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 4-8x11.5μm, 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 13h & 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 enzymaticbinding 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 mitogenactivated 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 flulike, 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 Gramnegative 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 0.21% 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 1315°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.

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