Weapons Grade Anthrax (The confusion continues - excepts from linked sources)Q. If anthrax is used as a weapon, isn't it "weaponized?" A. No.
Q. If I test positive for anthrax, am I "infected." A. Yes. Infected: "To contaminate, or cause to be diseased with a germ or virus." - Webster's New World Dictionary.
Q. What liquid would be used for spraying weaponized anthrax? A. Probably saltwater in a saline concentration similar to blood with anthrax spores suspended to a concentration of approximately 1.5 X 109 CFU/ml. (See "How Anthrax is made for aerosol challenge at Fort Detrick" below).
Iraq's Trademark Bentonite Additive
WASHINGTON, Oct. 28 — ABCNEWS has been told by four well-placed and separate sources that initial tests on an anthrax-laced letter sent to Senate Majority Leader Tom Daschle have detected a troubling chemical additive that authorities consider their first significant clue. An urgent series of tests conducted on the letter at Ft. Detrick, Md., and elsewhere discovered the anthrax spores were treated with bentonite, a substance that keeps the tiny particles floating in the air by preventing them from sticking together. The easier the particles are to inhale, the more deadly they are.
As far as is known, only one country, Iraq, has used bentonite to produce biological weapons. White House spokesman Ari Fleischer on Saturday flatly denied bentonite was found on the letters. Moments later, another senior White House official backed off Fleischer's comments, saying it does not appear to be bentonite "at this point." The official said the Ft. Detrick findings represented an "opinionated analysis," that three other labs are conducting tests, and that one of those labs had contradicted the bentonite finding. But, the official added, "tests continue."
Fleischer added today that no test or analysis has concluded that bentonite is present in the Daschle anthrax, and "no other finding contradicts or calls into question" that conclusion. Reading from what he said was a sentence from the report prepared by scientists at Fort Detrick, he told ABCNEWS, "It is interesting to note there is no evidence of aluminum in the sample." Aluminum, Fleischer said, would also be present if bentonite was.
While it's possible countries other than Iraq may be using the [bentonite] additive, it is a trademark of Iraqi leader Saddam Hussein's biological weapons program. "It means to me that Iraq becomes the prime suspect as the source of the anthrax used in these letters," former U.N. weapons inspector Timothy Trevan told ABCNEWS. In the process of destroying much of Iraq's biological arsenal, U.N. teams first discovered Iraq was using bentonite, which is found in soil around the world, including the United States and Iraq. "That discovery was proof positive of how they were using bentonite to make small particles," former U.N. weapons inspector Richard Spertzel told ABCNEWS.
http://abcnews.go.com/sections/us/DailyNews/WTC_Investigation.html
Unresponsiveness to Penicillin?
Dan Rather (CBS News anchor) said in a live TV and radio interview carried on all major networks on Thursday, October 18, 2001, that the CBS employee in his office who contracted anthrax on her chin or cheek had been given penicillin by her doctor, and he further stated that the cutaneous anthrax infection was, "unresponsive to penicillin." This unresponsiveness to penicillin can be a positive indicator of "weapons grade" anthrax. The U.S. press and media missed this comment for some unknown reason. Spore size, spore composition, VNTR, modification for aerosol challenge, and atmospheric mobility are other positive indicators.
Daschle Anthrax is Weapons Grade
"Two top government bioterror experts are convinced the anthrax sent to Senate Majority Leader Tom Daschle's office was “professionally made” and stems, directly or indirectly, from another nation's bioweapons program. The key, they say, lies not in the anthrax itself but in its formulation how the agent is prepared, its ability to become airborne, the structure of the particles and size of the spores. That over 30 people became exposed from a single envelope is telling, they add. Tom Ridge, director of the new Office of Homeland Security, said that the anthrax in the Daschle letter had not been “weaponized.” At least one outside expert concurs [with the former group]. “The empirical evidence suggests that's weapons grade material,” says Richard Spertzel, former head of the United Nations biological inspection team in Iraq. Ridge “does not know what he’s talking about,” Spertzel told U.S. News." - http://www.drudgereport.com/flash1.htm
"The letters displayed a "certain amount of expertise" in the type of anthrax they used which authorities believe was professionally produced, finely milled and electrostatically-charged to guarantee it would become airborne. Authorities said the use of electrostatically-charged anthrax in the letters meant that those who sent the bacteria sought to guarantee it would not remain stuck to the envelopes but spread into the air when opened. Keeping the bacteria together in small clumps, to enhance the chance of an airborne infection, would require an electrostatic charge ensuring that thousands of spores would cling to themselves as an airborne powder. Authorities are not sure how the letters were electrostatically-charged, but experts said several methods exist and the necessary equipment can be found in pharmaceutical or biology labs." - NY Post, Oct 24 2001
Sverdlovsk accident (USSR)
"In the years since the Sverdlovsk accident, [Ken] Alibek and a research team had taken the Soviet military's anthrax and made it even more deadly. He developed a process to take ground up anthrax spores and coat each particle in plastic and resin. It keeps the anthrax aloft four times longer, increasing its ability to infect people."
