ANTHRAX
ETIOLOGY
Bacillus
anthracis is the specific cause of the disease, and pathogenic strains have plasmid-encoded
virulent factors: a poly- glutamic capsule, which aids in resistance to
phagocytosis and is encoded by virulence plasmid pX02, and a tripartite toxin comprised
of oedema (factor D lethal (factor II) and protective antigen (factor III)
encoded by plasmid pool. Both plasmids are required for full virulence and avirulent
strains occur
There is little
diversity among isolates of B. anthracis but genotyping can be used for
epidemiological studies.
The organism
forms spores that persist in the environment for decades.
EPIDEMIOLOGY
Occurrence
The disease probably originated in sub-Saharan Africa4,5 and has spread to have a worldwide distribution, although the area prevalence varies with the soil, the climate and the efforts put into suppressing its occurrence. It is often restricted to particular areas, the so-called 'anthrax belts', where it is enzootic. Currently, the characteristic epidemiology of anthrax in developed countries is the occurrence of multicentric foci of infection. Many sudden deaths occur without observed illness, in areas that have recently had appropriate climatic conditions and in which the disease has occurred as long ago as 30 years previously. In tropical and subtropical climates with high annual rainfalls, the infection persists in the soil, so frequent, serious outbreaks of anthrax are commonly encountered. In some African countries, the disease occurs every summer and reaches a devastating occurrence rate in years with heavy rainfall. Wild fauna - including hippos, cape buffalo, and elephants - die in large numbers. Predators probably act as inert carriers of the infection.5, C, Intemperate, cool climates only sporadic outbreaks derive from the soil-borne infection. Accidental ingestion of a contaminated bone meal or pasture contaminated by tannery effluent is a more common source. In this circumstance, outbreaks are few and the number of animals affected is small. The development of an effective livestock vaccine coupled with the use of penicillin and the implementation of quarantine regulations has caused a marked decline in the occurrence of anthrax in most countries compared to its historical incidence.
Source
of the infection
B. anthracis can
infect animals directly from the soil or from fodder grown on infected soil,
from a contaminated bone meal or protein concentrates, or from infected excreta, blood,
or other discharges from infected animals. The initial source is often from old anthrax
graves where the soil has been disturbed. The spread of the organism within an area may
be accomplished by streams, insects, dogs, feral pigs, and other carnivores,
and by faecal contamination from infected animals and birds. Avian scavengers
such as gulls, vultures, and ravens can carry spores over
considerable distances and the faeces of carrion-eating birds can contaminate waterholes.
Infected wildlife is also a source for domestic animals on common grazing land.
Introduction of
infection into a new area is usually through contaminated animal products such
as bone meal, fertilizers, hides, hair and wool, or by contaminated concentrates
or forage. In recent times as many as 50% of consignments of bone meal imported
into the UK are contaminated with the anthrax bacillus.
Outbreaks in pigs can usually be traced to the ingestion of infected bone meal
or carcasses. Water can be contaminated by the effluent from tanneries, infected carcasses and by flooding and the deposition of anthrax infected soil.
An outbreak of
anthrax has been recorded following the injection of infected blood for the
purpose of immunization against anaplasmosis. There have been several
reports of the occurrence of anthrax after vaccination, probably as a result of
inadequately attenuated spores. Wound infection occurs occasionally.
Transmission
of the infection
Infection gains
entrance to the body by ingestion, inhalation, or through the skin. while the
exact mode of infection is often in doubt, it is generally considered that most
animals are infected by the ingestion of contaminated food or water. Indeed, experimental transmission by this means has not always been successful.
Injury to the mucous membrane of the digestive tract will facilitate infection
but there is little doubt that infection can take place without such injury.
The increased incidence of the disease on sparse pasture is probably due both
to the ingestion of contaminated soil and to injury to the oral mucosa facilitating
invasion by the organism. Inhalation infection is thought to be of minor
importance in animals, although the possibility of infection through
contaminated dust must always be considered.
