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 [1539].

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[1539]

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.

Signs of the disease:

The first symptoms of gastrointestinal anthrax are nausea, loss of appetite, bloody diarrhea, and fever, followed by severe stomach pain. One-fourth to more than half of GI anthrax cases lead to death.

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.[1666]

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. [1667] [1668] [1669] [1670]

Revocation of the Lather Brushes Regulation in 1998 [1671]

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) [1689].

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[1690]:

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.

Host cell invasion strategy of Bacillus antracis discovered

[]
Lethal toxin is a virulence factor of Bacillus anthracis in the pulmonary anthrax form. This disease is fatal when not treated. The host responds with inflammatory process of the lung. Raymond and colleagues 2009 found that Bacillus anthracis strain expressing active lethal toxin represses the inflammation in part by altering chromatin accessibility of IL-8 promoter to NF-κB in epithelial cells. The authors concluded that the epigenetic reprogramming of the lethal toxin is an efficient strategy for host invasion used by Bacillus anthracis.

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

Security of BioSafety Level 4 laboratories

[1691]
LeDuc and colleagues report that BioSafety Level 4 (BSL-4) laboratories directors in the United States reviewed in 2008 the current status of biocontainment laboratory operations. Various approaches to strengthen the safety and security of maximum-containment laboratory workers and their environment have been proposed:

Having 2 persons physically present within the BSL-4 facility any time that work is being performed (the 2-person rule). This was discarded as being to expensive, challenged its value and considered the risks that would result from a requirement that 2 persons be physically present in the laboratory at all times.
Use of surveillance cameras to monitor workers in the BSL-4 facility; and 3) use of radios or other means of communication between workers inside the laboratory and qualified contact persons outside the actual BSL-4 environment. This was found to present the best results, because the video material can be achieved for years and video cameras are already present in these departments, presenting no additional expenses.
All BSL-4 laboratories today are equipped with remote-controlled surveillance cameras that can track a person within the maximum-containment laboratory.
Use of radios or other means of communication between workers inside the laboratory and qualified contact persons outside the actual BSL-4 environment. All BSL-4 laboratories have telephones, and most BSL-4 facilities in the United States have some form of radio communication available to persons working in the suite.

Personnel screening:

According to CDC persons who have gained the right of independent access to maximum-containment facilities are highly trained professionals who have earned the confidence of the laboratory director and are generally well respected and trusted by their colleagues. In addition, all such persons have had satisfactory background investigations and have obtained Department of Justice numbers in compliance with the Centers for Disease Control and Prevention Select Agent Program requirements. Furthermore, several BSL-4 laboratories are enacting some form of enhanced psychological screening and formal periodic monitoring of these persons.

US Biosafety Level 4 (BSL-4) maximum-containment laboratories work with biologic war organisms like Anthrax, plague, smallpox, botulism, Viral Hemorrhagic Fevers, tularemie. Although no clinical infections, or major incidents in operation of the physical facilities were publicly known, a serious lack of security of these laboratories is its own personal which can steal powerful bacteria and viruses from their working place to use it as blackmail, avenge or even selling it to terrorists. The anthax terror attacks 2001/2002 were caused by a security leak of a government laboratory. [1692] [1693]
Such a lack of security has been crucially demonstrated by Dr. Bruve Ivins working for years at Fort Detrick, Maryland, known for military research and development of infectious pathogens and biological weapons. The anthrax bacteria of this laboratory killed five people and sickened 17 others. This incidence demonstrates the eminent public threat of keeping, multiplying and potentiating virulence of disease agents for biologic weapons.

The anthrax spores used in the 2001/2002 attacks came from a single flask of spores known as Bacillus anthracis RMR-1029 that was created and solely maintained by Dr. Ivins. While working inordinate hours alone at night and on the weekend in the lab where the flask of spores and production equipment were stored. Ivins had a history of mental health problems, including paranoid delusions. The government closed the case after Dr. Ivins committed suicide in 2008.

The threat is still existent and it comes from the evil created by the US public paranoia of self defence building up an arsenal of fire arms scattered at households all over the country, together with government activities building up biologic chemical and nuclear weapons. The USA must now be protect against itself, so as happened in 2001 with Anthrax bacteria from Fort Detrick.

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.
[1541]
The Pentagon sent a team of 113 people to the island to neutralize between 100 and 200 tons of anthrax in 2002. [1542]

Cities Readiness Initiative CRI-compliant mass prophylaxis campaigns not efficient in case of a large scale anthrax attack

[1543]
Hupert and colleagues 2009 in a study on the a large-scale anthrax attack on a large city found that such an attack would overwhelm hospital resources even with an extremely effective public health response. The authors found that such a breakdown of the health service would result primarily of expected delays in detecting the attack and initiating a response to it varying from 2.4% to 6.5% of the population which would need hospitalisation, depending on how many days the campaign would be delayed to start. The authors concluded that Cities Readiness Initiative CRI-compliant mass prophylaxis campaigns may not cope with aerosol anthrax releases in major cities. They urge for more attention on such subject.

Baccam and Boechler in a study of came to the conclusion that all protective measures and medical assistance activities are influenced by a rapid and effective post-attack activity. Uncertainty in medical efficacy and the time to initiate a post-exposure prophylaxis campaign were found to have the greatest impact on the number of predicted deaths. [1544]