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BACILLUS | Bacillus anthracis
Bacillus anthracis L Baillie, DERA, Salisbury, Wiltshire, UK EW Rice, US Environmental Protection Agency, Cincinnati, OH, USA Ó 2014 Elsevier Ltd. All rights reserved. This article is a revision of the previous edition article by Les Baillie, volume 1, pp 129–135, Ó 1999, Elsevier Ltd.
Characteristics of the Species Bacillus anthracis, the causative agent of the disease anthrax, is the only obligate pathogen within the genus Bacillus. The genus includes Gram-positive aerobic or facultatively anaerobic spore-forming, rod-shaped bacteria. The ability to form resistant spores accounts for its reported persistence in the environment over many years (accounts vary from 60 to 200 years) and resistance to physical agents, such as heat and chemical disinfectants. The spores can withstand temperatures of 70 C and, depending on the conditions, exposure to acids, alkalis, alcohols, phenolics, hypochlorite, quaternary ammonium compounds, and surfactants. They usually are destroyed by boiling for 10 min and by dry heat at 140 C for 3 h. They are susceptible to sporicidal agents, such as formaldehyde, and are inactivated by gamma radiation, an approach that has been used to decontaminate animal hides. Conditions conducive to the germination of B. anthracis are not well characterized. Germination is influenced by temperature, pH, moisture, and the presence of oxygen and carbon dioxide. Spores will germinate at temperatures of 8–45 C, pH values of 5–9, relative humidity >95%, and adequate nutrition. Optimum germination conditions for the Vollum strain of B. anthracis have been shown to be 22 C in the presence of the germinant L-alanine. Frequently, it is convenient to classify B. anthracis informally within the Bacillus cereus group, which includes B. cereus, B. anthracis, Bacillus thuringiensis, and Bacillus mycoides on the basis of phenotypic reactions. Genetic techniques have provided clear evidence, however, that B. anthracis can be distinguished reliably from other members of the bacilli. In practical terms, the demonstration of virulence constitutes the principal point of difference between typical strains of B. anthracis and those of other anthrax-like organisms. Although primarily a disease of herbivores, particularly the human food animals, cattle, sheep, and goats, the organism can infect humans, frequently with fatal consequences if untreated. In herbivores, the disease usually runs a hyperacute course, and signs of illness can be absent until shortly before death. At death, the blood of the animal generally contains >108 bacilli per milliliter. Bacillus anthracis is regarded as an obligate pathogen; its continued existence in the ecosystem appears to depend on a multiplication phase within an animal host. Spores of anthrax reach the environment either from infected animals and their products or as a consequence of the actions of humans. In the wild, it is thought that the release of spores from infected animals plays an important part of the infective cycle; the spores contaminate the soil, and healthy animals that graze on contaminated land are exposed to the spores and subsequently may develop infection. The disease largely has been eradicated from the western world due to mass animal vaccination programs and the
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maintenance of stringent veterinary control measures. In other parts of the world where vaccination is not available or routinely administered, the organism is still a significant cause of animal mortality and human disease. In the United Kingdom, the sudden death of a food animal is investigated by the veterinary authorities, and, if death is due to anthrax, the animal and its products are destroyed. In countries, with less–well-developed public health systems, the meat of an infected animal may be considered too valuable to ‘waste’ and, subsequently, the flesh is likely to be consumed or sold. In Zambia, custom dictates that an animal that has died from unknown causes cannot be disposed of, but it is opened up, shared among relatives and friends, and eaten. Efforts to advise local communities on the dangers of such behavior meet resistance due to the economic loss caused by burying or burning. Three forms of the disease are recognized in humans: cutaneous, pulmonary, and gastrointestinal infection. Development of meningitis is possible in all three forms of anthrax. The gastrointestinal tract and pulmonary forms are regarded as being most frequently fatal due to the fact that they can go unrecognized until it is too late to instigate effective treatment. The cutaneous form accounts for the majority of human cases (>95%). It is generally believed that B. anthracis is noninvasive and thus requires a break in the skin to gain access to the body. Infection is normally caused by spores of the organism colonizing cuts or abrasions of the skin (Figure 1). Workers who carry contaminated hides or carcasses on their shoulders are liable to infection on the back of their necks, while handlers of other food materials or products tend to be infected on the hands, arms, or wrists. Most carbuncular cases recover without treatment, but in 20% of the cases, the infection will progress into a generalized septicemia, which is invariably fatal. Pulmonary anthrax is caused by the inhalation of spores of B. anthracis that is aerosolized during the processing of
Figure 1
Cutaneous anthrax lesion.
Encyclopedia of Food Microbiology, Volume 1
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BACILLUS j Bacillus anthracis contaminated animal products, such as hides, wool, and hair. The onset of illness is abrupt. The early clinical signs are of a mild respiratory tract infection with mild fever and malaise, but acute symptoms may appear within a few hours with dyspnea, cyanosis, and fever. Death usually follows within 2–3 days with acute splenomegaly and circulatory collapse as terminal events. This form of infection has an associated mortality rate of >80%. Gastrointestinal anthrax occurs mainly 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. Most cases of intestinal anthrax result from eating insufficiently cooked meat from anthrax-infected animals. Gastrointestinal anthrax is probably greatly underreported in many of these rural disease-endemic areas. Although most cases have been reported from adults, children tend to have a more fulminate course of infection. Due to the rareness of the conditions, there are no figures for the number of organisms that need to be ingested to cause disease. Two clinical forms of gastrointestinal anthrax may occur following the ingestion of contaminated food or drink: l
Intestinal anthrax: the symptoms include nausea, vomiting, fever, abdominal pain, hematemesis, bloody diarrhea, and massive ascites. Toxemia and shock develop and death results. l Oropharyngeal anthrax: the main clinical features are sore throat, dysphagia, fever, regional lymphadenopathy in the neck, and toxemia. Even with treatment, the mortality is about 50%. It is extremely important that effective treatment is started early as the prognosis is often death. Suspicion of the case being anthrax depends very greatly on the awareness and alertness on the part of the physician as to the patient’s history and the likelihood that he or she had consumed contaminated food and drink. The two major virulence factors of B. anthracis are the ability to form an antiphagocytic capsule and a toxin expression. Both of these factors are carried on different plasmids, with the loss of either resulting in a reduction in the virulence of the organism.
