Anthrax

Introduction

Anthrax is a zoonosis, a pick it up from contaminated soil by either eating or inhaling the spores.

Animals which suffer a sudden death caused by Anthrax show blood and bloody fluids coming out of body orifices. Anthrax bacteria which are present in this blood and fluids draining into the soil form spores which were proved to be alive after 70 years. Burying infected materials such as carcasses, blood into pits is a menace to further generations as memory of the site goes lost and spores are liberated because of earth work such as construction of roads, new plantations or even installing a new grave.

The anthrax bacteria in the carcass of died animals are likely to be killed by the bacteria of putrefaction. The main danger lies therefore on blood and fluids draining into soil as due to specific conditions of environment and temperatures sporulation can take place [901].

History of Anthrax

The Bible as deep source of ancient knowledge of food, veterinary, and human medicine cites at Exodus the Sixth Plague killing livestock and affecting people with black spots.

Homer in his Iliad refers to what probably was a plague of Anthrax as a punishment imposed by Apollo.

Virgil in his Georgics writes about a disease spreading from animal to human[901]

Bacillus anthracis Cohn 1872,177.Al

Anthrax bacteria belong to the Bacillus genus which has only one aerobic form. This form is Bacillus anthracis which causes skin anthrax or if inhaled the serious form of pulmonary anthraxThe spores are ellipsoidal, located in the middle of the vegetative form, without enlarging the original form. After a certain time the vegetative form decays due to autolysis. The spores remain in union with the previous spores through a thin layer. In this manner log chains of spores are formed.

This organism was seen hundred years ago in the blood of animals ill with anthrax. Robert Koch 1877 proved it to be the cause of the disease by inoculating pure cultures into susceptible cattle:

Characteristics: Gram-positive rods with square end shape, tending to form long chains (Bamboo cane like), which is very specific. The germ is not motile. This is important to distinguish it from other sporulating aerobic bacteria.
The rods measure 1-1,2 micra in width and 3,5 micra in length. The vegetative form are destroyed by chemical and physical agents but the spores can survive for decades in dust or soil and on other objects. The spores survive 5 minutes boiling and ordinary disinfectants.

Sterilisation and disinfection: The spores are sterile in dry heat of 150 $^{o}$ C only after 60 minutes.
In humid atmosphere anthrax spores die in 5 to 10 minutes at 100 $^{o}$ C.

In threads of silk the anthrax spores are sterilised at 121

C in 15 minutes. This has been used to test the function of autoclaves using dried threads of silk or hair which had been inoculated with anthrax spores. Because of the danger of handling such pathogen germ anthrax spores are now substituted by not pathogenic earth bacteria.

Disinfection of hands: Wash carefully with water and soap. Do not use nail brushes as small damage to the skin may happen permitting anthrax spores to get into deeper layers of the skin.
Hands may be disinfected using 0,2% peracetic acid (or 0,5% Wolfasteril 2 X 1 minute.

Disinfection is made with 10% formalin for 2 hours. The spores are resistant to 5% solution of phenol.
Surfaces can be disinfected with 1% peracetic acid (or 2,5% Wolfasteril for 20 minutes)

The spores are not formed in living animals or even mankind, but are present in large number in agar culture and in dead organism.

Culture of Bacillus anthracis

Media: Anthrax bacteria grow well in standard media. For diagnostic blood-agar is often used with little or no hemolysis. The bacilli have capsules in the animal body but are not capsulated in artificial culture.

Morphology of the colonies:On agar plates the colonies are large, white and rough and have "curled hair" edges.

Identification of Bacillus anthracis

Direct sputum, smears of wounds, stool, from hairs, hides, feedstuffs,fertilisers, soil or enrichment broth are plated on blood-trimethoprim-agar plates ( Art nr. 1611e Heipha Diagnostika), incubation for 18 to 24 hours at 36+-1 $^{o}$ C

Morphology of colonies on BTP-Agar:Most of Bacillus spp can grow rapidly on this Agar. The selective supplement Trimethoprim/sulfamethoxazol inhibits completely Bacillus subtilis and to a high degree the Bacillus licheniformis is suppressed. The growth of Gram-positive cocci such as Staphylococcus and Enterococcus as well as Enterobactericeae is very reduced. Sheep blood supports the growth. A strong hemolysis is typical for other Bacillus spp. such as Bacillus cereus, Bacillus thuringiensis, Bacillus mycoides.

All colonies of Bacillus anthracis present definitely no hemolysis at Blood-Trimethoprim-Agar.

Bacillus anthracis grows as white, great colonies (2-6 mm in diameter) with characteristic dull surface.
Gram-positive rods,spores, smooth ends, no beta-hemolysis, capsules.

Substance	gram/litre
Peptone from casein	14,0 g
Peptone from beef	4,5 g
Yeast extract	4,5 g
NaCl	5,0 g
Agar	16,0 g
Sheep blood	50 ml
Trimethoprim	1,6 mg
Sulfamethoxasol	6,4 mg
pH 7,3 +- 0,2 light-red colour

Identification on Cereus-Ident-Agar should be made of the suspected colonies:

Substance	gram/litre
Special peptone	21,0 g
Growth supplement	17,0 g
Chromogenic selective
supplement	2,7 g
Agar
pH 7,3 +- 0,1 teh medium is clear and light-yellow

The chromogenic substrate X-myoinositol-1-phosphate can be broken by enzymes from Bacillus cereus to the chromophor X-(5-bromo-4-chloro-3-indoxyl) which precipitate in the inside of the colony with the characteristic turquoise colour.

