Anthrax
Kristin A. Clothier
Anthrax is a soil-borne bacterial disease primarily affecting herbivores and most often presenting as acute septicemia with high mortality. Other animal species, including humans, can be infected.
Anthrax occurs worldwide and is noteworthy because of both its impact on agriculture and potential to cause zoonotic infections. Bacillus anthracis, the etiologic agent of anthrax, has been classified by the Centers for Disease Control and Prevention (CDC) and USDA as a Tier 1 Select Agent because of its potential to be used as a bioterrorism agent.1 Dissemination of genetically modified B. anthracis spores through the U.S. Postal Service (USPS) in 2001 demonstrated its usefulness in this capacity.2 Possession of this organism is under federal jurisdiction and is highly regulated.Etiology
B. anthracis is a member of the B. cereus group of genetically related Bacillus sp.2,3 In the vegetative form found predominantly in tissues of infected animals, B. anthracis is a large, rectangularshaped, gram-positive rod. Under nutritionally limiting circumstances it produces spores, facilitating survival in harsh environmental conditions including heat, drying, and chemical treatments.3,4 Sporulation is an oxygen-dependent process and does not occur under anaerobic conditions. Although B. anthracis was previously thought to be an obligate pathogen, recent research demonstrates that vegetative cells can survive and replicate in soil, plant rhizospheres, and amoeba in the environment, which may enhance transmission.4,5 Genetic transfer between members of this group has been demonstrated by the recent identification of B. cereus biovar anthracis, which contains molecular features of B. anthracis and B. cereus.6
Pathogenesis
Entry into mammalian hosts can occur via inhalation, ingestion, or cutaneous routes, although ingestion is most common in animal species.
Grazing herbivores are often exposed via contaminated pastures or forage grown on anthrax-positive fields. Although considered less susceptible, carnivores and omnivores can be infected by ingesting meat from infected animals or from processed food products.4,7 Once spores are introduced, they may be phagocytosed by macrophages and trafficked to regional lymph nodes or enter the circulation. After conversion to the vegetative state, bacteria replicate rapidly, lyse host cells, and escape to the extracellular environment. Plasmid-encoded virulence factors, particularly a poly- D-glutamic acid capsule encoded on pXO1 and a three-component toxin encoded on pXO2, are keys to bacterial survival in the host.2,8 Presence of the capsule enables the bacterium to resist phagocytosis and mask surface antigens, resulting in evasion of the host's immune system. Toxin production is enhanced by increases in CO2 and HCO3- present when the bacteria moves from the environment into the host.2,3 Lethal factor and edema factor are secreted proteins that, when bound to the common receptor binding component (protective antigen [PA]), form lethal toxin (LT) and edema toxin (ET.)8 Upon binding to host cells, PA oligeromizes to induce endocytosis into the host cell. In the acidic endosome, PA creates a stable pore through which LF and EF enter the cytoplasm. Both toxins interfere with cell signal transduction pathways by different mechanisms: EF increases cyclic adenosine monophosphate (cAMP) production while LF cleaves and inactivates cellular proteins. In early stages of infection, enzymatic effects of EF and LF disrupt innate immune cell functions and block inflammatory cytokine responses, particularly in neutrophils, macrophages, and dendritic cells, resulting in proliferation and dissemination of B. anthracis.2'3 As septicemia progresses, toxins affect endothelial cells resulting in edema, hemorrhagic shock with hypotension, and multiorgan failure.7,8Epidemiology
Anthrax occurs worldwide.
Frequency of outbreaks depends on geographic location with some regions of the world (Africa, Central Asia, the Middle East, South America) considered endemic for the disease. Animal outbreaks impact humans both by disrupting livelihoods and through exposure to B. anthracis-affected carcasses and products.7,9 In the United States, anthrax occurs more frequently in established “zones” of certain states (Texas, Minnesota, Montana, North and South Dakota), although sporadic cases have been reported from other locations.10 Some areas with greater incidence of disease follow old cattle trails, which may have been the primary source for contaminating those environments.11Environments favorable for maintaining anthrax spores include soils with elevated pH, high concentrations of selected cations (Ca++, Mn++), and rich in organic material.5,8,11 Droughts followed by heavy rainfall or flooding often precede outbreaks, as do earth-disturbing activities like excavations. Topography that favors collection of runoff and establishment of temporary water storage areas (seasonal ponds) may promote the concentration of spores.7,10 Outbreaks usually occur during warmer months of the year.
