The Carbon Cycle

Carbon is the fourth most abundant element in the universe, and essential to life on earth. Carbon is the element that provides the backbone of all organic molecules (see Chapter 2, Chemistry of Life). All living organisms from microorganisms, to plants and animals, contain a large number of organic compounds and therefore a large amount of carbon. Thus the carbon cycle is considered to be the primary biogeochemical cycle.

For the carbon cycle to start, autotrophic organisms are necessary, either photoautotrophs or chemoautotrophs—organisms that use carbon dioxide as their primary source of carbon. Photoautotrophs are the primary organisms in the carbon cycle and include cyanobacteria, green and purple bacteria (sulfur and nonsulfur), algae, photosynthetic protozoa, and plants. All these organisms convert carbon dioxide (CO₂) to organic molecules via carbon fixation during photosynthesis. Photoautotrophs require sunlight and for that reason are restricted to soil and water surfaces. Chemoautotrophs can also fix carbon dioxide into organic matter, but because they cannot use the energy of the sun, they need to metabolize compounds such as hydrogen sulfide for energy.

The next step in the carbon cycle involves the consumption of autotrophs by chemoheterotrophs such as animals and protozoans, which then catabolize the organic molecules for energy, resulting in the release of CO₂. Some organic molecules produced by the autotrophs may be incorporated into the tissues of heterotrophs, where they remain until that organism dies. Dead organisms are decomposed with the help of microorganisms, metabolizing organic molecules and releasing CO₂ in the process. The release of CO₂ into the atmosphere starts the cycle over again (Figure 24.2).

The Nitrogen Cycle

Nitrogen gas is the most abundant gas in the earth’s atmosphere, representing about 79% of all the gases in air. Unfortunately, the nitrogen in the atmosphere is unusable by most organisms. Only about 0.03% of the earth’s nitrogen is fixed in the form of nitrates, nitrites, ammonium ion, and organic compounds. Nitrogen is necessary for all living organisms for the synthesis of proteins, nucleic acids, and other nitrogen-containing compounds. The nitrogen cycle consists of several steps as illustrated in Figure 24.3.

Nitrogen fixation is a process by which nitrogen gas (N₂) is reduced to ammonia (NH₃), a process catalyzed by the enzyme nitrogenase. Only a limited number of prokaryotes are capable of performing this process. Nitrogenase functions only in the complete absence of oxygen, which does not present a problem for anaerobic microbes. Aerobic nitrogen fixers must protect nitrogenase from oxygen, which is done in several ways. Some of these microbes use oxygen at such a high rate that it never enters the cell and therefore does not come in contact with the enzyme. Some cyanobacteria fix nitrogen only at night, when the oxygen-producing cycle of photosynthesis does not occur; other cyanobacteria form nonphotosynthetic cells referred to as heterocysts to protect the enzyme from oxygen in the atmosphere as well as from the oxygen produced during photosynthesis. Nitrogen fixers can be free-living or symbiotic.

Free-living, nitrogen-fixing bacteria include aerobic species of Azotobacter, aerobic cyanobacteria, and anaerobic species of Bacillus and Clostridium. They are found in particularly high concentrations in the rhizosphere, a region about 2 mm in diameter around the roots of plants. As these organisms die, they release the fixed nitrogen into the upper layers of the soil, where it then becomes available to plants and to the animals that graze on the plants.

Symbiotic nitrogen-fixing bacteria live in direct symbiosis with plants. The main organism in this category is Rhizobium, which is especially adapted to leguminous plant species, on which the organism forms root nodules. Nitrogen is fixed in the nodules, the plant provides an anaerobic environment and nutrients to the bacteria, and the bacteria then provides the plant with usable nitrogen. The nitrogen fixers provide enough nitrogen to the leguminous plants so that the farmer does not have to apply nitrogen fertilizers.

Lichens, which are a symbiotic (mutualistic) combination of a fungus and an algae or cyanobacteria, also play an important role in nitrogen fixation in the forest environment. If the symbiont is a nitrogen-fixing cyanobacterium, the fixed nitrogen will eventually enrich the forest soil.

Ammonification is the process by which amino groups are converted to ammonia. Almost all nitrogen in the soil is present in organic molecules of organisms. Bacteria and fungi decompose wastes and dead organisms and break down proteins into amino acids, which are then transported into the microbial cells. Next, in the process of deamination the amino groups of amino acids are removed and converted to ammonia. In dry or alkaline soil ammonia will escape as gas; however, in moist soil it is converted to ammonium ion, which can be absorbed by bacteria and plants and used for amino acid synthesis.

