Source: http://www.asmscience.org/content/book/10.1128/9781555818364
Timestamp: 2019-04-25 06:24:00+00:00

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During the past two decades, strains of nontoxigenic (NT) Vibrio cholerae O1 have been isolated in several countries, including Bangladesh, Guam, Brazil, Peru, Japan, England, and the United States. This chapter summarizes the available epidemiologic, microbiologic, and clinical information on NT V. cholerae O1 isolates from humans in the United States received by the Centers for Disease Control and Prevention (CDC) between 1977 and 1991. NT V. cholerae of O group 1 is biochemically and serologically indistinguishable from toxigenic V. cholerae O1, the cause of epidemic cholera, but does not produce cholera toxin (CT). The elevated levels of cyclic AMP in the mucosal cells of the small bowel cause a cascade that ultimately results in hypersecretion of chloride and bicarbonate into the intestinal lumen and hence a net osmotic efflux of water. NT V. cholerae O1 isolates were associated with a broad spectrum of illnesses. Symptoms recorded in medical records and epidemiologic reporting forms showed that 6 (43%) of 14 patients were hospitalized but that none died. Public health practitioners should regard NT V. cholerae O1 as a potential human pathogen but not as a pathogen with the same lethality and epidemic potential as toxigenic V. cholerae O1.
Vibrio cholerae is divided into more than 130 O serogroups; however, only organisms of the O1 serogroup have so far been associated with cholera in humans. V. cholerae O1 strains of both biotypes have been further subdivided into three serotypes, designated Inaba, Ogawa, and Hikojima, grouped according to the structure of the O antigens on the lipopolysaccharide (LPS). The LPS of gram-negative bacteria is the most abundant molecule on the cell surface, where it provides a protective barrier to hydrophobic agents and detergents. The most common sugars found in the O polysaccharide are perosamine and quinovosamine. A number of studies designed to correlate the various O-antigen polysaccharides with particular antigenic specificities have been carried out. The genes involved in O-antigen biosynthesis in V. cholerae O1 strains 569B (Inaba, classical) and O17 (Ogawa, EI Tor) have been cloned and expressed in Escherichia coli K-12. The E. coli RfaD is an ADP-L-glycero-D-mannoheptose epimerase and is one of the critical proteins involved in the synthesis of the core oligosaccharide of the LPS in E. coli. Genetic complementation studies have suggested that the determinant responsible for Ogawa specificity lies at the distal end of the rfb region, an area in which no readily detectable differences could be discerned.
Beginning in October 1992, cases of cholera-like disease associated with a Vibrio cholerae strain that did not agglutinate with O1 antisera were noted in Madras, India. The emergence of V. cholerae O139 demonstrates that the O1 antigen is not an exclusive marker for epidemic potential and underscores the need to better understand the interplay of genetic and epidemiologic factors that allow strains to spread rapidly through populations. V. cholerae O139 Bengal does not agglutinate with monoclonal or polyclonal antisera directed against the O1 antigen. DNA sequences of 16S rRNA (positions 330 to 615) from these isolates are typical for V. cholerae O1 strains. V. cholerae O139 carries the gene for and expresses cholera toxin. Monoclonal and polyclonal antisera directed against the O139 antigen are available on a limited, experimental basis; such antisera should be available commercially in the near future. As with V. cholerae O1, the mainstay of therapy for diarrheal disease is oral rehydration. Since resistance to antimicrobial agents is in most instances plasmid mediated, it may be only a matter of time before tetracycline resistance is acquired by O139 strains.
Author: J. Glenn Morris, Jr.
