recovered, the virus becomes
latent, probably in the dorsal root ganglia. Later in life, frequently at times of reduced cell-mediated
immunity or local trauma, the virus is activated and causes the vesicular skin lesions and nerve pain of
zoster.
Clinical findings
Varicella: After an incubation period of 14-21 days, brief prodromal symptoms of fever and
more severe in adults. Varicella pneumonia and encephalitis are major rare complications. Reye,s
syndrome, characterized by encephalopathy and liver degeneration, is associated with VZV and
influenza B virus infection, especially in children given aspirin. It pathogenesis is unknown.

128
 Immunity: immunity following varicella is lifelong; a person gets varicella only once, but
zoster can occur despite this immunity to varicella. Zoster usually occurs only once. The frequency of
zoster increases with advancing age, perhaps as a consequence of waning immunity.
Laboratory diagnosis: 1.Virological: Isolation of the virus in cell culture(multinucleated giant
cells are seen) and identification with specific antiserum 2.Serological test - arise in antibody titer can
be used to diagnose varicella but is less useful in the diagnosis of zoster, since antibody is already
present.
Prevention: Live, attenuated VZV (Varivax) vaccine is used. One dose is recommended for
children 1-12years of age. Teenagers and adults who have not had the disease should receive two
doses. Because it is a live vaccine, it should not be given to immunocompromised people or to
pregnant women. The vaccine is effective in preventing varicella, but zoster can still occur in those
previously infected because the vaccine does not eliminate the latent state.
Zoster: The occurrence of painful vesicles along the course of a sensory nerve of the head or
trunk is the usual picture. The pain can last for weeks, and postzoster neuralgia can be debilitating. In
immunocopromisd patients, life-threatening disseminated infections such as pneumonia can occur.

Cytomegalovirus (CMV): CMV is structurally and morphologically identical to other

herpesviruses but antigenically different. Human are the natural hosts. Giant cells are formed, hence
the name”cytomegalo”. It is the most common cause of congenital abnormalities. It also causes
pneumonia and other diseases in immunocompromised patients.
Transmission and epidemiology: CMV is transmitted by a variety of modes. Early in life it is
transmitted across the placenta, within the birth canal, and quite commonly in breast milk. In young
children, its most common mode of transmission is via saliva. Later in life it is transmitted sexually; it
is present in both semen and cervical secretions. It can also be transmitted during blood transfusion
and organ transplants.
Pathogenesis: Infection of the fetus can cause cytomegalic inclusion disease, characterized by
multinucleated giant cells with prominent intranuclear inclusions. Many organs are affected, and
widespread congenital abnormalities result. Infection of the fetus occurs mainly when primary
infection occurs in the pregnant woman, ie, when she has no antibodies that will neutralize the virus
before it can infect the fetus. The fetus usually will not be infected if the pregnant woman has
antibodies against the virus. Congenital abnormalities are more common when a fetus is infected
during the first trimester than later in gestation, because the first trimester is when development of
organs occurs and the death of any precursor cells can result in congenital defects.
In the immunocompromised host, CMV can cause severe and even fatal infections. This occurs in
transplant recipients, cancer patients on chemotherapy and, more particularly, in the HIV infected.
CMV is an important pathogen in AIDS. In AIDS patients, the already weakened immune response is
further damaged by the neospecific CMI-inhibiting effect of CMV. One of the glycoproteins on the
surface of CMVC acts as a receptor for the Fc portion of immunoglobulin molecules. This leads to
masking of the virus by attachment of irrelevant immunoglobulin molecules, preventing access to
specific anti-CMV antibody.
Prevention and treatment: Prevention is indicated only in high risk cases such as organ
transplants, immunodeficient persons and in premature infants. Screening of blood and organ donors
and administration of CMV immunoglobulins have been employed in prevention.
No vaccine is available. Experimentally, live attenuated vaccines and purified CMV
polypeptide vaccine have been found to be immunogenic but not effective in protecting
immunodificient subjects from CMV infection.
For treatment acyclovir (more effective in prophylaxis); Ganciclovir and foscarnet (causes
more side effects) are used for the treatment of CMV disease in AIDS patients.
129
 Epstein- Barr virus(EBV) - Causes infectious mononucleosis. It is associated with Burkitt’s
lymphoma. Other B cell lymphomas, and nasopharyngeal carcinoma. EBV is also associated with
hairy leukoplakia, a whitish, nonmalignant lesion on the tongue seen especially in AIDS patients.
EBV is structurally and morphologically identical to other herpesviruses but antigenically
different. The most important antigen is viral capsid antigen (VCA), because it is used most often in
diagnostic tests.
Humans are the natural hosts. EBV infects mainly lymphoid cells, primarily B lymphocytes. In latently
infected cells, multiple copies of EBV DNA are found in the cytoplasm of infected B lymphocytes.
Some, but not all, genes are transcribed, and only a subset of those is translated into proteins.
Replicative cycle is similar to that of HSV. EBV enters B lymphocytes at the site of the
receptor for the C3 component of complement.
Transmission and Epidemiology: EBV is transmitted primarily by the exchange of saliva, eg.
during kissing. The saliva of people with a reactivation of a latent infection as well as people with an
active infection can serve as a source of the virus. In contrast to CMV, blood transmission of EBV is
very rare.
Pathogenesis and Clinical findings: The infection first occurs in the oropharynx and then
spreads to the blood, where it infects B lymphocytes. Cytotoxic T lymphocytes react against the
infected B cells. EBV remains latent within B lymphocytes.
Infectious mononucleosis is characterized primarily by fever, sore throat, lymphadenopathy,
and splenomegaly. Anorexia and lethargy are prominent. Hepatitis is frequent, encephalitis occurs in
some patients. Spontaneous recovery usually occurs in 2-3 weeks. In certain immunocompressed
patients, a severe, often fatal EBV infection called X-linked immunoproliferative syndrome occurs.
Laboratory diagnosis: Virological: Saliva or throat washing and peripheral blood leucocytes
can be inoculated on to umbilical cord lymphocytes. If the specimen contains EBV, it leads to
immortalization of the cells to produce a lymphoblastoid cell line. EBV can also be detected by PCR.
In biopsy of affected tumors, viral DNA can be demonstrated by staining with specific antibodies.

