Chapter 2 The development of techniques for examining the nucleic acids of microorganisms has seen major changes in which we classify and group related microorganisms. This is especially true for the study of ribosomal ribonucleic acid (rRNA), which is a highly conserved sequence involved in the synthesis of proteins in all living organisms. Based on this analysis, the classification of living things has been placed into a three-domain system consisting of Archaea, Eukarya, and Bacteria. Of these, the Bacteria and Archaea are termed prokaryotes (no organized cell nucleus), and the Eukarya are known as eukaryotes (an organized cell nucleus). Eukaryotic microbes other than algae and fungi are collectively called protists. Within the Eukarya are fungi, protozoa, helminth worms, algae, plants, animals, and humans. Archaea are microbes that look somewhat similar to bacteria in size and shape under the light microscope, but they are actually genetically and biochemically quite different. They appear to be a simpler form of life and may in fact be the oldest form of life on earth. Viruses are obligate intercellular parasites and are not a member of any group. They are usually composed only of a nucleic acid surrounded by a protein coat. Eukaryotes are much more complex than prokaryotic cells in structure and nutritional requirements. Prokaryotes divide by simple binary fission, whereas eukaryotes divide by a more complex process called mitosis. Fungi, protozoa, and helminth worms are all eukaryotes. All contain members that are pathogenic to man and animals. While algae do not infect humans, they can produce toxins that are harmful to animals and man. Fungi obtain nutrients solely by adsorption of organic matter from dead organisms. Even when they invade living tissues, they typically kill cells and then adsorb the nutrients from them. Some fungi are capable of developing environmentally resistant spores, which can be transported through the air. Fungal diseases in humans are referred to as mycoses. Airborne fungal spores are responsible for some allergies in humans. Yeasts are classified with the fungi and some are pathogenic to humans (Candida albicans). Certain fungi produce toxins (i.e., aflatoxins) when growing in foods that are mutagenic and carcinogenic in animals. Inhalation is an important route of transmission for some fungal diseases, such as aspergillosis, blastomycosis, coccidioidomycosis, and histoplasmosis. These fungi are found growing in animal feces, soil, or decaying organic matter (compost, manure) and may be transmitted through the air when this matter is disturbed. Some fungi may be transmitted by direct contact with the skin. Fungi are often found in drinking water distribution systems, and they have been linked to infections in immunocompromised individuals [1]. Food is not believed to be a significant route of transmission of pathogenic fungi. Protozoans are unicellular organisms that are surrounded by a cytoplasmic membrane that is covered by a protective structure called a pellicle. Most are free living feeding off of bacteria. Some of these can cause disease, while others are obligate parasites. They are capable of forming structures called cysts, oocysts, or spores (depending on the life cycle of the organism) resistant to adverse environmental conditions such as desiccation, starvation, high temperatures, and disinfectants. Some pathogenic protozoa are found mainly in the soil (Naegleria) or water (Acanthamoeba). Others, such a Giardia and Cryptosporidium, whose natural habitat is the intestines of warm-blooded animals, are capable of causing disease in both humans and animals. Others, such as Entamoeba histolytica, are only capable of causing disease in humans. Pathogenic enteric (replicate in the intestines) protozoans are usually excreted in the feces or urine and can be transmitted by fecally contaminated food and water. Some protozoa may be transmitted through the air (Acanthamoeba) or through fomites (inanimate objects). Some important environmentally transmitted protozoa are listed in Table 2.1. The clinically important protozoa are divided taxonomically into the amoebas (Entamoeba spp.), flagellates (Giardia), and coccidian, meaning “globose in shape” (Cryptosporidium and Cyclospora). Microsporidia are also capable of causing intestinal infections, but their classification is uncertain, because they have many characteristics associated with fungi. They are all obligate parasites and are transmitted via infectious cysts, oocysts, or spores by the fecal–oral route. The life cycles of these protozoa are similar. The initial stage occurs during ingestion of the cyst, oocyst, or spore. After ingestion, the body temperature and passage through the stomach cause these infective stages to open up in a process called excystation. Table 2.1 Some Characteristics of Environmentally Transmitted Parasites The Giardia cysts house two trophozoites, which is the stage that attaches to intestinal cells and grows in the intestinal tract through asexual reproduction. As the trophozoites grow and begin to cover the wall of the intestinal tract, diarrhea can result. As the trophozoites are released, some form cysts as they pass through the bowels. Cryptosporidium and Cyclospora both produce oocysts. The life cycle of Cyclospora is still not well known, except that its oocysts require some period of time in the environment before they are infective. Cryptosporidium oocysts contain four sporozoites, which enter the intestinal cells and begin the infection process. As more cells become infected, the