Nitrification involves oxidation of nitrogen in the ammonium ion to produce nitrate (NO₃⁻). This is a two-step process involving two genera of autotrophic nitrifying bacteria, which oxidize ammonia or nitrite (NO₂⁻) to obtain energy. The first step involves the oxidation of ammonia to nitrites, performed by Nitrosomonas spp. Nitrites are toxic to plants, but in the second stage Nitrobacter oxidize nitrites into nitrates, which are soluble and can be used by plants.

Denitrification is the process by which nitrate is reduced to nitrogen (N₂) by anaerobic cellular respiration. The gas then escapes into the atmosphere. Denitrification takes place in waterlogged soils where little oxygen is available. Pseudomonas species seem to be the most prevalent group of bacteria involved in the denitrification of soil. This conversion of valuable nitrate into nitrogen gas represents a considerable economic loss in agriculture.

Anammox is the acronym for anaerobic ammonium oxidation and is a recent addition to knowledge of the nitrogen cycle. In this process, performed by anammox bacteria (previously thought to be denitrifying bacteria), nitrite and ammonium are directly converted to dinitrogen gas. This process accounts for up to 50% of the dinitrogen gas produced in the oceans. Ammonia is the major source of fixed nitrogen in the oceans and because the anammox bacteria remove ammonia efficiently from those bodies of water, these organisms limit oceanic primary productivity. On the positive side, the ability of anammox bacteria to convert ammonium and nitrite directly to dinitrogen gas provides a promising alternative to current methods of removal of nitrogen from wastewater (see Life Application: Anammox and Wastewater Treatment).

The Sulfur Cycle

Sulfur is the tenth most abundant element in the universe and is present in vitamins, proteins, and hormones. Sulfur is important for the appropriate functioning of proteins and enzymes in plants, as well as in animals that depend on plants for sulfur. Plants absorb sulfur when it is dissolved in water, and animals consume the plants for their sulfur needs. The majority of earth’s sulfur is stored underground in rocks and minerals, and as sulfate salts deep within the ocean sediments.

The sulfur cycle is similar to the nitrogen cycle, except that sulfur originates from natural sedimentary deposits. The sulfur cycle includes both atmospheric and terrestrial processes (Figure 24.4). The terrestrial process begins with the weathering of rocks, resulting in the release of stored sulfur. Subsequently, the sulfur comes in contact with air and is then converted into sulfate (SO₄). Plants and microorganisms will take up the sulfate and convert it into organic forms. Animals will consume the organic forms via food they consume, thereby moving the sulfur through the food chain. As organisms die, bacteria decompose them, releasing sulfur-containing amino acids into the environment. The sulfur released from the amino acids is converted by microorganisms to its most reduced form, hydrogen sulfide (H₂S). This process is called sulfur dissimulation. H₂S is then oxidized to elemental sulfur and then to sulfate by various microorganisms under various conditions. These organisms include nonphotosynthetic autotrophs such as Thiobacillus and Beggiatoa and photoautotrophic green and purple sulfur bacteria. The cycle starts again with the uptake of sulfate by plants and algae that animals eat. Anaerobic respiration by the bacterium Desulfovibrio reduces sulfate back to H₂S.

Sulfur can also be found in the atmosphere. A variety of natural sources emit sulfur directly into the atmosphere, including volcanic eruptions, bacterial processes, decaying organisms, and the evaporation of water. When sulfur dioxide enters the atmosphere it will react with oxygen to produce sulfur trioxide gas, or it can react with other chemicals in the atmosphere to produce sulfur salts. Furthermore, sulfur dioxide can react with water to produce sulfuric acid. All these particles will eventually settle back onto the earth, or react with rain and fall back to earth as acid deposition. The particles will then be absorbed by plants, and the sulfur cycle will start over again.

The Phosphorus Cycle

Phosphorus is an essential nutrient for plants, animals, and microorganisms. It is part of nucleic acids (DNA, RNA, ATP, and ADP) and of the plasma membranes (phospholipids). Phosphorus can be found in water, soil, and sediments. Unlike nitrogen and sulfur, phosphorus cannot be found in the atmosphere in the gaseous state. Phosphorus exists in the form of phosphate ion and undergoes little change in its oxidation state. The phosphorus cycle (Figure 24.5) involves changes of phosphorus from insoluble to soluble forms that are available for uptake by organisms, and from organic to inorganic forms by pH-dependent processes. Because no gaseous form of phosphorus exists it has the tendency to accumulate in the seas and other bodies of water.

