Causative agents of zoonoses:

B.anthracis and L.interrogans

In zoonoses a nonhuman vertebrate host is

the reservoir of infection and humans are
involved only incidentally. The human
infection follows contact with the reservoir
host, but is not essential for the microbe’s
life cycle, or for its maintenance in nature.
Only few zoonotic infections are transmitted
effectively form human to human.
Classification:
2. Infections of viral nature (rabies, tick-borne encephalitis
etc.)
3. Infections of bacterial nature (plaque, tularemia, anthrax,
brucellosis etc.)
4. Infections of rickettsial nature (Q fever, tsutsugamushi
fever)
5. Infections of chlamydial nature (psittacosis)
6. Infections, caused by fungi
7. Infections, caused by protozoa
8. Infections, caused by spirochetes (leptospirosis, Lyme
disease)
 Causative agent of anthrax

Kingdom:Bacteria
Phylum:Firmicutes
Class:Bacilli
Order:Bacillales
Family:Bacillaceae
Genus:Bacillus
Species:B. anthracis
Morphology: the typical large cells, measuring 4-8x1-
1.5μm, have square ends and are arranged in long chains;
spore is located in the center of nonmotile bacilli. Bacilli are
Gram-positive. Microorganisms can form capsule into hosts
or on serum agar.
B.anthracis in pus (leucocytes are seen) , stained
by methylene blue
B.anthracis in tissue (capsule can be revealed
around some bacilli), stained by Gram method
B.anthracis in tissue (stained by
immunofluorescent method)
B.anthracis in pure culture, stained by Oscheshko
method (blue vegetative cells & red spores)
When conditions for growth are good, with plentiful
nutrients and water available (e.g., in the host),
B.anthracis are rod-shaped vegetative organisms that
grow and divide. When conditions are unfavorable, each
forms a very resistant dormant spore that is able to
survive extreme environmental conditions.
The spore is a dehydrated cell with thick walls and
additional layers that form inside the cell membrane. It
can remain inactive for many years, but if it comes into a
favorable environment, it begins to grow again. It is
sometimes called an endospore, because it initially
develops inside the rod-shaped form. The endospores of
B.anthracis are oval. They are highly refractile and
contain dipicolinic acid. Electron micrograph sections
show that they have a thin outer spore coat, a thick
spore cortex, and an inner spore membrane surrounding
the spore contents. The spores resist heat, drying, and
many disinfectants (including 95% ethanol).
• The ability of Bacillus anthracis to form spores
makes it a difficult organism to control. Spores
can exist in the soil for decades. They can drift
gently in the wind, dormant until they find a place
that has the temperature, nutrients, and other
conditions to allow growth. When they find their
new host (an animal or human) they change to
the rod-like form and begin to multiply rapidly.
While they are in the spore form they can
survive boiling, freezing, or even suspension in
alcohol. They can resist dry heat at 140 C for 1-
3h & boiling or steam at 100 C for 5-10 min. It
takes special measures to kill them, such as
steam under pressure (autoclaving at 121 C
destroys them in 15 min.), or chemicals known
as sporicides (5% formaldehyde). The most
effective measure is burning of animal
carcasses, dead from anthrax.
• This ability to survive extreme conditions for long
periods of time is one of the major reasons
Bacillus anthracis has been used by terrorists.
The organism multiplies in soil when the soil
pH>6 and when early rain has been followed by
a long dry spell, supporting the existance of soil
source of infection for decades (now it is proved
than even the whole century).
• For experienced microbiologist, growing Bacillus
anthracis in the laboratory and causing it to form
spores is an easy task. Putting a culture
containing millions of Bacillus anthracis spores
into a form (envelope scandal in USA in 2001)
can produce an effective weapon.
 Cultural properties: colonies of B.anthracis are round, it
grow on all ordinary media as typical colonies with a wavy
margin, the so called “medusa head” appearance.
Hemolysis is uncommon with B.anthracis. Gelatin is
liquefied (“inverted fir tree” on gelatin stab culture).
 Antigenic structure

B.anthracis has three types of antigens:

1. Capsular antigen
2. Polysaccharide antigen of cell wall
3. Protective antigen (component of anthrax
toxin complex)
 Virulence factors
B.anthracis has only two virulence factors:
2. The poly-D-glutamic acid capsule
3. The toxin complex comprising three
proteins: the protective antigen, oedema
factor & lethal factor
Bacillus anthracis forms a single antigenic type of capsule consisting
of a poly-D-glutamate polypeptide. All virulent strains of B. anthracis
form this capsule. Production of capsular material is associated with
the formation of a characteristic mucoid or "smooth" colony type.
"Smooth" (S) to "rough" (R) colonial variants occur, which is correlated
with ability to produce the capsule. R variants are relatively avirulent.