http://www.tv.cbc.ca/national/pgminfo/redlies/redlies3.html
VNTR (Variable Number Tandem Repeat) region separates all known strains into five categories
How Anthrax is made for aerosol challenge at Fort Detrick (U.S. Army study from the internet)
Spore challenge - The virulent Ames strain of B. anthracis was obtained from the U.S. Department of Agriculture, Ames, Iowa. It was grown in Leighton-Doi medium, and spores were harvested and washed in sterile, distilled water as previously described9. The spores were purified by centrifugation through 58% Renografin-76, washed again, then resuspended in 1% phenol and stored at 4°C. For aerosol challenge, spores were suspended to a concentration of approximately 1.5 X 109 CFU/ml, then heat-shocked at 60°C for 45 min. Eight-ml aliquots of the spores were used for aerosol challenge with a three-jet Collison nebulizer as previously described3,4,11. The concentration of spores in the aerosol (sampled in water in an all-glass impinger and the aerosol inhaled dose expressed as LD50) were also determined as previously described3,4,11. An aerosol inhaled dose of 5.5 X 104 spores of the B. anthracis Ames strain was previously determined to be 1 LD50 in rhesus monkeys (B. Ivins, unpublished observations).
Note: The above is not genetically modified anthrax, another indicator of weapons grade anthrax, but it is "weapons grade anthrax" because of its modification for aerosol challenge.
http://www.anthrax.osd.mil/Site_Files/articles/INDEXclinical/anthraxlibrary/efficacystandard.pdf
2.3 Strains
B. anthracis appears to be one of the most monomorphic species known. That is to say, isolates from whatever type of source or geographical location are almost identical phenotypically and genotypically. Phenotypically, strain differences are only apparent in non–quantifiable or semi–quantifiable characteristics, such as colonial morphology, flocculation in broth culture, cell size, LD50 in animal tests, and so on. The biochemical, serological or phage typing methods available in the case of other pathogens have proved of no value for identifying different strains of B. anthracis. At the molecular level, gnomic differences have also proved hard to detect, although, some progress is being made in attempts to devise chromosomally–based strain differentiation systems Henderson 1996; Anderson et al. 1996; Keim et al. 1997; Jackson et al. 1997.
3.2 Incidence of anthrax in animals
In the USA it is confined to a few persistent pockets with sporadic cases in South Dakota, Nebraska, and Oklahoma but probably a hyperendemic situation persists throughout the south western quadrant of Texas.
3.5 Diagnosis in animals
At death in most susceptible species (the pig being a notable exception), the blood contains 107 to 108 bacilli per ml provided the animal has not been treated (numbers may also be lower in immunized animals which succumb to the disease). For reasons unknown, numbers of B. anthracis at death are very low in pigs (hundreds per milliliter or less).
4 Anthrax in humans
4.1 Incidence
The major sources of human anthrax infection are direct or indirect contact with infected animals, or occupational exposure to infected or contaminated animal products. Other possible sources are rare and epidemiologically trivial. Human anthrax incidence is dependent on the level of exposure to affected animals and national incidence data for non–industrial cases reflect the national livestock situation. Historical analysis of epidemiological data globally reveals the following approximate ratios: (a) one human cutaneous anthrax case to ten anthrax livestock carcasses; (b) one incident of enteric human anthrax to 30–60 anthrax–infected animals eaten; (c) in humans, 100–200 cutaneous cases for each enteric case that occurs.
Industrial anthrax incidence data can be inferred from the volume and weight of potentially affected materials handled or imported, taking into account the quality of prevention, such as vaccination of personnel and forced ventilation of the workplace. These relationships are essentially all that can be used for many countries where human anthrax is infrequently, erratically or incompletely reported. In addition, certain countries suppress anthrax reporting at the local or national levels.
Human case rates for anthrax are highest in Africa, the Middle East and central and southern Asia. Where the disease is infrequent or rare in livestock, it is rarely seen in humans.
4.2 Susceptibility. Data for risk assessments
4.2.1 Historical information
Circumstantial evidence indicates that man is moderately resistant to anthrax. Before vaccines and antibiotics became available, and at a time when understanding of industrial hygiene was relatively basic, workers in at–risk industrial occupations processing animal products were exposed to significant numbers of anthrax spores on a daily basis. In Britain, 354 cases of anthrax in such industries were notified during the 13–year period 1899–1912 (Anon, 1918). Although the numbers of persons exposed is not known, it must have been many thousands, and the number of cases clearly represented only a very small proportion of the number exposed.