Excess phosphorus can present a problem in a habitat. Agricultural fertilizers rich in phosphate and nitrogen are easily leached from the fields by rain. The resulting runoff into rivers and lakes can cause eutrophication, the overgrowth of microorganisms in nutrient-rich waters. The organisms involved include mainly algae and cyanobacteria. Such overgrowth, referred to as a bloom, depletes oxygen from the water, resulting in the death of aerobic organisms such as fish. Anaerobic organisms then take over, leading to increased production of H₂S and resulting in the release of foul odors.

Microbial Populations in Soil

The microbial populations in soil vary depending on the type of soil. Bacteria are the most abundant and diverse soil inhabitants; they are found in all layers and often form biofilms, especially around plant roots. Bacteria are highly adaptable to changing environments, and therefore are plentiful in most soils. Fungi are next in numbers. Both free-living and symbiotic fungi are present only in topsoil, where they can form large mycelia (mats of fibrous hyphae) that potentially cover several acres. Algae and protozoa are also present in soil but in smaller numbers than bacteria and fungi. Soil algae are photoautotrophs and therefore exist on or near the soil surface. Protozoans are mobile and move around the soil feeding on other microbes, especially bacteria. Although viruses are present within microorganisms, they generally are not found free.

Freshwater Ecosystems

About 3% of the world’s water is freshwater, 99% of which is either frozen in glaciers and pack ice, or is buried in aquifers. The remainder of the freshwater is found in lakes, ponds, rivers, and streams. Microorganisms are dispersed vertically within lake and pond systems, according to the availability of oxygen, light intensity, and temperature. Surface waters are higher in oxygen and light intensity, and warmer than deeper waters. In contrast to stagnant waters where oxygen is readily depleted, in larger lakes the wave action continuously mixes nutrients, oxygen, and organisms, resulting in efficient use of its resources. In stagnant waters, because of the depletion of oxygen, anaerobic organism will dominate and a lesser water quality will be the result. Four distinct vertical zones can be observed in lakes and ponds (Figure 24.6): a littoral zone, a limnetic zone, a profundal zone, and a benthic zone:

• The littoral zone (Figure 24.7) is the area along the shoreline with considerable rooted vegetation and good light source. This is the zone where nutrients enter the lake or pond, providing an abundance of food and promoting a high diversity of animals and bacteria in the biotope.

• The limnetic zone is the well-lit, upper layer of water away from the shore. This area is occupied by phytoplankton consisting of algae and cyanobacteria, and zooplankton, small crustaceans, and fish. Areas of the limnetic zone with sufficient oxygen supply also contain Pseudomonas spp., Cytophaga, Caulobacter, and Hyphomicrobium.

• The profundal zone is the deeper water located below the limnetic zone. It is characterized by limited light penetration (diffuse light), lower oxygen content, and lower temperature. The organisms of this zone depend on import of organic matter drifting down from the littoral and limnetic zones. This zone is inhabited mainly by primary consumers that are either attached to or crawl along the sediments at the bottom of the lake. These bottom-dwelling organisms are referred to as benthos. Purple and green sulfur bacteria can also be found in this zone.

• The benthic zone consists of the sediments at the bottom of a lake; it includes the sediment surface as well as some subsurface layers. This layer also contains benthos, which generally live in close relationship with the substrate bottom. These life forms can tolerate cool temperatures and low oxygen levels. Bacteria found in this zone include Desulfovibrio, methane-producing bacteria, and Clostridium species.

Contrary to the vertical stratification of lakes and ponds, rivers and streams lack this stratification because the organisms and nutrients are continuously swept along and mixed. Many organisms live toward the edges, where currents are low and organic material enters the water. Furthermore, biofilms can readily be found on rocks of rivers and streams.

Marine Ecosystems

Marine (saltwater) ecosystems are part of the largest aquatic system on earth, covering more than 70% of the earth’s surface. Marine ecosystems host many different species ranging from planktonic organisms that comprise the base of the marine food web, to fish, and large marine mammals. These ecosystems have some unique qualities, specifically the presence of dissolved compounds, particularly salts. As in freshwater systems the oceans can also be divided into layers, but they do have a fifth zone, the abyssal zone, which encompasses the deep ocean trenches (Figure 24.8). The majority of marine microorganisms are present in the littoral zone, where light is readily available and nutrient levels are high. The upper 200 m of ocean contains an unseen population of microbes that supports oceanic life. These are photosynthetic organisms called the marine phytoplankton. The many kinds of different bacteria among the phytoplankton are a food source for protozoa, which in turn are prey for the multicellular organisms among the zooplankton. The zooplankton is a source of food for the fish. The carbon dioxide and mineral nutrients released as by-products of the metabolic activities of bacteria, protozoa, and zooplankton are recycled into the photosynthetic phytoplankton.

