Cholera, Vibrio cholerae

O1 and O139, and Other

Pathogenic Vibrios
Richard A. Finkelstein

General Concepts
Cholera and Vibrio cholerae

Clinical Manifestations

Cholera is a potentially epidemic and life-threatening secretory diarrhea characterized

by numerous, voluminous watery stools, often accompanied by vomiting, and resulting
in hypovolemic shock and acidosis. It is caused by certain members of the species
Vibrio cholerae which can also cause mild or inapparent infections. Other members of
the species may occasionally cause isolated outbreaks of milder diarrhea whereas
othersthe vast majorityare free-living and not associated with disease.

Structure, Classification, and Antigenic Types

Vibrios are Gram-negative, highly motile curved rods with a single polar flagellum.
They tolerate alkaline media that kill most intestinal commensals, but they are sensitive
to acid. Numerous free-living vibrios are known, some potentially pathogenic. Until
1992, cholera was caused by only two serotypes, Inaba (AC) and Ogawa (AB), and two
biotypes, classical and El Tor, of toxigenic O group 1 V cholerae. These organisms may
be identified by agglutination in O group 1-specific antiserum directed against the
lipopolysaccharide component of the cell wall and by demonstration of their
enterotoxigenicity. In 1992, cholera caused by serogroup O139 (synonym "Bengal"; the
139th and latest serogroup of V cholerae to be identified) emerged in epidemic
proportions in India and Bangladesh. This serovar is identified by 1) absence of
agglutination in O group 1 specific antiserum; 2) by agglutination in O group 139
specific antiserum; and 3) by the presence of a capsule.

Pathogenesis

Cholera is transmitted by the fecal-oral route. Vibrios are sensitive to acid, and most die
in the stomach. Surviving virulent organisms may adhere to and colonize the small
bowel, where they secrete the potent cholera enterotoxin (CT, also called "choleragen").
This toxin binds to the plasma membrane of intestinal epithelial cells and releases an
enzymatically active subunit that causes a rise in cyclic adenosine 51-monophosphate
(cAMP) production. The resulting high intracellular cAMP level causes massive
secretion of electrolytes and water into the intestinal lumen.

Host Defenses

Gastric acid, mucus secretion, and intestinal motility are the prime nonspecific defenses
against V cholerae. Breastfeeding in endemic areas is important in protecting infants
from disease. Disease results in effective specific immunity, involving primarily
secretory immunoglobulin (IgA), as well as IgG antibodies, against vibrios, somatic
antigen, outer membrane protein, and/or the enterotoxin and other products.

Epidemiology

Cholera is endemic or epidemic in areas with poor sanitation; it occurs sporadically or

as limited outbreaks in developed countries. In coastal regions it may persist in shellfish
and plankton. Long-term convalescent carriers are rare. Enteritis caused by the
halophile V parahaemolyticus is associated with raw or improperly cooked seafood.

Diagnosis

The diagnosis is suggested by strikingly severe, watery diarrhea. For rapid diagnosis, a
wet mount of liquid stool is examined microscopically. The characteristic motility of
vibrios is stopped by specific antisomatic antibody. Other methods are culture of stool
or rectal swab samples on TCBS agar and other selective and nonselective media; the
slide agglutination test of colonies with specific antiserum; fermentation tests (oxidase
positive); and enrichment in peptone broth followed by fluorescent antibody tests,
culture, or retrospective serologic diagnosis. More recently the polymerase chain
reaction (PCR) and additional genetically-based rapid techniques have been
recommended for use in specialized laboratories.

Control

Control by sanitation is effective but not feasible in endemic areas. A good vaccine has
not yet been developed. A parenteral vaccine of whole killed bacteria has been used
widely, but is relatively ineffective and is not generally recommended. An experimental
oral vaccine of killed whole cells and toxin B-subunit protein is less than ideal. Living
attenuated genetically engineered mutants are promising, but such strains can cause
limited diarrhea as a side effect. Antibiotic prophylaxis is feasible for small groups over
short periods.

Other Vibrio Infections

Other serogroups of V cholerae may cause diarrheal disease and other infections but are
not associated with epidemic cholera. Vibrio parahaemolyticus is an important cause of
enteritis associated with the ingestion of raw or improperly prepared seafood. Other
Vibrio species, including V vulnificus, can cause infections of humans and other
animals including fish. Campylobacter species (formerly included with vibrios) can
cause enteritis. C pylori, now known as Helicobacter pylori, is associated with gastric
and duodenal ulcers (see Ch. 23).