In 4 mills in the USA, in which unvaccinated workforces, varying in size from 148 to 655, were "chronically exposed to anthrax", annual case rates were only 0.6 to 1.4% (Brachman et al., 1962). In one mill, workers were found to be inhaling 600 to 1300 anthrax spores over an 8–hour shift without ill effect (Dahlgren et al., 1960) and in two goat–hair mills, B. anthracis was recovered from the nose and pharynx of 14 of 101 healthy persons. Despite extensive exposure to anthrax, cases among workers in wildlife reserves are exceedingly rare (Quinn and Turnbull, 1998).
Nevertheless, outbreaks and epidemics do occur in humans; sometimes these are sizable, such as the epidemic in Zimbabwe which began in 1979, was still smoldering in 1984–5 and had by that time affected many thousands of persons, albeit with a low case fatality rate (Turner, 1980; Davies 1982; Kobuch et al. 1990). Occasionally, the case fatality rates are substantial, such as in the Sverdlovsk incident in Russia in 1979 (Abramova et al., 1993; Meselson et al., 1994). The outbreak in a mill in New Hampshire, USA, in 1957 was not associated with any unusual change in occupational exposure but seems to have been an isolated event within a prolonged period of exposure (Brachman et al., 1960).
4.2.2 Infectious dose
Infectious doses, which have not been established for man, and the severity of the resulting infection clearly depend on several factors such as route of infection, nutritional and other states of health on the part of the infected person, and probably on the relative virulence of the infecting strain. For the purpose of risk assessments, dependency on information from animal tests is unavoidable. The published data on infectious and lethal doses in animals have been collated elsewhere (Watson and Keir, 1994).
Cutaneous infections. It probably does not take many spores to initiate a cutaneous infection, but it is generally accepted that the spores must gain access to subepidermal tissue through a cut or abrasion before this can occur and risk of infection reflects the chance of this happening. This risk is greatly reduced in at–risk occupations by appropriate clothing and gloves, dressing of wounds, and other hygienic practices.
Pulmonary (inhalation) infections. Recorded inhalation LD50s in non–human primates range from 2500 to 760 000 spores (Meselson et al., 1994; Watson and Keir, 1994). The US Department of Defence bases its strategies on an estimate that the LD50 for humans is 8000 to 10,000 spores (Meselson et al., 1994). However the only hard data on inhalation infectious doses in humans come from the studies in goat hair processing mills referred to in 4.2.1. In any event, substantial exposure is evidently necessary before the risk of inhalation anthrax becomes significant. In a recent study (Turnbull et al., 1998) the highest levels found in air sampled 3 to 9 m downwind from disturbed dry, dusty anthrax carcass sites in Namibia were 20 to 40 colony–forming units of spores per cubic metre. This corresponds to the conservative estimate that it would require about 2.5 minutes for an average human undergoing moderate activity to inhale 1 spore. It is, furthermore, well established that, at sizes above 5 µm, particles face increasing difficulty in reaching the alveoli of the lungs. The likelihood of inhaled spores penetrating far enough to induce inhalation anthrax therefore depends greatly on the size of the particles to which they are attached.
The overall conclusion from the available evidence is that the risks of pulmonary anthrax outside industrial situations are very low.
Oral route infections. There is very little information on infectious doses by the oral route, but what is true for the skin is probably largely true for the oropharyngeal and gastrointestinal epithelium. The chance of infection is likely to be enhanced by, if not dependent on, the existence of a lesion in the epithelium through which spores can gain entry and establish an infection.
Treatability. The fact that anthrax is readily treated if diagnosed at a sufficiently early stage of infection also needs to be taken into account when assessing risks. Awareness of the likelihood of exposure having taken place is clearly an important part of the equation.
4.2.3 Biological warfare associations
In the developed parts of the world where it is now seen rarely, anthrax has developed something of a "doomsday bug" status in the mind of the public, and the name frequently engenders unnecessary fear, for example, in relation to contaminated burial or industrial (e.g. tannery) sites. This anxiety results from the association of anthrax with the topic of biological warfare.