Capsule production depends on plasmid; its transfer to
nonencapsulated B. anthracis via transduction produces the
encapsulated phenotype.
• The poly-D-glutamyl capsule is itself nontoxic,
but functions to protect the organism against the
bactericidal components of serum and
phagocytes, and against phagocytic engulfment.
The capsule plays its most important role during
the establishment of the infection, and a less
significant role in the terminal phases of the
disease, which are mediated by the anthrax
toxin.
• The poly-D-glutamyl capsule is formed in vivo or
in the laboratory when the bacterium is grown on
serum plates in a 5% CO2 atmosphere.
 Anthrax toxin complex
• Production of the anthrax toxin is mediated by plasmid. The toxin
consists of three distinct antigenic components. Each component of
the toxin is a thermolabile protein with a mw of approximately
80kDa.
• Factor I is the edema factor (EF) which is necessary for the edema
producing activity of the toxin. EF is known to be an inherent
adenylate cyclase, similar to the Bordetella pertussis adenylate
cyclase toxin.
• Factor II is the protective antigen (PA), because it induces
protective antitoxic antibodies in guinea pigs. PA is the binding (B)
domain of the anthrax toxin which has two active (A) domains, EF
(above) and LF (below).
• Factor III is known as the lethal factor (LF) because it is essential
for the lethal effects of the anthrax toxin. Apart from their
antigenicity, each of the three factors exhibits no significant
biological activity in an animal. However, combinations of two or
three of the toxin components yield the following results in
experimental animals.
• Anthrax toxin has the familiar A-B enzymatic-
binding structure of bacterial exotoxins with PA
acting as the B fragment and either EF or LF
acting as the active A fragment.
• EF+PA has been shown to elevate cyclic AMP to
extraordinary levels in susceptible cells.
Changes in intracellular cAMP are known to
affect changes in membrane permeability and
may account for edema. In macrophages and
neutrophils an additional effect is the depletion
of ATP reserves which are needed for the
engulfment process. Hence, one effect of the
toxin may be to impair the activity of regional
phagocytes during the infectious process.
LF+PA have combined lethal activity as stated
above. The lethal factor is a Zn++ dependent
protease that induces cytokine production in
macrophages and lymphocytes, and its
mechanism of action is slowly becoming
understood. The crystal structure of lethal factor
is known to to be a member of the mitogen-
activated protein kinase (MAPKK) family of
enzymes that disrupts cellular signaling.
Furthermore, the identity of the human receptor
for anthrax PA, named anthrax toxin receptor,
has been demonstrated to be a type I
membrane protein that binds directly to PA.
The effects of EF and LF on neutrophils have been
studied in some detail. Phagocytosis by
opsonized or heat-killed Bacillus anthracis cells
is not inhibited by either EF or LF, but a
combination of EF + LF inhibits engulfment of
the bacteria and the oxidative burst in the pmns.
The two toxin components also increased levels
of cAMP in the neutrophils. These studies
suggest that the two active components of the
toxin, EF + LF, together increase host
susceptibility to infection by suppressing
neutrophil function and impairing host
resistance.
Transmission of anthrax
 Pathogenesis of anthrax
The human is usually a dead-end-host. In humans
there are four possible forms of the disease
anthrax. Historically, the most common form has
been cutaneous anthrax, in which the organism
enters through a break in the skin. The
cutaneous form begins as a papule at the entry
site that progresses over several days to a
vesicle and then ulcerates. Edema, sometimes
massive, surrounds the lesions, which then
develop a characteristic black eschar. The
patient may have fever, malaise and headache.
A small percentage of cutaneous infections
become systemic, and these can be fatal.
a characteristic black eschar
A more serious form is inhalation anthrax. Here the victim
breathes in the organism and develops a severe
respiratory disease. Systemic infection resulting from
inhalation of Bacillus anthracis has a mortality rate
approaching 100%. Initial symptoms are vague and flu-
like, progressing to hypotension, shock and massive
bacteremia and toxemia. The severe symptoms are
believed to be the result of the bacillis exotoxins. Early
antibiotic treatment is an absolute necessity and should
be started during the incubation period if a person has
been exposed. After acute symptoms have appeared,
antibiotics can kill the organisms, but will not destroy the
powerful toxins that have already been formed, and the
person commonly dies in 2-3 days from respiratory
failure, sepsis and shock.
• The third form, intestinal anthrax, is
contracted from the consumption of
contaminated meat.Oropharyngeal
anthrax begins with severe sore throat or
with an ulcer in the oropharyngeal cavity,
accompanied by neck swelling and fever.
Gastrointestinal anthrax begins with
anorexia, nausea, vomiting and abdominal
pain. There may be hemorrhagic diarrhea.
