The ecology of Bacillus anthracis

Molecular Aspects of Medicine 30 (2009) 356–367

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Molecular Aspects of Medicine journal homepage: www.elsevier.com/locate/mam

Review

The ecology of Bacillus anthracis Martin Hugh-Jones a,*, Jason Blackburn b a b

Department of Environmental Science, School of the Coast and Environment, Louisiana State University, Baton Rouge, LA 70803-5705, USA Spatial Epidemiology and Ecology Research Laboratory, Department of Geography, California State University-Fullerton, Fullerton, CA 92834-6846, USA

a r t i c l e

i n f o

Article history: Received 24 August 2009 Accepted 24 August 2009

Keywords: Soil pH Exosporium Insects Rainfall Carcass disposal Landscape ecology

a b s t r a c t The global distribution of anthrax is largely determined by soils with high calcium levels and a pH above 6.1, which foster spore survival. It is speculated that the spore exosporium probably plays a key part by restricting dispersal and thereby increasing the probability of a grazing animal acquiring a lethal dose. ‘Anthrax Seasons’ are characterized by hot-dry weather which stresses animals and reduces their innate resistance to infection allowing low doses of spores to be infective. Necrophagic flies act as case-multipliers and haemophagic flies as space-multipliers; the latter are aided by climatic factors which play a key part in whether epidemics occur. Host death is a function of species sensitivity to the toxins. The major function of scavengers is to open the carcass, spill fluids, and thereby aid bacilli dispersal and initiate sporulation. In the context of landscape ecology viable spore distribution is a function of mean annual temperature, annual precipitation, elevation, mean NDVI, annual NDVI amplitude, soil moisture content, and soil pH. Ó 2009 Elsevier Ltd. All rights reserved.

Contents 1. 2.

3.

4.

5.

6. 7. 8.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Soils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Soil germination and vegetative cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. Rain, up & down, and sideways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. Index case infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Climate/hot-dry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Pathogen genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Insects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1. Blow flies: case multipliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.2. Biting flies: space multipliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.3. Ticks, flies and mosquitoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Host death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.1. Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5.2. Sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carcass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Landscape ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusion and discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

* Corresponding author. E-mail address: [email protected] (M. Hugh-Jones). 0098-2997/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.mam.2009.08.003

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Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 365

1. Introduction The term ecology or oekologie was coined by the German biologist Ernst Haeckel in 1866, which he defined as ‘‘the comprehensive science of the relationship of the organism to the environment” or the study of the relationships between living organisms and their environments (Haeckel, 1866). This differs from the definition of epidemiology, a discipline which is ultimately concerned with improved disease control. So we will be examining not the livestock problem but how this pathogen interacts with the soil environment, with its various hosts, primarily wildlife, and mechanical vectors with disease as an incidental, though it is important for its long time persistence, multiplication, and spread.

2. Soils Anthrax spores survive best in black steppe soils rich in organic matter and calcium. The persistence of anthrax was commented upon by Higgins in 1916 in that ‘‘a suitable soil must be slightly alkaline.” Citing the work of Minett and Dhanda (1941) and of Whitworth (1924), Van Ness and Stein (1956) and Van Ness (1971), with the results of their own studies of the geographic distribution of outbreaks in the US, put forward an hypothesis that ‘‘anthrax occurs in livestock that live upon a soil with a pH higher than 6.0, and in an ambient temperature above 15.5 °C.” The ‘Map of Soils of the World’ (Fanning and Fanning, 1989) shows that Van Ness’s high risk soils are contained within the mollisol and aridisol soils of North America. This is still true today when we compare the spatial distribution of naturally occurring outbreaks and soil or other environmental variables (Blackburn et al., 2007). Russian contemporaries of Van Ness reported the same patchy distribution of anthrax mortality in the steppes though their interpretation was and is different in favouring multiplication of the organism outside the host. This association of high anthrax mortalities with dark steppe soils, specifically chernozem and kastanozem soils, rich in organic matter, with a calcareous or gypsum-rich subsoil, and above neutral pH still applies in Russia and Central Asia though the present incidence is much reduced (Cherkasski, 2002; Kasianenko et al., 1984; Kolonin 1969). These Russian soil definitions when applied to North American soils are equally predictive there of enzootic risk. In South America cattle are intensively grazed on the related thick phaeozem soils in Argentina at risk of anthrax; in North America this soil is used for grain crops. In the Kruger National Park (KNP), South Africa the areas with a soil calcium of >150 milliequivalents and a pH >7.0 had anthrax death rates in the wildlife more than seven times higher than for those areas with lower soil values (Smith et al., 1999). We have noted (Hugh-Jones, unpublished data) that depressions or ‘‘pot-holes”, 0.2–0.3 Ha, in South Texas will through rainfall accumulate minerals and humus from surrounding sandy-loam soils. In this way a pot-hole will contain x2 to x3 more calcium, x6 to x10 of phosphorus, >x2 of magnesium, even increased levels of sodium. The end result is a locus friendly to spore survival in an area that would otherwise be inimical. Such a place will have grass and, after rain, water and longer grazing; sick animals will find shade in the margin scrub and cool themselves in the water, and die; and fulfill Dragon and Renie’s criteria (1995) for a spore ‘‘storage area.” Humus particles are positively charged at a neutral pH and act as chelators, collecting and holding bacteria. In the moist state anthrax spores carry a negative surface charge. The negative surface charge on the exosporium of Bacillus megaterium SG-1 spores varies with pH and is zero at pH 4.5 and increases rapidly until pH 6.0 to level off at pH values greater than 8 (He and Tebo, 1998). It is likely that Bacillus anthracis spores have broadly similar properties. Therefore, if so, in alkaline soils the negatively charged spores would also be attracting positively charged calcium and other divalent cations which would tip the diffusion equilibrium inside the spores to favor maintaining calcium in the spore core matrix and extend spore viability and germinative ability until the next grazing host happens (Himsworth, 2008). Conversely in acidic soils of less than pH 4.5, the now positive charge on the surface of the spores may tip the equilibrium so that more cations leach out of the core and result in an accelerated loss of viability (Dragon and Renie, 1995) and release its humus grasp. The disappearance of anthrax from areas with soils pH <6.1 would suggest that this leaching process for B. anthracis may start above pH 4.5. For example, Vilas and Gonzalez (1950) found that spores could not be recovered from soils with a pH of 5.1 or less after 108 days. Studies on the surface charge characteristics of anthrax spores in soil are long overdue and it would not be surprising if the genomics of the various A and B clones in some way matched their survival preferred soils. The loose nature of the exosporium and its hirsute nap coat (Beaman et al., 1972; Driks, 2002; Sylvestre et al., 2002) will allow it to efficiently adhere through its negative charge to humus and limestone particles in the soil and to stop dispersal in alkaline soils. In this way spores will collect where they form and enter the soil, thereby maintaining a very high concentration of spores potentially lethal at that place to a subsequent grazing animal. Contaminated carcass sites in the dry, dusty soils of the Etosha National Park, Namibia, are noteworthy both for their discreteness and the high levels of contamination over many years (Lindeque and Turnbull, 1994; Turnbull et al., 1998). The same has been noted in the Wood Bison National Park area, Canada, with repeated outbreaks at Hook Lake, Grand Detour, elsewhere in the park and the Peace River Delta dating back to the index outbreak in 1962 (Choquette et al., 1972; Broughton, 1992; Dragon