illness begins and oocysts are excreted in high numbers in the feces (about 100,000/g). Worms and helminths are small multicellular animals that are parasitic to humans and animals. They include flukes, tapeworms, and roundworms (Ascaris lumbricoides). The ova (eggs) are excreted in the feces of an infected animal or human and spread by wastewater, soil, or food. Some major environmentally transmitted helminths are listed in Table 2.1. Bacteria occur in three basic shapes—spherical (cocci), rod (bacilli), and spirilla (corkscrews)—and generally range in size from 0.3 to 2 µm. The Gram stain is used to classify most bacteria into Gram-positive bacteria that stain purple and the Gram-negative bacteria that stain pink. Gram-negative bacteria have a lipid–polysaccharide in their cell wall, which is responsible for fever production during infection. Many pathogenic bacteria are capable of producing a wide range of toxins that may be responsible for the symptoms associated with the disease they cause. Some forms of bacteria produce spores (Bacillus, Clostridium) that are very heat and disinfectant resistant and are capable of surviving for prolonged periods of time in the environment (years). Some of the most important environmentally transmitted bacteria are shown in Table 2.2. Table 2.2 Selected Bacteria of Medical Importance Transmitted through the Environment Many human pathogenic bacteria infect both animals and humans and are capable of growth in the environment outside the host organism. Examples are common among the enteric bacteria such as Salmonella, which causes diarrhea in chickens, cattle, and humans. It is also cable of growing to large numbers in contaminated food (i.e., eggs, meat). In contrast, some human enteric pathogens only infect humans (e.g., Shigella). In some cases, only certain strains of common enteric bacteria produce illness. Escherichia coli is a common inhabitant of all warm-blooded animals, but only certain strains are capable of producing illness in humans (E. coli O157:H7). Most human enteric bacterial pathogens do not survive long in water or soil. There are exceptions such as Vibrio cholerae, whose natural habitat is the marine environment. It is introduced into the human population through seafood and under conditions of poor sanitation is readily spread by the water route. Rickettsia and chlamydia are bacteria that are obligate intracellular parasites. They often infect both animals and arthropods (ticks and lice). They can be transmitted to humans by ticks or food products such milk from an infected cow. They are associated with illnesses such as Q fever and Rocky Mountain spotted fever. Mycoplasmas are small bacteria (0.3–0.8 µm) that are pleomorphic in shape and lack a cell wall like most bacteria. Some are parasitic to plants and animals, while others live on decaying organic matter. Mycoplasma pneumonia is a leading cause of pneumonia among college students and is common among military recruits. It does not spread quickly among populations, and contact with droplets generated by sneezing and coughing may be the primary route of transmission [2]. Opportunistic pathogens are organisms (usually bacteria and fungi) that ordinarily do not cause disease in their normal habitat in normal healthy persons [3]. For example, microbes that gain access to the broken skin can cause opportunistic infections. Opportunistic infections are, however, of greatest concern in persons already weakened (e.g., severe burns on the skin, diabetes, cystic fibrosis, existing infection, etc.) or those that have an impaired immune system. Opportunistic infections are the greatest concern in hospitals (nosocomial infections), where there is a risk of serious illness that may be life threatening because of the weakened condition of the patient due to preexisting illness. Many types of organisms in our environment have the ability to cause opportunistic infections, particularly in seriously debilitated persons. In hospitals, the situation can be particularly acute because of the use of antibiotics that selects for bacteria that are resistant to many common antibiotics. Thus, bacteria like Staphylococus aureus, Clostridium difficile, enterococcus, and enteric bacteria have become major problems in health care facilities. Sources of opportunistic infections include patients and their contacts, medical devices, water, food, and other commonly touched surfaces. Water is now well documented as a source of Mycobacterium avium, Pseudomonas aeruginosa, Legionella pneumophila, and fungal infections within health care facilities [1, 4]. Viruses infecting humans are usually 200 nm in size or smaller and cannot be seen with a light microscope. Viruses are obligate intracellular parasites; they only replicate inside a living host. Whereas cells contain both RNA and DNA and both grow and divide, viruses contain only RNA or DNA, do not grow, and never divide. Viruses multiply by directing syntheses and assembly of viral components inside cells to form new viruses. When not in a host cell, a virus is an inert macromolecule (i.e., nucleic acid and proteins). The major components of viruses are a central core nucleic acid and a protein coat or capsid. Certain viruses contain enzymes, and some have a lipid coat. Viruses die or become inactivated (no longer cable of replicating in a host cell) by damage to the structural integrity of the protein and nucleic acid, including the lipid envelope. A complete virus particle, including the lipid envelope if it has one, is called a virion. The most widely used taxonomic criteria