Intestinal anthrax can become systemic
and lead to death.
The fourth form, septic anthrax, is acquired
by bites of insects (fleas etc.) or as
consequence of generalization of previous
three forms. The outcome is lethal.
The special equipment for work with anthrax
Scientific work with anthrax in the laboratory
 Diagnosis of anthrax
Methods of diagnosis:
Bacterioscopical
Bacteriological
Biological
Serological
Skin test
Molecular-genetic
 Specific prophylaxis of anthrax
In Russia live vaccine is used. It contains viable spores of
nonencapsulated strain of B.anthracis. Two injections
protect for 1 year. Annual booster injections of the
vaccine are required to maintain a protective level of
immunity.
The anthrax vaccine for humans, which is used in the
U.S., is a preparation of the protective antigen
recovered from the culture filtrate of an avirulent,
nonencapsulated strain of Bacillus anthracis that
produces PA during active growth. Anthrax immunization
consists of three subcutaneous injections given two
weeks apart followed by three additional subcutaneous
injections given at 6, 12, and 18 months. Annual booster
injections of the vaccine are required to maintain a
protective level of immunity.
 Treatment of anthrax
Antibiotics should be given to unvaccinated
individuals exposed to anthrax. Penicillin,
tetracyclines and fluoroquinolones are effective if
administered before the onset of lymphatic
spread or septicemia, estimated to be about 24
hours. Antibiotic treatment is also known to
lessen the severity of disease in individuals who
acquire anthrax through the skin. Inhalation
anthrax was formerly thought to be nearly 100%
fatal despite antibiotic treatment, particularly if
treatment is started after symptoms appear.
Anti-anthrax immunoglobulin is used for
prophylaxis and treatment of anthrax.
Leptospira interrogans – causative agent of
leptospirosis
Taxonomy
Kingdom:Monera
Phylum:Spirochaetes
Class:Spirochaetes
Order:Spirochaetales
Family:Leptospiraceae
Genus:Leptospira
Species:Leptospira interrogans
Consists of 32 serogroups and more than 250 serovars
Morphology:
Leptospira are spiral-shaped bacteria that are 6-20
μm long and 0.1 μm in diameter with a wavelength
of about 0.5 μm. One or both ends of the
spirochete are usually hooked. Because they are
so thin, live Leptospira are best observed by
darkfield microscopy.
The bacteria have a number of degrees of
freedom; when ready to proliferate via
binary fission, the bacterium noticeably bends in
the place of the future split.
L.interrogans by darkfield microscopy &
silver impregnation
L.interrogans by immunofluorescent test
• Leptospira have a Gram-negative-like cell envelope consisting of a
cytoplasmic and outer membrane. However, the peptidoglycan layer
is associated with the cytoplasmic rather than the outer membrane,
an arrangement that is unique to spirochetes. The two flagella of
Leptospira extend from the cytoplasmic membrane at the ends of
the bacteria into the periplasmic space and are necessary for the
motility of Leptospira.
• The outer membrane contains a variety of lipoproteins and
transmembrane outer membrane proteins. As expected, the protein
composition of the outer membrane differs when comparing
Leptospira growing in artificial medium with Leptospira present in an
infected animal. These proteins may be important for adhesion of
Leptospira to host tissues and in resisting complement, respectively.
• The outer membrane of Leptospira, like those of most other Gram-
negative bacteria, contains lipopolysaccharide (LPS). Differences in
the highly immunogenic LPS structure account for the numerous
serovars of Leptospira.Consequently, immunity is serovar specific;
current leptospiral vaccines, which consist of one or several
serovars of Leptospira endemic in the population to be immunized,
protect only against the serovars contained in the vaccine
preparation. Leptospiral LPS has low endotoxin activity.
 Cultural properties of L.interrogans
• Leptospira are typically cultivated at 30°C in liquid and
semisolid media, which can be supplemented with 0.2-
1% rabbit serum to enhance growth of fastidious strains.
Growth of pathogenic Leptospira interrogans in an
artificial liquid media becomes noticeable in 4-7 days;
growth of saprophytic strains occur within 2-3 days. The
minimal growth temperature of pathogenic species is 13-
15°C. Because the minimal growth temperature of the
saprophytes is 5-10°C, the ability of Leptospira to grow
at 13°C can be used to distinguish saprophytic from
pathogenic Leptospira species. The optimal pH for
growth of Leptospira is 7.2-7.6.
• Leptospira are aerobes whose major carbon and energy
source during in vitro growth is long-chain fatty acids,
which are metabolized by beta-oxidation. Fatty acid
molecules are bound by albumin and are released
slowly into the medium to prevent its toxic accumulation.