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and Elkin, 2001; Novakowski et al., 1963). In the almost 40 years between the trials on Gruinard Island and predecontamination sampling there had been a negligible spread from the original detonation and testing sites (Manchee et al., 1990). While the exosporium with its hirsute nap is an excellent large surface area structure with which to have anchoring interactions with soil substrates, experiments with B. anthracis and other members of the Bacillus cereus have shown that the loose fitting exosporium is easily sheared from the rest of the spore by a minor mechanical force (Faille et al., 2007). This means that the exosporium could be a weak link in the chain holding the spores in place but the fact that it remains indicates a Darwinian advantage and probably strain differences reflecting different selection processes (Tauveron et al., 2006). While the exosporium may help to maintain a spore concentration in organically rich soil under natural conditions but on a slope and in poor soil the high energy of a spring run-off or a seasonal flood could rip or tear the exosporia freeing the spores and either wash them away or carry them to concentrate elsewhere in low lying areas as the floods regress. There the spores would latch on to a second soil substrate through their remaining exosporial tags or by their spore coats. Of course in the absence of detailed electron microscope studies we do not know the condition of spores in concentrator sites. Hypothetically it may be possible that when animals churn up the mud at the bottom of pans (pot-holes) a loose exosporium would allow some of the spores to break loose, be available for drinking, and penetrate deeper into the host’s body through micro-abrasions or across the intestinal mucosa. This would be more efficient than depending on spores clumped in a mud particle and gastric acidity to free them. Repeated soil samplings from sites with a past history of confirmed anthrax spore contamination eventually reveal apathogenic isolates within 5–8 years (Cherkasski, 2002). Such isolates usually lack the capsular plasmid pX02 and less frequently both pX01 and pX02 (Turnbull et al., 1992). Plasmid loss is not an uncommon laboratory artifact but in the absence of strong evidence for soil germination another explanation must be sought. It is possible that under harsh environmental conditions either or both plasmids may be spontaneously lost. Dragon has commented (personal communication) that as an obligate pathogen it is probably to the advantage of B. anthracis to have the plasmid virulence genes operational from the moment of germination. Very speculatively, to achieve this, the plasmids are possibly packaged differently from the chromosomal DNA. The trade off for this accessibility is that the plasmids are not as well protected as the chromosomal genes from ultraviolet inactivation and other stresses during the spore survival phase. But if effective, sufficient fully pathogenic organisms will successfully germinate when the opportunity occurs to infect the next host and multiply accordingly. This would also provide a Darwinian pressure for higher plasmid numbers and higher virulence (Coker et al., 2003). With multiple copies the better the chances of at least one or more sets unpacking correctly. As evidence in support of this speculation, it is noted that the initiation of capsule formation is an early event following spore germination with Ezzell and Welkos (1999) observing capsular material blebbing out from cracks in the spore coat prior to sloughing of the exosporium or any sign of outgrowth of the vegetative cell. 2.1. Soil germination and vegetative cycles The question as to whether spores in soil will germinate, multiply as vegetative bacilli and then resporulate is a matter of discussion. The keystone paper is Minett and Dhanda (1941), who achieved it using sterilized, wet, alkaline soil at 25–30 °C but not with unsterilized soil. Multiplication did not occur in acid soils; nor does germination (Manchee et al., 1981). Saile and Koehler (2006) have demonstrated that spores will germinate and establish stable populations of vegetative cells in the rhizosphere of fescue (Festuca arundinacea) grass in the laboratory in an otherwise sterile environment; they were also able to demonstrate horizontal gene transfer. The optimum germination temperature is 39 °C and falls off with higher and lower temperatures to virtually nothing at 18 °C (Davies, 1960). In natural circumstances the vegetative cells are fragile and die even in simple environments such as water or milk (Turnbull et al., 1989, 1991; Bowen and Turnbull 1992) or from an innate inability to compete with other microflora (Sterne, 1959; Zarubkinskii, 1960; Vasil’eva, 1960) plus the amount of nutrients that they need (Sussmann and Halvorson, 1966; Titball et al., 1991) is demanding and would soon be used up without replenishment – blood appears to be ideal but is obviously rarely available. That it will increase the pH is temporary and incidental (Manchee et al., 1981). On the other hand Cherkasski (2002) quotes some 30 years of successful Soviet research where multiyear sowing with a wide variety of crop plants – winter wheat, rye, maize, vetch, garlic, clover, alfalfa, various grasses – was effective in making these sites safe for grazing, and even a single sowing of alfalfa with yard grass, or vetch with oats. Hanna, Bergman and Thomason (Anon, 2004) sterilised a local Michigan soil, added water and anthrax spores and found that germination is a relatively rare event involving about 2% of spores, but those that germinated replicated very well until the soil nutrients were soon exhausted when they sporulated. While in total germination and replication were poor with an overall increase of only some 20- to 250-fold, sporulation was extremely efficient. Soil encourages sporulation, not germination, which would explain why vegetative bacilli are not found in nature (Hanna, 2008). Others have proposed that environmental cycling is not rare, primarily Van Ness (1971) with the concept of ‘‘incubator areas”. This was based on his studies and those of Van Ness and Stein (1956) that ‘‘anthrax occurs in livestock that live upon a soil with a pH above 6.0, and in an ambient temperature above 15.5 °C.” Specifically these ‘‘incubator areas” are depressions which collect water, dead vegetation, calcium and other salts washed in from the surrounding slightly higher ground and thus provide a medium suitable for germination and multiplication. However the major part of Van Ness’s argument is based on the 1957 epidemic in Craig County, Oklahoma, and adjoining Kansas during a decade of frequent outbreaks due to contaminated bone meals (Blackburn et al., 2007) and the prior meteorological scenario is typical of Tabanid based spread