O-group 1 and non-O1 Vibrio cholerae give identical reactions in standard biochemical assays; differentiation is based on expression of the O1 antigen and agglutination with O1 antisera. The O1 biosynthetic gene cluster in V. cholerae (VcRfb) contains ca. 20 kb of DNA. V. cholerae strains can also be classified on the basis of enzyme electrophoretic patterns (zymovar analysis). Non- O1 strains can produce a polysaccharide capsule, and some preliminary data suggest that a number of different capsular types exist; in the future, capsular typing may provide a useful means of classifying strains. It is also possible that typing systems for non-O1 V. cholerae could be based on the analysis of outer membrane protein profiles or chromosomal restriction patterns for rRNA or other critical genes. While seafood remains an important vehicle of infection for sporadic non-O1 disease outside of the United States, it is clear that transmission can also occur through other routes. The gastroenteritis associated with non-O1 V. cholerae can range from mild illness to profuse, watery diarrhea comparable to that seen in patients with epidemic cholera. Current data suggest that there are subsets of strains that are able to cause sporadic human disease: strains that colonize and produce either cholera toxin (CT) or NAG-ST have been associated with diarrhea, while heavy encapsulation appears to be a prerequisite for septicemia.
The anecdotal information that persisted for 100 years maintained that the only reservoir of toxigenic Vibrio cholerae O1, the epidemic strain of cholera, was the human intestinal tract and that this strain was taxonomically separate from V. cholerae non-O1, also known as “nonagglutinable vibrios” and found in aquatic, predominantly estuarine environments. The autochthonous nature of V. cholerae Ol is an important factor in the epidemiology of cholera and is significantly so in endemic areas. The first extensive studies of salinity and temperature relationships using microcosms were reported by Singleton and coworkers who used V. cholerae O1 strain LA4808, isolated during the 1978 El Tor cholera outbreak in Louisiana, and nine other clinical or environmental strains, including both O1 and non-O1 V. cholerae. Anwarul Huq and coworkers demonstrated that V. cholerae associated with planktonic live copepods survived longer, i.e., remained culturable, in laboratory microcosms than V. cholerae exposed to dead copepods or to Pseudoisochrysis sp., a blue-green alga used to feed live copepods. In this study, cells of V. cholerae were enumerated by plate count, and it was found that the organisms could be cultured on plates for up to 336 h. The study was carried out with divers in the Chesapeake Bay region of the United States and at the Black Sea off the coasts of Russia and Ukraine. V. cholerae O1 was isolated by culture and detected in seawater and from swabs of the ears, noses, and throats of divers by the fluorescent-antibody method when culture was unsuccessful.
This chapter reviews new information about cholera enterotoxin (CT) and about recently discovered toxins produced by Vibrio cholerae. The single tryptophan residue (Trp-88) in each subunit is essential for binding, as shown by chemical modification and site-directed mutagenesis studies. The majority of the experiments described in the chapter have been performed in vivo, and the results often differ from those obtained in in vitro experiments examining the role of the enteric nervous system (ENS). Several additional toxins produced by V. cholerae are discussed, such as hemolysin-cytolysin, zonula occludens toxin (Zot), accessory cholera enterotoxin and miscellaneous toxins. Stable enterotoxin (ST) and thermostable direct hemolysin (TDH), have well-established mechanisms of enterotoxicity but are rarely produced by V. cholerae. Two of the toxins, the sodium channel inhibitor and the "new cholera toxin" (NCT) of Sanyal and coworkers, are insufficiently characterized to access their potential roles in disease. The three non-CT V. cholerae toxins that are the best characterized are the hemolysin-cytolysin, Zot, and Ace. The study of CT has produced profound insights into the pathogenesis, treatment, and prevention of cholera. Furthermore, it has yielded knowledge on basic cellular functions and structures such as intracellular messengers and neurological pathways. The study of V. cholerae toxins other than CT has demonstrated new potential mechanisms of diarrhea and may also produce new insights into basic cellular functions such as the assembly and regulation of tight junctions.