for animal viruses are based on four characteristics: Beyond these physical characteristics, other criteria such as immunological typing and effects on host cells are used. Based on these criteria, animal viruses are divided into a number of families whose names end in “-viridae,” for example, Picornaviridae. Each family contains numerous genera and species names ending in “-virus,” for example, rotavirus. The viruses may be further divided into types based on serology or genotyping. The characteristics of the major groups of animal viruses are shown in Table 2.3. Table 2.3 Classification of Animal Viruses Viruses are fairly host specific: for example, many humans and primates are the only hosts for many viruses (e.g., norovirus). Other viruses, such as foot-and-mouth disease, infect both cattle and humans. Other viruses, such as togaviruses, are capable of infecting arthropods, which in turn bite humans, thus transmitting several kinds of encephalitis and tropical fevers such as dengue and yellow fever. Genera of the same virus may infect both animals and humans but seldom, if ever, cross species barriers. For example, rotaviruses are a common cause of diarrhea in almost all infant animals, including humans. However, the rotavirus types that infect animals usually do not infect humans in the natural environment. Other viruses, such as reoviruses, may cause a serious illness in one species (e.g., chickens) and infect humans without definable or reproducible ill effects. The virus may infect the host, but no apparent illness is observed. Viruses that infect bacteria are known as bacteriophages or simply phages. Almost all groups of bacteria have phages. Phages that infect coliform bacteria are called coliphages. Coliphages have been used extensively as models (surrogates) for studies of virus transport and behavior in the environment because they are inexpensive to grow and assay compared to animal viruses. Also, they can be grown in much higher numbers and are not infectious to humans. The most environmentally significant viruses are the enteric viruses, transmitted by the fecal–oral route, and the respiratory viruses, transmitted by inhalation or contact of the virus with the nose, eye, or mouth. There are now more than 200 known enteric viruses, with new types being recognized every year. They cause a wide range of illnesses (Table 2.4). Some may be transmitted by both fecal matter and aerosols (Coxsackievirus B3). A large number of respiratory viruses are also known to exist (Table 2.5). Table 2.4 Some Human Enteric Viruses Source: From Ref. [5]. Table 2.5 Some Respiratory Viruses A group of exceedingly small infectious agents known as prions are responsible for neurological diseases in humans and animals [7]. Prions are resistant to enzymes that digest nucleic acids, cooking, and normal autoclaving. Several animal diseases fall into this category, including mad cow disease. All are neurological diseases called spongiform encephalopathies because of the formation of large vacuoles that develop in the brain. The human diseases are kuru, Creutzfeldt–Jakob disease (CJD), and fatal insomnia. They are transmitted by ingestion of infected food. Kuru was discovered to be transmitted among cannibals in New Guinea. An association with CJD and consumption of certain foods (e.g., sheep, oxen, cattle, and hog brains) has been observed. The epidemic of bovine spongiform encephalopathy (BSE) or mad cow disease was caused by the feeding of scrapie (a prion infection)-infected sheep meat to cattle [8]. This resulted in an outbreak of a new variant of CJD in humans in the United Kingdom from consumption of BSE-infected meat [9]. These diseases are characterized by a progressive dementia, leading to death. The course of the illness may take many years or decades to develop. Although humans are continually exposed to a vast array of microorganisms in the environment, only a small proportion are capable of causing disease in the host in such a manner to cause infection and disease. Infection is the process in which a microorganism multiplies or grows in or on a host. Infection does not necessarily result in disease. In the case of most enteric infections, usually only half of those infected develop clinical disease. In the case of measles virus, almost 100% of those infected develop the disease. A person who is infected but does not develop clinical signs of disease is referred to as asymptomatic (Fig. 2.1). Although not ill, these persons can still excrete or secrete large numbers of pathogens into the environment. The ability to produce symptomatic disease (a clinical illness) is related to a number of factors, such as the type and strain of microorganism and the age of the host. For example, hepatitis A infections are generally asymptomatic in small children (<10% are symptomatic), while 75% or more of the adults are symptomatic. Only a small proportion of all common infectious diseases get reported because not everyone seeks medical attention and clinical tests are often performed or not required to be reported to health agencies (Fig. 2.2). Figure 2.1 Outcomes of exposure to a microbial infection. Figure 2.2 Detection or notification of Cryptosporidium cases represents only a small proportion of all cases. Although infection usually precedes the causation of disease, some microbes can produce toxins in the environment, and when exposure occurs through ingestion or other routes, illness can result. Examples are Clostridium perfringens and S. aureus, which produce toxins when growing in food. Ingestion of food results