There is, in fact, no conflict between the statements and evidence given in 4.2.1 and 4.2.2 that humans are fairly resistant to fatal anthrax infection and the possible aggressive use of anthrax spores. The "worst case" natural contamination in the environment is found at the carcass sites of animals that have died of anthrax. In a study in Namibia of 106 such sites, the highest contamination level found was just over 1 million anthrax spores per gram of soil, but 79% had less than 1000 per gram and 25% less than 10 per gram (Lindeque and Turnbull, 1994). Levels in other types of inadvertently contaminated environments (soils at tannery sites, horsehair plaster, etc.) rarely exceed a few units or tens of spores per gram (Turnbull, 1996). Natural environmental exposure to infectious doses in the normal course of human life and endeavour is, therefore, a fairly unlikely event.
Aggressive scenarios, in contrast, envisage exposures to overwhelmingly massive doses (many millions of spores) which can only be created artificially. The Figure of 100 kg of dried anthrax spores, given in one article (Taylor, 1996) on the subject as technically feasible for aggressive delivery, represents dose levels in the order of 1013 human LD50s. It must be supposed that this could cause substantial devastation to human and animal communities within selectively targeted areas. The public health implication of deliberately induced anthrax outbreaks and its use as a biological weapon have been reviewed elsewhere (WHO, 1970).
4.3 Epidemiology and transmission: the forms of anthrax
Anthrax in humans is classically divided in two ways. The first type of classification, which reflects how the occupation of the individual led to exposure, differentiates between non–industrial anthrax, occurring in farmers, butchers, knackers, veterinarians and so on, and industrial anthrax, occurring in those employed in the processing of bones, hides, wool and other animal products. The second type of classification, reflecting the route by which the disease was acquired, distinguishes between cutaneous anthrax acquired through a skin lesion, gastrointestinal tract anthrax contracted from ingestion of contaminated food, primarily meat from an animal that died of the disease, or conceivably from ingestion of contaminated water and pulmonary (inhalation) anthrax from breathing in airborne anthrax spores.
Non–industrial anthrax, resulting from handling infected carcasses, usually manifests itself as the cutaneous form; it tends to be seasonal and parallels the seasonal incidence in the animals from which it is contracted. Cutaneous anthrax transmitted by insect bites and intestinal anthrax from eating infected meat are also non–industrial forms of the disease. Industrial anthrax also usually takes the cutaneous form but has a far higher probability than non–industrial anthrax of taking the pulmonary form through inhalation of spore–laden dust.
Humans almost invariably contract anthrax directly or indirectly from infected animals. Records of person–to–person spread or laboratory–acquired anthrax are rare (Heyworth et al., 1975; Collins 1988; Lalitha et al., 1988; Quinn and Turnbull, 1998).
It is generally believed that B. anthracis is non–invasive and cutaneous and gastrointestinal tract anthrax infection require entry through a small cut, abrasion or other lesion (insect bite, ulcer, etc.). Thus anthrax eschars are generally seen on exposed regions of the body, mostly on the face, neck, hands and wrists.
As inferred earlier, in some countries mechanical transmission by biting insects is believed to be at least an occasional mechanism by which anthrax is contracted by humans (Rao and Mohiyudeen, 1958; Davies, 1983); that this can happen has been demonstrated experimentally (Sen and Minett, 1944; Turell and Knudson, 1987).
4.4 The clinical disease
Cutaneous anthrax is said to account for 95% or more of human cases globally. All three forms, cutaneous, gastro–intestinal tract and pulmonary, are potentially fatal if untreated, but the cutaneous form is often self–limiting. Data from pre–antibiotic and vaccine days indicate that 10–20% of untreated cutaneous cases might be expected to result in death (Anon, 1918). With treatment, less than 1% are fatal.
Overt gastrointestinal tract and pulmonary cases are more often fatal, largely because they go unrecognized until it is too late for effective treatment. However, serological and epidemiological evidence suggests that undiagnosed low–grade gastrointestinal tract or pulmonary anthrax with recovery can also occur, and may not be infrequent, among exposed groups (Brachman et al., 1960; Norman et al., 1960; Sirisanthana et al., 1988; Van den Bosch, 1996).
Development of meningitis is a dangerous possibility in all three forms of anthrax.
4.4.1 Cutaneous anthrax
The incubation period ranges from as little as 9 hours to 2 weeks, mostly 2 to 6 or 7 days.
The general scenario is as follows:
Day 0 Entry of the infecting B. anthracis (usually as spores) through a skin lesion
(cut, abrasion, insect bite, etc.).Days 2–3 A small pimple or papule appears (see Figures M–R).
Days 3–4 A ring of vesicles develops around the papule. Vesicular fluid may be exuded. Unless the patient was treated, capsulated B. anthracis can be identified in polychrome methylene blue–stained (M'Fadyean stain) smears of this fluid and isolated on conventional agars, preferably blood agar (see Appendix I). Marked oedema starts to develop. Unless there is secondary infection, there is no pus and the lesion is not painful, although painful lymphadenitis may occur in the regional lymph nodes.