The capsule of B. anthracis is composed of a polypeptide (poly-D-glutamic acid), which inhibits phagocytosis and opsonization of the bacilli. The genes controlling capsule synthesis, CapA, CapB, and CapC are organized in an operon that is located on the plasmid, pXO2. Capsule expression is subject to regulation by CO2 and bicarbonate via an, as yet, unclear mechanism involving the regulator atxA. This regulator also controls the level of expression of the anthrax toxin genes (Figure 2). Why the expression of virulence factors should be linked to CO2 and bicarbonate levels is unclear. It could be that the bacteria ‘monitor’ the level of these agents in the host as an indication of the nutrient availability. The tripartite anthrax toxin is considered to be the major virulence factor. The three proteins of the exotoxin are protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxins follow the A–B model with the A moiety being the catalytic part and the B moiety being the receptor-binding part. PA acts as the B moiety and binds to the cell surface receptor, where LF and EF complete for binding to PA. EF is an inactive adenyl cyclase that is transported into the target cell by PA. Once in contact with the cytoplasm, EF binds calmodulin (a eukaryotic calcium-binding protein) and becomes enzymatically active, converting adenosine triphosphate into cyclic adenosine monophosphate (cAMP). The resulting effects are the same as those caused by cholera toxin with the affected cells secreting large amounts of fluid. The contribution of EF to the infective process is ill defined. In general, bacterial toxins that increase cAMP dampen the innate immune responses of phagocytes and there is some evidence that this may be true for edema toxin. It is generally considered that the pathological changes seen in infected animals are due to the lethal factor combined with PA. In the only studies directly implicating EF as a virulence factor, mice were found to be killed by lower doses of the lethal toxin when EF was administered simultaneously. Lethal toxin is the central effector of shock and death from anthrax. Animals injected intravenously with purified lethal toxin succumb in a manner that closely mimics the natural systemic infection. Lethal toxin appears to be a zinc-dependent metalloprotease, but its substrate and mode of action have yet to be defined. It affects most types of eukaryotic cells.
CO 2 /HCO 3 lef
Cap A C
ap
dep
C
B
CO 2 /HCO 3 g
Cap
pa
CO 2 /HCO 3 acp A
pXO21 90 kbp
cya
atxA
pXO1 185 kbp
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CO 2 /HCO 3 temp.
Figure 2 Coordinate regulation of virulence factors. The production of the capsule and anthrax toxin genes are enhanced by CO2/bicarbonate and temperature. The molecular mechanism of enhanced virulence has not been elucidated.
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BACILLUS j Bacillus anthracis
Macrophages, which play a key role in combating infection, are particularly sensitive to the toxin. At low levels, the toxin appears to interfere with the ability of macrophages to kill bacteria. The toxin also stimulates the production of cytokines within the macrophage. As the level of toxin increases in the blood, more cytokines accumulate within the macrophage until the cell is finally lysed. It is proposed that this sudden release of cytokine leads to shock and would explain the rapid death seen in animals. In addition to the major factors previously outlined, B. anthracis expresses a number of other factors that may contribute to virulence. These ‘minor factors’ could account for the difference in virulence between strains. Like many other pathogenic organisms, B. anthracis produces an S-layer composed of two proteins called Eal and Sap. S-layers are proteinaceous paracrystalline sheaths present on the surface of many Archaebacteria and Eubacteria. S-layers have been found on many bacterial pathogens, including Campylobacter spp. and Clostridium spp. Various functions have been proposed for S-layers, including shape maintenance, molecular sieving, or phage fixation. The S-layer may be a virulence factor, protecting pathogenic bacteria against complement killing. It has been demonstrated that B. anthracis can produce a number of chromosomally encoded extracellular proteases that, like lethal toxin, kill macrophages. The presence of similar, if not identical, toxin genes in a number of members of the B. cereus, B. thuringiensis, and B. mycoides group raises the possibility that these genes also may be present in B. anthracis. A homolog to the cereolysin gene of B. cereus has been detected in B. anthracis. Although functionally inactive in the majority of strains, spontaneously occurring low-level activity has been demonstrated. It would not be surprising if homologs to other bacillus virulence factors were not detected.
Detection Given the scarcity of anthrax in the industrial world it is unlikely that many routine diagnostic laboratories would have the experience or access to the materials required, to identify the organism correctly. The main problem is the differentiation of B. anthracis from the phenotypically similar B. cereus/thuringiensis group, which may also be present in many of the samples examined for anthrax. Direct detection of the organism in the field is relatively simple in animals that have died suddenly of the disease. At death the blood of an animal generally contains >108 bacilli per milliliter. Blood films are dried, fixed immediately by heat or immersion for 1 min in absolute alcohol, and stained with polychrome methylene blue, which after 20 s is washed off. When the slide is dry, it is examined for characteristic deep blue, squareended bacilli surrounded by a well-demarcated pink capsule (McFadyean’s reaction) (Figure 3). In some animal species, such as pigs, the terminal bacteremia is limited, and the bacilli are unlikely to be seen in McFadyean-stained blood smears. Antigen-based direct detection methods have been developed that are more sensitive than staining. A highly specific immunochromatographic assay has been developed utilizing
Figure 3
Capsule stain.