in diarrhea due to the toxin in the food, not the growth of the organism in the host. The capacity of a microorganism to cause disease is determined by the production of a variety of virulence factors possessed by the infecting microorganism. Virulence is the degree of intensity of the disease produced by a pathogen. Factors that determine virulence include the ability of the organism to adhere to certain cells within the body, to produce toxins, to penetrate living cells, to produce substances that attack the host immune system, and to produce enzymes that help the organism spread through tissues. Since virulence factors are controlled by genetic traits, they vary between types and strains of microorganisms. Thus, the virulence of influenza may vary from one season to the next, depending on the genetic characteristics that govern virulence. A number of host factors may also influence the outcome of infection and the severity of disease. These are listed in Table 2.6. Generally, infections are more severe in the elderly, the very young, the immunocompromised, and those with poor nutrition. Table 2.6 Host Factors That Can Influence Susceptibility and Severity of Disease To cause illness, the pathogen must first grow in the host. The time between infection and appearance of clinical signs and symptoms (diarrhea, fever, rash, etc.) is the incubation time (Table 2.7). This time may be as short as 6–12 h in the cases of norovirus gastroenteritis to 30–60 days in the case of hepatitis A. At any time during infection, the pathogen may be excreted into the environment by the host in the feces, urine, respiratory secretions, and so on. Although maximum release may occur during the height of the clinical illness, it may also precede the first signs of clinical illness. In the case of hepatitis A virus (HAV), maximum excretion in the feces occurs before the onset of illness. The concentration of organisms released into the environment varies with the type of organism and the route of transmission. The concentration of enteric viruses during diarrhea may be as high as 1010–1012/g (Table 2.8). Usually, lower concentrations of pathogens are found in nasal and respiratory secretions. Super shedders have also been documented for cattle infected with E. coli O157:H7, where certain infected individuals excrete very large numbers of a pathogen over a prolonged period of time [10]. It has been suggested that these super shedders play a significant role in the spread of disease among populations and contamination of food supplies [10]. Table 2.7 Incubation Time for Common Enteric Pathogens Table 2.8 Concentration of Enteric Pathogens in Feces Diseases are often described by their signs or symptoms. A sign is a characteristic of a disease that can be observed by examining a patient. A symptom is a characteristic of a disease that can be observed or felt only by the patient. Signs of disease include swelling, redness, coughing, runny nose, diarrhea, and fever. Symptoms include such things as pain, nausea, and malaise. Even after apparent recovery of some diseases, aftereffects called sequela can develop. For example, some bacterial infections can cause reactive arthritis following diarrhea. An acute infection or disease develops rapidly and runs its course quickly. Gastroenteritis and common colds are examples of acute disease. A chronic disease develops more slowly and persists for a longer period of time. Tuberculosis is an example of a chronic disease. The picornaviruses are single-strained RNA viruses and include several groups of important viruses transmitted by air, water, and food. These include the enteroviruses, hepatitis A, and foot-and-mouth disease. The enteroviruses are perhaps the most diverse in terms of the wide range of clinical disease they cause. Diseases caused by enteroviruses range from severe paralysis (paralytic poliomyelitis) to aseptic meningitis, hepatitis, myocarditis, skin rashes, and the common cold. The initial site of enterovirus replication is usually the intestinal tract, although it may quickly spread to other organs, particularly nerve tissue (Fig. 2.3). Fortunately, serious outcomes represent only a small proportion of all infections. The best-known members of this group are the polioviruses, which before the development of a vaccine in the 1950s were considered a major public health problem because poliomyelitis is a crippling disease. Widespread use of the vaccine has resulted in the eradication of the disease from most of the world. Figure 2.3 Infections of enteric viruses. Enteroviruses represent a group now of more than 100 types including polio, Coxsackie, echo, and parechovirus [11]. Enteroviruses are divided into five groups (Table 2.9). Because enteroviruses were the first human viruses easily grown in cell culture, a great deal has been learned about their epidemiology and occurrence in the environment. Infections are most common in childhood. Isolation of echo- and Coxsackieviruses from stool of children may be as high as 8–10% during the summer months [12]. The fecal–oral route is believed to be the main route of transmission, although respiratory transmission may also be significant for some types [13]. It is believed that almost all enteroviruses (except possibly enterovirus type 70, which causes eye infections) can be transmitted by the fecal–oral route. Secondary transmission in households is very high and may exceed 90%, although it is typically lower (18–75%), often depending upon the type and strain of virus and sanitary conditions [13]. Incubation times vary greatly with