Days 5–7 The original papule ulcerates to form the characteristic eschar. Topical swabs will not pick up B. anthracis. Detection by polychrome methylene blue–stained smears or isolation requires lifting the edge of the eschar with tweezers (this gives no pain unless there is secondary infection) and obtaining fluid from underneath. The fluid will probably be sterile if the patient has been treated appropriately. Oedema extends some distance from the lesion. Clinical symptoms may be more severe if the lesion is located in the face, neck or chest. In these more severe forms, clinical findings are high fever, toxaemia, regional painful adenomegaly and extensive oedema; shock and death may ensue.
Day 10 (approximately). The eschar begins to resolve; resolution takes almost six weeks and is not hastened by treatment. A small proportion of cases, if untreated, develop systemic anthrax with hyperacute symptoms.
4.4.1.1 Differential diagnosis
Boil (early lesion), orf, vaccinia, glanders, syphilitic chancre, erysipelas, ulcer (especially tropical). These lack the characteristic oedema of anthrax. The absence of pus, the lack of pain, and the patient's occupation may provide further diagnostic pointers. The outbreak of Rift Valley Fever, referred to in 3.5.1 and initially thought to be anthrax, also affected numerous humans.
In differential diagnosis of the severe forms, orbital cellulitis, dacrocystitis and deep tissue infection of the neck should be considered in the case of severe anthrax lesions involving the face, neck and anterior chest wall. Necrotising soft tissue infections, particularly group A streptococcal infections and gas gangrene, and severe cellulitis due to staphylococci, should also be considered in the differential diagnosis of severe forms of cutaneous anthrax.
4.4.1.2 Immunological tests
Subject to certain provisos, serology can, on occasion, be useful in supportive or retrospective diagnosis of anthrax (see A.I.5). The practical aspects have been covered in 3.5.2.
Similarly, in the Russian sphere of influence, a skin test utilising AnthraxinT (Antiplague Research Institute, Sovetskaya St., 13/15, Stavropol, 355106 Russian Federation; Fax: +7 8652 260312), first licensed in the former USSR in 1962, has become widely used for retrospective diagnosis of human and animal anthrax and for vaccine evaluation (Shylakhov et al., 1997). This is a commercially produced heat–stable protein–polysaccharide–nucleic acid complex, derived from oedematous fluid of animals injected with the vaccine STI or the Zenkowsky strains of B. anthracis. The test involves intradermal injection of 0.1 ml of Anthraxin and inspection after 24 h for erythema and induration at the site lasting for 48 hours after the injection. This delayed type hypersensitivity is seen as reflecting anthrax cell mediated immunity and was reportedly able to diagnose anthrax retrospectively some 31 years after primary infection in up to 72 % of cases (Shlyakhov et al., 1997). It was used with success in a retrospective investigation of a series of cases occurring in a spinning mill in Switzerland where synthetic fibres were combined with goat hair from Pakistan (Pfisterer, 1990). The diagnostic reliability of Anthraxin, like Ascoli test antigen (A.I.3.1), depends on the nature of anthrax rather than on the specificity of the antigens involved.
4.4.1.3 Precautions
Surgical tools should be sterilized without delay after use, and dressings should be incinerated. The wearing of surgical gloves by medical staff and orderlies is recommended but risks to these staff are NOT high. Direct human–to–human transmission is exceedingly rare (see 4.3). Vaccination of medical staff and orderlies is not necessary.
4.4.2 Gastrointestinal anthrax
There are two clinical forms of gastrointestinal anthrax which may present following ingestion of B. anthracis in contaminated food or drink.
Intestinal anthrax: Symptoms include nausea, vomiting, fever, abdominal pain, haematemesis, bloody diarrhoea and massive ascites. Unless treatment commences early enough, toxaemia and shock develop, followed by death. There is evidence that mild, undiagnosed cases with recovery occur.
Oropharyngeal anthrax: The main clinical features are sore throat, dysphagia, fever, regional lymphadenopathy in the neck and toxaemia. Even with treatment, the mortality is about 50% (Doganay et al., 1986).
The suspicion of anthrax depends largely on awareness and alertness on the part of the physician as to the patient's history and to the likelihood that he/she had consumed contaminated food or drink.4.4.2.1 Confirmation of diagnosis. See 4.4.3.1
4.4.2.2 Differential diagnosis (gastrointestinal anthrax)
The differential diagnosis in intestinal anthrax includes food poisoning (in the early stages of intestinal anthrax), acute abdomen due to other reasons, and haemhorragic gastroenteritis due to other microorganisms, particularly necrotising enteritis due to Clostridium perfringens.