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– Craig County, where most outbreaks were, had 35 in. of rain from April through June followed by ‘‘a prolonged drought with hot weather.” Index outbreaks occurred on high ground in north central Craig County, and in southern Craig County and to the northward in nearby Kansas (Van Ness, 1959), all with non-alkaline soils generally about 5.0 pH. And there have been no outbreaks in that part of Oklahoma/Kansas since 1957, which of itself would argue against ‘‘incubator areas”. Latterly Kaufman (1990) has claimed that the frequent association of outbreaks following rain during a drought or the onset of a rainy season is best explained by a concomitant growth spurt by B. anthracis in the soil. However both are hypothetical and never confirmed by scientific study. An alternative simple explanation would be that the rain initiates grass growth when the surviving grass will be extremely short from repeated grazing during the previous dry period, and the water – especially if allowed to stand as in a depression – loosens the soil such that, when grazed, soil and lurking spores are ingested along with the grass and other vegetation. Genetically B. anthracis is extremely conservative with only 3% of the genome with any changes (Keim et al., 1997), which would belie frequent environmental cycling. Similarly, if it were common the historic multitudes of buried carcasses and bloody soils would have engendered many permanently contaminated sites worldwide, allowing for soil type and pH and interpreting ‘permanent’ as 100 years. The reality is that such sites are truly extraordinarily rare, though well documented, and probably merely reflect extremely high initial spore counts. For practical purposes B. anthracis is better perceived as an obligate pathogen with the motto ‘‘sporulate or die” (Turnbull et al., 2008a).

2.2. Rain, up & down, and sideways It has been proposed that water may collect and concentrate spores in ‘storage areas’ (Dragon and Renie, 1995). Spores have a high surface hydrophobicity and so could be carried during a rain runoff in clumps of humus and organic matter to collect and coconcentrate in standing pools or puddles. As they have a high buoyant density this would result in them and their organic matter clumps remaining suspended in the standing water to be further concentrated as the water evaporated. Thus theoretically ‘storage areas’ may collect more spores from extended areas to reach increasing spore concentrations over time and be lethally available to incidental grazing potential hosts. At the same time there are probably inverse distance factors that could as well disperse spore-humus clumps in many diverse directions over extended distances, just as others probably converge and concentrate after a few metres. The former final dilution could well be far beyond any possible acquisition of an LD50; the latter might do the reverse and allow a secondary storage (concentration) site, albeit smaller than its sources, to be dangerous longer. However there have been cattle outbreaks in animals grazing water meadows subject to spring flooding, e.g., Turner et al., 1999. It will depend on the specific soil topology and character. Just as rain will move spores down into the dry soil as it drains and away from sunlight and U/V light, standing water will have the capacity to move hydrophobic buoyant spores upwards into the grazed vegetation.

3. Infection 3.1. Index case infection For multiplication B. anthracis is an obligate pathogen lurking in the soil. While the oral minimum infective oral dose is largely unknown for wildlife species it was noted (de Vos, 1990; de Vos and Scheepers, 1996) that while 100–250 spores parenterally administered consistently killed kudu in the Kruger National Park, the oral LD50 with same strain was approximately 15 million spores. In healthy unstressed sheep, horses and cattle the lethal oral dose is of the order of 1.5–5  108 spores; parenterally the minimum dose for sheep is 75 spores, killing in 108 h, but only 36 h with 55,000 spores (Turnbull et al., 2008a). Browsers graze. Once one grazing animal has been brought down, others can be infected from it, from licking the blood spilt or seeping from the carcass, from spores deposited on surrounding browse by necrophilic blow flies, and via the contaminated mouthparts of haemophagic biting flies. These initial index cases are sporadic, seemingly random, and at low grazing densities in a relative absence of insect vectors they may be singular, certainly limited in secondary cases, and unappreciated. For example in the analysis of the 2008 plains bison (Bison bison bison) epidemic in SW Montana no cases had been reported in that part of the state since the mid-1950s (David Hunter, personal communication). In the intervening years sporadic summer deaths had been put down to hemlock poisoning and catarrhal fever. Also elk (Alces alces) died on the mountain ridges away from casual discovery. Mummified carcasses and scattered bones from infected carcasses can present diagnostic problems to many laboratories. However both wood (Bison bison athabascae) and plains bison will gather around fallen colleagues, nudging, bunting, nuzzling, even using their horns to try to get the fallen bison up. Bison bulls will aggressively horn and stomp on other fallen bulls, even on patently dead bulls. This probably, more than from display-wallowing in a wallow where its previous owner had died and disintegrated, explains why bulls can and do form the majority of cases; bulls are infrequently found dead in wallows but more usually in the cool shade of the nearby trees or on the open meadow. On the third or fourth day bison will leave the area where an animal has fallen.