Transmission can occur by access to blood or tissues from infected animals. Bites from blood-sucking insects have been shown to contribute to anthrax spread during outbreaks.7 Carcasses of affected wildlife are an additional source for contaminating environments because they are unlikely to be disposed of properly and scavenging animals feeding on carcasses can disseminate organisms more widely.10,11
Clinical Presentation
Susceptibility to infection depends on the individual animal species and the route of infection.10 Ruminants (cattle, sheep, and goats) are most susceptible to peracute infections, and clinical signs before death may not be observed. If present, nonspecific clinical signs include fever, depression, respiratory distress, congestion of mucous membranes, and convulsions.
In more protracted cases, bloody diarrhea, hematuria, and localized tissue swelling may be observed. Cutaneous infections in animals are uncommon.7,10Horses may develop an acute intestinal form and present with colic, diarrhea, cyanosis, fever, and depression followed by a fatal septicemia. A more localized form, possibly initiated by insect transmission, typically involves massive edema in the neck region, which may progress to dependent edema involving the thorax, abdomen, prepuce, or mammary gland.7
Swine are less susceptible to infection. Oropharyngeal involvement with swelling of the head and neck is the most common presentation; edematous tissue may obstruct breathing and swallowing. Cervical lymph nodes are prominently involved.12 An intestinal form may present as dysentery. Pregnant animals may abort.7,10
Pathology
Animals that die of anthrax sometimes produce bloody exudates from body orifices and show incomplete rigor mortis. Blood often will not clot. Carcasses undergo rapid decomposition. If a carcass is opened, the spleen is frequently enlarged and has a “blackberry jam” consistency. Lymph nodes and internal organs may be edematous and hemorrhagic. Localized exposure may result in ulceration, regional lymph node involvement, or peripheral tissue edema.12 Histologically, large numbers of bacilli are observed in affected tissues. Spleen and lymph node architectures are obscured by massive hemorrhages. The presence of intravascular organisms is typical for septicemic anthrax.12
Diagnosis
Initially, anthrax may be confused with other conditions that cause acute death (clostridial infections, lightning strikes, toxic or plant poisoning) and may not be recognized. If anthrax is suspected, carcasses should not be opened to prevent sporulation and further contamination of the environment. Definitive diagnosis is based on microscopic examination of blood or tissue smears (preferably collected within 24 hours after death) and bacteriologic cultures performed in the laboratory, particularly for index cases.10 Samples that minimize potential for environmental contamination and maximize diagnostic accuracy include unclotted blood collected from superficial veins or ocular fluid collected in a sterile syringe using a large-gauge needle. An intact eye can also be removed and submitted to the laboratory for diagnostic evaluation.
In the more localized form of anthrax, samples from regional lymph nodes may be required. Tissue samples containing normal bacterial flora (e.g., ear, skin, intestinal content) are not recommended for testing because recovery of normal flora may obscure the presence of B. anthracis in culture. Anthrax carcasses undergo rapid putrefaction and vegetative B. anthracis cells are destroyed during decomposition, so the time between death and sample collection can substantially affect the recovery rate of B. anthracis.v°n To ensure use of specialized diagnostic and safety procedures, always notify laboratory personnel when a case of anthrax is suspected.8Gram stains performed on direct smears demonstrate typical large, gram-positive, squared-ended rods found singly or in chains of two to four cells. A polychrome methylene blue (M'Faydean) stain confirms the presence of the B. anthracis capsule surrounding organisms.8 Anthrax bacilli must be distinguished from postmortem-invading clostridial species, which have more rounded ends and are acapsular when examined with M'Faydean stain. B. anthracis also stains well with other common laboratory stains, including Wright and Giemsa.
Isolation of B. anthracis is readily made on standard sheep blood agar.8,10 Many organisms grow on this media, so cultures of samples from sterile sites are more likely to identify B. anthracis if it is present. With a doubling time of 30 to 60 minutes, colonies are apparent after as little as 6 hours of incubation.13 Typical B. anthracis colonies after 24 hours of incubation are large, gray-white in color, have a rough, dry texture, and are usually nonhemolytic.8 Gram stains of young colonies show long chains (10 or more cells) of large, square- ended, gram-positive rods often described as having the appearance of a row of “railway boxcars.”