the type of virus and may be as short as 12 h for Coxsackievirus type A24 eye infections to 35 days for poliovirus. The presentation of symptomatic illness is also highly type and strain dependent. Some infections may pass through the community [12]. The only evidence of infection may be the isolation of the virus in the stool or an antibody response. However, typically half of those infected will be symptomatic [12]. Echoviruses usually cause milder infections than those caused by Coxsackievirus. The case–fatality ratio in recognized cases of enterovirus illness has been reported to range from 0.01% to 0.94% [14]. Table 2.9 Some Human Enteroviruses and Parechoviruses and Clinical Illness Coxsackieviruses are named after Coxsackie, New York, where they were first isolated. They have been associated with several water recreational outbreaks. Most infections due to Coxsackieviruses are symptomatic; according to Cherry [15], 50% of infections of Coxsackievirus type A and 80% of type B result in the development of symptoms in the host; however, asymptomatic epidemics, with little or no acute illness, can occur. Table 2.10 lists some of the various diseases caused by Coxsackieviruses. The most common diseases associated with Coxsackieviruses are cardiovascular disease, respiratory disease, gastroenteritis, and central nervous system disorders. Table 2.10 Diseases/Outcomes Caused by Coxsackievirus Coxsackievirus type B is a common viral agent associated with heart disease [18, 19]. Lasting chronic heart damage can result and in children less than 3 months of age from Coxsackievirus infection can lead to death. More than 90% of viral meningitis cases are caused by enteroviruses particularly the Coxsackieviruses [19]. The role of Coxsackieviruses in insulin-dependent diabetes mellitus has been the subject of many studies but is still controversial and appears to be associated with infection in genetically predisposed persons [19]. Coxsackie B viruses have been associated with several recreational outbreaks of aseptic meningitis [20–22]. Echovirus 30 was associated with an outbreak of gastroenteritis from a swimming pool [23]. Recent advances in molecular biology have made it easier to associate outbreaks of waterborne disease with enteroviruses. In recent years, 11 outbreaks of nondisinfected surface or groundwaters have been documented (Table 2.11). Most of these outbreaks have been associated with meningitis. While enteroviruses have been detected in foods (particularly shellfish), very few foodborne outbreaks have been documented [34]. In two reported foodborne outbreaks associated with echoviruses in the United States, the source of the virus was not identified [35]. Table 2.11 Drinking Water Outbreaks Associated with Enteroviruses HAV or infectious hepatitis (to differentiate it from serum hepatitis or hepatitis B) has been responsible for foodborne diseases and outbreaks. It is one of the more thermally resistant enteric viruses and can survive for prolonged periods in the environment. Hepatitis A is common throughout the world, but the incidence in developed countries has decreased significantly since the introduction of a vaccine. Humans are the only known host for the virus with an incubation period averaging 30 days. Secondary transmission among susceptible household contacts is 10–20% from a family member with acute illness [36]. Hepatitis A is usually a mild illness, which almost always results in complete recovery. Severity and disease manifestation are age related. An estimated 80–95% of infected children younger than 5 years of age do not develop overt disease, whereas clinical manifestations are observed in approximately 75–90% of infected adults [37]. The mortality rate in children 14 years old or younger is 0.1%; this rate rises to 0.3% in individuals between the ages of 15 and 39 years and 2.1% in those older than 40 years. Hepatitis A is characterized by sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed in several days by jaundice. In contrast to hepatitis B, HAV infection is not chronic. HAV is excreted in feces of infected people and can produce clinical disease when susceptible individuals consume contaminated water or foods. Rhinoviruses are now considered members of enterovirus genus since there is no significant difference in genome organization or particle structure [38]. The main difference is the sensitivity of the rhinoviruses to inactivation by low-pH characteristic of the human stomach. There are currently more than 250 types of rhinoviruses recognized. Rhinoviruses produce a typical common cold characterized by nasal obstruction, sneezing, pharyngeal discomfort, and cough. The length of infection in young adults is about 7 days, with peak symptomatology occurring on the second and third days. Symptoms last up to 2 weeks and in some cases as long as a month. Rhinovirus infections are less severe than those of influenza and do not result in mortality. Infections are most common in children under 1 year of age with 1.2 infections per year compared to adults at 0.2 infections per year [39]. In temperate climates, rhinoviruses reach their peak incidence in September and early fall. In the tropics, infections are associated with the rainy season. The majority of rhinovirus infections are associated with symptomatic respiratory illness. The percentage of rhinovirus infection associated with clinical disease ranges from 63% in families to 74–88% in military recruits and medical students [17, 40]. Secondary attack rates are related to the titer of preexisting antibodies and have been