In the differential diagnosis of oropharyngeal anthrax, streptococcal pharyngitis, Vincent's angina, Ludwig's angina, parapharyngeal abscess, and deep tissue infection of the neck should be considered.
4.4.3 Pulmonary (inhalation) anthrax
Symptoms prior to the onset of the final hyperacute phase are non–specific and suspicion of anthrax depends on the knowledge of the patient's history. In probably the best–documented set of five case reports of inhalation anthrax (Plotkin et al., 1960), the illnesses began insidiously with mild fever, fatigue and malaise lasting one to several days. Headache, muscle aches, chills and fever were recorded in all four patients with development of a cough in four and mild pain in the chest in one. This mild initial phase was followed by the sudden development of dyspnoea, cyanosis, disorientation with coma and death in four of the patients, in whom treatment was unsuccessful. Death occurred within 24 hours of onset of the hyperacute phase.
4.4.3.1 Confirmation of diagnosis (pulmonary and intestinal anthrax)
As indicated, clinical diagnosis is dependent on a knowledge of the patient's history; early symptoms are non–specific and "flu–like" with mild upper respiratory tract signs in pulmonary anthrax or resembling mild food poisoning in intestinal anthrax. In fact, in pulmonary anthrax, the X–ray picture of the lung is very characteristic, with extremely enlarged mediastinal lymph nodes. Frequently, however, confirmatory diagnosis of pulmonary or gastrointestinal anthrax will usually take place after the patient has died or, if correct treatment is initiated early enough, when he or she is well recovered.
The definitive diagnosis is made by the isolation of B. anthracis from sputum in pulmonary anthrax and from vomitus, faeces and ascites in intestinal anthrax. Blood cultures may be positive in either form of the disease.
Depending on the treatment administered and the stage the disease has reached at the time of collection of specimens, smears stained for demonstration of the capsule (Appendix I) may be positive, or the specimens may be positive by culture. B. anthracis may be visualized in or isolated from sputum (pulmonary anthrax) or faeces (intestinal anthrax) but this cannot be relied upon. Specialized laboratories may be able to demonstrate anthrax toxin in fluid specimens (serum or oedematous fluid) or, in the case of patients who survive, anti–toxin and anti–capsular antibodies may be demonstrable in convalescent sera (Appendix I [A.I.5]). The Anthraxin hypersensitivity test referred to in 4.4.1.2 may also be applicable.
Death being due to the toxin, belated treatment can sterilize the blood and tissue fluids while still not preventing death. If this sterilising effect has not occurred, the capsulated B. anthracis may be visible in capsule–stained smears of these fluids and should be easily isolated from them by bacteriological culture.
Where anthrax has not been suspected prior to postmortem, characteristic signs are dark haemolysed unclotting blood, enlarged haemorrhagic spleen, petechial haemorrhages throughout the organs, and a dark oedematous intestinal tract, ulcerated or with areas of necrosis. In pneumonic anthrax, the mediastinal lymph nodes are always affected with haemorrhagic necrotizing lymphadenitis. Nevertheless, it may be hard to differentiate between pulmonary and intestinal anthrax at autopsy and the decision as to how the disease was contracted may have to be based on the patient's history.
4.4.4 Anthrax meningitis
Meningitis due to anthrax is a serious clinical development which may follow any of the other three forms of anthrax. The case fatality rate is almost 100%; the clinical signs of meningitis with intense inflammation of the meninges, markedly elevated CSF pressure and the appearance of blood in the CSF (the meningitis of anthrax is a haemorrhagic meningitis) are followed rapidly by loss of consciousness and death (Levy et al., 1981; Koshi et al., 1981; Lalitha et al., 1990; George et al., 1994; Lalitha et al., 1996). Only a few instances of survival as a result of early recognition of the problem and prompt treatment are on record (Khanne et al., 1989; Lalitha et al., 1996).
Differential diagnosis should take into account acute meningitis of other bacterial aetiology. The definitive diagnosis is obtained by visualisation of the capsulated bacilli in the CSF and/or by culture.
4.4.5 Anthrax sepsis
Sepsis develops after the lymphohematogenous spread of B. anthracis from a primary lesion (cutaneous, gastrointestinal or pulmonary). Clinical features are high fever, toxaemia and shock, with death following in a short time.
In the differential diagnosis, sepsis due to other bacteria should be considered. Definitive diagnosis is made by the isolation of B. anthracis from the primary lesion and from blood cultures.
5 Pathogenesis and pathology
5.1 Toxin as the cause of death
The events occurring between entry of infecting B. anthracis into a lesion or uptake from the lungs and death were covered in 3.4. At one time it was held that death from anthrax was due to capillary blockage, hypoxia and depletion of nutrients by the exceedingly large numbers of bacilli. Subsequently it was shown that death is attributable to a toxin (Keppie et al., 1955).