This chapter focuses on composition and molecular mechanism of control of ToxR regulon. Early studies of the in vitro production of cholera toxin defined certain conditions that increased overall levels of cholera toxin. Early work on the genetics of cholera toxin expression relied heavily on the use of chemical mutagenesis to produce mutants altered in toxinogenesis. The gene product is a 32-kDa integral inner membrane protein. The amino-terminal two-thirds of the protein is cytoplasmic, and the carboxyterminal one-third is periplasmic. ToxR-alkaline phosphatase fusions that replaced the majority of the periplasmic domain no longer responded to high osmolality. The cytoplasmic portion of ToxR contains a region of homology to OmpR. The function of this region has been characterized by taking advantage of this homology to indicate which amino acid residues may be important for function. Amino acid substitution of glutamic acid 51 with lysine resulted in inactive protein that was dominant over wild type. Vibrio cholerae possesses an array of virulence factors that are highly regulated. Among the factors necessary for a successful infection are cholera toxin, the toxin-coregulated pilus, and other accessory colonization factors.
The best characterized pilus of Vibrio cholerae is the toxin-coregulated pilus (TCP) described by Taylor and coworkers. The pilus is designated TCP, for toxin-coregulated pilus, and the pilin subunit is designated TcpA. Fusions to genes tcpBCDEF show reduced levels of pilin and lack pili on the cell surface. Therefore, these genes probably encode assembly and transport proteins required for pilus biogenesis. Further analyses of TcpE, TcpF, and TcpC function are discussed. Amino acid sequencing of gel-purified TcpA reveals an amino-terminal region that bears striking homology to type IV pilins, formerly referred to as N-methylphenylalanine pilins because of the modification present on their amino-terminal residues. Secretion of bacterial cell surface proteins composing macromolecular complexes occurs by a mechanism only recently elucidated in comparison to the initial stages of general prokaryotic protein export. The pathway for TCP biogenesis provides a model system for investigating these novel secretion processes. A number of the phoA fusions initially isolated on the basis of loss of TCP expression still express the TcpA pilin but fail to assemble or secrete pili. Thus, these genes are postulated to encode proteins with biogenesis functions. The Neisseria gonorrhoeae proteins PilF, PilT, and PilD, presumed to play a role in pilus biogenesis, share substantial homology to pilus assembly proteins of the same name from Pseudomonas aeruginosa. Recent data suggest that the TcpC protein may contain novel functions independent of its hypothesized role in pilus biogenesis.
Our current knowledge of cholera and the highly effective oral treatment of the disease could not have been attained without the use of animal models and human volunteers. Ever since the discovery of the etiologic agent of cholera, there have been attempts to develop suitable animal models for studying the interactions of Vibrio cholerae with its accidental human niche. The major animal models that have been employed in cholera research and some examples of the valuable information provided by these models have been discussed in this chapter. Finkelstein clearly demonstrated the presence of classical exo-enterotoxin (CT) (choleragen) and proceeded to work on purification of the material from Syncase medium, using infant rabbits as his biological assay. Concomitant with toxin purification, attempts were made to derive mutants of V. cholerae that would produce altered toxins or natural toxoids. Colonization studies are largely dependent on animal models, because the complex interactions between the two dynamic living systems are impossible to duplicate in vitro. The standard measure of vaccine efficacy was usually determined by the mouse protection assay. For all of the remarkable advances that have been made in cholera research at the bench, the real answers will have to be provided by V. cholerae in its normal environmental niche and in its transient home away from home, the intestines of humans and experimental animals.
This chapter outlines present knowledge on the immune mechanisms operating against cholera based on studies of experimental animals infected with Vibrio cholerae as well as of humans living in areas where cholera is endemic or convalescing from cholera disease. Owing to the critical role of cholera toxin (CT) in the pathogenesis of V. cholerae O1, much interest has been focused on the importance of antitoxic immunity for protection against cholera. Although lipopolysaccharide (LPS) apparently is responsible for much of the antibacterial cholera immunity induced, both by cholera vaccines and by V. cholerae infection, additional antigens, some of which may be expressed only during certain culture conditions or during growth in the intestine, may contribute to antibacterial protective immunity. The immune responses induced by clinical cholera in humans appear to be very efficient in providing prolonged protective immunity against subsequent infection with V. cholera O1 bacteria. Symptomatic or asymptomatic infections with V. cholerae in early childhood may result in partial or complete immunity to cholera disease or even infection. Studies of the incidence of diarrhea in breast-fed children also suggest a protective effect of antitoxic as well as antibacterial antibodies in milk against cholera. The protective role of these antibodies has been difficult to assess, e.g., by comparing diarrhea morbidity in breast-fed and bottle-fed children, owing to several confounding factors.