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There is limited evidence that a carrier state of latent infection can develop in individual animals and some species. Dormant spores can circulate in the blood of black rats (Rattus norwegicus) for 30 days (Walker et al., 1967). B. anthracis has been recovered from abdominal lymph nodes of apparently healthy Ascoli-negative Zebu cattle in Chad where the disease is hyperenzootic (Provost and Trouette, 1957). Healthy pigs slaughtered months after an outbreak have been found to have chronic infections in the enlarged tonsils, cervical, submandibular and mesenteric lymph nodes (Hutyra et al., 1946; Stein, 1948); this was also noticed in healthy pigs going to slaughter nine months after the 1952 epidemic in Ohio, Indiana and Illinois. This hypothesises the potential for these latent infections to be prolonged, in spite of the occasional spore germinating and handled successfully by circulating macrophages, but when the host is subjected to environmental stress convert to the peracute disease, distant in time and space from where the spores were first acquired. The global distribution of the A strains but very limited distributions of the B strains would suggest that the A strains may have this capacity. 3.2. Climate/hot-dry It is a consistent worldwide observation that anthrax is a hot season disease and especially of hot-dry climates. The resistance of animals to infectious diseases is adversely affected by extremes of temperature. Exposure to hot weather alters the host resistance in a number of ways; by altering nonspecific local resistance of the skin and mucous membranes and thus facilitating the entry of pathogenic organisms; it will affect the clearance of infected cells such as macrophages; and can directly impact the physiologic and metabolic control systems that modulate specific immune responses. Heat exposure may indirectly affect host resistance by inducing changes in nutrition, behavior, and management (WMO, 1989; Webster, 1981). While graze and browse may be abundant and highly nutritious in the wet season it can become sparse and of low nutritional value during the dry season and increasingly abrasive (Starr, 1988). The end result is to markedly reduce the necessary ID50 and thus LD50 which will result in one or more index animals succumbing to disease and thus initiate the train of events that facilitate further cases. Similarly animals tend to congregate where there is grazing increasing the probability of spread. Some ranchers have noted that in hot dry weather there will be occasional brief showers and cervid cases are seen ten days later, presumably from grazing the fresh grass in a moist spore-laden locus. On the other hand hotter temperatures will increase the probability of sporulation, but also of drying and the putrefaction rate of infected carcasses. Similarly they will kill unsheltered spores in surface soils. 3.3. Pathogen genomics Repeated epidemics narrow the choice of genetic strains available in an area to subsequent outbreaks. For example in the Kruger National Park between 1970 and 1997 there were a series of epidemics and out of 98 isolates archived from that period 21 were B1 (genotype 87), 74 A3 (genotype 67), and 1 A3 genotype 39 and 2 A3 genotype 45, with B1 concentrated in the north of the park close to the Parfuri river, A strains in the body of the park. The 1990 epidemic started in the central regions and progressed north and was all but entirely A strains. This followed torrential rains that had scoured the northern regions of the park and probably physically removed lurking B1 spores (Smith et al., 2000). Similarly present outbreaks in Alberta, North & South Dakota, Minnesota, Manitoba, and Saskatchewan involve very largely, especially recently, the ‘Western North American’ strain A1a genotype 2 and reflect the repeated epidemics across those black soil prairies (Van Ert et al., 2007). As we get better at genetically identifying outbreak strains and field collections expand minor variants appear. For example in a 2004 epidemic in Basilicata affecting 41 farms and 124 animals – cattle, sheep, goats, horses and deer – the 52 isolates were a single genetic strain (sgt-eB) in the A1a cluster, but two minor variants were found in one animal (sgt-eB,m2 reflecting a single mutation in the CL12 fragment) and four animals (sgt-eB, m1 reflecting a single mutation in the CL33 fragment), all on separate farms (Fassanella et al., 2009). This points up that this epidemic included minimally three sporadic outbreaks; the epidemic initiator of 25th August; and the two initiating the sgt-eB, m1 and sgt-eB, m2 series outbreaks. On the 28th July there was a single sporadic outbreak involving one bovine and genetically separate from the events of 25th August and after. As commented elsewhere initial outbreaks are sporadic and can occur throughout the ‘‘anthrax season.” When circumstances are right some will initiate epidemics, and within such may be separate sporadic outbreaks with or without related subsequent cases. At this time there is only one group of strains that is species related, those associated with wood bison. Why is unclear but it may be related to the large biomass of wood bison bulls, 1150 kg. If that is so, a similar differentiation may occur in the Etosha National Park where elephants are regularly affected. But it should be noted that there is a variety of species of wide ranging size in Etosha, and thus many anthrax susceptible species smaller than elephants, whereas in the wood bison habitats in northern Canada there is only the occasional moose and so the pathogen must be bison targeted to survive.