reported to vary from 21% to 70% [41]. Maximum virus excretion occurs 2–3 days after infection. Evidence suggests that transmission of the virus is due to direct contact with nasal secretions and not by inhalation [42]. Hand contact with the nose and eyes is believed to be the major route of exposure (Fig. 2.4). Figure 2.4 Routes of acquisition of rhinovirus infections modified from Ref. [39]. The hepatitis E virus (HEV) is the leading cause of acute viral hepatitis among young and middle-aged adults in developing countries. HEV has a diameter of 32–34 nm and a single-stranded RNA (ssRNA) genome. Currently, HEV is placed in a sole genus Hepevirus within a new family Hepeviridae. It has been suggested that the higher prevalence of HEV in adults may be due to a silent infection early in life, with subsequent waning of immunity after 10–20 years, when they again become susceptible to infection by HEV [43]. Important epidemiological features of HEV infection are the frequent occurrence of outbreaks associated with consumption of sewage-polluted water and its severity, particularly among pregnant women, in whom the case–fatality rate may be as high as 25% [44]. Secondary attack rates are low and reported to be 0.7–8.0% in households [45, 46]. HEV also infects swine and other animals and has been transmitted by the consumption of undercooked meat from infected animals [47]. To date, no outbreak has occurred in the United States. Hepatitis E is clinically indistinguishable from hepatitis. The incubation period for hepatitis E varies from 2 to 9 weeks. The first member of these genera of viruses was first identified during an outbreak of gastroenteritis in the town of Norwalk, Ohio, and was first known as the Norwalk virus. Since then, it was found that they are the major cause of viral gastroenteritis worldwide. This group of viruses has been placed in the Caliciviridae family and is divided into two genera that infect humans: Noroviruses and Sapoviruses. There are also several other genera of caliciviruses that only infect animals. Man is the only known host for these two genera, which are further divided into several genotypes. Genotypes GI and GII are the most common types identified causing illness. Noroviruses are major causes of both food- and waterborne disease. It is estimated that 67% of the foodborne illnesses in the United States are caused by noroviruses [48]. Caliciviruses are nonenveloped viruses with a diameter of approximately 26–35 nm and a positive-sense ssRNA genome. Infectivity studies with volunteers have shown that individual susceptibility is more important than acquired immunity. It has been suggested that genetically determined factors are the primary determinants of resistance to Norovirus infection, perhaps at the level of cellular receptor sites (i.e., blood group antigens expressed in the gut). Norovirus and related viruses usually produce a mild and brief illness, lasting 1–2 days. It is characterized by nausea and abdominal cramps, followed commonly by vomiting in children and diarrhea in adults. Studies in human volunteers suggest that reinfection occurs, indicating no long-term protection against symptomatic infection. Mortality does occur but usually only in immunocompromised individuals or the elderly [49]. Human noroviruses cannot be grown in cell culture on a continuous basis, and the murine norovirus has been often used as a model for environmental studies. Rotaviruses have a genome consisting of 11 double-stranded RNA (dsRNA) segments surrounded by a distinctive two-layered protein capsid of 70 nm in diameter. Rotaviruses are the most important agents of infantile gastroenteritis around the world. Group A rotavirus is endemic worldwide. It is the leading cause of severe diarrhea among infants and children and accounts for about half of the cases requiring hospitalization. In temperate areas, it occurs primarily in the winter, but in the tropics, it occurs throughout the year. Group B rotavirus, also called adult diarrhea rotavirus, has caused major epidemics of severe diarrhea affecting thousands of persons of all ages in China. Group C rotavirus has been associated with rare and sporadic cases of diarrhea in children in many countries. Rotaviruses are shed in large numbers, up to 1010 viral particles per gram of feces [50]. Rotaviruses are transmitted by the fecal–oral route. Person-to-person spread by contaminated hands is probably one of the most important routes by which rotaviruses are transmitted.
Microbial Agents and Transmission
Microbial Taxonomy
Eukaryotes
Organism
Infective Form (Size)
Mechanism of Transmission
Distribution
Reservoirs
Giardia lamblia
Cyst (8–16 µm)
Person–person, waterborne, foodborne
Worldwide
Humans, beavers, muskrats, voles
Cryptosporidium parvum
Oocysts (3–6 µm)
Person–person, waterborne, foodborne
Worldwide
Many vertebrates, especially cattle
Entamoeba histolytica
Cyst (10–16 µm)
Person–person, waterborne, foodborne
Areas of poor sanitation
Usually humans (potentially pigs, primates, and dogs)
Naegleria fowleri
Trophozoite (10–15 µm)
Trophozoite swims up nasal cavity
Worldwide
None: free living in aquatic or soil environment
Cyclospora cayetanensis
Sporulated oocysts (8–10 µm)
Waterborne, foodborne
Asia, Caribbean, Mexico, and Peru
Not known
Enterocytozoon bieneusi
Spore (1–2 µm)
Fecal–oral
Unknown
Mammals and birds?
Encephalitozoon hellem
Spore
Urine–oral
Unknown
Not known
Encephalitozoon cuniculi
Spore
Fecal/urine–oral
Unknown
Laboratory rabbits, rodents, and dogs
Encephalitozoon intestinalis
Spore
Fecal/urine–oral
Unknown
Dogs, mammals?