INTRODUCTION
Vibrios are highly motile, gram-negative, curved or comma-shaped rods with a single
polar flagellum. Of the vibrios that are clinically significant to humans, Vibrio cholerae
O group 1, the agent of cholera, is the most important. Vibrio cholerae was first isolated
in pure culture by Robert Koch in 1883, although it had been seen by other
investigators, including Pacini, who is credited with describing it first in Florence, Italy,
in 1854.

Cholera is a life-threatening secretory diarrhea induced by an enterotoxin secreted by V

cholerae. Cholera and the cholera enterotoxin are increasingly recognized as the
prototypes for a wide variety of non-invasive diarrheal diseases, collectively known as
the enterotoxic enteropathies; of these, diarrhea due to enterotoxigenic strains of
Escherichia coli (see Ch. 26) is the most important. Cholera remains a major epidemic
disease. There have been seven great pandemics. The latest, which started in 1961,
invaded the Western Hemisphere (for the first time this century) with a massive
outbreak in Peru in 1991. There have since been more than a million cases in Central
and South America as well as a few imported cases in the U.S. and Canada. V cholerae
serogroup O139, which arose in October of 1992 in India and Bangladesh, may become
the cause of the 8th great pandemic of cholera.

Other vibrios may also be clinically significant in humans, and some are known to cause
diseases in domestic animals. Nonpathogenic vibrios are widely distributed in the
environment, particularly in estuarine waters and seafoods. For this reason, isolation of
a vibrio from a patient with diarrheal disease does not necessarily indicate an etiologic
relationship.

Vibrio Cholerae

Clinical Manifestations

Following an incubation period of 6 to 48 hours, cholera begins with the abrupt onset of
watery diarrhea (Fig. 24-1). The initial stool may exceed 1 L, and several liters of fluid
may be secreted within hours, leading to hypovolemic shock. Vomiting usually
accompanies the diarrheal episodes. Muscle cramps may occur as water and electrolytes
are lost from body tissues. Loss of skin turgor, scaphoid abdomen, and weak pulse are
characteristic of cholera. Various degrees of fluid and electrolyte loss are observed,
including mild and subclinical cases. The disease runs its course in 2 to 7 days; the
outcome depends upon the extent of water and electrolyte loss and the adequacy of
water and electrolyte repletion therapy. Death can occur from hypovolemic shock,
metabolic acidosis, and uremia resulting from acute tubular necrosis.
FIGURE 24-1 Pathophysiology of cholera.

Structure, Classification, and Antigenic Types

The cholera vibrios are Gram-negative, slightly curved rods whose motility depends on
a single polar flagellum. Their nutritional requirements are simple. Fresh isolates are
prototrophic (i.e., they grow in media containing an inorganic nitrogen source, a
utilizable carbohydrate, and appropriate minerals). In adequate media, they grow rapidly
with a generation time of less than 30 minutes. Although they reach higher population
densities when grown with vigorous aeration, they can also grow anaerobically. Vibrios
are sensitive to low pH and die rapidly in solutions below pH 6; however, they are quite
tolerant of alkaline conditions. This tolerance has been exploited in the choice of media
used for their isolation and diagnosis.

Until 1992, the vibrios that caused epidemic cholera were subdivided into two biotypes:
classical and El Tor. Classical V cholerae was first isolated by Koch in 1883.
Subsequently, in the early 1900s, some vibrios resembling V cholerae were isolated
from Mecca-bound pilgrims at the quarantine station at El Tor, in the Sinai peninsula,
that had been established to try to control cholera associated with pilgrimages to Mecca.
These vibrios resembled classical V cholerae in many ways but caused lysis of goat or
sheep erythrocytes in a test known as the Greig test. Because the pilgrims from whom
they were isolated did not have cholera, these hemolytic El Tor vibrios were regarded as
relatively insignificant except for the possibility of confusion with true cholera vibrios.
In the 1930s, similar hemolytic vibrios were associated with relatively restricted
outbreaks of diarrheal disease, called paracholera, in the Celebes. In 1961, cholera
caused by El Tor vibrios erupted in Hong Kong and spread virtually worldwide.
Although in the course of this pandemic most V cholerae biotype El Tor strains lost
their hemolytic activity, a number of ancillary tests differentiate them from vibrios of
the classical biotype.

The operational serology of the cholera vibrios which belong in O antigen group 1 is
relatively simple. Both biotypes (El Tor and classical) contain two major serotypes,
Inaba and Ogawa (Fig. 24-2). These serotypes are differentiated in agglutination and
vibriocidal antibody tests on the basis of their dominant heat-stable lipopolysaccharide
somatic antigens. The cholera group has a common antigen, A, and the serotypes are
differentiated by the type-specific antigens, B (Ogawa) and C (Inaba). An additional
serotype, Hikojima, which has both specific antigens, is rare. V cholerae O139 appears
to have been derived from the pandemic El Tor biotype but has lost the characteristic
O1 somatic antigen; it has gained the ability to produce a polysaccharide capsule; it
produces the same cholera enterotoxin; and it seems to have retained the epidemic
potential of O1 strains.