Sample tests
1.Herpes viruses:
a)are none-enveloped, large viruses
b)can penetrate by air-droplet route
c)contain linear double stranded RNA
d)form eosinophilic cytoplasmic inclusions
2.Which of the following are typical of Herpesviruses:
1.are DNA containing enveloped viruses
2.are transmitted by transmissive route
3.genome is single stranded RNA like in influenza virus
4.form eosinophilic intranuclear inclusions
a)2.4 .b)1.4. c)1.2. d)2.3.
3.Herpesviruses:
a)have different serotypes by different tropism
b)possess expressed hepatotoxic action
c)cause parotitis
d)penetrate by alimentary route like hepatitis A virus
4.Herpes simple viruses:
a)can penetrate by transmissive route
b) the source of infection are animals
c)can reproduce in nerve ganglia
d)has spiral symmetry

130
 HEPATITIS VIRUSES
Viral hepatitis is a systemic disease primarily involving the liver. Medically important hepatitis
viruses are: Hepatitis A virus (HAV); Hepatitis B virus (HBV); non-A, non-B viruses, of which
Hepatitis C virus (HCV); Hepatitis D virus (HDV, delta agent) and Hepatitis E virus (HEV), Hepatitis
G virus (table). Hepatitis viruses are taxonomically unrelated. Except for type B, which is a DNA
virus, all the others are RNA viruses. The features common to them are their hepatotropism and ability
to cause a similar icteric illness, ranging in severity from the unapparent to the fulminate fatal forms.
As all types of hepatitis viruses cause a clinically indistinguishable acute illness, their differentiation is
based on their serological and molecular markers. By epidemiological and clinical criteria, two types
of viral hepatitis had been recognized for long:
1.One type occurred sporadically or as epidemics, affecting mainly children and young adults, and
transmitted by the fecal- oral route. This was called infectious hepatitis (Hepatitis A).
2.A second type of viral hepatitis, transmitted mainly by inoculation, was originally observed in
persons receiving serum inoculation or blood transfusion. This had been given various names such as
homologous serum jaundice, serum hepatitis and transfusion hepatitis (Hepatitis B).

HEPATITIS A VIRUS
Hepatitis A (infectious hepatitis) is a subacute disease of global distribution, affecting mainly
children and young adults.
HAV is a distinct member of the Picornavirus family. HAV is a 27-32nm, non-enveloped,
spherical virus. It has single stranded RNA positive polarity genome, icosahedral nucleocapsid and
replicates in the cytoplasm of the cell. Only one serotype is known. HAV is stable to treatment with 20
per cent ether, acid and heat (60C for one hour) and its infectivity can be preserved for at least one
month after being dried and stored at 250 C and 42% relative humidity or for years at -20 C. The virus
is destroyed by autoclaving (121C for 20 minutes), by boiling in water for 5 minutes.