4. Insects Anthrax has long been associated with insects, primarily necrophilic and haemophagic flies. B. anthracis has also been recovered from Musca domestica and from ticks. It is unlikely that the latter reflect any meaningful epidemiologic risk. It is probable that vector ecological and feeding preferences, land cover, and host species feeding-habits and densities explain

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why browsing kudu are the major affected species in one area, e.g., Kruger National Park, and grazing antelope in another, e.g., Etosha National Park, and some species essentially ignored, e.g., goats in the Texas Edwards Plateau. 4.1. Blow flies: case multipliers Blowflies, and their larvae, feed on the body fluids of anthrax carcasses in great numbers and, when replete, fly to adjoining vegetation usually in the immediate vicinity to vomit up excess fluid from their stomachs so that they can then digest their meal and defaecate, both teeming with bacilli and soon spores. This results in browse in proximity to and facing anthrax cases being severely contaminated. Braack and de Vos (1990) monitoring Chrysops albiceps and Chrysops marginalis found an average of 19 droplets per leaf between 1 and 3 m from the ground in near proximity to the target carcass. While blowflies may be life-long carriers vegetative cells disappear from their digestive tract within two weeks (de Vos and Turnbull, 2004). The geometry of dispersal is such that the browsing risk decreases exponentially and is very soon only theoretical. On the other hand browsers, whether kudu (Tragelaphus strepsiceros), white tailed deer (Odocoileus virginianus), or goats, enjoy crinkly leaves, such as would be available in hot dry weather and likely to abrade their throats and oesophagi, facilitating the ingress of spores. Thus these sites have the real potential to significantly increase the incidence among browsers. The spores will remain on this vegetation until it rains, which is why deaths cease with the onset of the seasonal rains whether in KNP, Tanzania, or West Texas. 4.2. Biting flies: space multipliers There is a long tradition of anthrax being spread by haemophagic flies. In west Texas the ranchers call horse-flies charbon flies even though they themselves are frequently of German extraction. The Texas paradigm is that they are especially dangerous after a wet spring and/or early summer and are responsible for significant spread outwards from sporadic outbreaks. Historically Budd (1863) first indicated a fly risk and Henning (1893) first specifically incriminated ‘horse flies’. Dalrymple (1900) noted their significant involvement in the 1899 widespread epidemic in Louisiana. In 1912, Schuberg and Kuhn demonstrated that infections could be transmitted between sick or dead animals to healthy ones using mice and guinea pigs and Stomoxys calcitrans. Later Schuberg and Boing (1914) were able to infect sheep and goats using S. calcitrans. Tabanus rubidus is effective transmitters for horses and buffalo (Kraneveld and Djaenodin, 1940). Others have pointed out the risks presented by biting flies, specifically the hippoboscids and tabanids (Mitzmain, 1914; Kehoe, 1917; Frey, 1919; Morris, 1918, 1920; Nieschulz, 1928; Viljoen et al., 1928; Olsufev and Leler, 1935; Sen and Minett, 1944; Sterne, 1959). Davies (1983) argued that it was tabanids that enabled the severe widespread Zimbabwe epidemic of 1978–79 following heavy rains that would have supported large hatches of tabanids. The spread of anthrax in India has also been ascribed to biting flies (Krishna Rao and Mohiyudeen, 1958) when 90% of affected cattle had cutaneous infections, incidence was a function of fly density, and ceased with the disappearance of the flies and onset of the monsoon. The mesoscale distribution of cervid anthrax in West Texas can also be defined by fly density and is missing where tabanid flies are essentially absent in areas with average wind speeds over 3.3 m/s, especially on higher elevations (Blackburn, 2006). This probably explains why Boer sheep grazing windswept ridge tops were spared while white-tailed deer (Odocoileus virginianus) in sheltered draws and valleys suffer enzootic outbreaks. While the conceptual risk of tabanids is clear the actual mechanics are less so starting with what proportion of flies caught >100 m from a moribund/dead animal will have contaminated mouthparts and the levels of contamination, feeding patterns and risk, minimal lethal doses for different target species exposed to biting flies, and the possibility of defining distant risk – empirical data repeatedly has secondary outbreaks 5–10 km from the index outbreak, occasionally significantly further. Kraneveld and Mansjoer (1939) found that T. rudidus that had fed on an infected animal had anthrax bacilli in their faeces until fly death (up to 18 days); the number of bacilli was very variable and sporulation delayed. Bacilli and spores could be recovered from their mouthparts for a week after feeding even when flies were allowed to feed repeatedly on healthy animals after the single primary feed on a sick animal. Kraneveld and Djaenodin (1940) fed these flies on septicaemic animals but not to engorgement. Transferred within 10 s to 14 target horses, 12 were infected and 11 died; two of the latter dead horses had been fed upon by just a single fly each and the rest by larger numbers. Six buffaloes (carabao) were exposed but transmission only occurred when 75 flies were allowed to feed on the target animal; whether the surviving five developed antibodies is unknown though not unlikely. When flies were allowed to fully feed and then held for 48 h – the normal interval between feeds – before feeding on six target horses, only two sickened, one of which died having been fed on by 40 flies, the maximum number used; the sick survivor had been fed on by only 10 flies; the apparently unaffected were fed upon by 1, 2, 5 and 20 flies. Olsufev and Leler (1935) hypothesized that the success of individual flies feeding impacts on subsequent risk. Those able to successfully fully feed, even if from repeated visits to the same animal, will delay feeding again and thus may be of less risk but successfully result in distant secondary cases. On the other hand those feeding on newly dead animals with maximum bacteraemias but acquiring less blood will need to feed again sooner and thus be of higher risk to nearby animals. The Texas paradigm of summer anthrax epidemics following heavy rains in the spring or early summer is consistent, and has many echoes – Alberta in 1999 (ProMED-mail, 1999), Saskatchewan in 2006 (ProMED-mail, 2006), North and South Dakota in 2005 (ProMED-mail, 2005a,b), western Edwards Plateau in Texas in 2001 (ProMED-mail, 2001), as well as in Northern