a monoclonal capture antibody to the anthrax toxin component, PA. This assay can detect as little as 25 ng ml1 of PA and can be performed in a few minutes without the need for special reagents. This test could be used in addition to staining to screen animal blood and tissue and confirm the presence, or absence, of the organism. DNA-based detection using polymerase chain reaction (PCR) methodologies have been used successfully to detect the presence of B. anthracis in environmental samples. Once the problem of PCR inhibitors in blood and animal tissue have been overcome, it should be possible to detect the organism in animal samples. Unless there is an index of suspicion, it is unlikely that animal products would be examined routinely for the presence of B. anthracis. In cases in which contamination with B. anthracis is suspected, the World Health Organization (WHO) in their Comprehensive and Practical Guidelines on Anthrax propose the isolation protocol shown in Figure 4. The sensitivity limit of this technique is approximately five spores per gram of the starting material. The number of bacteria isolated very much depends on the distribution of the organism within the sample. The polymyxin-lysozyme-EDTA-thallous acetate (PLET) agar described in the method is a semiselective medium for B. anthracis, which contains polymyxin (30 000 units l1), lysozyme (300 000 units l1), ethylenediaminetetraacetic acid (0.3 g l1), and thallous acetate (0.04 g l1). Once colonies have been isolated, further testing is required to confirm their identity (Table 1). Many saprobic species of aerobic spore-forming bacilli are hard to distinguish from
BACILLUS j Bacillus anthracis
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Blend to suspend in 2 volumes of sterile distilled /deionized water (buffered if specimen is likely to have a low / high pH)
ROUTE A (When there is reason to believe some or all of the B. anthracis will be in vegetative forms)
ROUTE B (When B. anthracis is only likely to be present as spores)
Decant ±10 ml into a tube / bottle
Prepare ±10 ml volumes of undilute and 1:10, 1:100 and 1:1000 dilutions of the suspension in sterile distilled /deionized water
Place in 62.5 °C water bath for 15 min (‘heat shock’) or (‘alcohol shock’ by adding equal volume of 95 –100% ethanol and hold 1 h)
Second
First
Place in 62.5 °C water bath for 15 min (‘heat shock’) or (‘alcohol shock’ by adding equal volume of 95 –100% ethanol and hold 1h)
Spread 100 l of each dilution on polymyxin blood agar (BAP) and 200–250 l on PLET agar
Prepare ±10 ml volumes of 1:10 and 1:100 dilutions of the suspension in sterile distilled /deionized water
Spread 100 l of each dilution on blood agar (BA) and 200–250 l on PLET agar
Incubate BAP / BA overnight at 37 °C and PLET for 36 – 48 h at 37 °C
Figure 4
WHO protocol for the isolation of anthrax.
B. anthracis except on the basis of pathogenicity. The most commonly encountered are B. cereus/thuringiensis/mycoides, Bacillus subtilis, and Bacillus licheniformis. The preliminary tests shown in Table 1 are used routinely by the Anthrax Section, Centre for Emergency Preparedness and Response (CEPR), Porton Down, Salisbury, United Kingdom, and allow the presumptive identification of an isolate as B. anthracis. Similar tests are conducted under the auspices of the US Centers for Disease Control and Prevention’s Laboratory Response Network. l
Lack of motility: Log phase cultures of the organism grown in nutrient broth at 22 C and 30 C are examined for motile
organisms by phase contrast microscopy. Unlike the other closely related bacilli, B. anthracis is nonmotile. l Lack of hemolysis: When cultured on 7% defibrinated horse blood agar, colonies of B. anthracis are large, opaque, and white, and have a very rough surface and an irregular edge. They are normally nonhemolytic, although the occurrence of hemolytic colonies has been reported. l Sensitivity to diagnostic gamma phage: Sensitivity to B. anthracis–specific phage is determined by spreading 200 ml of a log phase culture over the surface of a blood agar plate. After incubation for 1 h at 37 C, 20 ml of B. anthracis-specific gamma phage suspension is spotted on the plate. After overnight incubation at 37 C, the plate is examined for
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Table 1
BACILLUS j Bacillus anthracis Detection and identification methods for B. anthracis
Direct Microscopy (McFadyean’s stain) Antigen detection Polymerase chain reaction Preliminary tests Lack of motility Lack of hemolysis Sensitivity to diagnostic gamma phage Sensitivity to penicillin Commercial biochemical kits: API 50CHB, Biolog Confirmatory tests (specialist lab) Virulence in animals – guinea pig Capsule formation – McFadyean’s stain Toxin detection – immunoassays Virulence gene detection – polymerase chain reaction
plaques. On rare occasions phage-negative B. anthracis and phage-positive B. cereus may be encountered (Figure 5). l Sensitivity to penicillin: The test organism is subcultured to a blood agar plate; a 10 unit penicillin disk is spotted on the culture and the plate is incubated overnight at 37 C. Bacillus anthracis is sensitive to penicillin, whereas B. cereus is resistant. Very rarely penicillin-resistant B. anthracis isolates are encountered. Commercially available biochemical screening systems such as API 50CHB (bioMerieux, France) and Biolog (Biolog Inc., Hayward, United States) have been evaluated for their ability to identify B. anthracis. These systems offer the advantage of being easy to use and show promise as simple, first-line, one-step screening tests for the presumptive identification of B. anthracis. These tests are called presumptive tests as other strains of bacilli can give similar reactions to B. anthracis. The demonstration of virulence constitutes the principal point of difference between typical strains of B. anthracis and those of other anthraxlike organisms. Traditionally, the guinea pig has been the model used to demonstrate virulence. The animal is injected with the sample, and if it dies, the cause of death is confirmed by the isolation of B. anthracis from blood. Although this traditional technique is
sensitive, it is likely to be replaced by more sensitive in vitro tests. Virulent isolates of B. anthracis produce both a capsule and exotoxins. Detection of capsule formation is relatively simple. Capsule-forming organisms, when grown on medium containing bicarbonate and in the presence of CO2, produce colonies that are raised and mucoid in appearance, whereas noncapsule-forming organisms produce flat, dull colonies. In addition, the presence of the capsule can be confirmed by McFadyean’s stain. Detection of active toxin production is not as straightforward and requires either an animal system, a tissue culture assay using toxin-sensitive cell lines, or an immunological technique, such as an enzyme-linked immunosorbent assay. PCR allows for the detection of the genes encoding the virulence factors without the need for their expression. Specific DNA primers have been developed for the detection of capsule and toxin genes. Primers have been developed specific to the genome of the organism allowing the detection of atypical, nonvirulent strains of B. anthracis. A rapid-viability PCR method, incorporating primers and probes specific for the chromosome and each of the two virulence plasmids, has been developed to detect viable, virulent B. anthracis in environmental samples.