'Woolsorter's
disease' in humans is due to the inhalation of anthrax spores by workers in the
wool and hair industries, but even in these industries, cutaneous anthrax is much
more common. Biting flies, mosquitoes, ticks, and other insects have often been
found to harbour anthrax organisms and the ability of some to transmit the
infection has been demonstrated experimentally.5,8,9 However, there is little
evidence that they are important in the spread of naturally occurring diseases,
except tabanid flies The transmission is mechanical only, and a
local inflammatory reaction is evident at the site of the bite. The tendency,
in infected districts, for the heaviest incidence to occur in the late summer
and autumn may be due to the increase in the fly population at that time but an
effect of higher temperature on the vegetative proliferation of B. anthracis in the
soil is more likely.
Risk factors
Host risk factors
The disease
occurs in all vertebrates but is most common in cattle and sheep and less
frequent in goats and horses. Humans occupy an intermediate position between this
group and the relatively resistant pigs, dogs, and cats. In farm animals, the disease
is almost invariably fatal, except in pigs, and even in this species, the case fatality
rate is high. Algerian sheep are said to be resistant and, within all species,
certain individuals seem to possess sufficient immunity to resist natural
exposure. Whether or not this immunity has a genetic basis has not been
determined. The most interesting example of natural resistance is the dwarf pig,
in which it is impossible to establish the disease. Spores remain in tissues
ungerminated and there is complete clearance from all organs within 48 hours. The
ability to prevent spore germination appears to be inherited in this species.
Environment risk
factors
Outbreaks
originating from a soil-borne infection always occur after a major climate change,
for example, heavy rain after a prolonged drought, or dry summer months after
prolonged rain, and always in warm weather when the environmental temperature is
over 15°C (600P). The hypothesis that these climatic conditions lead to sporulation
and vegetative proliferation with the production of incubator areas for anthrax
in the soil appears improbable, but spores have a high buoyant density and in
wet soils could become concentrated and remain suspended in standing water with a further concentration on the soil surface as the water evaporates. This relationship
to climate has made it possible to predict 'anthrax years'. Other risk factors in the environment include
close grazing of tough, scratchy feed in dry times, which results in abrasions
of the oral mucosa, and confined grazing on heavily contaminated areas around
water holes. Some genotypes appear to persist better in calcium-rich soils and organic
soils and poorly drained soils have risk in endemic areas. Pathogen risk
factors When material containing anthrax bacilli is exposed to the air, spores
are formed that protract the infectivity of the environment for very long
periods. The spores are resistant to most external influences including the
salting of hides, normal environmental temperatures and standard disinfectants.
Anthrax bacilli have remained viable in soil stored for 60 years in a rubber- Stoppard
bottle, and field observations indicate a similar duration of viability in exposed
soil, particularly in the presence of organic matter, in untrained alkaline soil
and in a warm climate. Acid soils reduce the survival of B. anthemic.
Economic importance
In most
developed countries vaccination of susceptible animals in enzootic areas has reduced
the prevalence of the disease to negligible proportions on a national basis, but
heavy losses may still occur in individual herds. Loss occurs due to mortality
but also from withholding of milk in infected dairy herds and for a period
following vaccination.
Zoonotic potential
Anthrax has
been an important cause of fatal human illness in most parts of the world, but
in developed countries, it is no longer a significant cause of human or livestock
wastage because of appropriate control measures. However, it still holds an important
position because of its potential as a zoonosis and it is still an important zoonosis
in developing countries. It is a major concern as an agent of bioterrorism and
is listed as a category A agent by the US Centers for Disease
Control and Prevention.
An account of
an outbreak in a piggery in the UK should be compulsory reading for veterinary
students as an example of the responsibilities of veterinarians in a modern
public -health-conscious and litigation-minded community. 13,1􀄙 In developing
countries anthrax can still be a major
cause of livestock losses and a serious cause of mortality amongst humans who
eat meat from infected animals and develop the alimentary form of this disease, or
who handle infected carcasses.15 Cutaneous anthrax has occurred in veterinarians
following postmortem examination of anthrax carcasses. The areas at particular
risk for infection are the forearm above the glove line and the neck. Infection
begins as a pruritic papule or vesicle that enlarges and erodes in 1-2 days
leaving a necrotic ulcer with subsequent formation of a central black eschar.