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Russia (Olsufev and Leler, 1935) and India (Krishna Rao and Mohiyudeen, 1958). The reverse also applies – drought in the winter and spring will be followed by only sporadic singular, not multiple, deaths during the subsequent hot-dry summer. In enzootic areas seemingly random sporadic deaths is the usual summer pattern. It is the biting flies that turn them into epidemics. As tabanid flies have prolonged larval development of some 9–10 months the spring rains will not affect the numbers of eggs laid but will impact the number of larvae successfully pupating and emerging. Another factor for successful spread is not just large numbers of flies but also delay in recognition and initiating an appropriate control response. For cattle it is usually some 5–10 animals in one herd dead but in Saskatchewan in 2006 it started with 50 cattle dead in adjoining farms and went province-wide; in the 2008 plains bison epidemic in SW Montana ten deaths were noted but 20 had died before control was initiated for an epidemic that killed some 298 bison, 80 elk and two white-tailed deer; in South Dakota the index outbreak initially involved 38 plains bison and two rodeo bulls, and it spread NW, NE and south through 11 counties. The third factor is an absence of vaccination or of herd immunity arising from a previous epidemic. In this context it is also clear that annual area vaccination must be enforced about known affected herds to prevent both sporadic cases from occurring and to negate the pathogen dispersal capabilities of biting flies; empirically it needs to be within a radius of 5– 10 km of known high risk herds, i.e. any herd with an outbreak in the previous 5 years. Following the 1993 epidemic in the MacKenzie Sanctuary when some 10% of the 1800 bison died, the following March 39/42 sampled adult animals had significant antibody titres (Turnbull et al., 2001). This widespread subclinical exposure will explain why epidemics two summers running are not seen. It also suggests that bison may be like cattle in being easy to infect but hard to kill and thus develop high antibody titres to the circulating but successfully resisted toxins. While with livestock the secondary attack rate will usually involve only a few animals per distant affected farm, with wildlife it can be significant and is a function of density. For example in West Texas in 2001 mortality rates on the various deer ranches ranged from 25% to 100%, commonly 80%, as the stocking rates are inflated, sometimes grossly, by feeding stations to facilitate commercial ‘‘hunting”. 4.3. Ticks, flies and mosquitoes As a result of the anti-plague activities in the Central Asian states B. anthracis is sometimes recovered from ticks collected during the routine summer field safaris collecting ticks and rodents. They are also recovered off moribund animals (Stiles, 1944; Buriro, 1980; Akhmerov et al., 1982). As yet there is no experimental evidence to indicate risk. Sen and Minett (1944) were able to transfer infection when Musca domestica and Calliphora erythrocephala were separately fed on incisions in the sides of goats dead from anthrax and then transferred to cauterized skin of healthy goats; infection transmission did not occur when they were put in contact with eyes of healthy goats. So while there is a potential it is probably meaningless in reality. Turell and Knudson (1987) were able to transfer infections between infected and healthy guinea pigs using Aedes aegypti and Aedes taeniorhynchus. The success rate between ingestion of bacteraemic blood and target guinea pig exposure was only 12% of the exposed guinea pigs plus all were within a 64 h interval. As these mosquitoes do not fly significant distances – traditionally female A. aegypti fly only some 30 m after feeding and then to incubate and lay eggs – the risk, if any, is very localized and would be hard to differentiate from that due to coincident biting flies. Also potential transmission by these mosquito species and others will depend on the numbers and density of mosquitoes feeding with already bloody mouthparts. 5. Host death 5.1. Mortality It has long been noted that ‘‘In certain outbreaks a single species of animal may show a more marked susceptibility than others which are apparently similarly exposed.” (Higgins, 1916). An inverse relationship exists between resistance to infection and susceptibility to the toxin complex as reflected in the level of the terminal bacteraemia (Lincoln et al., 1967). Cattle are very prone to natural infection but die with high bacteraemias indicative of a toxin resistance, thus high protective antigen titres, and a rapid response to the US Navy field immunochromatographic test (ICT) while the titre stays high (Muller et al., 2004). On the other hand in spite of dozens of attempts we have yet to have a white-tailed deer laboratory confirmed to have died from anthrax be ICT test-positive; apropos we have also learnt that laboratory confirmation is not easy with whitetailed deer, which would reinforce the conclusion that they are hypersensitive to the toxins, and die with low titres and bacteraemias. Then there are regional differences in species incidence. For example in northern New South Wales sheep and cattle are affected with equal frequency but southern NSW cattle are four times more likely to be affected than sheep and bovine mortality rates can be 13 times higher (Wise and Kennedy, 1980). Both roan (Hippotragus equinus) and sable (Hippotragus niger) antelope are found in the KNP but mortality in the former is very significantly higher than for the latter. The difference appears to be not in susceptibility but that when grazing is sparse roan switch to browsing and a much higher exposure to risk from blow fly contaminated browse (de Vos and Bryden, 1998). This also puts the browsing KNP kudus (Tragelaphus strepsiceros) at high risk where they form >50% of all recorded anthrax cases with zebras (Equus burchelli) merely noted among the miscellaneous cases in the park. On the other hand in the Etosha