Regulations Most countries have regulations concerning the handling and disposal of infected food animals and their products. Concerns about the importation of contaminated animal products into the United Kingdom at the beginning of the twentieth century led the government to set up disinfection stations to treat all animal hair and leather goods. The United Kingdom Anthrax Order (1991) prescribes the steps that should be taken to deal with an animal that has, or is suspected of having, anthrax. This measure calls for the infected animals and its products, such as milk, to be destroyed, thus removing them from the food chain. The WHO has produced detailed comprehensive and practical guidelines on anthrax, detailing best practices on all aspects of the disease. In many areas where anthrax is endemic, particularly Africa, the problem is not the lack of regulations but rather the will and the means to enforce them in the face of local customs.
Importance to the Food Industry
Figure 5
Gamma phage lysis.
The number of reported cases of foodborne illness involving B. anthracis is extremely small compared with other traditional food-poisoning organisms. To date, there has never been a documented case in the United States. In countries with welldeveloped veterinary and public health systems, infected animals will be identified and removed from the food chain. In countries where such systems are not in place, the potential exists for contaminated animals and their products to be processed and consumed. A survey of animals in a slaughterhouse in eastern Nigeria revealed that 5% of cattle and 3.3% of sheep were positive for anthrax. These infected animals not only pose a risk to the people consuming the meat but also an
BACILLUS j Bacillus anthracis occupational risk to workers exposed to the carcasses. In the same survey, it was found that 13% of butchers and skinners had acquired cutaneous anthrax. Slaughterhouse waste in the form of offal for animal feed, and slurry discharged into the environment, represents a further source of potential infection. A study of uncut anthrax-contaminated slaughterhouse waste showed that viable anthrax still could be recovered after the offal had been heat treated for 30 min at 130 C. The use of bone charcoal by the food industry in the production of sugar products presents an avenue for anthrax contamination. The bones normally are obtained from areas of the world in which anthrax is endemic and, on occasion, B. anthracis has been isolated. For this reason, the bones must be sterilized, usually by gamma irradiation, before use. It is also important to note the potential of gastrointestinal anthrax occurring as a result of a bioterrorism event. Intentional contamination of food products may result in disease that would differ from naturally occurring infections associated with the consumption of meat from an infected animal.
Importance to the Consumer Due to the scarcity of the disease, there are few published records of human infection. The cases that are published mainly originate from Africa, the Middle East, and central and southern Asia. Figures for human anthrax in China showed that of 593 recorded cases, 384 were linked to the dismembering and processing of infected animals and only 192 cases were due to the consumption of contaminated meat. In neighboring Korea, sporadic outbreaks of human anthrax have been reported. From 1992 to 1995, three outbreaks occurred, a total of 43 cases, all linked to the consumption of contaminated beef or bovine brain and liver. An outbreak in India was centered on an infected sheep. Of the five individuals who skinned and cut up its meat for human consumption, four developed fatal anthrax meningitis. Another person who wrapped the meat in a cloth and carried it
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home on his head developed a malignant pustule on his forehead and went on to develop meningitis. A large number of people who cooked or ate the cooked meat of the dead sheep remained well.
See also: Bacillus: Bacillus cereus; Bacterial Endospores; Classification of the Bacteria: Traditional; Bacteria: Classification of the Bacteria – Phylogenetic Approach; Nucleic Acid–Based Assays: Overview.
Further Reading Anthrax Order, 1991. Statutory Instruments 1991. No. 1824, Animals. HMSO, London. Beatty, M.E., Ashford, D.A., Griffin, P.M., Tauxe, R.V., Sobel, J., 2003. Gastrointestinal anthrax. Archives of Internal Medicine 163 (10), 2527–2531. Bravata, D.M., Holty, J.-E.C., Wang, E., Lewis, R., Wise, P.H., McDonald, K.M., Owens, D.K., 2007. Inhalational, gastrointestinal, and cutaneous anthrax in children. Archives of Pediatrics & Adolescent Medicine 161 (9), 896–905. George, S., Mathai, D., Balraj, V., Lalitha, M.K., John, T.J., 1994. An outbreak of anthrax meningoencephalitis. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 206–207. Kanafani, Z.A., Ghossain, A., Sharara, A.I., Hatem, J.M., Kanj, S.S., 2003. Endemic gastrointestinal anthrax in 1960s Lebanon: clinical manifestations and surgical findings. Emerging Infectious Disease 9 (5), 520–525. Letant, S.E., Murphy, G.A., Alfaro, T.M., Avila, J.R., Kane, S.R., Raber, E., Blunt, T.M., Shah, S.R., 2011. Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples. Applied and Environmental Microbiology 77 (18), 6570–6578. Okolo, M.I., 1985. Studies on anthrax in food animals and persons occupationally exposed to the zoonoses in Eastern Nigeria. International Journal of Zoonoses 12, 276–282. Reiddinger, O., Strauch, D., 1978. Some hygienic problems in the production of meat and bone meal from slaughterhouse offal and animal carcasses. Annali Dell’lstituto Superiore di Sanità 14, 213–219. Sirisanthana, T., Brown, A.E., 2002. Anthrax of the gastrointestinal tract. Emerging Infectious Diseases 8 (7), 649–651. Turnbull, P.C.B. (Ed.), 1996. In: Proceedings of the International Workshop on Anthrax, Winchester, UK, 1995. Salisbury Medical Bulletin, Special Suppl. No. 87. World Health Organization, 2008. In: Turnbull, P.C.B. (Ed.), World Organization for Animal Health, Food and Agriculture Organization of the United Nations Anthrax in Humans and Animals, fourth ed. World Health Organization, Geneva, Switzerland, p. 207.