Verification of isolates as B. anthracis is based on lack of motility, capsule production on specialized media, lysis by a specific bacteriophage (gammaphage), and confirmatory PCR for toxin and capsule genes.8,14 Gram stains from cultures incubated for approximately 48 hours show bacteria with centrally located spores, which do not cause the cells to bulge.
Cultures must be handled using at least biosafety level 2 conditions to protect laboratory personnel during confirmatory procedures.8,14 Care should be taken to prevent exposure to air and inadvertent spore dissemination from culture plates. Other available molecular- and antigen-based rapid screening tests are primarily used by first responders in potential bioterrorist events and to discriminate between vaccine and wild-type strains. Definitive diagnosis of a B. anthracis isolate must be made at a CDC Laboratory Response Network (LRN) confirmatory laboratory. All positive samples must be destroyed within 7 days of diagnosis (LRN).14Treatment, Control, and Prevention
Any suspected anthrax case should be reported to appropriate local or state veterinary regulatory officials as soon as possible. The peracute nature of many anthrax infections can make early intervention impossible and treatment unrewarding. Field isolates are generally susceptible to penicillin and tetracycline drugs.10,15 If attempted, treatment should be continued for a minimum of 5 days. B. anthracis strains resistant to these antibiotics have been reported. Antibiotic treatment will also interfere with detection of B. anthracis in laboratory specimens.13
Carcass management is critical in controlling anthrax outbreaks. Carcasses should not be opened or moved from the area where they are discovered and should be destroyed by either burning or deep burial. Burning carcasses is preferred to burial because it is more effective at destroying spores, but it may not be permitted in certain areas. Any contaminated bedding or soil should be handled similarly.8,9
In the face of an outbreak, regulatory officials will likely quarantine affected premises. All potentially exposed animals should be closely observed for signs of infection. Clinically normal animals should be vaccinated using a commercially available live acapsular vaccine (Sterne) strain (Anthrax Spore Vaccine [Colorado Serum Co., Denver, Colo.]).8,9 Prophylactic antibiotic treatment of unaffected herdmates is sometimes practiced; however, therapy will interfere with the efficacy of attenuated vaccines and should be considered carefully in outbreak management. Vaccination should be delayed for at least 7 days after antimicrobial administration.8,10 Treatment decisions should be undertaken only after consultation with local veterinary public health or regulatory officials. Vaccination of neighboring herds should be initiated to prevent new infections due to either exposure to similar predisposing environmental conditions or through spread from initial outbreak sources. Controlling insects and both avian and mammalian scavengers will aid in preventing dissemination to neighboring premises.7,10
Prophylactic vaccination of animals in endemic areas should be performed annually 4 weeks before turning animals onto pastures where outbreaks have occurred. A single dose of vaccine is given subcutaneously in the neck. In heavily contaminated areas, a second dose may be given 2 to 3 weeks later to enhance protection. Follow the vaccine manufacturer’s recommendations or local regulatory officials’ instructions regarding postvaccination market withholding time for meat and discard time for milk.9 Localized swelling may occur at the site of vaccination, particularly in horses. Goats and llamas may exhibit serious adverse reactions to the vaccine.10
Ensuring that pastures are of good quality so that grazing close to the soil is avoided will minimize exposure to spores. Avoiding rough feed that might traumatize mucosal surfaces will also decrease incidence rates. Use of lime or other calcium-containing products that alkalinize the soil should not be recommended.8 If possible, avoid grazing animals on contaminated pastures altogether and encourage alternate uses for the land.
Public Health
Naturally occurring anthrax cases in humans in the United States are rare, and when they occur, the cutaneous form is most common.11,13 Veterinarians are included among at-risk individuals because of the potential for contact with organisms while handling infected carcasses. If anthrax is suspected, avoid activities that result in bacterial contact. Do not open carcasses for necropsy to prevent sporulation and bacterial exposure. Use proper personal protective equipment, including physical barriers (e.g., gloves) and respiratory protection, when collecting diagnostic samples and during carcass disposal. Naturally acquired inhalational anthrax in humans is rare because of the relatively high LD50 (8000 to 10,000 spores) required for infection. Spores greater than 5 mm in diameter will be cleared by the mucociliary escalator. The size restriction along with static charges on spores make aerosolization and inhalation to terminal airways less likely.13 A case report from 2009 identified exposure drums made from goat hides as the likely route of human contact, indicating that animal products can be a source of infection.16 Exposure to B. anthracis should always be taken seriously, and owners of affected animals should be advised to consult with their physician for advice on medical care.