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National Park zebra are 45% of recorded cases and kudu are only 0.8%, which evades a ready explanation other than speculation that one area, KNP, is blow fly dominated and the other, Etosha, with tabanides. In contrast to herbivores, pigs and carnivores are highly resistant to anthrax and the ingestion of large numbers of organisms, as are found in infected carcasses, is generally required to induce infection in them. Even so severe mortalities have been noted in wild dogs, lions, leopards, cheetahs in spite of their innate resistance (Hugh-Jones and de Vos, 2002). Not unexpectedly some 98% of lion, hyaena and jackal sera from the Etosha National Park demonstrated very high antibody titres against protective antigen (PA) but only 7% of herbivores, suggesting that few herbivores in the park survive infection (Lindeque et al., 1996). These differences may be explained by host species occupying different ecological niches but not of equiprobable risk; of different grazing behaviors; of vector species availability and density subject to different climates, ecologies, and host-target potentials; and strain virulence differences. Although textbooks describe extensive blood extravasation from anthrax cases after death, in fact it is infrequent and if it occurs modest. For example it did not occur with any of the 178 wood bison carcasses in the 1993 epidemic in the MacKenzie Bison Sanctuary (Cormack Gates, personal communication). The senior author saw it during his first outbreak investigation in 1972 but not since. During a large outbreak of 2005 in west Texas, extravasation was not documented in over 40 whitetailed deer sampled (Blackburn JC, unpublished data). 5.2. Sporulation Overall sporulation in the field is a complex question needing better quantification. If sporulation times are prolonged there is increasing risk of vegetative cell non-survival intervening. For example Turnbull et al. (2008a) quotes a water table bath experiment in which he and his colleagues successfully held three B. anthracis strains on agar slants at 12 °C but they required up to 2 weeks before sporulating; two other strains failed to sporulate. None grew, sporulated, or survived at 9 °C. The upper limit for sporulation is 45 °C (Mitscherlich and Marth, 1984). Davies (1960) showed the sensitive interaction of temperature, 37 °C, and relative humidity (R.H.) such that at 100% R.H. it took 12 h to complete, but at 650% R.H. only 7/20 (35%) cultures had sporulated by 34 h. But at 26 °C sporulation was delayed and took 28 h at 100–90% R.H. and 60 h at 20–50% R.H. though 19/20 cultures sporulated. The latter is slow but overall more successful, if unchallenged, than at the higher temperatures. It would appear that the vegetative cells were less subject to drying at the lower temperature. Minett (1950) noted that sporulation in an opened carcass was largely dependent on the ambient temperature. At 32.2 °C spores formed in the blood exuding from severed neck vessels (goats and guinea pigs) within 17–24 h, whereas at 15.6 and 21.1 °C the bacilli gradually disintegrated with the growth of contaminants; in blood removed from the cooler carcasses and protected from gross contamination spores were present by 44 h but in small numbers. Minett noted that spores survived in the bone marrow of goat carcasses for a week at 17.8–23.3 °C, but the frequency with which anthrax is associated with sun-dried bones would suggest that survival within bones is not uncommon. Enzootic anthrax areas are found generally in warmer latitudes but there are a number of well known places near or above the Arctic Circle, albeit in the summer when the days approach 24-h daylight and the cumulative sunlight provides adequate heat; e.g., wood bison in the Wood Bison National Park, Alberta, and in the MacKenzie Sanctuary, Northwest Territories, Canada, and in the caribou of the Taymyr Peninsula, northern Siberia. The cooler climate may explain why anthrax is not seen in the Andes above 3000 m. 6. Carcass To quote Sterne (1959) on sporulation, a high oxygen tension is not necessary as a reduction in partial pressure does not materially affect sporulation but a high partial pressure of CO2 diminishes sporulation, which is why sporulation only occurs after the carcass has be opened. Thus the major function of scavengers is to open the carcass to spill bloody fluids and allow sporulation. If there was blood extravasation after death, spores will form in this spilt blood before it acidifies but it is a race. However the major production is from the opened carcass especially as it is dismembered and parts dragged away to be consumed elsewhere. If the carcass is not opened the anaerobic decomposition and acidification will kill the contained vegetative cells within 4 days (Minett, 1950) resulting in minimal environmental contamination. Sporadic deaths are quickly found by carrion feeders. However in an epidemic they can be soon satiated and will ignore the increasing numbers of carcasses. Speculatively the most dangerous carcasses are those in a shaded area (minimal U/V light) with a deep moist humus-rich calcareous soil when the vultures and other scavengers are modestly hungry – enough to merely open the carcass – and it can lie undisturbed, seeping, generating and shedding spores. On the whole soil contamination is not extraordinary but modest. Lindeque and Turnbull (1994) found that 25% sites associated with anthrax carcasses of antelope had only 1–10 spores/g soil, 29% 11–100 spores/g, 25% 101–1000 spores/g, 10% 1001–10,000 spores/g, 7% 10,001–100,000 spores/g, and 4% over a million spores. Even when the initial spilt blood count is well over 106 cells/ml only in a small proportion of occasions does the soil get substantially contaminated and then all with a rapid decline with time. However the spore counts in the alkaline karstveld soils were significantly higher than those in sandy soils. Similar findings were reported by Dragon et al. (2005) for dead wood bison in the Wood Bison National Park in 2001 but spatially much more extensive as befits a 1135 kg animal. While there are some spectacular accounts of spore