In this chapter, the term cholera gravis will denote the most severe life-threatening form of the disease, which can be rapidly fatal without treatment. One of the clear messages about cholera is that if the environment is made safer by sound sanitary practices and if good personal hygiene is put in place, the disease largely goes away. Anti-cholera toxin binding (CTB) antibody titers in contact with children&apos;s saliva were used to correlate their immune responses to CTB vaccination with nutritional status as part of the intervention arm of the family study mentioned in the chapter. The cholera attack rates were 88% in the heavy users of cannabis and 37% in light users. Because of the extensive use of cannabis-containing products in many areas where cholera is endemic, habitual usage may be a factor in host susceptibility where the risk of intake of contaminated food and water is high. The molecular genetic basis for the group ABO system was elucidated by Yamamoto, who suggested that the ABO markers be designated a histo-blood system, since the genes and their products are also found in many other cell types, e.g., epithelial cells. At present, two major attachment systems are known to be involved in Vibrio cholerae adherence and colonization of animals and humans. The toxin-coregulated pilis (TCPs) share 80% amino acid homology at the amino acid level.
This chapter traces the epidemic in Africa, describes epidemiologic investigations that determined prevalent modes of transmission, and discusses efforts to control the epidemic and prevent deaths. During the 20th century, cases of cholera were mostly limited to areas in Asia and the Indian subcontinent where cholera is endemic. Between 1972 and 1991, cholera spread throughout much of the remainder of Africa. Both foodborne and waterborne transmission of cholera have been documented in Africa. Seafood, implicated as a vehicle of cholera transmission in other parts of the world, was associated with illness in Guinea-Bissau in 1987. The survival of Vibrio cholerae O1, biotype E1 Tor, in the types of water storage containers used in Africa has been studied. Management of cholera case treatment was evaluated during an outbreak among Mozambican refugees in Malawi in 1990. The rapidity with which resistant isolates could become prevalent was demonstrated in 1977 in Tanzania. A strategy for the control and prevention of cholera in Africa was set forth in a declaration presented at the Forty-fifth World Health Assembly. Since cholera has become endemic throughout much of Africa, local and foreign governments and relief organizations must coordinate their efforts to prevent unnecessary loss of life throughout that continent.
During the last 2 decades, evidence has accumulated from Australia and the United States that shows that both of these countries have environmental reservoirs for toxigenic Vibrio cholerae O1. Australia recorded cholera for the first time in 1832, just 15 years after the first pandemic began in India. The association of cholera cases with river water led to extensive studies of rivers in Queensland. New methods that have become more and more capable of reproducibly subtyping V. cholerae O1 strains have been developed. Cholera was first recognized in the United States in June 1832, when it appeared in New York, having crossed the Atlantic soon after its extension from Asia into Europe in 1829. Several factors may contribute to the relative unimportance of oysters in causing cholera in the United States. The new subtyping methods used on the Australian strains have also been applied to strains from the United States. Endemic cholera in Australia and the United States is characterized in both countries by reservoirs in water, hemolytic strains that are unique to each country, occurrence during the summer and fall, and very few detected cases. Although Australia and the United States have the strongest evidence for environmental reservoirs of toxigenic V. cholerae O1, similar reservoirs may exist in many countries.