Transmission and Epidemiology:

Humans are the reservoir for HAV. Virus appears in the faeces roughly 2 weeks before the
appearance of symptoms. Children are the most frequently infected group, and outbreaks occur in
special living situations (kinder gardens, summer camps). Common-source outbreaks arise from faecal
contaminated water or food such as oysters grown in polluted water and eaten raw. HAV is rarely
transmitted via the blood, because the level of viremia is low and chronic infection does not occur.
Hepatitis A is an anthroponose infection. The source of infection is patient. HAV is transmitted
by the faecal-oral route (infection is by ingestion). The primary reproduction in small intestine is
occurs. Later the virus enters into the blood and spreads to the liver via the blood. For these viruses the
target cells are hepatocytes and these cells are infected. The incubation period is 21-28 days but can
last 50 days. The prodromal period lasts 5-7 days. Fever, anorexia, nausea, vomiting, and jaundice are
typical. After 2-4 days urine becomes dark (like beer), the faeces is pale (acholia), and elevated
transaminase levels are seen, increasing of Specific M immunoglobulins is detected which have
differential diagnostic meaning. During this period the enlargement of liver is detected. The next is
specific illness period, which lasts from 1 week to 1.5 months, jaundice develops, and the intoxication
becomes weak. Jaundice begins from the oral cavity mucous layer, palate, frenula, sclera and then
skin. The urine becomes darker (urobilinuria, choluria), acholia is intensive.
Disappearance of the jaundice shows convalesces and it lasts from 2 to 6 months.
Post infectious immunity is stable depends on virus neutralizing antibodies, memory cells, and
intestinal local immunity. There is no cross immunity between HAV and any of the other hepatitis
viruses.
Prevention: General prophylaxis consists of improved sanitary practices and prevention of
fecal contamination of food and water.
Specific prophylaxis: Specific passive prophylaxis by pooled normal human immunoglobulin
IM, before exposure or in the early incubation period, can prevent or attenuate clinical illness, while
not necessarily preventing infection and virus excretion. Specific active prophylaxis by inactivated
cultural vaccine and recombinative vaccine are used.
Treatment is symptomatic. No specific antiviral drug is available.

131
 Laboratory diagnosis: The detection of IgM antibody is the most important test. IgM and anti-
HAV antibody appears during the late incubation period, reaches peak levels in 2-3weeks and
disappears after 3-4 months. The IgG antibody appears at about the same time, peaks in 3-4 months
and persists much longer, perhaps for life. Demonstration of IgM antibody in serum indicates current
or recent infection, while the IgG antibody denotes recent or remote infection and immunity. ELISA
kits for detection of IgM and IgG antibodies are available.

HEPATITIS B VIRUS (HBV)