FIGURE 24-2 Vibrio cholerae (O group 1 antigen).

Other antigenic components of the vibrios, such as outer membrane protein antigens,
have not been extensively studied. The cholera vibrios also have common flagellar
antigens. Cross-reactions with Brucella and Citrobacter species have been reported.
Because of DNA relatedness and other similarities, other vibrios formerly called
"nonagglutinable" are now classified as V cholerae. The term nonagglutinable is a
misnomer because it implies that these vibrios are not agglutinable; in fact, they are not
agglutinable in antisera against the O antigen group 1 cholera vibrios, but they are
agglutinable in their own specific antisera. More than 139 serotypes are now
recognized. Some strains of non-O group 1 V cholerae cause diarrheal disease by means
of an enterotoxin related to the cholera enterotoxin and, perhaps, by other mechanisms,
but these strains have not been associated with devastating outbreaks like those caused
by the true cholera vibrios. Recently, vibrio strains that agglutinate in some O group 1
cholera diagnostic antisera but not in others have been isolated from environmental
sources. Volunteer feeding experiments have shown that these atypical O group 1
vibrios are not enteropathogenic in humans. Recent studies using specific toxin gene
probes indicate that these environmental isolates not only are nontoxigenic, but also do
not possess any of the genetic information encoding cholera toxin, although some
isolates from diarrheal stools do.

The cholera vibrios cause many distinctive reactions. They are oxidase positive. The O
group 1 cholera vibrios almost always fall into the Heiberg I fermentation pattern; that
is, they ferment sucrose and mannose but not arabinose, and they produce acid but not
gas. Vibrio cholerae also possesses lysine and ornithine decarboxylase, but not arginine
dihydrolase. Freshly isolated agar-grown vibrios of the El Tor biotype, in contrast to
classical V cholerae, produce a cell-associated mannose-sensitive hemagglutinin active
on chicken erythrocytes. This activity is readily detected in a rapid slide test. In addition
to hemagglutination, numerous tests have been proposed to differentiate the classical
and El Tor biotypes, including production of a hemolysin, sensitivity to selected
bacteriophages, sensitivity to polymyxin, and the Voges-Proskauer test for acetoin. El
Tor vibrios originally were defined as hemolytic. They differed in this characteristic
from classical cholera vibrios; however, during the most recent pandemic, most El Tor
vibrios (except for the recent isolates from Texas and Louisiana) had lost the capacity to
express the hemolysin. Most El Tor vibrios are Voges-Proskauer positive and resistant
to polymyxin and to bacteriophage IV, whereas classical vibrios are sensitive to them.
As both biotypes cause the same disease, these characteristics have only epidemiologic
significance. Strains of the El Tor biotype, however, produce less cholera enterotoxin,
but appear to colonize intestinal epithelium better than vibrios of the classical variety.
Also, they seem some what more resistant to environmental factors. Thus, El Tor strains
have a higher tendency to become endemic and exhibit a higher infection-to-case ratio
than the classical biotype.

Pathogenesis

Recent studies with laboratory animal models and human volunteers have provided a
detailed understanding of the pathogenesis of cholera. Initial attempts to infect healthy
American volunteers with cholera vibrios revealed that the oral administration of up to
1011 living cholera vibrios rarely had an effect; in fact, the organisms usually could not
be recovered from stools of the volunteers. After the administration of bicarbonate to
neutralize gastric acidity, however, cholera diarrhea developed in most volunteers given
104 cholera vibrios. Therefore, gastric acidity itself is a powerful natural resistance
mechanism. It also has been demonstrated that vibrios administered with food are much
more likely to cause infection.