5.2 The virulence factors of B. anthracis
The capsule and the toxin complex are the two known virulence factors of B. anthracis.
The poly–D–glutamic acid capsule is presumed to act by protecting the bacterium from phagocytosis.
The toxin complex, which consists of three synergistically acting proteins, Protective Antigen (PA, 83kDa), Lethal Factor (LF, 87 kDa) and Oedema Factor (EF, 89 kDa), is produced during the log phase of growth of B. anthracis. LF in combination with PA (lethal toxin) and EF in combination with PA (oedema toxin) are now regarded as responsible for the characteristics signs and symptoms of anthrax.
According to the currently accepted model, PA binds to receptors on the host's cells and is activated by a host protease which cleaves off a 20 kDa piece, thereby exposing a secondary receptor site for which LF and EF compete to bind. The PA+LF or PA+EF are then internalized and the LF and EF are released into the host cell cytosol.
EF is an adenylate cyclase which, by catalysing the abnormal production of cyclic–AMP (cAMP), produces the altered water and ion movements that lead to the characteristic oedema of anthrax. High intracellular cAMP concentrations are cytostatic but not lethal to host cells. EF is known to impair neutrophil function and its role in anthrax infection may be to prevent activation of the inflammatory process.
LF appears to be a calcium– and zinc–dependent metalloenzyme endopeptidase (Hammond and Hanna, 1998). It has recently been shown (Duesbery et al., 1998) that it cleaves the amino terminus of two mitogen–activated protein kinase kinases and thereby disrupts a pathway in the eukaryotic cell concerned with regulating the activity of other molecules by attaching phosphate groups to them. This signalling pathway is known to be involved in cell growth and maturation; the manner in which its disruption leads to the known effects of LF has yet to be elucidated. On the basis of mouse and tissue culture models, macrophages are a major target of lethal toxin which is cytolytic in these. The initial response of sensitive macrophages to lethal toxin is the synthesis of high levels of tumour necrosis factor and interleukin–1 cytokines and it seems probable that death in anthrax results from a septic shock type mechanism resulting from the release of these cytokines.
The endothelial cell linings of the capillary network may also be susceptible to lethal toxin and the resulting histologically visible necrosis of lymphatic elements and blood vessel walls is presumably responsible for systemic release of the bacilli and for the characteristic terminal haemorrhage from the nose, mouth and anus of the victim (see Figures on the front cover).
The detailed nature and mode of action of the toxin has been more thoroughly reviewed in various texts (Leppla, 1992; Quinn and Turnbull, 1998). Most of the recorded histopathological studies on anthrax were done between 1945 and 1970; these are reviewed elsewhere (Quinn and Turnbull, 1998).
6 Bacteriology
B. anthracis, the causative agent of anthrax, is a Gram positive, aerobic or facultatively anaerobic, endospore–forming, rod–shaped bacterium approximately 4 µm by 1 µm, although under the microscope, it frequently appears in chains of cells. In blood smears, smears of tissues or lesion fluid from diagnostic specimens, these chains are two to a few cells in length (see Figure B); in suspensions made from agar plate cultures, they can appear as endless strings of cells – responsible for the characteristic tackiness of the colonies (see Figure D). Also characteristic is the square–ended ("box–car" shaped) appearance traditionally associated with B. anthracis vegetative cells, although this is not always very clear. In the presence of oxygen, and towards the end of the exponential phase of growth, one ellipsoidal spore is formed in each cell; this does not swell the sporangium and is generally situated centrally, sometimes sub–terminally (see Figure A).
Under anaerobic conditions and in the presence of bicarbonate (HCO3), the vegetative cell secretes a polypeptide (poly–( – D–glutamic acid) capsule. As covered in section 5, the capsule is formed in vivo and is one of the two virulence factors of B. anthracis. It is also a primary diagnostic aid (see 3.5 and 4.4.1, Appendix I [A.I.2] and Figure B).
6.1 Detection and isolation
In appropriate blood or tissue specimens collected within a few hours of death from animals (see 3.5) or humans with anthrax, B. anthracis is readily detected in capsule–stained (M'Fadyean–stained) smears and readily isolated in pure culture on blood or nutrient agar plates. The same applies to smears of fluid from cutaneous lesions of humans prior to treatment (see 4.4.1).
In old or decomposed animal specimens, or processed products from animals that have died of anthrax, or in environmental samples, detection is likely to involve a search for relatively few B. anthracis within a background flora of other bacteria, many of which will probably be other Bacillus species, in particular, the closely–related B. cereus. In this case, selective techniques are necessary. A procedure for the isolation of B. anthracis from such specimens is given in Appendix I (A.I.1).