PATHOGENESIS
Upon ingestion
of the spores, the infection may occur through the intact mucous membrane, through
defects in the epithelium around erupting teeth, or through scratches from
tough, fibrous food materials. The organisms are resistant to phagocytosis, in
part due to the presence of the poly-o-glutamic acid capsule, and proliferate
in regional draining lymph nodes, subsequently passing via the lymphatic vessels
into the bloodstream; septicemia, with a massive invasion of all body tissues, follows. B. anthracis produces a lethal
toxin that causes oedema and tissue damage, death resulting from shock and acute
renal failure and terminal anoxia. In pigs, localization occurs in the lymph nodes
of the throat after invasion through the upper part of the digestive tract.
Local lesions usually eventually lead to fatal septicemia.
CLINICAL FINDINGS
The incubation
period after field infection is not easy to determine but is probably 1-2 weeks.
Cattle and sheep
Only two forms
of the disease occur in these species, the peracute and the acute. The peracute
form of the disease is most common at the beginning of an outbreak. The animals
are usually found dead without premonitory signs, the course is probably
only 1-2 hours, but fever, muscle tremor, dyspnea, and congestion of the
mucosae may be observed. The animal soon collapses and dies after terminal convulsions.
After death, discharges of blood from the nostrils, mouth, anus, and vulva are
common. The acute form runs for about 48 hours. Severe depression and
listlessness are usually observed first, although they are sometimes preceded
by a short period of excitement. The body temperature is high, up to 42°C (107°F),
the respiration is rapid and deep, the mucosa congested and hemorrhagic, and the
heart rate is much increased. No food is taken and ruminal stasis is evident.
Pregnant cows may abort. In milking cows, the yield is very much reduced and the milk may be
bloodstained or deep yellow in colour. Alimentary tract involvement is usual and
is characterized by diarrhoea and dysentery. Local oedema of the tongue and
edematous lesions in the region of the throat, sternum, perineum, and flanks
may occur.
Horses
Anthrax in the
horse is always acute but varies in its manifestations with the mode of
infection. When infection is by ingestion there is septicemia with enteritis
and colic. When infection is by insect transmission, hot, painful, edematous,
subcutaneous swellings appear about the throat, lower neck, the floor of the thorax
and abdomen, prepuce, and mammary gland. There is high fever and severe
depression and there may be dyspnea due to swelling of the throat or colic due
to intestinal irritation. The course is usually 48-96 hours.
CLI NICAL PATHOLOGY
Haematology and
blood chemistry examinations are not conducted because of the risk of human
exposure. In the living animal, the organism may be detected in a stained smear
of peripheral blood. The reference standard for diagnosis is the detection, by
microscopic examination, of
a clearly defined meta chromatic capsule on square-ended bacilli (often in chains) in a blood smear stained with aged polychrome methylene blue. The blood should be carefully collected in a syringe to avoid contamination of the environment. When local oedema is evident smears may be made from aspirated oedema fluid or from lymph nodes that drain that area. For a more certain diagnosis, especially in the early stages when bacilli may not be present in the bloodstream in great numbers, blood culture or the injection of syringe-collected blood into guinea pigs is satisfactory. Fluorescent antibody techniques are available for use on blood smears and tissue sections. Monoclonal antibodies are also used to provide specific identification of anthrax organisms. As the carcass decomposes and the vegetative forms of B. anthemic die, diagnosis by smear is more difficult and an immunochromatographic test for protective antigen has been developed that has high specificity and does not give positive results in recently vaccinated cattle In cases where antibiotic therapy has been used, the identification from blood smears or culture may be difficult and animal passage may be necessary. Isolation of anthrax bacilli from infected soil is difficult, but a real-time quantitative PCR with reported high sensitivity has been described.
NECROPSY FINDINGS
There is a
striking absence of rigour Mortis and the carcass undergoes gaseous
decomposition, quickly assuming the characteristic 'sawhorse' posture. All-natural orifices usually exude dark, tarry blood that does not clot. If there
is a good reason to suspect the existence of anthrax the carcass should not be
opened. If necropsy is carried out, the failure of the blood to clot, widespread
ecchymoses, bloodstained serous fluid in the body cavities, severe enteritis
and splenomegaly are strong indications of the presence of anthrax. The enlarged
spleen is soft, with a consistency likened to 'blackberry jam'. Subcutaneous
swellings containing gelatinous material, and enlargement of the local lymph
nodes are features of the disease in horses and pigs. Lesions are most
frequently seen in the soft tissues of the neck and pharynx in these species. To
confirm the diagnosis on an unopened carcass, peripheral blood or local oedema
fluid should be collected by needle puncture. Since the blood clots poorly, jugular
venipuncture may permit sample collection. Smears prepared from these fluids
should be stained with polychrome methylene blue and examined (see Clinical pathology,
above). These fluid samples can also be used for bacteriological culture if
smear results are equivocal. The smears should be prepared and interpreted by an experienced and
qualified microbiologist. If the decomposition of a carcass is advanced, a small
quantity of blood may be collected from the fresh surface of an amputated tail
or ear. A portion of the spleen is the specimen of choice for bacteriological
culture if the carcass has been opened. An immunofluorescence test is available
but cross-reactivity with other Bacillus spp. makes its use impractical. An immunochromatographic
test that has high specificity for protective antigens has been developed for
use in decomposed carcasses.