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recoveries, even after 250 years, spores persist best in dry soils where microbial activity is minimal. In moist soils it is usually in the range of 3 months to 3–4 years, and rarely longer (Sterne, 1959). Spores will survive passage through the scavenger intestinal tract but vegetative cells will not; for example we were unable to find any positive turkey vulture (Cathartes aura) faeces (n = 20) under a roost in the middle of a significant distribution of confirmed deer anthrax deaths in West Texas in 2005 (Hugh-Jones, unpublished data). Spores but not vegetative cells survive passage through the acid (pH 1–1.5) vulture stomach (Anon, 1979; Houston and Cooper, 1975). Anthrax spores were recovered from approximately half of the faeces from jackals (Canis mesomelas), vultures (Gyps africanus, Torgos tracheliotus, Trigonoceps occipitalis) and hyaenas (Crocuta crocuta) collected in the vicinity of carcasses in the Etosha National Park but not at a distance; the fecal spore density was extremely variable (Lindeque and Turnbull, 1994). In Argentina Saggese et al. (2007) recovered spores from 1/14 cloacal swab samples from Chimango caracaras (Milvago chimango). In general scavengers are fairly resistant to infection but cases are seen (Edebes, 1976; Hugh-Jones and de Vos, 2002; Kraneveld and Mansjoer, 1941) and antibodies noted, e.g., in lions (Turnbull et al., 1989), and in three species of northern Namibian vultures (Turnbull et al., 2008a). The problem with vultures and marabou storks is not the spores in them but the spores on them (Pienaar, 1967). After feeding they fly to a bathing site, a cattle water-trough, a pond, a pool, a ‘mini’ dam, which is normally nearby. Vegetative cells in the blood contaminated feathers will sporulate in the water within 15–68 h (Lindeque and Turnbull, 1994). The spore load from an individual bird might be slight (Turnbull et al., 2008b) but as 50–185 birds may feed on a zebra or 250 on an elephant carcass the cumulative spore off-load, say in a cattle water trough, can present a very real risk. This water contamination is transient. Vultures devour animals rapidly when hungry, for example impala soft tissues within one hour and the carcass within half a day, but sporulation occurs only after the carcass has been opened and exposed to the air for a number of hours. Thus vultures greatly reduce the total volume of infective material in the vegetative form while spreading spores to their bathing sites. 7. Landscape ecology To understand the macro-ecology of B. anthracis, and given that most research currently suggests that germination and multiplication occurs in the host, while spore survival occurs in the soil (vide supra), it is necessary to identify the geographic area where bacilli spores can thrive for long periods of time. Landscape ecology provides a useful perspective of scale for such analyses. Haines-Young et al. (1994) provide an overview of landscape ecology and the role that geographic information science (GIS) can play in testing hypotheses within this theoretical framework. Landscape ecology in this context can be defined as the study of relationships between the biological requirements of the bacilli in spore form and the ecological conditions that support spore survival, and the geographic areas where these requirements are met and may lead to subsequent outbreaks under appropriate seasonal climatic and weather events. This broad perspective is useful for understanding the broad-scale geographic distribution of the bacterium (Van Ness and Stein, 1956; Smith et al., 2000; Cherkasski, 2002; Parkinson, 2001; Parkinson et al., 2003) and identifying areas where wildlife or livestock may be at risk. Ecological niche modeling is a spatially-explicit modeling approach that pattern matches or statistically identifies (depending on the approach employed) non-random relationships between species occurrence data (here anthrax outbreak locations and confirmed spore recoveries from soil) and ecological conditions (such as temperature, precipitation and soil pH). These relationships between occurrences and variables are defined in ecological space and then applied to the geographic landscape to produce spatially explicit models of potential distributions for the target species. The modeling efforts of Blackburn et al. (2007) provided the first quantitative estimate of the US landscape that could support B. anthracis. This study was constructed using a 50-year record of culture-positive anthrax outbreaks in the US that could be spatially identified to the nearest 1 km and combinations of either 1 km or 8 km environmental variables including mean annual temperature, annual precipitation, elevation, mean normalized difference vegetation index (NDVI), annual NDVI amplitude, soil moisture content, and soil pH. Briefly, this landscape encompasses a north–south corridor from Southwestern Texas northward into the Dakotas, where it then extends westward through Montana into the Snake River Basin. There is a westward expansion of suitable habitat from southwest Texas through New Mexico and Arizona into Nevada and California. While recent outbreaks in western Montana have validated these predictions eastward (Blackburn et al., in review), less data are available to confirm the predictions in New Mexico and Arizona. These modeling efforts confirmed that B. anthracis has an established natural ecology in the American landscape and illustrated a spatially-explicit decline in outbreaks in the eastern states associated with the collapse of imported hair, wool and hide based industries (Blackburn et al., 2007). 8. Conclusion and discussion Although in this paper the interactions between the various factors have to be presented in a relatively simple way, the reality is that they are complex, not unidirectional, and exist in a multidimensional space. For example rain impacts spore survival and movements, insect numbers, grazing and browse availability and quality, animal nutrition, health, and fertility, which goes to animal density and the probability of haematophagous insect vectors finding the next animal to feed upon. There are many ongoing research questions but selecting only four topics:

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1. Soils: There is a happenstance series of studies of spore survival in soil. These need to be done in a structured manner with known soils of different pHs and different strains of B. anthracis. The variance in survival robustness of the Kruger A group strains in a wide range of soil pH and calcium contents vs. B group survival in a narrow but higher range of pH and calcium needs to be confirmed outside of the KNP. That we do not see enzootic anthrax in regions with soils below a pH of 6.1 needs confirmation of spore non-survival in those lower pH soils. 2. Sporulation in reality in the field: Over the years we have taken soil samples from underneath countless white tailed deer carcasses in an enzootic area in West Texas and have yet to have one produce a positive culture. Other investigators have had the same negative experience. From the repeated outbreaks most summers the spores must be being successfully deposited and surviving somewhere. Hypothetically it is in a shady moist spot, maybe involving a significant loss of blood, but until we find one or more spore positive sites it is only an hypothesis. White tails are small, 41–91 kg (does) to 68– 136 kg (bucks), and yet their spore shedding carcasses can maintain enzoonicity. How? Infected bison bulls at 1140 kg have no problems, which is logical considering their bulk. 3. Tabanid haematophagous flies: Experimentally it is clear that Tabanid flies have the potential to spread anthrax. And we have seen enough examples of the Texas Paradigm to know that it is true. But someone has yet to catch flies with contaminated mouthparts at a meaningful distance from a moribund or dead animal. The prevalence of such pathogen bearing flies in an epidemic is unknown. That secondary outbreaks occur not just on neighbouring ranches but in counties up to 16–24 km away would suggest that a single fly may carry sufficient spores or vegetative cells to infect a second animal but we do not know the spore-carriage capacity of a female fly, nor its variance as a product of the species fed upon, nor by how much intervening blood meals will dilute the load. We have the theory; now we need the practicality. 4. Latent infections: The evidence, such as it is, for latent infections is limited to laboratory rodents, to cattle and pigs, and in unusual circumstances. That it may be a not uncommon event is unknown and even whether it can occur in wildlife species. Logically it could be soon explored if, for example, shot deer passing through dressing stations were sampled – retropharyngeal & mesenteric lymph nodes and sera – during the hunting season after an epidemic with controls after a normal season. But when one considers very long incubation periods in aerosol-exposed humans and Cercopithicus monkeys it does seem that maybe it is something this pathogen is hiding in plain view. There are two questions: (1) do latent B. anthracis infections exist and, if they do and (2) is there any difference in prevalence between the major strain groups, i.e. A vs. B?

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