bacillis anthracis

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Review

The ecology of Bacillus anthracis

Martin Hugh-Jones a,*, Jason Blackburn b

a Department of Environmental Science, School of the Coast and Environment, Louisiana State University, Baton Rouge, LA 70803-5705, USAb 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 calciumlevels 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.

2009 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

2. Soils. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

2.1. Soil germination and vegetative cycles . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 358

2.2. Rain, up & down, and sideways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

3. Infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

3.1. Index case infection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 359

3.2. Climate/hot-dry. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

3.3. Pathogen genomics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

4. Insects. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 360

4.1. Blow flies: case multipliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

4.2. Biting flies: space multipliers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 361

4.3. Ticks, flies and mosquitoes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

5. Host death . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

5.1. Mortality . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 362

5.2. Sporulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

6. Carcass . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 363

7. Landscape ecology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

8. Conclusion and discussion. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 364

0098-2997/$ - see front matter 2009 Elsevier Ltd. All rights reserved.doi:10.1016/j.mam.2009.08.003

* Corresponding author.

E-mail address: [email protected] (M. Hugh-Jones).

Molecular Aspects of Medicine 30 (2009) 356367

Contents lists available at ScienceDirect

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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 com-prehensive 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 path-

ogen 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 com-

mented 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 Nesss 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 environ-

mental variables (Blackburn et al., 2007). Russian contemporaries of Van Ness reported the same patchy distribution of an-

thrax 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 Cen-

tral Asia though the present incidence is much reduced (Cherkasski, 2002; Kasianenko et al., 1984; Kolonin 1969). These Rus-

sian 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 val-

ues (Smith et al., 1999).

We have noted (Hugh-Jones, unpublished data) that depressions or pot-holes,$0.20.3 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

Renies 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


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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. Lat-

terly 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 sec-

ondary 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 100250 spores

parenterally administered consistently killed kudu in the Kruger National Park, the oral LD50 with same strain was approx-

imately 15 million spores. In healthy unstressed sheep, horses and cattle the lethal oral dose is of the order of 1.55 108

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 con-

taminated 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 unap-

preciated. 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 spo-

radic summer deaths had been put down to hemlock poisoning and catarrhal fever. Also elk (Alces alces) died on the moun-

tain 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, nuz-

zling, 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. Dor-

mant 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 germinat-

ing 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 resis-

tance 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 di-

rectly 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 nutri-

tional value during the dry season and increasingly abrasive (Starr, 1988). The end result is to markedly reduce the necessaryID50 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 per-

iod 21 were B1 (genotype 87), 74 A3 (genotype 67), and 1 A3 genotype 39 and 2 A3 genotype 45, with B1 concentrated in thenorth 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 exam-

ple in a 2004 epidemic in Basilicata affecting 41 farms and 124 animals cattle, sheep, goats, horses and deer the 52 iso-

lates 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 frag-

ment), 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 sea-son. 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 hab-

itats 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 fromMusca 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 adjoin-

ing 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 an-

thrax 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 Turn-

bull, 2004). The geometry of dispersal is such that the browsing risk decreases exponentially and is very soon only theoret-

ical. 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, facil-

itating 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 dan-

gerous 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 dem-

onstrated 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 197879 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 wind-

swept 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 dis-

tant risk empirical data repeatedly has secondary outbreaks 510 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 ani-

mals 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 devel-

oped 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 max-

imum 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 Da-

kota 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 910 months the spring rains will not affect the num-

bers 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 appro-

priate control response. For cattle it is usually some 510 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 sig-

nificant antibody titres (Turnbull et al., 2001). This widespread subclinical exposure will explain why epidemics two sum-

mers 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 den-

sity. 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 sepa-

rately fed on incisions in the sides of goats dead from anthrax and then transferred to cauterized skin of healthy goats; infec-

tion 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

andAedes taeniorhynchus. The success rate between ingestion of bacteraemic blood and target guinea pig exposure was only12% 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 infec-

tion 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 anti-

gen 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 white-

tailed deer, which would reinforce the conclusion that they are hypersensitive to the toxins, and die with low titres and bac-

teraemias. 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 spec-

ulation 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 organ-

isms, 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 unex-

pectedly 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 (Linde-

que 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 white-

tailed 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 Turn-bull 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 at650% R.H. only 7/20 (35%) cultures had sporulated by 34 h. But at 26 C sporulation was de-

layed and took 28 h at 10090% R.H. and 60 h at 2050% 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) with-

in 1724 h, whereas at 15.6 and 21.1 C the bacilli gradually disintegrated with the growth of contaminants; in blood re-

moved 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 17.823.3 C, but the fre-

quency 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 Ter-

ritories, 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 con-

sumed elsewhere. If the carcass is not opened the anaerobic decomposition and acidification will kill the contained vegeta-

tive 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 110 spores/g soil, 29% 11100 spores/g, 25% 1011000 spores/g,

10% 100110,000 spores/g, 7% 10,001100,000 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 usu-

ally in the range of 3 months to 34 years, and rarely longer ( Sterne, 1959).

Spores will survive passage through the scavenger intestinal tract but vegetative cells will not; for example we were un-

able to find any positive turkey vulture (Cathartes aura) faeces (n = 20) under a roost in the middle of a significant distribu-

tion 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 11.5) 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 1568 h (Lindeque and Turnbull, 1994). The spore

load from an individual bird might be slight (Turnbull et al., 2008b) but as $50185 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 contam-

ination 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 sci-

ence (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 out-

breaks 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 geo-

graphic 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 northsouth corridor from South-

western 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 Nevadaand 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 ofB. 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 cal-

cium 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, 4191 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 con-

taminated mouthparts at a meaningful distance from a moribund or dead animal. The prevalence of such pathogen bear-

ing flies in an epidemic is unknown. That secondary outbreaks occur not just on neighbouring ranches but in counties up

to 1624 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 spe-

cies. Logically it could be soon explored if, for example, shot deer passing through dressing stations were sampled ret-

ropharyngeal & 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 mon-

keys 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?

Acknowledgements

We are very thankful for the help of many colleagues but especially Dan Dragon, Phil Hanna, and Peter Turnbull for their

shared insights, experience, and critical comments in assembling this review; also Denise Westphal, whose research librar-

ian skills were invaluable and never ceased to amaze.

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