Differentiation between Bacillus anthracis and Bacillus cereus
The suspected colonies from the Blood-Trimethoprim-agar plate should be transfered to a Cereus-Ident-Agar (Art Nr. 174e Heipha Diagnostika).

Bacillus cereus and Bacillus thuringiensis as colonies with up to 4 mm as diameter and turquoise colour. Bacillus anthracis colonies have the same size ( up to 4 mm ) but they are white. Biochemical reactions and PCR should be performed with these colonies.[961]

Disease

Anthrax disease is found in sheep,cattle and pigs.
Before terrorists disseminated spores of anthrax in America infection in human was rare and occurred by handling products or eating meat from infected animals. Today the disease is still common in South and Central America, Southern and Eastern Europe, Asia, Africa and the Caribbean

Intestinal anthrax:The spores in animal feed, in dust of infected soil with excreta or carcasses of diseased animals are ingested and can cause an intestinal infection.

Contaminated food can also bear anthrax bacteria. The disease can turn out to be fatal if not treated.

Skin anthrax: Man can get skin anthrax handling infected animals, their hides, wool, hair and bristles.
Diagnosis is done by gram-colouring and culture of exudates or smear.

Pulmonary anthrax: Is acquired by inhaling anthrax spores from wool or hair of diseased animals.The disease can turn out to be fatal if not treated
Diagnosis is made from culture of blood. The culture of the sputum is often negative.

The source of animal epidemics is animal feed.
The source of infections of man is wool, hair or hides. Contaminated bristles of shaving brushes causing facial pustules have been reported. [962] [963] [964] [965]

Revocation of the Lather Brushes Regulation in 1998 [966]

All these reported cases of human anthrax infection with hairs and brushes date before 1930. FDA states that no case of cutaneous anthrax in the United States has been associated with lather brushes since 1930, and the continuation of existing requirements is unnecessary to protect the public health. And CDC revoked the Lather Brushes Regulation pertaining to the treatment, sterilization, handling, storage, marking, and inspection of lather brushes. 42 CFR 71.151 on January 11, 1985 (50 FR 1516), in 1998.

Therapy: Beta-lactame, Penicillin and other antibiotics such as Ciprofloxacin and Penicillin V Fluorochinolone, Doxycyline and macrolide (erytromicin, spiramycin, josamycin and azalide) [978].

Pathogenesis of Bacillus anthracis

Death is most likely due to O $_{2}$ depletion, secondary shock, increased vascular permeability followed by respiratory and cardiac failure.
The pathogenesis depends on two virulence factors[979]:

Poly-D-glutamic acid capsule
tripartite toxin

Poly-D-glutamic acid capsule: is encoded by the plasmid pX02, which can be transferred to a nonencapsulated Bacillus anthracis by transduction resulting in a capsulated phenotype. The capsule is non toxic but protects the bacterium from bactericidal components and from phagocytosis.

Tripartite toxin is mediated by the temperature sensitive plasmid pX01. The toxin consists of three parts:
82.7 kDa protective antigen (PA)
90.2 kDa lethal factor (LF)
88.9 kDa oedema faktor (OF)
-Remark: "Da" stands for Dalton- other findings of Bradley et al and Andrew Panifer et al published in Nature 2001,414 225-233 give hope to the development of new therapeutics against anthrax and the tumours caused, being interesting for farmers of the regions where anthrax is prevailing.

Control of animal anthrax: The spread of the disease is being controlled by use of a vaccine of avirulent spores and safe disposal of carcasses.
The disease may be imported from other countries by means of hides, leather and wool with origin from anthrax epidemic regions.

Diagnosis

The diagnosis is positive when large gram-positive rods are found in smears of pus from a typical malignant pustule with black edges giving the skin anthrax disease the name of "carbuncle" which means "charcoal". Further cultivation may be necessary, showing characteristic colonies on nutrient agar. Genetic identification follows.
In the mid 70th a 48 years old German died of intestinal anthrax after eating meat and sausages from an animal from emergency slaughtering.
A sever intestinal disease caused by Anthrax took place in April 1979 in the city Sverdloosk causing 64 human death resulting from tainted meat.
In 1994 one case of skin Anthrax was known in Germany.

Bacillus anthracis is similar to Bacillus cereus

Virulent and avirulent strains can be differentiated from Bacillus cereus using the API system.

Characteristics	B. anthracis	B. cereus
Motility	-	+
Lysis by gama phage	+	-
String of perl test(10 U	+	-
ml penicillin G)
Growth on
Chloralhydrate agar	-	+
2-Phenylethanol agar	d	+
Polymyxin-lysozyme-	+	-
EDTA-thallous acetate agar
Phosphatase	-	d
Degradation of tyrosine	-	d

War reminescence:

During the cold war several tons of Anthrax bacteria were stock piled in the city of Sverdlovsk as a weapon. Later on, Mikhail Gorbachev decided the Anthrax should be destroyed. The whole stockpile was bleached and poured into the ground of Vosrozhdeniya (Renaissance) island in the Aral Sea.

Tests of soil samples from six of 11 vast burial pits show that some of the spores were still alive in 1999.

The spores are highly resistant to inactivation and may be present in soil for decades. They may infect animals that ingest the spores while grazing. Uzbek and Kazakh experts fear the buried anthrax spores could escape their sandy tomb, stirred up by carriers like gophers and other rodents, lizards and birds, and be brought to Uzbek and Kazak territory.

Central Asian and U.S. officials fear that, as access to the island eases, the buried anthrax could be used by terrorists to make more of the deadly agent.
[903]
The Pentagon sent a team of 113 people to the island to neutralize between 100 and 200 tons of anthrax in 2002. [904]