HBV is the cause of serum hepatitis. HBV is a member of the Hepadnaviridae family. Hepatitis
B is an anthroponose, viral infection, which infects especially liver and causes development of liver
disease and hepatocellular carcinoma. Type B hepatitis is the most widespread and the most important
type of viral hepatitis.
Structure: It is enveloped virion (known as a Dane particle named after the scientist, who first
discovered the virus-under the electron microscope, sera from type B hepatitis patients show three
types of particles: spherical, filamentous, double –walled spherical structure), with an icosahedral
nucleocapsid. The size is 35-42 nm in diameter. The viral genome consists of double stranded circular
DNA. One of the strands is incomplete (15-60 defective genome), so that the DNA appears partially
double stranded and partially single stranded. This part doesn’t have infectious properties. In
nucleocapsid viral DNA polymerase is present, which has both DNA-dependent DNA polymerase and
RNA-dependent reverse transcriptase functions. This polymerase can repair the gap strand and render
the genome fully double stranded. The genome contains four genes that encode the following
properties: surface (envelope) protein, core (nucleocapsid) protein, DNA polymerase, antigens.
Antigenic structure: They contain four antigens:
HBs antigen (HBsAg was known as Australian antigen, because it was first found in the serum
of an Australian aborigine): It is glycoprotein-lipid complex. This antigen is on the surface of virion.
HBsAg consists of two parts: preS2 which is poly-globulin receptor by which the virus is absorbed on
the host cell receptors (on the hepatocytes). PRES1 ensures immunogenic properties. This antigen is
discovered in blood.
HBc antigen: it is a nucleoprotein, which is in the core of virion. They can be found in the
hepatocyte’s nuclei, but they do not enter into the blood.
HBe antigen: This antigen is separated from HBc antigen, when HBc antigen crosses
hepatocytes and HBe antigen is detected in the blood.
HBx antigen: This antigen takes part in cancer transformation of hepatocytes.
In patient’s organism immunoglobulins against HBc, HBe, HBs antigens are synthesized.
Reproduction (picture1): After entry of the virion into the cell and its uncoating, the virion
DNA polymerase synthesizes the missing portion of DNA and a double-stranded closed circular DNA
is formed in the nucleus. This DNA is a template for mRNA synthesis by cellular RNA polymerase.
Hepadnaviruses are the only viruses that produce genome DNA by reverse transcription with mRNA
as the template.
Pathogenesis: The portal of entry for this virus: blood, sexual and perinatally from mother to
newborn. The virus enters into the blood, and dissemination of the virus in the organism occurs.
Primary HBV fixes on the hepotocytes and reproduced, but reproduction of the virus in the
hepatocytes is not accompanied by cytolysis of these cells. It shows that virus doesn’t have direct cyto-
pathic effect and pathogenic process occurs when immunocytes recognize the antigens of the HBV on
the surface of the hepatocytes. So, the damaging of the liver cells is immune dependent. The
pathological variety of infection (acute, subacute, chronic, and persistent) depends on the antigens of
the virus and interaction with host cell. In acute forms suppression of T helpers occurs. Inhibition of T
helpers means disturbing of recognition of viral antigens and at least inhibition of immunoglobulin
synthesis occurs.
In chronic forms inhibition of T suppressors occurs. It develops autoimmune reactions against
hepatocytes. HBV can interact with macrophages. In normal immune response macrophage presents
viral antigen and induced normal humoral immune response (HBs, HBc, HBe immunoglobulins
synthesised). It means postinfectious immunity is of high duration and life- long. But HBV can infect
macrophages and in antigen recognition system defects occur and it develops immunodeficiency,
development of persistency results and HBV DNA is integrated into cell DNA. A high rate of
132
hepatocellular carcinoma occurs. The HBV genome has no oncogene, and hepatocellular carcinoma
appears to be the result of persistent cellular regeneration that attempts to replace the dead hepatocytes.
Alternatively, malignant transformation could be the result of insertional mutagenesis, which could
occur when the HBV genome integrates into the hepatocyte DNA. Integration of the HBV DNA could
activate a cellular oncogene, leading to a loss of growth control. Chronic infection is associated with a
high risk of hepatocellular carcinoma.
Clinical findings: The incubation period 50-180 days. The prodromal period lasts 4-10 days and
the clinical appearance of acute hepatitis B is similar to that of hepatitis A. However, with hepatitis B,
symptoms tend to be more severe, and life- threatening hepatitis can occur.
Many HBV infections are asymptomatic and are detected only by the presence of antibody to
HBsAg.
Epidemiology: The source of infection is patient. Approximately 5 percent of humanity are
carriers, in their blood HBs antigen is present. The routes of transmission are parenteral (blood
transfusion, syringe-intravenous injections), sexual, prenatally. Persons who had hepatitis probably
should not be used as blood donors.
HBV is sensitive to high temperatures (100 C for 1 min.; 60 C for 10 min). They are sensitive
to formalin. HBsAg is not destroyed by ultraviolet irradiation of plasma or other blood products.
Laboratory diagnosis: Specific diagnosis of hepatitis B rests on the serological demonstration of
the viral markers. It is therefore necessary to understand the sequence of their appearance in blood.
HBsAg is the first marker to appear in blood after infection, being detectable even before elevation of
transaminases and onset of clinical illness.
Immunity: depends on humoral and cellular mechanisms. Immunoglobulins participate in
elimination of viruses from the host organism and protect intact hepatocytes.T8 cells ensure
elimination of viruses from the host organism too.
Prevention: Prevention involves vaccine. Vaccine can be prepared by purifying HBsAg from
healthy carriers (treating these particles with virus-inactivating agents-formalin, heat). Now vaccine
can be prepared by gene engineering (recombinative HBs vaccine).
Treatment: No specific antiviral treatment is available for acute HBV infection. Interferon alpha
alone or in combination with other antiviral agents such as lamivudine and famcyclovir, has been
beneficial in some cases of chronic hepatitis. There is no effective treatment for the carrier state,
though spontaneous resolution takes place in some of them.

Non-A, Non-B hepatitis viruses

Hepatitis D virus (Delta virus): This virus was discovered in 1977. HDV is spherical virus with
single stranded RNA genome of negative polarity. The genome of HDV is very small and encodes
only one protein, internal cor protein D (delta) antigen. HDV is defective virus; it cannot be replicated
by itself because it does not have the genes for its envelope protein. This virus can be replicated only
in cells also infected with HBV, because HDV uses the surface antigen of HBV (HBsAg) as its
envelope protein. HBV is helper virus for HDV and this connection occurs by two ways: 1. When,
both (HBV and HDV) viruses infect the host organism together (at the same time). 2. When HBV
infects the organism and then HDV enters (superinfection with HDV). The mechanism of action isn’t
discovered closely, but there is some evidence that HDV is directed cytopathic for hepatocytes. Portal
of entry is similar to HBV. Hepatitis in patients co-infected with HDV and HBV is more severe than in
those infected with HBV alone.
Laboratory diagnosis: The diagnosis is made by detecting either delta antigen or IgM antibody
to delta antigen in the patient’s serum.
No specific prophylaxis exists, but immunisation with the HBV vaccine is effective as HDV
cannot infect persons immune to HBV. Screening of blood donors for HBsAg automatically limits
bloodborne HDV infection.