The benthic abyssal zones have sparse nutrients, but they still support microbial growth, especially around hydrothermal vents. These vents release superheated, nutrient-rich water, providing nutrients and energy for thermophilic chemoautotrophic anaerobes. These organisms in turn support a variety of invertebrate and vertebrate animals.

Waterborne Diseases

Waterborne diseases that can occur after floods include typhoid fever, cholera, leptospirosis, and hepatitis A (see Chapter 1 [Scope of Microbiology] and Chapter 12 [Infections of the Gastrointestinal System]). Although flooding is associated with an increased risk of infection, the risk is low unless there is a significant population displacement and/or major contamination of the water supply. Therefore a major risk factor for outbreaks associated with floods is the contamination of drinking water facilities. The risk can be minimized once the threat has been recognized and disaster response addresses the provision of safe drinking water as a priority.

Furthermore, an increased risk for waterborne infection and disease exists through direct contact with polluted waters. Such infections include wound infections, dermatitis, conjunctivitis, and ear, nose, and throat infections. However, these conditions generally do not cause epidemics.

A disease that can reach epidemic proportions is leptospirosis, which can be transmitted directly from contaminated water. Transmission occurs by contact of the skin and mucous membranes with water, damp soil or vegetation, or mud contaminated with rodent urine. Flooding facilitates the spread of the organism because of the proliferation of rodents, which shed large amounts of leptospira in their urine.

Vector-borne Diseases

Vector-borne diseases may be indirectly increased after floods because of the increase in the number and range of vector habitats. Standing water caused by overflow of rivers and/or heavy rain are infamous breading sites for mosquitoes. An increase in the mosquito population enhances the potential for exposure of the disaster-affected population and the emergency personnel. Possible vector-borne infections and disease include dengue, malaria, and West Nile fever. Although flooding initially may flush out mosquito breeding grounds, they will be reestablishing as the floodwaters recede. Malaria epidemics after flooding are a well-known phenomenon in malaria-endemic areas worldwide.

Dead Humans and Animals

Risk posed by corpses exists only for workers who routinely handle corpses. There is no concrete evidence that corpses pose a risk of epidemics after natural disasters. However, workers may have a risk of contracting tuberculosis, blood-borne infections such as hepatitis B/C and HIV, as well as gastrointestinal infections such as rotavirus diarrhea, salmonellosis, Escherichia coli infections, typhoid/paratyphoid fevers, hepatitis A, shigellosis, and cholera.

Other Health Risks

Other health risks associated with flooding include injuries that make people more susceptible to infections. Tetanus boosters may be indicated for people who sustain open wounds. Hypothermia may also be a problem, which increases the risk of respiratory tract infections due to exposure to floodwaters and rain. Furthermore, respiratory diseases are associated with crowded conditions in temporary emergency shelters after flooding.

Bacterial Agents

Although many microorganisms can serve as biological weapons, most bacteria are easy to grow and therefore are generally most dreaded. These bacterial agents include Bacillus anthracis, Yersinia pestis, Clostridium botulinum, and Francisella tularensis.

Anthrax is caused by Bacillus anthracis, a gram-positive, spore-forming rod. It is a zoonotic disease that can cause three forms of anthrax: cutaneous, gastrointestinal, and respiratory (see section on inhalation anthrax in Chapter 11, Infections of the Respiratory System). Although all forms can lead to dangerous and potentially fatal infections, the forms that best lend themselves to use as bioweapons are the inhalation and cutaneous forms.

Inhalation anthrax is the most lethal form of anthrax. Beginning on September 18, 2001, letters containing anthrax spores were mailed to several news media offices and to two Democratic U.S. Senators (Figure 24.9). In this process, postal workers were also endangered. Among those who developed anthrax during this attack, all who presented the full symptoms of inhalation anthrax before antibiotic therapy was applied, died. The weaponized form of anthrax involved in both inhalation and cutaneous forms of the disease consists of spores that are typically maintained as a powder or are aerosolized. These spores are much more potent than those that occur naturally in the environment. The lethal dose for inhalation anthrax has now been reestimated as several hundred spores instead of earlier estimates of 2500 to 55,000.