Characteristics of the Species Bacillus anthracis, the causative agent of the disease anthrax, is the only obligate pathogen within the genus Bacillus. The genus includes Gram-positive aerobic or facultatively anaerobic spore-forming, rod-shaped bacteria. The ability to form resistant spores accounts for its reported persistence in the environment over many years (accounts vary from 60 to 200 years) and resistance to physical agents, such as heat and chemical disinfectants. The spores can withstand temperatures of 70 C and, depending on the conditions, exposure to acids, alkalis, alcohols, phenolics, hypochlorite, quaternary ammonium compounds, and surfactants. They usually are destroyed by boiling for 10 min and by dry heat at 140 C for 3 h. They are susceptible to sporicidal agents, such as formaldehyde, and are inactivated by gamma radiation, an approach that has been used to decontaminate animal hides. Conditions conducive to the germination of B. anthracis are not well characterized. Germination is influenced by temperature, pH, moisture, and the presence of oxygen and carbon dioxide. Spores will germinate at temperatures of 8–45 C, pH values of 5–9, relative humidity >95%, and adequate nutrition. Optimum germination conditions for the Vollum strain of B. anthracis have been shown to be 22 C in the presence of the germinant L-alanine. Frequently, it is convenient to classify B. anthracis informally within the Bacillus cereus group, which includes B. cereus, B. anthracis, Bacillus thuringiensis, and Bacillus mycoides on the basis of phenotypic reactions. Genetic techniques have provided clear evidence, however, that B. anthracis can be distinguished reliably from other members of the bacilli. In practical terms, the demonstration of virulence constitutes the principal point of difference between typical strains of B. anthracis and those of other anthrax-like organisms. Although primarily a disease of herbivores, particularly the human food animals, cattle, sheep, and goats, the organism can infect humans, frequently with fatal consequences if untreated. In herbivores, the disease usually runs a hyperacute course, and signs of illness can be absent until shortly before death. At death, the blood of the animal generally contains >108 bacilli per milliliter. Bacillus anthracis is regarded as an obligate pathogen; its continued existence in the ecosystem appears to depend on a multiplication phase within an animal host. Spores of anthrax reach the environment either from infected animals and their products or as a consequence of the actions of humans. In the wild, it is thought that the release of spores from infected animals plays an important part of the infective cycle; the spores contaminate the soil, and healthy animals that graze on contaminated land are exposed to the spores and subsequently may develop infection. The disease largely has been eradicated from the western world due to mass animal vaccination programs and the
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maintenance of stringent veterinary control measures. In other parts of the world where vaccination is not available or routinely administered, the organism is still a significant cause of animal mortality and human disease. In the United Kingdom, the sudden death of a food animal is investigated by the veterinary authorities, and, if death is due to anthrax, the animal and its products are destroyed. In countries, with less–well-developed public health systems, the meat of an infected animal may be considered too valuable to ‘waste’ and, subsequently, the flesh is likely to be consumed or sold. In Zambia, custom dictates that an animal that has died from unknown causes cannot be disposed of, but it is opened up, shared among relatives and friends, and eaten. Efforts to advise local communities on the dangers of such behavior meet resistance due to the economic loss caused by burying or burning. Three forms of the disease are recognized in humans: cutaneous, pulmonary, and gastrointestinal infection. Development of meningitis is possible in all three forms of anthrax. The gastrointestinal tract and pulmonary forms are regarded as being most frequently fatal due to the fact that they can go unrecognized until it is too late to instigate effective treatment. The cutaneous form accounts for the majority of human cases (>95%). It is generally believed that B. anthracis is noninvasive and thus requires a break in the skin to gain access to the body. Infection is normally caused by spores of the organism colonizing cuts or abrasions of the skin (Figure 1). Workers who carry contaminated hides or carcasses on their shoulders are liable to infection on the back of their necks, while handlers of other food materials or products tend to be infected on the hands, arms, or wrists. Most carbuncular cases recover without treatment, but in 20% of the cases, the infection will progress into a generalized septicemia, which is invariably fatal. Pulmonary anthrax is caused by the inhalation of spores of B. anthracis that is aerosolized during the processing of
Figure 1
Cutaneous anthrax lesion.
Encyclopedia of Food Microbiology, Volume 1
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BACILLUS j Bacillus anthracis contaminated animal products, such as hides, wool, and hair. The onset of illness is abrupt. The early clinical signs are of a mild respiratory tract infection with mild fever and malaise, but acute symptoms may appear within a few hours with dyspnea, cyanosis, and fever. Death usually follows within 2–3 days with acute splenomegaly and circulatory collapse as terminal events. This form of infection has an associated mortality rate of >80%. Gastrointestinal anthrax occurs mainly 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. Most cases of intestinal anthrax result from eating insufficiently cooked meat from anthrax-infected animals. Gastrointestinal anthrax is probably greatly underreported in many of these rural disease-endemic areas. Although most cases have been reported from adults, children tend to have a more fulminate course of infection. Due to the rareness of the conditions, there are no figures for the number of organisms that need to be ingested to cause disease. Two clinical forms of gastrointestinal anthrax may occur following the ingestion of contaminated food or drink: l
Intestinal anthrax: the symptoms include nausea, vomiting, fever, abdominal pain, hematemesis, bloody diarrhea, and massive ascites. Toxemia and shock develop and death results. l Oropharyngeal anthrax: the main clinical features are sore throat, dysphagia, fever, regional lymphadenopathy in the neck, and toxemia. Even with treatment, the mortality is about 50%. It is extremely important that effective treatment is started early as the prognosis is often death. Suspicion of the case being anthrax depends very greatly on the awareness and alertness on the part of the physician as to the patient’s history and the likelihood that he or she had consumed contaminated food and drink. The two major virulence factors of B. anthracis are the ability to form an antiphagocytic capsule and a toxin expression. Both of these factors are carried on different plasmids, with the loss of either resulting in a reduction in the virulence of the organism.