Hepatitis C virus (HCV)

HCV is in the family Flaviviridae, Hepacivirus genus. It is enveloped virus. The size is 50-70
nm. The genome is linear, single stranded RNA, positive polarity, enclosed within a core and
surrounded by an envelope, carrying glycoprotein spokes. Humans are the reservoir for HCV. The
transmission of the HCV into the host organism is mainly by blood or blood products. Sexual
133
transmission is probably less important. Vertical transmission from mother to baby may take place.
HCV infects hepatocytes primarily, but there is no evidence for a virus-induced cytopathic effect on
the liver cells. The cytopathic effect is immune mediated by cytotoxic T cells.
Clinical findings are similar to HBV (fever, anorexia, nausea, vomiting, and jaundice, dark urine,
pale faeces). HCV can ensure chronic liver disease, cirrhosis and hepatocellular carcinoma. The
incubation period is long 15-160 days with a mean 50 days. The acute illness is usually mild or
anicteric. Overt jaundice is seen in about 5% of patients only. The important part in type C hepatitis is
the chronic illness. About 50-80% of patients progress tom chronic hepatitis, with some developing
cirrhosis and hepatocellular carcinoma.
Prophylaxis: Only general prophylaxis, such as blood screening, is possible. No specific active
or passive immunising agent available. There is no vaccine.
Treatment: Prolonged treatment with interferon alpha, either alone or in combination with
antiviral agents like ribavirin has been reported to be useful in some cases.
Laboratory diagnosis: HCV infection is diagnosed by detecting antibodies to HCV by ELISA.

Hepatitis E virus (HEV)

HEV is spherical, non-enveloped virus. The genome is single stranded RNA positive polarity.
The size is 32-34 nm in diameter. They are in
Calciviridae family. HEV enters into organism
alimentary (transmitted enterically) like HAV.
Clinically the disease resembles hepatitis A, with
the exception of a high mortality rate in pregnant
women. Chronic liver disease does not occur, and
there is no prolonged carrier state. There is no
antiviral treatment and vaccine. The test for HEV
antibody is not available. The diagnosis is typically
made by excluding HAV and other causes.

Hepatitis G virus (HGV)

In 1996, HGV was isolated, from patients with post-transfusion hepatitis. HGV is in the
Flavivirus family. The role of HGV in the causation of liver disease has yet to be established.

134
 Sample tests

1.The following are typical of Hepatitis C virus, Except:

a)is a simple, small sized DNA containing virus
b)transmission by sexual, vertical routs
c)can be integrated in genome of the target cell
d)is provided by oncogenic activity

2.The following are typical of Hepatitis C virus, Except:

a)can cause productive and integrative types of infection
b)the genome is single stranded RNA of positive polarity
c)the cytopathic effect is immune mediated by cytotoxic T cells
d)the genome is single stranded RNA of negative polarity

3.Dain’s particles are typical of:

a)HAV
b)HCV
c)HBV
d)HGV

4.The following are typical of Hepatitis B virus, Except:

a)is spherical
b)is small, RNA containing simple virus
c)the genome is circular DNA
d)can be transmitted by sexual route

5.Hepatitis D virus:
a)contains single-stranded DNA
b)affects only persons infected with Hepatitis C
c)is provided by immune dependent cytopathic effect
d)is defective

6.Hepatitis C virus:
1.is small sized virus
2.is transmitted by alimentary route
3.is transmitted by intravenous administration of infected blood or serum
4.is single-stranded DNA virus
a)1.3. b)1.2. c)2.3. d)2.4.