Cholera is exclusively a disease of the small bowel. To establish residence and multiply
in the human small bowel (normally relatively free of bacteria because of the effective
clearance mechanisms of peristalsis and mucus secretion), the cholera vibrios have one
or more adherence factors that enable them to adhere to the microvilli (Fig. 24-3).
Several hemagglutinins and the toxin-coregulated pili have been suggested to be
involved in adherence but the actual mechanism has not been defined. In fact, there may
be multiple mechanisms. The motility of the vibrios may affect virulence by enabling
them to penetrate the mucus layer. They also produce mucinolytic enzymes,
neuraminidase, and proteases. The growing cholera vibrios elaborate the cholera
enterotoxin (CT or choleragen), a polymeric protein (Mr 84,000) consisting of two
major domains or regions. The A region (Mr 28,000), responsible for biologic activity
of the enterotoxin, is linked by noncovalent interactions with the B region (Mr 56,000),
which is composed of five identical noncovalently associated peptide chains of Mr
11,500. The B region, also known as choleragenoid, binds the toxin to its receptors on
host cell membranes. It is also the immunologically dominant portion of the holotoxin.
The structural genes that encode the synthesis of CT reside on a transposon-like element
in the V cholerae chromosome, in contrast to those for the heat-labile enterotoxins (LTs)
of E coli (Ch. 25), which are encoded by plasmids. The amino acid sequences of these
structurally, functionally, and immunologically related enterotoxins are very similar.
Their differences account for the differences in physicochemical behavior and the
antigenic distinctions that have been noted. There are at least two antigenically related
but distinct forms of cholera enterotoxin, called CT-1 and CT-2. Classical O1 V
cholerae and the Gulf Coast El Tor strains produce CT-1 whereas most other El Tor
strains and O139 produce CT-2. Vibrio cholerae exports its enterotoxin, whereas the E
coli LTs occur primarily in the periplasmic space. This may account for the reported
differences in severity of the diarrheas caused by these organisms.

Studies in adult American volunteers have shown that 5µ g of CT, administered orally
with bicarbonate, causes 1 to 6 L of diarrhea; 25µg causes more than 20 L.
FIGURE 24-3 Vibrio cholerae attachment and colonization in experimental
rabbits. The events are assumed to be similar in human cholera. (A) Scanning electron
microscopy during early infection. Curved vibrios adhering to epithelial surface.
(Approximately X 4,000.) (B) Higher power scanning electron micrograph showing
single polar flagellum of the cholera vibrios. (C) Transmission electron microscopy of
vibrios in both end-on and horizontal modes close to tips of microvilli. (From Nelson
ET, Clements JD, Finkelstein RA: Vibrio cholerae adherence and colonization in
experimental cholera: electron microscopic studies. Infect Immun 14:527, 1976, with
permission.)

Synthesis of CT and other virulence-associated factors such as toxin-coregulated pili are

believed to be regulated by a transcriptional activator, Tox R, a transmembrane DNA-
binding protein.

The molecular events in these diarrheal diseases involve an interaction between the
enterotoxins and intestinal epithelial cell membranes (Fig. 24-4). The toxins bind
through region B to a glycolipid, the GM1 ganglioside, which is practically ubiquitous
in eukaryotic cell membranes. Following this binding, the A region, or a major portion
of it known as the A1 peptide (Mr 21,000), penetrates the host cell and enzymatically
transfers ADP-ribose from nicotinamide adenine dinucleotide (NAD) to a target protein,
the guanosine 5'-triphosphate (GTP)-binding regulatory protein associated with
membrane-bound adenylate cyclase. Thus, CT (and LT) resembles diphtheria toxin in
causing transfer of ADP-ribose to a substrate. With diphtheria toxin, however, the
substrate is elongation factor 2 and the result is cessation of host cell protein synthesis.
With CT, the ADP-ribosylation reaction essentially locks adenylate cyclase in its "on
mode" and leads to excessive production of cyclic adenosine 51-monophosphate
(cAMP). Pertussis toxin, another ADP-ribosyl transferase, also increases cAMP levels,
but by its effect on another G-protein, Gi (Fig. 24-5). The subsequent cAMP-mediated
cascade of events has not yet been delineated, but the final effect is hypersecretion of
chloride and bicarbonate followed by water, resulting in the characteristic isotonic
voluminous cholera stool. In hospitalized patients, this can result in losses of 20 L or
more of fluid per day. The stool of an actively purging, severely ill cholera patient can
resemble rice waterthe supernatant of boiled rice. Because the stool can contain 108
viable vibrios per ml, such a patient could shed 2 X 1012 cholera vibrios per day into
the environment. Perhaps by production of CT, the cholera vibrios thus ensure their
survival by increasing the likelihood of finding another human host. Recent evidence
suggests that prostaglandins may also play a role in the secretory effects of cholera
enterotoxin. Recent studies in volunteers using genetically-engineered Tox- strains of V
cholerae have revealed that the vibrios have putative mechanisms in addition to CT for
causing (milder) diarrheal disease. These include Zot (for Zonula occludens toxin) and
Ace (for accessory cholera enterotoxin), and perhaps others, but their role has not been
established conclusively. Certainly CT is the major virulence factor and the act of
colonization of the small bowel may itself elicit an altered host response (e.g., mild
diarrhea), perhaps by a trans-membrane signaling mechanism.
FIGURE 24-4 Mechanism of action of cholera enterotoxin. Cholera toxin
approaches target cell surface. B subunits bind to oligosaccharide of GM1 ganglioside.
Conformational alteration of holotoxin occurs, allowing the presentation of the A
subunit to cell surface. The A subunit enters the cell. The disulfide bond of the A
subunit is reduced by intracellular glutathione, freeing A1 and A2. NAD is hydrolyzed
by A1, yielding ADP-ribose and nicotinamide. One of the G proteins of adenylate
cyclase is ADP-ribosylated, inhibiting the action of GTPase and locking adenylate
cyclase in the "on" mode (Modified from Fishman PH: Mechanism of action of cholera
toxin: events on the cell surface. p. 85. In Field M, Fordtran JS, Schultz SG (eds):
Secretory Diarrhea. Waverly Press, Baltimore, 1980, with permission.)
FIGURE 24-5 Comparison of activities of cholera enterotoxin (CT) with pertussis
toxin (PT). The a-subunits of Gs and Gi, with GTP-binding sites, are ADP-ribosylated,
respectively, by A1 peptide of CT or by the A subunit of PT, preventing, respectively,
the hydrolysis of Gs-GTP to GDP or the responsiveness of Gi to inhibitory hormones,
both effectively producing increases in adenylate cyclase activity. (Modified from Gill
DM, Woolkalis M: Toxins which activate adenylate cyclase. CIBA Found Symp
112:57, 1985, with permission.)