The infection process begins when the spores are inhaled into the lungs, where they reach the alveolar spaces. Most are destroyed by alveolar macrophages (Figure 24.10). Surviving spores can move into the lymphatic system and travel to the mediastinal lymph nodes to undergo germination into the vegetative bacillus form. Once germination begins toxins are released, causing both local and systemic symptoms. These symptoms include hemorrhagic damage to the lungs and a systemic inflammatory response. The severity of the infection is proportional to the level of toxins in the blood and not the number of bacteria in the blood culture. In other words, a culture negative for the bacterium does not necessarily indicate that the danger of anthrax does not exist. The symptoms of inhalation anthrax change with progression of the disease. Early symptoms and symptoms of late stages (2 to 3 d after the onset of the early stages) of the infection are illustrated in Box 24.2.

Many symptoms of inhalation anthrax are difficult to distinguish from ordinary pneumonias or influenza. Differences that specifically point to anthrax are abnormal lung examination results, dyspnea (labored, difficult breathing), nausea or vomiting, and chest pain or pleurisy. Furthermore, anthrax typically does not present the symptoms of headache, sore throat, or rhinorrhea (runny nose). Because this form of anthrax is difficult to distinguish from other respiratory infections, a definitive diagnosis will require the use of microscopy, sample culturing, and lung radiography. Because early diagnosis is critical for the survival and recovery of the infected patient, anthrax must always be considered when presented with the symptoms previously listed.

The treatment for inhalation anthrax (which is also the treatment for cases of gastrointestinal anthrax) is usually penicillin G procaine, ciprofloxacin or doxycycline, or both. These antibiotics have proven effective if administered in the early stages of the disease. The anthrax vaccination in use in the United States is relatively new and has few clinical data in terms of its effectiveness against inhalation anthrax.

Cutaneous anthrax accounts for 95% of cases of the naturally occurring form of anthrax. Eleven of the 22 cases involved in the anthrax powder attacks after 9/11 were cutaneous forms of the disease. The infection results from spore contact with the skin, particularly in areas where there are abrasions or breaks in the skin. The mechanism of the infection is much the same as for the inhalation form, differing primarily on the tissue being affected. At the infection site, painless, pruritic, papular lesions will form within 1 week of exposure to the spores. Within 2 days fluid vesicles around the papules form, containing numerous bacilli and some white blood cells. These vesicles will enlarge and the site will become edematous (swollen with fluid). Eventually the lesions rupture and become a necrotic ulcer covered by a black eschar (Figure 24.11). Within 2 weeks the eschar will dry up and fall off. On occasion a patient may experience a secondary infection with Staphylococcus aureus if the organism is present at the lesion site. In rare cases a systemic infection may occur as a late manifestation of a cutaneous infection. This systemic infection can be manifested as bacteremia, possibly resulting in renal failure, anemia, bleeding, and ecchymoses.

Plague is caused by the bacterium Yersinia pestis, a gram-negative rod (see Chapter 14, Infections of the Circulatory System). The plague can take two forms, bubonic and pneumonic, with the latter having a higher mortality rate. One of the natural methods of transmission of the organism to humans is through the bite of infected fleas that have in turn become infected by biting infected rodents, the reservoirs of the pathogen. This infection route usually results in development of the bubonic form of plague. Another route of transmission is through the inhalation of aerosolized droplets containing the pathogen. In a bioterrorism attack using Y. pestis, the most likely means of transmission would be through aerosolization of the pathogen.

Deliberate infection of the natural animal reservoirs in densely populated areas is another possible but rather inefficient means of attempting to spread the plague. The incubation time for the Y. pestis organism is between 2 and 7 days. General symptoms for the pneumonic plague and advanced stage symptoms with sepsis are illustrated in Box 24.3.

Diagnosis of plague begins with the confirmation of clinical symptoms, followed by a Gram stain and a Wright, Giemsa, or Wayson stain, which reveals the Y. pestis “safety pin” appearance. If plague is suspected at this point, all tests should then be conducted under Biosafety Level (BSL)-2 protocols. Chest x-rays of patients with suspected pneumonic plague will likely show a bilateral pneumonia. A rapid direct fluorescent antibody (DFA) immunoassay is available that can detect in vivo antigens associated with the pathogen in a blood sample.