The capsule of B. anthracis is composed of a polypeptide (poly-D-glutamic acid), which inhibits phagocytosis and opsonization of the bacilli. The genes controlling capsule synthesis, CapA, CapB, and CapC are organized in an operon that is located on the plasmid, pXO2. Capsule expression is subject to regulation by CO2 and bicarbonate via an, as yet, unclear mechanism involving the regulator atxA. This regulator also controls the level of expression of the anthrax toxin genes (Figure 2). Why the expression of virulence factors should be linked to CO2 and bicarbonate levels is unclear. It could be that the bacteria ‘monitor’ the level of these agents in the host as an indication of the nutrient availability. The tripartite anthrax toxin is considered to be the major virulence factor. The three proteins of the exotoxin are protective antigen (PA), lethal factor (LF), and edema factor (EF). The toxins follow the A–B model with the A moiety being the catalytic part and the B moiety being the receptor-binding part. PA acts as the B moiety and binds to the cell surface receptor, where LF and EF complete for binding to PA. EF is an inactive adenyl cyclase that is transported into the target cell by PA. Once in contact with the cytoplasm, EF binds calmodulin (a eukaryotic calcium-binding protein) and becomes enzymatically active, converting adenosine triphosphate into cyclic adenosine monophosphate (cAMP). The resulting effects are the same as those caused by cholera toxin with the affected cells secreting large amounts of fluid. The contribution of EF to the infective process is ill defined. In general, bacterial toxins that increase cAMP dampen the innate immune responses of phagocytes and there is some evidence that this may be true for edema toxin. It is generally considered that the pathological changes seen in infected animals are due to the lethal factor combined with PA. In the only studies directly implicating EF as a virulence factor, mice were found to be killed by lower doses of the lethal toxin when EF was administered simultaneously. Lethal toxin is the central effector of shock and death from anthrax. Animals injected intravenously with purified lethal toxin succumb in a manner that closely mimics the natural systemic infection. Lethal toxin appears to be a zinc-dependent metalloprotease, but its substrate and mode of action have yet to be defined. It affects most types of eukaryotic cells.
CO 2 /HCO 3 lef
Cap A C
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dep
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pXO21 90 kbp
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CO 2 /HCO 3 temp.
Figure 2 Coordinate regulation of virulence factors. The production of the capsule and anthrax toxin genes are enhanced by CO2/bicarbonate and temperature. The molecular mechanism of enhanced virulence has not been elucidated.
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Macrophages, which play a key role in combating infection, are particularly sensitive to the toxin. At low levels, the toxin appears to interfere with the ability of macrophages to kill bacteria. The toxin also stimulates the production of cytokines within the macrophage. As the level of toxin increases in the blood, more cytokines accumulate within the macrophage until the cell is finally lysed. It is proposed that this sudden release of cytokine leads to shock and would explain the rapid death seen in animals. In addition to the major factors previously outlined, B. anthracis expresses a number of other factors that may contribute to virulence. These ‘minor factors’ could account for the difference in virulence between strains. Like many other pathogenic organisms, B. anthracis produces an S-layer composed of two proteins called Eal and Sap. S-layers are proteinaceous paracrystalline sheaths present on the surface of many Archaebacteria and Eubacteria. S-layers have been found on many bacterial pathogens, including Campylobacter spp. and Clostridium spp. Various functions have been proposed for S-layers, including shape maintenance, molecular sieving, or phage fixation. The S-layer may be a virulence factor, protecting pathogenic bacteria against complement killing. It has been demonstrated that B. anthracis can produce a number of chromosomally encoded extracellular proteases that, like lethal toxin, kill macrophages. The presence of similar, if not identical, toxin genes in a number of members of the B. cereus, B. thuringiensis, and B. mycoides group raises the possibility that these genes also may be present in B. anthracis. A homolog to the cereolysin gene of B. cereus has been detected in B. anthracis. Although functionally inactive in the majority of strains, spontaneously occurring low-level activity has been demonstrated. It would not be surprising if homologs to other bacillus virulence factors were not detected.
Detection Given the scarcity of anthrax in the industrial world it is unlikely that many routine diagnostic laboratories would have the experience or access to the materials required, to identify the organism correctly. The main problem is the differentiation of B. anthracis from the phenotypically similar B. cereus/thuringiensis group, which may also be present in many of the samples examined for anthrax. Direct detection of the organism in the field is relatively simple in animals that have died suddenly of the disease. At death the blood of an animal generally contains >108 bacilli per milliliter. Blood films are dried, fixed immediately by heat or immersion for 1 min in absolute alcohol, and stained with polychrome methylene blue, which after 20 s is washed off. When the slide is dry, it is examined for characteristic deep blue, squareended bacilli surrounded by a well-demarcated pink capsule (McFadyean’s reaction) (Figure 3). In some animal species, such as pigs, the terminal bacteremia is limited, and the bacilli are unlikely to be seen in McFadyean-stained blood smears. Antigen-based direct detection methods have been developed that are more sensitive than staining. A highly specific immunochromatographic assay has been developed utilizing
Figure 3
Capsule stain.