Toxoplasma gondii
Sporulated oocysts (11–12 µm)
Oral ingestion from soil or litter box (oocyst)
Worldwide
Cats are definitive host: humans, sheep, goats, pigs, cattle, and birds are intermediate hosts
Undercooked meats (tissue cysts)
Ascaris lumbricoides
Embryonated egg
Oral from soil contact
Worldwide
Humans
Trichuris trichiura
Embryonated egg
Waterborne, foodborne
Worldwide, especially tropics
Humans
Necator americanus
Filariform larva
Skin penetration
Tropical Africa, Asia, Central and South America, and Caribbean
Humans
Ancylostoma duodenale
Filariform larva
Skin penetration, oral from soil contact
South America and Caribbean
Humans
Taenia saginata
Cysticercus
Ingestion of undercooked beef containing cysticerci, waterborne
Worldwide
Intermediate host: cattle
Prokaryotes
Family, Genus, and Species
Clinical Infection
Route of Transmission (Source)
Spirochetes, Gram-negative helical bacteria
Leptospiraceae
Leptospira interrogans
Leptospirosis
Water (urine)
Aerobic/microaerophilic, helical Gram-negative bacteria
Gastroenteritis
Water, food
Campylobacter jejuni
Gram-negative aerobic rods and cocci
Pseudomonodaceae
Pseudomonas aeruginosa
Wound, burn, urinary tract infections
Water, food, air
Legionellaceae
Legionella pneumophila
Pneumonia (Legionnaire’s disease)
Air
Other genera
Brucella abortus
Brucellosis
Food, air
Bordetella pertussis
Pertussis (whooping cough)
Air
Francisella tularensis
Tularemia
Food, water, direct contact, insects
Facultative anaerobic Gram-negative rods
Enterobacteriaceae
Opportunistic infections; some strains cause diarrhea
Water, food
Escherichia coli
Opportunistic infections
Water, food
Shigella dysenteriae
Dysentery
Water, food
Salmonella typhi
Typhoid fever
Klebsiella pneumoniae
Opportunistic infections
Serratia marcescens
Opportunistic infections
Yersinia enterocolitica
Gastroenteritis
Water, food
Vibrionaceae
Vibrio cholerae
Cholera
Water, food
Vibrio parahaemolyticus
Gastroenteritis from seafood
Pasteurellaceae
Haemophilus influenzae
Meningitis, other pediatric diseases
Unknown
Rickettsias and chlamydias, Gram negative
Coxiella burnetii
Q fever
Air, food
Chlamydia psittaci
Psittacosis
Air
Gram-positive cocci
Micrococcaceae
Staphylococcus aureus
Food intoxication, skin infections
Food, direct contact, fomite
Endospore-forming Gram-positive rods and cocci
Bacillus anthracis
Anthrax
Air, water, food, soil
Clostridium botulinum
Botulism, food intoxications
Soil, food
Cl. perfringens
Gas gangrene, food intoxications
Food, soil
Cl. difficile
Gastroenteritis, colitis
Fomites, water
Regular, nonsporing, Gram-positive rods
Listeria monocytogenes
Meningitis
Food, air
Irregular, nonsporing, Gram-negative rods
Corynebacterium diphtheriae
Diphtheria
Air?
Actinomyces israelii
Actinomycosis
Air, soil?
Mycobacteria (acid-fast rods)
Mycobacterium tuberculosis
Tuberculosis
Food, air, water
M. avium
Pulmonary disease, disseminated disease in immunocompromised
Opportunistic Pathogens
Viruses
Nature of Nucleic Acid
Envelope and Shape
Typical Size (nm)
Example
Disease
RNA Viruses
Positive ssRNA
Naked, polyhedral
30
Picornaviruses
Paralysis, common cold, myocarditis
Positive ssRNA
Enveloped, polyhedral
40–70
Togaviruses
Encephalitis, yellow fever
Negative ssRNA
Enveloped, helical
12–15 × 150–300
Paramyxoviruses
Measles, mumps, rabies
70–85 × 130–380
Rhabdoviruses
Negative, segmented ssRNA
Enveloped, helical
90–120
Orthomyxoviruses
Influenza, hemorrhagic fever
50–300
Arenaviruses
Segmented dsRNA
Naked, polyhedral
60–80
Rotaviruses
Respiratory and gastrointestinal infections
Positive, single stranded
Enveloped, helical
80–130
Retroviruses
Leukemia, tumors, AIDS
DNA Viruses
Double-stranded linear DNA
Naked, polyhedral
70–90
Adenoviruses
Respiratory infections, gastroenteritis
Double-stranded linear DNA
Enveloped, polyhedral
150–200
Herpesviruses
Oral and genital herpes, chicken pox, shingles, mononucleosis
Double-stranded linear DNA
Enveloped, complex shape
160–260 × 250–450
Poxviruses
Smallpox, cowpox
Double-stranded circular DNA
Naked, polyhedral
45–55
Papovaviruses
Warts
Single-stranded linear DNA
Naked, polyhedral
18–26
Parvoviruses
Roseola in children, aggravates sickle cell anemia
Enteroviruses
HAV (hepatitis A virus)
Reoviruses
Rotaviruses
Adenoviruses
Astroviruses
Torovirus
Human caliciviruses
Norovirus
Sapprovirus
HEV (hepatitis E virus)
Picobirnaviruses
Bocaviruses
Coronaviruses
Virus Group
Serotypes
Some Diseases Caused
Rhinoviruses
>100
Common cold
Parainfluenza
4
Flulike illness
Influenza
4
Flu
RSV (Respiratory Syncytial Virus)
1
Severe respiratory illness
Coronaviruses
?
Illness in young children
Adenoviruses
56 [6]
Nose, throat, and eye infection
Bocaviruses
?