The Ascoli test
can be used to demonstrate antigen in severely decayed tissue samples and a
nested PCR technique has been used to demonstrate antigen in environmental
samples PCR methods can also be used to confirm the identity of bacterial
isolates. If other detection methods
fail,
experimental animal inoculation can be attempted. Immunohistochemical detection
of the bacilli in skin biopsies of cutaneous anthrax in humans has been described.20
This technique may be useful in retrospective analyses of suspect cases if
suitable fresh tissue samples were not collected. Anthrax is a reportable
disease in many countries, requiring the involvement of government regulatory
agencies when the disease is suspected or when the diagnosis is confirmed.
Representatives of these agencies can often facilitate sample collection and
transportation to an appropriate laboratory. If anthrax is suspected, then
shipping diagnostic samples via the mail or courier systems is strongly
discouraged. Instead, samples I should be appropriately packed, labelled and transported
directly to the laboratory by one of the staff members of the veterinary
clinic. In some countries, it may be illegal to send material such as anthrax through
the mail system. Samples for confirmation of diagnosis Bacteriology - unopened
carcass: blood or oedema fluid in a sealed, leakproof container; opened carcass: above
samples plus spleen (local lymph nodes in horses, pigs) in sealed, leakproof
containers (direct smear, CULT, bioassay)
• Histology -
formalin-fixed spleenllocal lymph nodes if the carcass has been opened (LM). Note
the zoonotic potential of this organism when handling carcasses and submitting specimens.
TREATMENT
Severely ill
animals are unlikely to recover but in the early stages, particularly when fever
is detected before other signs are evident, recovery can be anticipated if the correct
treatment is provided. Penicillin (20 000 IU/kg BW twice daily) has had considerable
vogue, but streptomycin (8-10g/d in two doses intramuscularly for cattle) is
much more effective. Oxytetracycline (5 mg/kg BW per day) parenterally has also
proved superior to penicillin in the treatment of clinical cases after
vaccination in cattle and sheep. Antiserum, if available, should also be
administered for at least 5 days in doses of 100-250 mL daily but it is
expensive. In vitro studies show all isolates to be susceptible to ampicillin,
streptomycin, erythromycin, tetracycline, methicillin, and netilmicin.21 It is
desirable to prolong treatment to at least 5 days to avoid a recrudescence of
the disease.
CONTROL
The control of
meat- and milk-producing animals in infected herds in such a way as to avoid
any risk to the human population is a special aspect of the control of anthrax.