Various animal models have been used to investigate pathogenic mechanisms,

virulence, and immunity. Ten-day-old suckling rabbits develop a fulminating diarrheal
disease after intraintestinal inoculation with virulent V cholerae or CT. Adult rabbits are
relatively resistant to colonization by cholera vibrios; however, they do respond, with
characteristic out pouring of fluid, to the intraluminal inoculation of live vibrios or
enterotoxin in surgically isolated ileal loops. Suckling mice are susceptible to
intragastric inoculation of vibrios and to orally administered toxin. Adult conventional
mice are also susceptible to orally administered toxin, but resist colonization except in
isolated intestinal loops. Interestingly, however, germ-free mice can be colonized for
months with cholera vibrios. They rarely show adverse effects, although they are
susceptible to cholera enterotoxin. Dogs have been used experimentally, although they
are relatively refractory and require enormous inocula to elicit choleraic manifestations.
Chinchillas also are susceptible to diarrhea following intraintestinal inoculation with
moderate numbers of cholera vibrios. Infections initiated by extraintestinal routes of
inoculation (e.g., intraperitoneal) largely reflect the toxicity of the lipopolysaccharide
endotoxin. The intraperitoneal infection in mice has been used to assay the protective
effect of conventional killed vibrio vaccines (no longer widely used).

Various animals, including humans, rabbits, and guinea pigs, also respond to
intradermal inoculation of relatively minute amounts of CT with a characteristic delayed
(maximum response at 24 hours), sustained (visible up to 1 week or more),
erythematous, edematous induration associated with a localized alteration of vascular
permeability. In laboratory animals, this response can be measured after injecting a
protein-binding dye, such as trypan blue, that extravasates to produce a zone of bluing
at the site of intracutaneous inoculation of toxin. This observation has been exploited in
the assay of CT and its antibody and in the detection of other enterotoxins.

In addition, because of the broad spectrum of activity of CT on cells and tissues that it
never contacts in nature, various in vitro systems can be used to assay the enterotoxin
and its antibody. In each, the toxin causes a characteristically delayed, but sustained,
activation of adenylate cyclase and increased production of cAMP, and it may cause
additional, readily recognizable, morphologic alterations of certain cultured cell lines.
The cells most widely used for this purpose are Chinese hamster ovary (CHO) cells,
which elongate in response to picogram doses of the toxin, and mouse Y-l adrenal
tumor cells, which round up. Cholera toxin has become an extremely valuable
experimental probe to identify other cAMP-mediated responses. It also activates
adenylate cyclase in pigeon erythrocytes, a procedure that was used by D. Michael Gill
to define its mode of action.