Lower respiratory infections
Prions
Clinical Characterization
Age (often most severe in the very young and the elderly)
Alcoholism
Chronic diseases (e.g., diabetes)
Double infection (i.e., infection by more than one organism)
Immune state
Nutritional status (poor nutrition increases severity)
Presence of receptors on cells for attachment or entry of organisms
Agent
Incubation Period
Modes of Transmission
Duration of Illness
Adenovirus
8–10 days
Water ingestion, direct contact, aerosol?
8 days
Campylobacter jejuni
3–5 days
Food ingestion, direct contact
2–10 days
Cryptosporidium
2–14 days
Food/water ingestion, direct and indirect contact
Weeks–months (immunocompromised)
E. histolytica
7–14 days
Food/water ingestion, direct and indirect contact
Variable (weeks–months)
E. coli
ETEC
16–72 h
Food/water ingestion
3–5 days
EPEC
16–48 h
Food/water ingestion, direct and indirect contact
5–15 days
EIEC
16–48 h
Food/water ingestion?
2–7 days
EHEC
72–120 h
Food ingestion, direct or indirect contact?
2–12 days
G. lamblia
7–14 days
Food/water ingestion, direct and indirect contact
Weeks–months
Hepatitis A
30–60 days
Hepatitis
2–4 weeks
L. monocytogenes
3–70 days
Food digestion, direct or indirect contact?
Variable
Norovirus
24–48 h
Food/water ingestion, direct and indirect contact, aerosol?
1–2 days
Rotavirus
24–72 h
Direct and indirect contact, aerosol?
4–6 days
Salmonellae
16–72 h
Food ingestion, direct and indirect contact
2–7 days
Shigellae
16–72 h
Food/water ingestion, direct and indirect contact
2–7 days
Yersina enterocolitica
3–7 days
Food ingestion, direct contact
1–3 weeks
Organism
Per Gram of Feces
Protozoan parasites
106–107
Helminths
Ascaris
104–105
Enteric viruses
Enteroviruses
103–107
Rotavirus
1010
Adenovirus
1011
Norovirus
1011
Enteric bacteria
Salmonella spp.
104–1010
Shigella
105–109
Indicator bacteria
Coliform
107–109
Fecal coliform
106–109
Microorganisms of Interest
Viruses
Picornaviruses
Enteroviruses
Virus
Serotypes
Clinical Illness
Poliovirus
3 types
Paralysis, aseptic meningitis, febrile illness
Enterovirus A
12 types
Paralysis, aseptic meningitis
Coxsackievirus
A 2–7
Hand, foot, and mouth disease (HFMD)
A 8–16
Encephalitis
Enterovirus
71
Herpangina
76
Exanthema, diarrhea
Enterovirus B
37 types
Aseptic meningitis, paralysis
Coxsackievirus
B1–B6, A9
Exanthems, respiratory diseases
Echovirus
1–9, 11–21, 24–33
Diarrhea
Enterovirus
69, 73–91
Pericarditis, myocarditis, febrile illness
Enterovirus C
11 types
Paralysis, aseptic meningitis
Coxsackievirus A
1, 11, 13, 15, 17–22, 24
Myocarditis, encephalitis
Enterovirus D
Pneumonia, acute hemorrhagic conjunctivitis
Enterovirus
68, 70
Parechoviruses
1–6
Pericarditis, herpangina, respiratory disease
Type
Disease/Outcome
A
Common cold, fever, herpangina, paralysis, aseptic meningitis
A10
HFMD [16]
A21
Respiratory illness
B
Pericarditis, pleurodynia, meningitis, myalgia, orchitis, fever
B
Systemic infection of infants, fever, paralysis, aseptic meningitis, pleurodynia
B
Myocarditis
B
Neonatal myocarditis
B
Encephalitis, myocarditis, fulminant infection in newborns
B
Aseptic meningitis, myocarditis, diabetes, myalgia, respiratory illness
B
Spontaneous abortion, stillbirth [17]
B
Myopericarditis
B
Miscarriage
B 1–5
Myocarditis
B 1–5
Encephalitis, paralysis, sepsis, meningitis, carditis symptoms
B
Diabetes
Type
Location
Illness
Reference
Echo 30, Echo 6, Coxsackie B5
Russia
Meningitis
[24]
Echo 3
South Africa
Meningitis, other
[25]
Enterovirus (and other agents)
Italy
Gastroenteritis
[26]
Enterovirus 71
Korea
Hand–foot–mouth
[27]
Echo 30
China
Meningitis
[28]
Echo 30
Belarus
Meningitis
[29]
Coxsackie B4
Russia
Multiple
[30]
Echo 18
United States
Meningitis
[31]
Enterovirus 71
Taiwan
Hand–foot–mouth
[32]
Enteroviruses (and other viruses)
Finland
Gastroenteritis
[33]
Hepatitis A Virus
Rhinoviruses
Hepatitis E Virus
Norovirus and Sapovirus
Rotaviruses
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