135
136
 AIDS

HIV human immunodeficiency virus, a non-oncogenic retrovirus, is the primary etiologic agent
of acquired immunodeficiency syndrome (AIDS). The illness was first described in 1981, and the end
of 1983 was isolated the virus. Both HIV-1 and HIV-2 cause AIDS, but HIV-1 is found worldwide,
HIV-2 is found primarily in West Africa.
HIV is a retrovirus, a member of the Lentivirinae subfamily, which causes “slow” infections with
long incubation periods.
Structure: HIV virion is spherical, 100-200 nm in
diameter. HIV has bar-shaped (D) core surrounded by an
envelope containing virus specific glycolproteins (gp120
and gp41). The genome of HIV consists of two identical
molecules of single-stranded, positive polarity RNA and is
said to be diploid. Three structural gens: gag, pol and env,
which encode the structural proteins. The genome RNA has
six regulatory genes. Two of these tat and rev are required
for replication, and the other four nef, vif, vpr and vpu, are
not required for replication and are termed “accessory”
genes.
The gag gene encodes the internal “core” proteins, the
most important of which is p24, an antigen used in serologic tests. The pol gene encodes several
proteins, including the virion “reverse transcriptase” which synthesizes DNA by using the genome
RNA as a template, an integrase that integrates the viral DNA into cellular DNA, and a protease that
cleaves the various viral precursor proteins. The env gene encodes gp160, a precursor glycoprotein
that cleaved to form the two envelope (surface) glycoproteins, gp120 and gp41.
And there are three viral enzymes in nucleocapsid of the virion: reverse transcriptase, protease,
integrase. Reverse transcriptase is the RNA-dependent DNA polymerase that is the source of the
family name retroviruses. This enzyme transcribes the RNA genome into the proviral DNA. The viral
protease cleaves the precursor polyproteins into functional viral polypeptides.
The essential regulatory gene is the tat (transactivation of transcription) gene, which encodes a
protein that enhances viral (and cellular) gene transcription. Tat protein and another regulatory protein
Nef repress the synthesis of class I MHC proteins, thereby reducing the ability of cytotoxic T cells to
kill HIV-infected cells. Rev gene controls the passage of late mRNA from the nucleus into the
cytoplasm.
Antigenic structure: There are several important antigens:
1. gp120 and gp41 are the type-specific envelope glycoproteins, gp120 protrudes from the surface and
interacts with the CD4 receptor on the cell surface. Gp41 is embedded in the envelope and mediates
the fusion of the viral envelope with the cell membrane at the time of infection.
2. The group specific antigen, p24, is located in the core and is not known to vary. Antibodies against
p24 do not neutralize HIV infectivity but serve as important serologic markers of infection.
The natural host range of HIV infectivity is limited to humans, although certain primates can be
infected in the laboratory. HIV is not an endogenous virus of humans; ie, no HIV sequences are found
in normal cell DNA. The origin of HIV and how it enteres the human population remains uncertain.
There is an evidence that chimpanzees living in West Africa were the source of HIV-1.
Replication cycle: The initial step is attachment, when the virion gp120 binds the CD4 protein
of the cell surface. The virion gp120 protein then interacts with the second protein on the cell surface,
one of the chemokine receptors. Next the virion gp41 protein mediates fusion of the viral envelope
with the cell membrane, and the virion enters the cell. After uncoating, the virion RNA-dependent
DNA polymerase transcribes the genome RNA into double-stranded DNA, which integrates into the
host cell DNA. The viral DNA can integrate at different sites in the host cell DNA, and multiple copies
of viral DNA can integrate. Integration is mediated by a virus-encoded endonuclease (integrase). Viral
mRNA is transcribed from the proviral DNA by host cell RNA polymerase and translated into several
large polyproteins, which are then cleaved by the virus-encoded protease to form the virion structural
proteins. The Gag polyprotein is cleaved to form the main core protein (p24), the matrix protein (p17),
and several smaller proteins. The Pol polyprotein is cleaved to form the reverse transcriptase,
137
integrase, and protease. The immature virion containing the precursor polyproteins forms in the
cytoplasm, and cleavage by the viral protease occurs as the immature virion buds from the cell
membrane. It is this cleavage process that results in the mature, infectious virion.
Transmission and Pathogenesis: Transmission of HIV occurs primarily by sexual contact and
by transfer of infected blood. Perinatal transmission from infected mother to neonate also occurs (35-
50), either across the placenta, at birth, or via breast milk. Small amounts of virus have been found in
other fluids, eg, saliva and tears, there is no evidence that they play a role in infection.
HIV infects helper T cells and kills them, resulting in suppression of cell-mediated immunity.
This predisposes the host to various opportunistic infections and certain cancers such as Kaposi’s
sarcoma and lymphoma. However, HIV does not directly cause these tumours because HIV genes are
not found in these cancer cells. The initial infection of the genital tract occurs in dendritic cells that
line the mucosa (Langerhans cells), after which the local CD4 positive helper T cells become infected.
HIV is first found in the blood 4-11 days after infection.
HIV also infects brain monocytes and macrophages, producing multinucleated giant cells and
significant central nervous system symptoms. The fusion of HIV-infected cells in the brain and
elsewhere mediated by gp41 is one of the main pathologic findings. The cells recruited into the
syncytia ultimately die. The death of HIV-infected cells is also the result of immunologic attack by
cytotoxic CD8 lymphocytes. Effectiveness of the cytotoxic T cells may be limited by the ability of the
viral Tat and Nef proteins to reduce class I MHC protein synthesis.
Another mechanism which explained the death of helper T cells is that HIV acts as a “super
antigen”, which indiscriminately activates many helper T cells and leads to their demise.
A person infected with HIV is considered to be infected for life. This seems likely to be the result
of integration of viral DNA into the DNA of infected cells.
The main immune response to HIV infection consists of cytotoxic CD8-positive lymphocytes.
These cells respond to the initial infection and control it for many years Mutants of HIV, especially in
the env gene encoding gp120, arise, but new clones of cytotoxic T cells proliferate and control the
mutant strain. Cytotoxic T cells lose their effectiveness because so many CD4 helper T cells have died
that the supply of lymphokines, such as IL-2, required to activate the cytotoixic T cells is no longer
sufficient. There is also evidence that “escape” mutants of HIV are able to proliferate unchecked
because the patient has no clone of cytotoxic T cells capable of responding to the mutant strain.
Antibodies against various HIV proteins, such as p24, gp120, and gp41, are produced but they
neutralize the virus poorly in vivo and appear to have little effect on the course of the disease.