The cholera epidemic in Latin America entered its third year in early 1993. Before 1980, only three Latin American countries had national programs for promoting rehydration treatment of diarrheal illness. The low mortality figures may reflect relatively thorough case counting in countries like Peru, Ecuador, and Colombia, but they also indicate the growing experience and skill of the Latin American medical community in treating cholera. Development of an effective and inexpensive vaccine would be an important advance, and several experimental vaccines are being tested in Latin American populations. The general success of treatment for cholera means that the Latin American medical system is now more skilled in treating any dehydrating diarrhea and that the populace is more confident about the benefit of such treatment. Cholera will remain a risk where people live below a sanitary threshold, without safe water and food, sewage disposal, or elementary hygiene. The epidemic is a metaphor for underdevelopment, a marker for many diseases that are transmitted by contaminated food and bad water. The scope of the epidemic extends beyond health and clinical medicine to include the fisheries, agriculture, shipping, and tourist industries of many nations.
Infection with Vibrio cholerae O1 is thought to occur only in humans and only after ingestion of the bacterium. Most of one&apos;s knowledge about the vehicles of cholera transmission stems from case-control investigations. Statistical tests can identify exposures that are reported significantly more frequently by patients than by controls, and such exposures are considered risk factors for illness. Cholera is exclusively a human disease, and no animal species has been found to be consistently infected. Municipal water systems that lack well maintained facilities for water disinfection and distribution provide a ready means of disseminating V. cholerae O1 to a much larger population than is usually served by a single contaminated source. Fish and shellfish have long been recognized as frequent vehicles of cholera transmission. Cooked grains and legumes support the growth of V. cholerae O1 quite well and have been repeatedly implicated in cholera transmission. Transmission of cholera can occur through a wide variety of food and water vehicles, and sometimes the vehicles have been items that were thought to be highly unlikely at the time, such as cooked crabs in Louisiana or bottled water in Portugal.
This chapter describes the newer molecular subtyping approaches as they have evolved and been applied to investigations of cholera since the 1970s. It presents specific applications of subtyping in outbreak and epidemic settings and in retrospective studies of well-defined culture collections of Vibrio cholerae O1, including some preliminary information on the serogroup O139. The increasing knowledge of the molecular epidemiology of cholera has been stimulated by advances in technology and by unexpected occurrences of the disease from 1978 to 1993. In this chapter, the authors look at epidemiologically and phenotypically defined groups of V. cholerae isolates and discuss the use of their molecular genetic characteristics and relationships to address specific questions regarding the occurrence and spread of cholera, i.e., the molecular epidemiology of cholera. Robot-assisted DNA sequence analysis of a 460-bp polymerase chain reaction amplification product from ctxB indicated that classical biotype strains isolated from 1921 through 1970 had identical DNA sequences. The ctxB sequences of 45 V. cholerae O1 strains showed only three nucleotide differences among all strains, and there were three ctxB genotypes; the four classical biotype isolates belong to genotype. The multilocus enzyme electrophoresis (MEE) analysis of isolates indicated that both patient and food isolates belonged to the typical seventh-pandemic electrophoretic type (ET). Molecular subtyping of V. cholerae can identify specific outbreak strains and can often determine the geographic origins of strains.
Presently, cholera surveillance varies from country to country and is not always smooth during an epidemic. Cholera control can be enhanced if the benefit of rapid and accurate reporting of cases is recognized and if attention is given to optimizing national and international surveillance. Surveillance is a key component in an ideal plan for cholera control. Other integral components include health education, environmental sanitation, clinical management, laboratory diagnosis, and epidemiologic investigation. A confirmed cholera case should be laboratory-confirmed Vibrio cholerae O1 infection of any person who has diarrhea. The laboratory is a central component of cholera surveillance. It is essential for confirming the arrival of V. cholerae O1, monitoring its continued presence or disappearance, determining its antimicrobial susceptibilities, and identifying its presence in the environment. Surveillance of community sewage by using Moore swabs is useful for determining the presence of V. cholerae O1 in an area where no cases have been detected from human surveillance. Cholera is a global public health problem that requires international cooperation for effective control. Surveillance, both national and international, is an essential part of cholera control.