These assays and models also have been applied in the study of an expanding number of
CT-related and unrelated enterotoxins. These include the LTs of E coli, which are
structurally and immunologically similar to it and are effective in any model that is
responsive to CT. The family of small molecular weight heat-stable enterotoxins (ST) of
E coli, which activate guanylate cyclase, and which are rapidly active in the infant
mouse and certain other intestinal models, are clearly unrelated to CT. CT-related
enterotoxins have been reported from certain nonagglutinable (non-O group I) Vibrio
strains and a Salmonella enterotoxin was shown to be related immunologically to CT.
CT-like factors from Shigella and V parahaemolyticus have thus far been demonstrated
only in sensitive cell culture systems. Other enterotoxins and enterocytotoxins, which
elicit cytotoxic effects on intestinal epithelial cells, also have been described from
Escherichia, Klebsiella, Enterobacter, Citrobacter, Aeromonas, Pseudomonas, Shigella,
V parahaemolyticus, Campylobacter, Yersinia enterocolitica, Bacillus cereus,
Clostridium perfringens, C difficile, and staphylococci. Escherichia coli, some vibrio
strains, and some other enteric bacteria produce cytotoxins that, like Shiga toxin of
Shigella dysenteriae, act on Vero (African green monkey kidney) cells in vitro. These
toxins have been called Shiga-like toxins, Shiga toxin-like toxins, Vero toxins, and Vero
cytotoxins. The classic staphylococcal enterotoxins perhaps should more properly be
called neurotoxins, as they seem to affect the central nervous system rather than the gut
directly to cause fluid secretion or histopathologic effects.

Host Defenses

Infection with cholera vibrios results in a spectrum of responses. These range from no
observed manifestations except perhaps a serologic response ( the most common) to
acute purging, which must be treated by hospitalization and fluid replacement therapy;
this is the classic response. The reasons for these differences are not entirely clear,
although it is known that individuals differ in gastric acidity and that hypochlorhydric
individuals are most prone to cholera. Whether individuals differ in the availability of
intestinal receptors for cholera vibrios or for their toxin has not been established. Prior
immunologic experience of subjects at risk is certainly a major factor. For example, in
heavily endemic regions such as Bangladesh, the attack rate is relatively low among
adults in comparison with children. In neoepidemic areas, cholera is more frequent
among the working adult population. Resistance is related to the presence of circulating
antibody and, perhaps more importantly, local immunoglobulin A (IgA) antibody
against the cholera bacteria or the cholera enterotoxin or both. Intestinal IgA antibody
can prevent attachment of the vibrios to the mucosal surface and neutralize or prevent
binding of the cholera enterotoxin. For reasons that are not clear, individuals of blood
group O are slightly more susceptible to cholera. Breastfeeding is highly recommended
as a means of increasing immunity of infants to this and other diarrheal disease agents.

Recovery from cholera probably depends on two factors: elimination of the vibrios by
antibiotics or the patient's own immune response, and regeneration of the poisoned
intestinal epithelial cells. Treatment with a single 200-mg dose of doxycycline has been
recommended. As studies in volunteers demonstrated conclusively, the disease is an
immunizing process. Patients who have recovered from cholera are solidly immune for
at least 3 years.

Cholera vaccines consisting of killed cholera bacteria administered parenterally have

been used since the turn of the century. However, recent controlled field studies indicate
that little, if any, effective immunity is induced in immunologically virgin populations
by such vaccines, although they do stimulate preexisting immunity in the adult
population in heavily endemic regions. Controlled studies have likewise shown that a
cholera toxoid administered parenterally was ineffective in preventing cholera. Probably
the natural disease should be simulated to induce truly effective immunity although a
parenterally administered conjugate vaccine consisting of the polysaccharide of the
vibrio LPS covalently linked to cholera toxin has given promising results in preliminary
studies. Studies in volunteers have shown that orally administered, chemically
mutagenized or genetically engineered mutants which do not produce CT or produce
only its B subunit protein can induce immunity against subsequent challenge. However,
most of these candidate vaccines also produce unacceptable side effectsprimarily mild
to moderate diarrhea. An exception is strain CVD103-HgR (a mercury resistant A-B+
derivative of classical biotype Inaba serotype strain 569B). This strain has minimal
reactogenicity but does not colonize well and therefore has to be given in higher doses.
Field studies with this strain are in progress. Combined preparations of bacterial somatic
antigen and toxin antigen have been reported to act synergistically in stimulating
immunity in laboratory animals; that is, the combined protective effect is closer to the
product than to the sum of the individual protective effects. However, a large field study
evaluating such nonviable oral vaccines in Bangladesh revealed that neither the whole-
cell bacterin nor the killed vibrios supplemented with the B-subunit protein of the
cholera enterotoxin induced sufficient long term protection, especially in children, to
justify their recommendation for public health use. No clear-cut advantage of the
inclusion of the B-subunit was demonstrated.

In any case, even if these vaccines were effective, the requirement for large and
repeated doses would make them too expensive for use in the developing areas that are
usually afflicted with epidemic cholera. Moreover, they were clearly less effective in
childrenthe primary target population in heavily endemic areas. Neither the killed whole
cell vaccine nor strain CVD103-HgR could be expected to protect against the new O139
serovar.