HIV has three main mechanisms by which it evades the immune system:
1.Integration of viral DNA into host cell DNA, resulting in a persistent infection;
2.A high rate of mutation of the env gene;
3.The production of the Tat and Nef proteins that down-regulate class IMHC proteins required for
cytotoxic T cells to recognize and kill HIV-infected cells.
Clinical findings: The clinical picture of HIV infection can be divided into: 1. an early, 2. an
acute stage, 3. middle, 4. latent stage, 5. a late, and immunodeficiency stage. In the acute stage, which
usually begins 2-4 weeks after infection, mononucleosis like picture of fever, lethargy, sore throat, and
generalized lymphadenopathy occurs. A maculopapular rash on the trunk appears. Leukopenia occurs,
but the number of CD4 cells is usually normal. This acute phase typically resolves spontaneously in
about 2 weeks. Antibodies to HIV typically appear 3-4 weeks after infection. Note that the inability to
detect antibodies prior to that time can result in “false-negative” serologic tests; i.e., the person is
infected, but antibodies are not detectable at the time of the test. This has important implications
because HIV can be transmitted to others during this period.
After initial viremia, a viral “set point” occurs, which can differ from one person to another. The
set point represents the amount of virus produced, i.e., the “viral load”, and tends to remain “set” or
constant, for years. The higher set point, the more likely the individual is to progress to symptomatic
AIDS.
In the middle stage, a long latent period, measured in years, usually ensues. The patient is
asymptomatic during this period. Although the patient is asymptomatic and viremia is low or absent, a
large amount of HIV is being produced by lymph node cells but remains sequestered within the lymph
nodes. This indicates that during this period of clinical latency, the virus itself does not enter a latent
state.
138
 A syndrome called AIDS-related complex (ARC) can occur during the latent period. The most
frequent manifestations are persistent fevers, fatigue, weight loss, and lymphadenopathy. ARC often
progresses to AIDS.
The last stage of HIV infection is AIDS,
manifested by a decline in the number of CD4 cells
and an increase in the frequency and severity of
opportunistic infections. The two most
characteristic manifestations of AIDS are Pneumo-
cystis pneumonia and Kaposi’s sarcoma. However,
many other opportunistic infections occur too:
disseminated herpes simplex, herpes zoster,
cytomegalovirus infections, fungal infections, etc.
Immunity: HIV-infected persons develop both humoral and cell-mediated responses against
HIV-related antigens. Antibodies to a number of viral antigens develop soon after infection, but the
response pattern against specific viral antigens changes over time as patients progress to AIDS.
Antibodies to the envelope glycoproteins (gp41, gp120, gp160) are maintained, but those directed
against the core protein (p24) decline. The decline of anti-p24 may herald the beginning of clinical
signs and other immunologic markers of progression.
Most infected individuals make neutralizing antibodies against HIV. The envelope
glycoproteins appear to be the major targets for antibody neutralization. The neutralizing antibodies
can be measured in vitro by inhibiting HIV infection of susceptible lymphocyte cell lines. Viral
infection is quantified by (1) reverse transcriptase assay, which measures the enzyme activity of
released HIV particles; (2) indirect immunofluorescence assay, which measures the percentage of
infected cells; (3) reverse transcriptase-polymerase chain reaction (RT-PCR) or branched-chain DNA
amplification assays that measure HIV nucleic acids. Relatively low neutralizing activity is present in
the sera of both asymptomatic seropositive individuals and patients with AIDS.
Treatment: Zidovudine and Lamivudine, which are nucleoside inhibitors and Indinavir a
protease inhibitor. This combination is known as HAART, which is an acronym for “highly active
antiviral therapy”. It is very effective in prolonging life, improving quality of life, and reducing viral
load but does not cure the chronic HIV infection. Another highly effective regimen is the combination
of Zidovudine, lamivudine, and the non-nucleoside reverse transcriptase inhibitor, Efavirenz.
Azidothymidine (AZT) inhibits HIV replication by interfering with proviral DNA synthesis.
Dideoxynosine (DDI), Videx is recommended for patients who are intolerant to AZT.
Nevirapine (Viramune), Delaviridine, Efavirenz (Sustiva) are reverse transcriptase inhibitors.
Protease inhibitors, such as Saquinavir, Ritonavir, etc., when combined with nucleoside
analogues, such as Azidothymidine, are very effective in inhibiting viral replication and increasing
CD4 cell counts.
Treatment for acute HIV infection with two reverse transcriptase inhibitors and protease inhibitor
is recommended. With this regimen, the viral load drops below the level of detection; CD4 cell counts
rise, and CD8 activity increases.
In this complex treating of opportunistic infections is included.
Laboratory diagnoses:
1. Virus isolation: HIV can be cultured from lymphocytes in peripheral blood (and occasionally
from specimens from other sites). The number of circulating infected cells varies with the stage of
disease. Higher titers of virus are found in the plasma and in peripheral blood cells of the patients with
AIDS, as compared with asymptomatic individuals. The magnitude of plasma viremia appears to be a
better correlate of the clinical stage of HIV infection than the presence of any antibodies. The most
sensitive virus isolation technique is to cocultivate the test sample with uninfected, mitogen-stimulated
peripheral blood mononuclear cells. Primary isolates of HIV grow very slowly compared with
laboratory-adapted strains. Viral growth is detected by testing culture supernatant fluids after about 7-
14 days for viral reverse transcriptase activity or for virus-specific antigens.
2. Serology: Detection of antibodies by ELISA (enzyme-linked immunosorbent assay). Because
there are some false-positive results with this test, the definitive diagnosis is made by Western blot
analysis, in which the viral proteins are displayed by acrylamide gel electrophoresis, transferred to
nitrocellulose paper (the blot), and reacted with the patient’s serum. If antibodies are present, they will
139
be bound to the viral proteins (predominantly to the gp41 or p24 protein). Enzymatically labelled
antibody to human IgG is then added. A colour reaction reveals the presence of the HIV antibody in
the infected patient’s serum.
3. Detection of viral nucleic acid or antigens: The polymerase chain reaction (PCR) is very
sensitive and specific technique that can be used to detect HIV DNA (branched – chain DNA – bDNA
test). The HIV RNA levels are important predictive markers of disease progression and valuable tools
to monitor effectiveness of antiviral therapies. Low levels of circulating HIV-1 p24 antigen can be
detected in the plasma by ELISA soon after infection. The antigen often becomes undetectable after
antibodies develop and may reappear late in the course of infection, indicating a poor prognosis. The
test can be used to detect antigen in supernatant fluids from virus-infected tissue culture cells.
The immunobloting test is used for revealing viral proteins gp 24, gp41and others.