a monoclonal capture antibody to the anthrax toxin component, PA. This assay can detect as little as 25 ng ml1 of PA and can be performed in a few minutes without the need for special reagents. This test could be used in addition to staining to screen animal blood and tissue and confirm the presence, or absence, of the organism. DNA-based detection using polymerase chain reaction (PCR) methodologies have been used successfully to detect the presence of B. anthracis in environmental samples. Once the problem of PCR inhibitors in blood and animal tissue have been overcome, it should be possible to detect the organism in animal samples. Unless there is an index of suspicion, it is unlikely that animal products would be examined routinely for the presence of B. anthracis. In cases in which contamination with B. anthracis is suspected, the World Health Organization (WHO) in their Comprehensive and Practical Guidelines on Anthrax propose the isolation protocol shown in Figure 4. The sensitivity limit of this technique is approximately five spores per gram of the starting material. The number of bacteria isolated very much depends on the distribution of the organism within the sample. The polymyxin-lysozyme-EDTA-thallous acetate (PLET) agar described in the method is a semiselective medium for B. anthracis, which contains polymyxin (30 000 units l1), lysozyme (300 000 units l1), ethylenediaminetetraacetic acid (0.3 g l1), and thallous acetate (0.04 g l1). Once colonies have been isolated, further testing is required to confirm their identity (Table 1). Many saprobic species of aerobic spore-forming bacilli are hard to distinguish from
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Blend to suspend in 2 volumes of sterile distilled /deionized water (buffered if specimen is likely to have a low / high pH)
ROUTE A (When there is reason to believe some or all of the B. anthracis will be in vegetative forms)
ROUTE B (When B. anthracis is only likely to be present as spores)
Decant ±10 ml into a tube / bottle
Prepare ±10 ml volumes of undilute and 1:10, 1:100 and 1:1000 dilutions of the suspension in sterile distilled /deionized water
Place in 62.5 °C water bath for 15 min (‘heat shock’) or (‘alcohol shock’ by adding equal volume of 95 –100% ethanol and hold 1 h)
Second
First
Place in 62.5 °C water bath for 15 min (‘heat shock’) or (‘alcohol shock’ by adding equal volume of 95 –100% ethanol and hold 1h)
Spread 100 l of each dilution on polymyxin blood agar (BAP) and 200–250 l on PLET agar
Prepare ±10 ml volumes of 1:10 and 1:100 dilutions of the suspension in sterile distilled /deionized water
Spread 100 l of each dilution on blood agar (BA) and 200–250 l on PLET agar
Incubate BAP / BA overnight at 37 °C and PLET for 36 – 48 h at 37 °C
Figure 4
WHO protocol for the isolation of anthrax.
B. anthracis except on the basis of pathogenicity. The most commonly encountered are B. cereus/thuringiensis/mycoides, Bacillus subtilis, and Bacillus licheniformis. The preliminary tests shown in Table 1 are used routinely by the Anthrax Section, Centre for Emergency Preparedness and Response (CEPR), Porton Down, Salisbury, United Kingdom, and allow the presumptive identification of an isolate as B. anthracis. Similar tests are conducted under the auspices of the US Centers for Disease Control and Prevention’s Laboratory Response Network. l
Lack of motility: Log phase cultures of the organism grown in nutrient broth at 22 C and 30 C are examined for motile
organisms by phase contrast microscopy. Unlike the other closely related bacilli, B. anthracis is nonmotile. l Lack of hemolysis: When cultured on 7% defibrinated horse blood agar, colonies of B. anthracis are large, opaque, and white, and have a very rough surface and an irregular edge. They are normally nonhemolytic, although the occurrence of hemolytic colonies has been reported. l Sensitivity to diagnostic gamma phage: Sensitivity to B. anthracis–specific phage is determined by spreading 200 ml of a log phase culture over the surface of a blood agar plate. After incubation for 1 h at 37 C, 20 ml of B. anthracis-specific gamma phage suspension is spotted on the plate. After overnight incubation at 37 C, the plate is examined for
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Table 1
BACILLUS j Bacillus anthracis Detection and identification methods for B. anthracis
Direct Microscopy (McFadyean’s stain) Antigen detection Polymerase chain reaction Preliminary tests Lack of motility Lack of hemolysis Sensitivity to diagnostic gamma phage Sensitivity to penicillin Commercial biochemical kits: API 50CHB, Biolog Confirmatory tests (specialist lab) Virulence in animals – guinea pig Capsule formation – McFadyean’s stain Toxin detection – immunoassays Virulence gene detection – polymerase chain reaction
plaques. On rare occasions phage-negative B. anthracis and phage-positive B. cereus may be encountered (Figure 5). l Sensitivity to penicillin: The test organism is subcultured to a blood agar plate; a 10 unit penicillin disk is spotted on the culture and the plate is incubated overnight at 37 C. Bacillus anthracis is sensitive to penicillin, whereas B. cereus is resistant. Very rarely penicillin-resistant B. anthracis isolates are encountered. Commercially available biochemical screening systems such as API 50CHB (bioMerieux, France) and Biolog (Biolog Inc., Hayward, United States) have been evaluated for their ability to identify B. anthracis. These systems offer the advantage of being easy to use and show promise as simple, first-line, one-step screening tests for the presumptive identification of B. anthracis. These tests are called presumptive tests as other strains of bacilli can give similar reactions to B. anthracis. The demonstration of virulence constitutes the principal point of difference between typical strains of B. anthracis and those of other anthraxlike organisms. Traditionally, the guinea pig has been the model used to demonstrate virulence. The animal is injected with the sample, and if it dies, the cause of death is confirmed by the isolation of B. anthracis from blood. Although this traditional technique is
sensitive, it is likely to be replaced by more sensitive in vitro tests. Virulent isolates of B. anthracis produce both a capsule and exotoxins. Detection of capsule formation is relatively simple. Capsule-forming organisms, when grown on medium containing bicarbonate and in the presence of CO2, produce colonies that are raised and mucoid in appearance, whereas noncapsule-forming organisms produce flat, dull colonies. In addition, the presence of the capsule can be confirmed by McFadyean’s stain. Detection of active toxin production is not as straightforward and requires either an animal system, a tissue culture assay using toxin-sensitive cell lines, or an immunological technique, such as an enzyme-linked immunosorbent assay. PCR allows for the detection of the genes encoding the virulence factors without the need for their expression. Specific DNA primers have been developed for the detection of capsule and toxin genes. Primers have been developed specific to the genome of the organism allowing the detection of atypical, nonvirulent strains of B. anthracis. A rapid-viability PCR method, incorporating primers and probes specific for the chromosome and each of the two virulence plasmids, has been developed to detect viable, virulent B. anthracis in environmental samples.