The experimental evidence to date indicates that the level of serum vibriocidal antibodies is correlated with the resistance of humans to cholera. This chapter proposes that a critical level of serum vibriocidal antibodies alone can prevent cholera, and describes a mechanism by which this may occur. On the basis of this hypothesis, vaccines have been designed to elicit antibodies, especially of the immunoglobulin G (IgG) class, to the lipopolysaccharide (LPS) of V. cholerae. The strongest evidence that serum vibriocidal antibodies may confer protective immunity comes from clinical studies of cholera vaccines composed of inactivated V. cholera O1 (cellular vaccines). The authors have reconsidered the data that suggest that serum-neutralizing antibodies to cholera toxin (CT) (antitoxin) do not confer protection against cholera. As has been observed for many viral and bacterial diseases, serum antibodies are effective preventive but not therapeutic agents. This principle is probably also true for cholera. Clinical, epidemiologic, and experimental data support the hypothesis that serum vibriocidal antibodies confer protective immunity to cholera. The authors developed conjugate vaccines composed of the detoxified LPS of V. cholerae O1 serotype Inaba bound to CT that were designed to elicit protective levels of vibriocidal antibodies, especially in infants as part of their routine immunization. Cholera conjugate vaccines have the potential to elicit protection in infants when injected along with diphtheria-typhoid-pertussis vaccine and the Haemophilus. influenzae type b conjugate as part of routine immunization.
There is an increasing need for an effective cholera vaccine. Although cholera can be treated successfully by oral and intravenous rehydration and electrolyte replacement therapy, diarrhea treatment centers are still scarce in many areas where cholera is now endemic, and use of an effective vaccine may in many places be the most promising possibility for controlling the disease or at least be a valuable complement to other control strategies. The intense vaccine development and research efforts during the last 10 years have now provided several oral vaccine candidates based on either a combination of nonliving bacteria and purified B-subunit antigen, or on live attenuated mutants of Vibrio cholerae O1 that produce the B subunit. The clarification of the subunit structure of cholera toxin and the roles of different subunits in toxin action immediately suggested a way to prepare a safe and highly immunogenic "toxoid" consisting of the purified cholera B subunits. Studies of the immune mechanisms operating in cholera and the immune response to various cholera antigens, especially cholera toxin and its B-subunit moiety, have been central themes in much of the recent mucosal immunity research. The B-subunit component is completely nontoxic and has proved to be an exceptionally potent oral immunogen because of its stability and its ability to bind to the intestinal mucosa. The results with the oral B-subunit-whole cell vaccine represent marked improvements over those achieved previously with parenteral cholera vaccines.
The history of cholera provides a framework for assessing the potential for eradicating the disease. Although large cholera epidemics may become less likely to occur as effective control measures are put into place, persistence of cholera as an epidemic disease in some regions of the world and its periodic epidemic emergence from natural reservoirs is likely for the foreseeable future. The public health management of cholera requires a coordinated laboratory effort. In many countries, epidemic cholera has served as the focus for improving surveillance activities in general. The two groups of toxigenic El Tor Vibrio cholerae O1 best documented to be capable of long-term environmental persistence are both consistently serotype Inaba, raising the possibility that this serotype is better suited to persistence than is the Ogawa serotype. Strategies of control that put intense selective pressure on the organism during an ongoing epidemic may lead to other evolutionary developments. Defining the ecologic determinants of persistence of toxigenic V. cholerae O1 in the environment will require clarification of the potential roles of copepods and other plankton in providing a niche for V. cholerae O1 and of the mechanism by which shellfish become contaminated in sewage-free environments. Research on cholera vaccines had been accelerated by the emergence of cholera in Latin America. Eradication of choleras is unlikely, because it has environmental reservoirs that will probably continue to cause occasional cases of cholera indefinitely.

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