Specific treatment for pneumonic plague varies; however, early antibiotic therapy is necessary in all cases as any delay may increase mortality. The antibiotic of choice is streptomycin or gentamicin. Alternate antibiotics include chloramphenicol, ciprofloxacin, or doxycycline. No vaccine is presently available for the pneumonic form of plague but active research is underway. In cases of pneumonic plague, infection control, particularly in healthcare facilities, is focused primarily on precautions to isolate airborne droplets. Decontamination of surfaces exposed to the pathogen is typically unnecessary because the Y. pestis is not a particularly hardy organism and does not survive for long periods of time outside the host.

Tularemia is an infection caused by Francisella tularensis, a gram-negative, facultatively anaerobic coccobacillus. It is readily transmitted through direct contact with infected animals, ingestion of contaminated food or water, inhalation of aerosolized bacteria, and through arthropod bites such as those of ticks or mosquitoes. This organism is one of the most communicable bacterial pathogens known. The host reservoirs include rabbits, rats, and other small mammals. The most commonly occurring cases of tularemia are the result of tick bites. Tularemia is not transmitted by person-to-person contact. For use as a bioweapon the most effective method of transmission would be the use of aerosolized agents. There are six distinct manifestations of tularemia involving different symptoms and organ system targets:

The forms most likely to be encountered in a bioterrorism attack using aerosolized F. tularensis would be typhoidal, pneumonic, and oropharyngeal. Of these, the typhoidal form of tularemia would most likely be the dominant form of the disease encountered. Each of these forms has a specific set of clinical symptoms (Box 24.4). Typhoidal and pneumonic forms are the most dangerous, causing the highest mortality rates. These forms of tularemia are especially detrimental in persons with any impaired cellular immunity because this pathogen is an intracellular organism.

Diagnosis of tularemia includes microscopic examination of sputum or blood for the presence of this small coccobacillus. Standard biochemical tests are not always effective because the organism is rather fastidious and slow growing. No readily rapid test for detection is currently available. A more accurate diagnosis will require use of the polymerase chain reaction (PCR), fluorescent antibody testing, enzyme-linked immunosorbent assay (ELISA), and pulsed-field gel electrophoresis (see Chapter 25, Biotechnology) at BSL-2 or BSL-3 laboratories (see Chapter 5, Safety Issues).

The antibiotic of choice for treatment of typhoidal or pneumonic tularemia is streptomycin. Gentamicin is useful in patients with a compromised immune system. Tetracycline and chloramphenicol are alternatives that have proven to be effective. Ciprofloxacin and doxycycline are the preferred antibiotics when treating large-scale outbreaks, with oral administration being the established treatment method.

The U.S. Food and Drug Administration (FDA) is presently assessing the effectiveness of a live attenuated vaccine, but at present vaccination of the general public is not recommended. Strict isolation procedures are not necessary for infection control as tularemia is not transmitted person-to-person. However, the organism is a rather hardy pathogen and areas in contact with infected items should be disinfected. Adequate surface decontamination procedures include washing with 10% chlorine bleach for 10 minutes, followed by washing with a 70% alcohol solution.

Botulism is the result of a potent toxin produced by Clostridium botulinum, a gram-positive, spore-forming, anaerobic bacillus (Figure 24.12) (see Chapter 13, Infections of the Nervous System and the Senses). The botulinum toxin is the most potent natural neurotoxin in existence and if dispersed ideally a single gram of this toxin could kill over 1 million people! Botulism is distinct among the Category A agents in that it is the toxin and not the bacterium itself that causes the disease. The means of natural transmission of the organism include consuming contaminated food or to a lesser extent water, and wound-site contact with the organism. The bacterium cannot penetrate through intact skin but can be absorbed through the mucosal linings of the gastrointestinal or respiratory tracts.

In a bioterrorism attack the toxin or organism would most likely be transmitted in an aerosol form, or through contamination of food and water. A number of factors about the toxin limit its effectiveness as a bioweapon. The toxin can be denatured when food is properly cooked. The chlorine levels used in most water treatment plants are also sufficient to denature the toxin, rendering it harmless if used in food or water supplies. Furthermore, the fact that the cells and any aerosolized toxin can be filtered from the air by means of a couple of layers of cloth, and that the toxin itself is denatured by sunlight within 1 to 3 hours of exposure, limits its use in aerosol form. These limitations can be somewhat mitigated by using enormous quantities of the toxin but especially in the case of water supply contamination, the botulinum toxin would be an impractical bioweapon. As previously discussed in Chapter 13 (Infections of the Nervous System and Senses), the botulinum toxin causes a number of symptoms usually associated with the nervous system, with the final stage involving suffocation due to paralysis of the respiratory muscles.