Epidemiology
Humans apparently are the only natural host for the cholera vibrios. Cholera is acquired
by the ingestion of water or food contaminated with the feces of an infected individual.
Previously, the disease swept the world in six great pandemics and later receded into its
ancestral home in the Indo-Pakistani subcontinent. In 1961, the El Tor biotype (a subset
distinguished by physiologic characteristics) of V cholerae, not previously implicated in
widespread epidemics, emerged from the Celebes (now Sulawesi), causing the seventh
great cholera pandemic. In the course of their migration, the El Tor biotype cholera
vibrios virtually replaced V cholerae of the classic biotype that formerly was
responsible for the annual cholera epidemics in India and East Pakistan (now
Bangladesh). The pandemic that began in 1961 is now heavily seeded in Southeast Asia
and in Africa. It has also invaded Europe, North America, and Japan, where the
outbreaks have been relatively restricted and self-limited because of more highly
developed sanitation. Several new cases were reported in Texas in 1981 and sporadic
cases have since been reported in Louisiana and other Gulf Coast areas. This now
endemic focus appears to be due to a clone which is unique from the pandemic strain. In
1991, the pandemic strain hit Peru with massive force and has since spread through
most of the Western Hemisphere, causing more than a million cases. Fortunately,
mortality has been less than 1 percent because of the effectiveness of oral rehydration
therapy. The vibrios surprised us again, in 1992, with the emergence of O139 in India
and Bangladesh. For a while it appeared that O139 would replace O1 (both classical and
El Tor) but it has exhibited quiescent periods when O1 reemerges.

Cholera appears to exhibit three major epidemiologic patterns: heavily endemic,

neoepidemic (newly invaded, cholera-receptive areas), and, in developed countries with
good sanitation, occasional limited outbreaks. These patterns probably depend largely
on environmental factors (including sanitary and cultural aspects), the prior immune
status or antigenic experience of the population at risk, and the inherent properties of the
vibrios themselves, such as their resistance to gastric acidity, ability to colonize, and
toxigenicity. In the heavily endemic region of the Indian subcontinent, cholera exhibits
some periodicity; this may vary from year to year and seasonally, depending partly on
the amount of rain and degree of flooding. Because humans are the only reservoirs,
survival of the cholera vibrios during interepidemic periods probably depends on a
relatively constant availability of low-level undiagnosed cases and transiently infected,
asymptomatic individuals. Long-term carriers have been reported but are extremely
rare. The classic case occurred in the Philippines, where "cholera Dolores" harbored
cholera vibrios in her gallbladder for 12 years after her initial attack in 1962. Her carrier
state resolved spontaneously in 1973; no secondary cases had been associated with her
well-marked strain. Recent studies, however, have suggested that cholera vibrios can
persist for some time in shellfish, algae or plankton in coastal regions of infected areas
and it has been claimed that they can exist in "a viable but nonculturable state."

During epidemic periods, the incidence of infection in communities with poor sanitation
is high enough to frustrate the most vigorous epidemiologic control efforts. Although
transmission occurs primarily through water contaminated with human feces, infection
also may be spread within households and by contaminated foods. Thus, in heavily
endemic regions, adequate supplies of pure water may reduce but not eliminate the
threat of cholera.

In neoepidemic cholera-receptive areas, vigorous epidemiologic measures, including

rapid identification and treatment of symptomatic cases and asymptomatically infected
individuals, education in sanitary practices, and interruption of vehicles of transmission
(e.g., by water chlorination), may be most effective in containing the disease. In such
situations, spread of cholera usually depends on traffic of infected human beings,
although spread between adjacent communities can occur through bodies of water
contaminated by human feces. John Snow was credited with stopping an epidemic in
London, England, by the simple expedient of removing the handle of the "Broad Street
pump" (a contaminated water supply) in 1854, before acceptance of the "germ theory"
and before the first isolation of the "Kommabacillus" by Robert Koch.

In such developed areas as Japan, Northern Europe, and North America, cholera has
been introduced repeatedly in recent years, but has not caused devastating outbreaks;
however, Japan has reported secondary cases and, in 1978, the United State experienced
an outbreak of about 12 cases in Louisiana. In that outbreak, sewage was infected, and
infected shellfish apparently were involved. Interestingly, the hemolytic vibrio strain
implicated was identical to one that caused an unexplained isolated case in Texas in
1973.