Regulations Most countries have regulations concerning the handling and disposal of infected food animals and their products. Concerns about the importation of contaminated animal products into the United Kingdom at the beginning of the twentieth century led the government to set up disinfection stations to treat all animal hair and leather goods. The United Kingdom Anthrax Order (1991) prescribes the steps that should be taken to deal with an animal that has, or is suspected of having, anthrax. This measure calls for the infected animals and its products, such as milk, to be destroyed, thus removing them from the food chain. The WHO has produced detailed comprehensive and practical guidelines on anthrax, detailing best practices on all aspects of the disease. In many areas where anthrax is endemic, particularly Africa, the problem is not the lack of regulations but rather the will and the means to enforce them in the face of local customs.
Importance to the Food Industry
Figure 5
Gamma phage lysis.
The number of reported cases of foodborne illness involving B. anthracis is extremely small compared with other traditional food-poisoning organisms. To date, there has never been a documented case in the United States. In countries with welldeveloped veterinary and public health systems, infected animals will be identified and removed from the food chain. In countries where such systems are not in place, the potential exists for contaminated animals and their products to be processed and consumed. A survey of animals in a slaughterhouse in eastern Nigeria revealed that 5% of cattle and 3.3% of sheep were positive for anthrax. These infected animals not only pose a risk to the people consuming the meat but also an
BACILLUS j Bacillus anthracis occupational risk to workers exposed to the carcasses. In the same survey, it was found that 13% of butchers and skinners had acquired cutaneous anthrax. Slaughterhouse waste in the form of offal for animal feed, and slurry discharged into the environment, represents a further source of potential infection. A study of uncut anthrax-contaminated slaughterhouse waste showed that viable anthrax still could be recovered after the offal had been heat treated for 30 min at 130 C. The use of bone charcoal by the food industry in the production of sugar products presents an avenue for anthrax contamination. The bones normally are obtained from areas of the world in which anthrax is endemic and, on occasion, B. anthracis has been isolated. For this reason, the bones must be sterilized, usually by gamma irradiation, before use. It is also important to note the potential of gastrointestinal anthrax occurring as a result of a bioterrorism event. Intentional contamination of food products may result in disease that would differ from naturally occurring infections associated with the consumption of meat from an infected animal.
Importance to the Consumer Due to the scarcity of the disease, there are few published records of human infection. The cases that are published mainly originate from Africa, the Middle East, and central and southern Asia. Figures for human anthrax in China showed that of 593 recorded cases, 384 were linked to the dismembering and processing of infected animals and only 192 cases were due to the consumption of contaminated meat. In neighboring Korea, sporadic outbreaks of human anthrax have been reported. From 1992 to 1995, three outbreaks occurred, a total of 43 cases, all linked to the consumption of contaminated beef or bovine brain and liver. An outbreak in India was centered on an infected sheep. Of the five individuals who skinned and cut up its meat for human consumption, four developed fatal anthrax meningitis. Another person who wrapped the meat in a cloth and carried it
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home on his head developed a malignant pustule on his forehead and went on to develop meningitis. A large number of people who cooked or ate the cooked meat of the dead sheep remained well.
See also: Bacillus: Bacillus cereus; Bacterial Endospores; Classification of the Bacteria: Traditional; Bacteria: Classification of the Bacteria – Phylogenetic Approach; Nucleic Acid–Based Assays: Overview.
Further Reading Anthrax Order, 1991. Statutory Instruments 1991. No. 1824, Animals. HMSO, London. Beatty, M.E., Ashford, D.A., Griffin, P.M., Tauxe, R.V., Sobel, J., 2003. Gastrointestinal anthrax. Archives of Internal Medicine 163 (10), 2527–2531. Bravata, D.M., Holty, J.-E.C., Wang, E., Lewis, R., Wise, P.H., McDonald, K.M., Owens, D.K., 2007. Inhalational, gastrointestinal, and cutaneous anthrax in children. Archives of Pediatrics & Adolescent Medicine 161 (9), 896–905. George, S., Mathai, D., Balraj, V., Lalitha, M.K., John, T.J., 1994. An outbreak of anthrax meningoencephalitis. Transactions of the Royal Society of Tropical Medicine and Hygiene 88, 206–207. Kanafani, Z.A., Ghossain, A., Sharara, A.I., Hatem, J.M., Kanj, S.S., 2003. Endemic gastrointestinal anthrax in 1960s Lebanon: clinical manifestations and surgical findings. Emerging Infectious Disease 9 (5), 520–525. Letant, S.E., Murphy, G.A., Alfaro, T.M., Avila, J.R., Kane, S.R., Raber, E., Blunt, T.M., Shah, S.R., 2011. Rapid-viability PCR method for detection of live, virulent Bacillus anthracis in environmental samples. Applied and Environmental Microbiology 77 (18), 6570–6578. Okolo, M.I., 1985. Studies on anthrax in food animals and persons occupationally exposed to the zoonoses in Eastern Nigeria. International Journal of Zoonoses 12, 276–282. Reiddinger, O., Strauch, D., 1978. Some hygienic problems in the production of meat and bone meal from slaughterhouse offal and animal carcasses. Annali Dell’lstituto Superiore di Sanità 14, 213–219. Sirisanthana, T., Brown, A.E., 2002. Anthrax of the gastrointestinal tract. Emerging Infectious Diseases 8 (7), 649–651. Turnbull, P.C.B. (Ed.), 1996. In: Proceedings of the International Workshop on Anthrax, Winchester, UK, 1995. Salisbury Medical Bulletin, Special Suppl. No. 87. World Health Organization, 2008. In: Turnbull, P.C.B. (Ed.), World Organization for Animal Health, Food and Agriculture Organization of the United Nations Anthrax in Humans and Animals, fourth ed. World Health Organization, Geneva, Switzerland, p. 207.