Once symptoms point to botulism, the initial diagnosis involves microscopic examination of serum, stool, gastric juices, and, if possible, vomit and an examination of the patient history and circumstances before the onset of the disease. Because of the quick action of the neurotoxin, it will be impossible to conduct the necessary testing to positively confirm botulism in a laboratory setting. In treating botulism, as soon as the signs and symptoms become convincing, an antitoxin should be administered. The effectiveness of this antitoxin in the case of an aerosol bioterrorism attack is not known because the antitoxin has not yet been tested on inhalation botulism. Even after administration of the antitoxin after the onset of observable symptoms, the patient will still suffer respiratory failure. The antitoxin will prevent only the progression of toxic effects; it will not reverse symptoms that are already present. The antitoxin will be life-saving only if the patient is assisted by mechanical respiration. Improvements in advanced life support and the development of the antitoxin have greatly improved the survival chances of patients with botulism. Long-term problems associated with botulism after recovery are unknown at this time, but survivors often do complain of chronic fatigue and difficult or labored breathing.

Viral Agents

Viruses are true parasites that cannot live on their own; they need a host cell to reproduce and then cause disease (see Chapter 7, Viruses). Although viruses cannot be grown on a plate or in a test tube, their genomes are generally smaller than bacterial genomes, making them easier to manipulate (see Chapter 25, Biotechnology). Because of the available technology, an increasing commercial exploitation of genetic engineering in both agriculture and medicine has occurred. This trend also may have the potential to create viruses and bacteria that are more virulent than nature’s worst case. The production of synthetic viruses with the ability to multiply by the millions represents a bioterrorism threat as great as that with natural occurring viruses, or maybe even more.

In 1980 the World Health Organization (WHO, Geneva, Switzerland) declared the smallpox virus to be eradicated as a result of a global vaccination program (see Chapter 7, Why You Need to Know and Life Application: Smallpox as Biological Weapon). The smallpox virus is usually inhaled, then taken up by macrophages and transported to lymph nodes where the virus multiplies. This viral increase activates T and B cells and produces a host antibody response (see Chapter 20, The Immune System). Within about 4 days viremia develops and the virus spreads to the spleen, bone marrow, and other lymph nodes. As the infection proceeds the virus infects the dermis and oropharynx, causing the classic skin lesions associated with smallpox. The lesions in the oropharyngeal region form quickly, resulting in the saliva of this region containing large amounts of the virus. Transmission readily occurs through contact with skin lesions before they scab over, or via aerosolized droplets from the oropharynx. Infection can also occur through contact with objects contaminated with the virus including clothes, bedding, and body fluids such as urine, sweat, or sputum.

Symptoms of a smallpox infection include the following:

Diagnosis involves presentation of the symptoms, followed by PCR testing for the definitive diagnosis. Many of the symptoms closely resemble those of chickenpox, produced by the varicella-zoster virus. The main distinguishing features are that the smallpox rash rapidly develops to maturity (1–2 d), whereas the chickenpox rash develops over several days with new lesions appearing as the infection progresses. Furthermore, the lesions of smallpox tend to be deep and concentrated on the face and extremities, whereas chickenpox lesions are superficial and concentrated on the trunk.

At present no chemical agent is approved by the FDA to treat smallpox. Although cidofovir, an antiretroviral agent, has shown some activity against smallpox in the laboratory, including animal studies, there are no human clinical data to support its use in human disease. Prevention through vaccination is still effective and there are ongoing discussions on a potential plan for implementing a large-scale vaccination program. The vaccine is 95% effective and recovery from smallpox confers a lifelong immunity to the disease. Some negative side effects to vaccination include local pain, swelling, and a low-grade fever.

Healthcare workers coming in contact with infected patients should use proper protective equipment such as high-efficiency particulate air (HEPA) filter masks, gowns, gloves, and face shields.

At this time, no naturally occurring smallpox have been reported. There are fears that the virus has been sold on the black market as a result of security problems encountered with the dissolution of the former Soviet Union. Other than laboratory personnel and research scientists, the greatest exposure risk for the general population would be by deliberate release.