140
 Sample tests

1.Genome of AIDS virus is:

a)a linear DNA
b)single-stranded RNA of negative polarity
c)circular double-stranded RNA
d)single-stranded RNA of positive polarity, presented by two molecules

2.The following cell populations are sensitive to AIDS virus, Except:

a)hepatocytes
b) monocytes
c)macrophages
d)B –lymphocytes

3.Which of the following are typical of Retroviruses:

1.contain revertase enzyme
2.is simple virus
3.genome is presented by RNA and DNA
4.genome is presented by RNA
a)1.3. b)1.2. c)1.4. d)2.4.

4.Which is the function of reverse transcriptase enzyme for AIDS virus:

a)changes the antigenic structure of virus
b)enhances the penetration of virus into the cell
c)synthesizes DNA on template of RNA
d)provides release of virus from the cell

5.Which of the following are typical of AIDS virus, Except:

a)is RNA-containing, enveloped virus
b)is provided by lymphotropic activity
c)affects CD4cells
d)affects T-killer cells

141
 BIBLIOGRAPHY

1. Anathanarayan and Paniker’s “Textbook of Microbiology”, India, 2013.

2. Warren Levinson, Ernest Jawetz “Medical microbiology and immunology” (New-York,

Tokyo, Madrid), 2000.

3. Ivan Roitt, Jonatha Brostoff, David Male “ IMMUNOLOGY” (London, New York, Toronto),
2001

142