It is necessary at the same time to avoid unnecessary waste and the imposition
of unnecessarily harsh prohibitions on the farmer. When an outbreak occurs, the
placing of the farm in quarantine, the destruction of discharges and cadavers, and
the vaccination of survivors are part of the animal disease control program
and indirectly reduce human exposure. Prohibition of movement of milk and meat
from the farm during the quarantine period should prevent the entry of the
infection into the human food chain. Vaccination of animals, although the
vaccine is a live one, does not present a hazard to humans, although there is a
withholding period for meat and milk after its use. Disposal of infected
material is most important and hygiene is the biggest single factor in the
prevention of the spread of the disease. Infected carcasses should not be opened
but immediately burned in situ or buried, together with bedding and soil
contaminated by discharges. If this can not be done immediately, a liberal
application of 5% formaldehyde on the carcass and its immediate surroundings
will discourage scavengers. Burning is the preferred method of disposal. Approximately
one cord of wood ' is required to effectively incinerate the carcass of a
mature cow. Bags of charcoal briquettes have also been used Burial should be at least 2 m deep with an
ample supply of quicklime added. All suspected cases and in-contact animals must
be segregated until cases cease and for 2 weeks thereafter the affected farm must
be kept in quarantine to prevent the movement of livestock. The administration of
hyperimmune serum to in-contact animals may prevent further losses during the quarantine period, but prophylactic
administration of a single dose of long-acting tetracycline or penicillin is a
much more commoner tactic. Disinfection of premises, hides, bone meal, fertilizer,
wool, and hair requires special care. When disinfection can be carried out
immediately, before spore foundation can occur, ordinary disinfectants or heat
(60°C (1400P) for a few minutes) are sufficient to kill vegetative forms. This is
satisfactory when the necropsy room or abattoir floor is contaminated. When
spore formation occurs (i.e. within a few hours of exposure to the air),
disinfection is almost impossible by ordinary means. Strong disinfectants such
as 5% Lysol require to be in contact with spores for at least 2 days. Strong
solutions of formalin or sodium hydroxide (5-10%) are probably the most effective.
Peracetic acid (3% solution) is an effective sporicide and, applied to the soil
in appropriate amounts (8 Llm2), is an effective sterilant. Infected clothing
should be sterilized by soaking in 10% formaldehyde. Shoes may present difficulty and sterilization is most efficiently achieved by placing them in a
plastic bag and introducing ethylene oxide. Contaminated materials should be
damp and left in contact with the gas for 18 hours. Hides, wool, and mohair are
sterilized commercially by gamma-irradiation, usually from a radioactive cobalt
source. Special care must be taken to avoid human contact with infected material
and, if such contact does occur, the contaminated skin must be thoroughly disinfected.
The source of the .infection must be traced and steps are taken to prevent further
spread of the disease. Control of the disease in a feral animal population presents
major problems.
Immunization
Immunization of
animals as a control measure is extensively used and many types of vaccines are
available. Those vaccines that consist of live attenuated strains of the
organism with low virulence but capable of fonning spores have been most
successful. The sporulation character has the advantage of keeping the living vaccine
viable over long periods. These vaccines have the disadvantage that the various
animal species show varying susceptibility to the vaccine, and anthrax may
result in some cases from vaccination. This has been largely overcome by preparing
vaccines of differing degrees of virulence for use in different species and in
varying circumstances. Another method of overcoming virulence is the use of
saponin or
saturated saline solution in the vehicle to delay absorption. This is the basis
of the carbozoo vaccine.22 The Stern avirulent spore vaccine has overcome the
risk of causing anthrax by vaccination and produces a strong immunity that
lasts for at least 26 months in
sheep and 1 year
in cattle. It is the vaccine used in most countries. Although only one dose was
originally thought to be necessary, with cases ceasing about 8 days after vaccination,
it now appears that two vaccinations are necessary for some situations.7,23 A
febrile reaction does occur after vaccination; the milk yield of dairy cows
will be depressed and pregnant sows will probably abort. The injection of
penicillin and probably other antibiotics, at this time should be avoided as
it may interfere with the development of immunity. When the disease occurs for
the first time in a previously clean area, all in-contact animals should either
be treated with hyperimmune serum or be vaccinated. The measures used to
control outbreaks and the choice of a vaccine depends largely on local
legislation and experience. Ring vaccination has been used to contain outbreaks
of the disease7,24 and in enzootic areas annual revaccination of all stock is necessary.
Surface contamination of a pasture (as opposed to deep soil contamination) can
persist for 3 years and cattle grazing these pastures should be revaccinated
annually for this period In endemic areas cattle are routinely vaccinated yearly.
Milk from vaccinated cows is usually discarded for 72 hours after the injection
in case the organisms in the vaccine should be excreted in the milk. Ordinarily, the organisms of the Stern vaccine do not appear in the milk nor can they be
isolated from the blood for 10 and 7 days respectively after vaccination.
Vaccinated animals are usually withheld from slaughter for 45 days. Deaths due
to anthrax have occurred in 3-month-old llamas after vaccination with a Stern
vaccine2s and may occur in goats. Older crias and adults were unaffected. It was
assumed that the dose of the vaccine was excessive for such young animals. In’
these species two vaccinations 1 month apart with the first dose one-quarter of the standard dose can be
used.
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