Viral hemorrhagic fevers (VHFs) are caused by four families of RNA viruses:

With the exception of the filoviruses, all these viruses are zoonotic. The pathogenic mechanism of VHFs is not fully understood; however, all the families display similar symptoms, suggesting similar mechanisms. Several mechanisms including platelet deficiency, direct endothelial and platelet injury, and cytokine dysregulation have been identified and vary somewhat depending on the family. These mechanisms account for the significant bleeding common to all four viral families. Because of the high mortality rate and dangers inherent in working with these viruses, testing for VHFs must be done at a BSL-4 facility (see Chapter 5, Safety Issues).

Filoviridae: Ebola and Marburg viruses can be naturally transmitted in a number of ways including aerosolized animal feces, arthropod bites, contact with contaminated animal carcasses, and person-to-person contact with the skin or oral aerosols of an infected person. As a bioweapon the transmission could be through an aerosol or direct contact with a carrier. Incubation time for Ebola virus is 2 to 21 days, and for Marburg virus it is 2 to 14 days. Both infections include the following symptoms:

When shock occurs, multiorgan system failure and hemorrhagic complications set in and death follows soon thereafter.

Diagnosis of a specific viral infection is difficult. Healthcare professionals must often rely on patient history and clinical symptoms to determine the possibility of VHF. Nasal, stool, serum, or almost any body fluid of an infected individual will contain the virus and should be sent to a designated BSL-4 facility for definitive diagnosis. These laboratories will then use ELISA, PCR, and viral isolation to confirm a preliminary diagnosis. Through electron microscopy the presence of specific viruses can also be observed. Ebola virus is easily identified by its unique “shepherd’s hook” shape (Figure 24.13).

For both Ebola and Marburg viruses no drugs are available for treatment. Supportive management is the only response available, which may include actions such as fluid resuscitation, balancing of electrolytes, dialysis, or mechanical ventilation. Because this type of care will require a patient being placed in a critical care unit, the infection control procedures necessary could lead to significant challenges for the treatment facility regarding transmission management. Because these viruses can be transmitted through aerosols and contact, special precautions such as negative-pressure rooms, strict contact precautions, and patient isolation will be required. At present there are no vaccines for Ebola and Marburg viruses. Mortality rates for filovirus infections are high, ranging from 25% to 70% for the Marburg virus and from 50% to 90% for the Ebola virus.

Arenaviruses: Lassa and Machupo viruses are naturally transmitted through aerosols from infected rodent waste, direct contact via mucous membranes or open skin with the virus, direct person-to-person contact with infected body fluid, and possibly through airborne droplets. As with Ebola and Marburg viruses the most likely method for transmission as a bioweapon would be through person-to-person contact with an infected carrier and possibly through an aerosol. The incubation time for Lassa virus is 5 to 16 days, and for Machupo virus it is 7 to 14 days. Symptoms for these viruses are as follows:

Specific symptoms for Lassa infections include edema of the head and neck, and pleural and pericardial effusions; specific symptoms for Machupo infections include CNS problems, and local and generalized seizures. Diagnosis of these infections is the same as for all VHFs: initial diagnosis based on history and symptoms and final diagnosis by a designated BSL-4 laboratory through ELISA, PCR, and viral isolation. Lassa virus infection is also distinguished by an elevated white blood cell count.

As with patients infected with Ebola or Marburg virus, the main treatment is supportive care. In the case of an arenavirus infection, if the drug ribavirin is administered just before or just after the infection occurs, the drug appears to be effective by limiting viremia and subsequent liver damage. Mortality rates for arenavirus infections range from 15% to 30%.

Bunyaviridae: Rift Valley and Congo-Crimean fever viruses are transmitted naturally by infected mosquitoes, by inhalation of aerosol from an infected animal, and by direct physical contact with an infected carcass or animal tissue. The potential for infection through the consumption of infected animal milk also exists. At the present time there is no evidence that the viruses can be transmitted by person-to-person contact. As a bioweapon, dissemination would most likely be via an aerosol. The incubation time for these viruses is 2 to 6 days. Symptoms of a Bunyaviridae infection include the following:

Diagnosis for this virus family is the same as for the other VHF families. Treatment involves supportive care and ribavirin, and the mortality rate is low (<1%).

Flaviviridae: Yellow fever and dengue fever viruses are naturally transmitted by mosquito or tick bites. No person-to-person transmission has yet been reported. The incubation time for these viruses ranges from 2 to 9 days. Symptoms of infection include the following:

Diagnosis is accomplished by the same methods as for the other VHF families. Treatment for flavivirus infections is strictly supportive and the mortality rate ranges from less than 10% to 20%.