 Virulence factors of L.interrogans
Adhesive factors:
cytoadhesines to hepatocytes and
nephroepithelium
Invasive factors:
hyaluronidase, fibronolysin, lipases;
spiral structure of leptospiral cells and their
motility
Toxins:
Several hemolysins, protein toxic substance,
endotoxin
 Transmission of leptospirosis
Urinary shedding of organisms from infected animals is the most
important source of these bacterial pathogens. Contact with the
organism via infected urine or urine-contaminated media (e.g., water,
food, soil) results in human infection. The organism enters the
body via abraded skin or mucous membranes, such as the conjunctiva
or alimentary tract. Occasionally, the organism may even enter the
body through intact skin. Infection has occurred after animal and rodent
bites, after contact with abortion products of infected animals, and after
ingestion of contaminated food and water. The latter route of
infection is believed to occur via the mucosa of the mouth and the
esophagus because leptospires cannot survive in an acidic
environment. The human is usually a dead-end-host.
Leptospirosis (e.g., dogs) in animals is often subclinical. Leptospires
may persist for long periods in the renal tubules of animals by
establishing a symbiotic relationship with no evidence of disease or
pathological changes in the kidney. As a result, animals that serve as
reservoirs of host-adapted serovars can shed high concentrations of
the organism in their urine without showing clinical evidence of disease.
 Pathogenesis of leptospirosis
After the organism gains entry via intact skin or mucosa, it
multiplies in blood and tissue. The resulting leptospiremia
can spread to any part of the body but particularly affects
the liver and kidney.
After the organism gains access to the kidney, it migrates
to the interstilium, renal tubules, and tubular lumen causing
interstitial nephritis and tubular necrosis. When renal failure
develops, it is usually due to tubular damage, but
hypovolemia from dehydration and from altered capillary
permeability can also contribute to renal failure.
Liver involvement is seen as centrilobular necrosis with
proliferation of Kupffer cells. Jaundice may occur as a
result of hepatocellular dysfunction.
• Leptospires may also invade skeletal muscle, causing
edema, vacuolization of myofibrils, and focal necrosis.
Muscular microcirculation is impaired and capillary
permeability is increased, with resultant fluid leakage
and circulatory hypovolemia.
• In severe disease, a disseminated vasculitic syndrome
may result from damage to the capillary endothelium.
Great amount of endotoxin released from disrupted
leptospires cause such damage & induce fever via its
pyrogenic properties.
• Despite the possibility of severe complications, the
disease is most often self-limited and nonfatal. Over
time, a systemic immune response may eliminate the
organism from the body but may also lead to a
symptomatic inflammatory reaction that can produce
secondary end-organ injury.
• Immunity is type-specific. The patient can suffer from
another serotypes of leptospires.
Scheme: pathogenesis of leptospirosis
Skin rash typical to leptospirosis
 Stages of leptospirosis
• The natural course of leptospirosis falls into 2 distinct phases: septicemic
and immune. During a brief period of 1-3 days between the 2 phases, the
patient shows some improvement.
First stage: This stage is called the septicemic or leptospiremic
stage because the organism may be isolated from blood cultures,
cerebrospinal fluid (CSF), and most tissues. During this stage,
which lasts about 4-7 days, the patient develops a nonspecific
flulike illness of varying severity.It is characterized by fever, chills,
weakness, and myalgias, primarily affecting the calves, back, and
abdomen.
Second stage:This stage is called the immune or leptospiruric
stage because circulating antibodies may be detected or the
organism may be isolated from urine; it may not be recoverable
from blood or CSF.This stage occurs as a consequence of the
body's immunologic response to infection and lasts 0-30 days or
more. Disease referable to specific organs is seen. These organs
include the meninges, liver, eyes, and kidney.
 Diagnosis of leptospirosis
Methods of diagnosis:
Bacterioscopical
Bacteriological
Biological
Serological
Molecular-genetic
 Specific prophylaxis of leptospirosis
Specific immunization against leptospirosis
is based on usage of killed vaccine,
prepared from heat-inactivated leptospires
of the most widespread serogroups &
serovars. Vaccination is used for the
groups of high risk (sewage workers,
travellers (e.g. swimming in contaminated
water), farmers, veterinarians, rodent
control workers, and other occupations
with animals).
 Treatment of leptospirosis
Aetiotropic drugs are antibiotics, such as
cefotaxime, doxycycline, penicillin,
ampicillin, and amoxicillin. Also specific
anti-leptospirosis immunoglobulin is used.
It is prepared from the blood of bullocks
hyperimmunized by killed leptospirosis
vaccine. Such immunoglobulin is
especially effective if inoculated during 3-4
days after acquiring infection. It decreases
the severity of leptospirosis.