Such is the nature of the properties of B. anthracis that few agents which differentially select between B. anthracis and other Bacillus species do so in favour of B. anthracis and those that do only do so unconvincingly. Of the selective media that have been proposed, the most successful is polymyxin–lysozyme–EDTA–thallous acetate (PLET) agar (Knisely, 1966), although care has to be taken to prepare this correctly. As yet no selective enrichment broth system has been devised for B. anthracis and, pending development of such a system, the sensitivity of in vitro detection of B. anthracis by conventional means in environmental samples or specimens from old or decomposed animals or from processed animal products is limited to approximately 5 cfu/g or /ml (Manchee et al., 1981).
As covered in Appendix I (A.I.1.4 and A.I.4), the most sensitive method for isolating the organism is inoculation into a guinea pig or mouse. Although, in line with increasing aversion to the use of animals for scientific tests, this should strictly be a last resort, there are times when this may still be the necessary approach, for example, when confirming anthrax in individuals or animals that have been treated with antibiotics, or in essential tests on environmental samples that contain sporostatic chemicals.
Polymerase chain reaction (PCR) detection systems have been developed for B. anthracis (Beyer et al. 1996; Patra et al. 1996; Sjöstedt et al. 1996; 1997), but it will probably be a few years before they become fully reliable, adequately sensitive and robust, and generally available for use in the non–specialist laboratory.
6.2 Identification and confirmation
With occasional exceptions, it is generally easy to identify B. anthracis and to distinguish it from other Bacillus species, including B. cereus. For all practical purposes, an isolate with the characteristic colonial morphology (Parry et al., 1983) on nutrient or blood agar (matt appearance, fairly flat, similar to B. cereus but generally rather smaller, more tacky, white or grey–white on blood agar, and often having curly tailing at the edges), and which is non–haemolytic or only weakly haemolytic, non–motile, sensitive to the gamma–phage and penicillin, and able to produce the capsule in blood or on anaerobic culture on bicarbonate media is B. anthracis (see Figures D and E, and Appendix I [A.I.2]) .
The PCR is becoming more widely available as a means of confirming the presence of the virulence factor (capsule and toxin) genes, and hence that an isolate is, or is not, virulent B. anthracis. For routine purposes, primers to one of the toxin genes (usually the Protective Antigen gene) and to one of the enzymes mediating capsule formation are adequate (Appendix I [A.I.6] and Figure F). In laboratories not equipped for PCR tests, if doubt remains at the end of the procedures given in Appendix I (A.I.1 and 2) as to the definitive identity of a suspect B. anthracis isolate, inoculation into a mouse or guinea pig may necessary (Appendix I [A.I.4]). However, as stated in 6.1, this should be a last resort procedure and confined to situations where a definitive identity is essential.
Movement of infectious or contaminated materials from the site of origin to a diagnostic or reference laboratory obviously presents a risk of spread of diseases if the materials inadvertently escape into the environment during transit. Attention is drawn to the recommendation of the United Nations Committee of Experts on Transport of Dangerous Goods for packaging, labelling and documentation in relation to transport of infectious specimens, detailed in other publications (WHO, 1997b), excerpts of which one is reported in Appendix VIII.
7 Treatment
Prompt and timely antibiotic therapy usually results in dramatic recovery of the individual or animal infected with anthrax. Almost all isolates of B. anthracis can be expected to be highly sensitive to penicillin and, being cheap and readily available in most parts of the world, this remains the basis of treatment schedules, particularly in animals and in humans in developing countries. The organism is also sensitive to numerous other broad spectrum antibiotics; should the use of penicillin be contraindicated, a wide range of alternative choices exist from among the aminoglycosides, macrolides, quinolones and tetracyclines. Chloramphenicol is also a satisfactory alternative.
We are only aware of four reports of the isolation of penicillin–resistant strains (Anon, 1996). The molecular basis of susceptibility and resistance is complex (Lightfoot et al., 1990). It looks as if it may prove to be the case that all B. anthracis strains carry the ? –lactamase gene(s) on their cromosomes but, apart from the exceptions mentioned, these are not expressed.
In pulmonary or gastrointestinal anthrax in humans, symptomatic treatment in an intensive care unit in addition to antibiotic therapy may save the patient's life; as referred to in 7.2.8, if available, plasmaphoresed hyperimmune serum or gamma–globulin from vaccinated persons may be considered in life–threatening situations.
http://www.who.int/emc-documents/zoonoses/docs/whoemczdi986.html#_Hlk436471728
-- end of excerpts.