Diagnosis

Rapid bacteriologic diagnosis offers relatively little clinical advantage to the patient
with secretory diarrhea, because essentially the same treatment (fluid and electrolyte
replacement) is employed regardless of etiology. Nevertheless, rapid identification of
the agent can profoundly affect the subsequent course of a potential epidemic outbreak.
Because of their rapid growth and characteristic colonial morphology, V cholerae can
be easily isolated and identified in the bacteriology laboratory, provided, first, that the
presence of cholera is suspected and, second, that suitable specific diagnostic antisera
are available. The vibrios are completely inhibited or grow somewhat poorly on usual
enteric diagnostic media (MacConkey agar or eosin-methylene blue agar). An effective
selective medium is thiosulfate-citrate-bile salts-sucrose (TCBS) agar, on which the
sucrose-fermenting cholera vibrios produce a distinctive yellow colony. However, the
usefulness of this medium is limited because serologic testing of colonies grown on it
occasionally proves difficult, and different lots vary in their productivity. This medium
is also useful in isolating V parahaemolyticus. They can also be isolated from stool
samples or rectal swabs from cholera cases on simple meat extract (nutrient) agar or bile
salts agar at slightly alkaline pH values. Following observation of characteristic colonial
morphology with a stereoscopic microscope using transmitted oblique illumination,
microorganisms can be confirmed as cholera vibrios by a rapid slide agglutination test
with specific antiserum. Classic and El Tor biotypes can be differentiated at the same
time by performing a direct slide hemagglutination test with chicken erythrocytes: all
freshly isolated agar-grown El Tor vibrios exhibit hemagglutination; all freshly isolated
classic vibrios do not. In practice, this can be accomplished with material from patients
as early as 6 hours after streaking the specimen in which the cholera vibrios usually
predominate. However, to detect carriers (asymptomatically infected individuals) and to
isolate cholera vibrios from food and water, enrichment procedures and selective media
are recommended. Enrichment can be accomplished by inoculating alkaline (pH 8.5)
peptone broth with the specimen and then streaking for isolation after an approximate 6-
hour incubation period; this process both enables the rapidly growing vibrios to
multiply and suppresses much of the commensal microflora.
The classic case of cholera, which includes profound secretory diarrhea and should
evoke clinical suspicion, can be diagnosed within a few minutes in the prepared
laboratory by finding rapidly motile bacteria on direct, bright-field, or dark-field
microscopic examination of the liquid stool. The technician can then make a second
preparation to which a droplet of specific anti-V cholerae O group 1 antiserum is added.
This quickly stops vibrio motility. Another rapid technique is the use of fluorescein
isothiocyanate-labeled specific antiserum (fluorescent antibody technique) directly on
the stool or rectal swab smear or on the culture after enrichment in alkaline peptone
broth. For cultural diagnosis, both nonselective and selective (TCBS) media may be
used. Although demonstration of typical agglutination essentially confirms the
diagnosis, additional conventional tests such as oxidase reaction, indole reaction, sugar
fermentation reactions, gelatinase, lysine, arginine, and ornithine decarboxylase
reactions may be helpful. Tests for chicken cell hemagglutination, hemolysis,
polymyxin sensitivity, and susceptibility to phage IV are useful in differentiating the El
Tor biotype from classic V cholerae. Tests for toxigenesis may be indicated.

Diagnosis can be made retrospectively by confirming significant rises in specific serum

antibody titers in convalescents. For this purpose, conventional agglutination tests, tests
for rises in complement-dependent vibriocidal antibody, or tests for rises in antitoxic
antibody can be employed. Convenient microversions of these tests have been
developed. Passive hemagglutination tests and enzyme-linked immunosorption assays
(ELISAs) have also been proposed.

Cultures that resemble V cholerae but fail to agglutinate in diagnostic antisera

(nonagglutinable or non-O group 1 vibrios) present more of a problem and require
additional tests such as oxidase, decarboxylases, inhibition by the vibriostatic pteridine
compound 0/129, and the "string test." The string test demonstrates the property, shared
by most vibrios and relatively few other genera, of forming a mucus-like string when
colony material is emulsified in 0.5 percent aqueous sodium deoxycholate solution.
Additional tests for enteropathogenicity and toxigenesis may be useful. Genetically
based tests such as PCR are increasingly being used in specialized laboratories.

Control

Treatment of cholera consists essentially of replacing fluid and electrolytes. Formerly,

this was accomplished intravenously, using costly sterile pyrogen-free intravenous
solutions. The patient's fluid losses were conveniently measured by the use of buckets,
graduated in half-liter volumes, kept underneath an appropriate hole in an army-type cot
on which the patient was resting. Antibiotics such as tetracycline, to which the vibrios
are generally sensitive, are useful adjuncts in treatment. They shorten the period of
infection with the cholera vibrios, thus reducing the continuous source of cholera
enterotoxin; this results in a substantial saving of replacement fluids and a markedly
briefer hospitalization. Note, however, that fluid and electrolyte replacement is all-
important; patients who are adequately rehydrated and maintained will virtually always
survive, and antibiotic treatment alone is not sufficient.

Recently it has been recognized that almost all cholera patients and others with similar
severe secretory diarrheal disease can be maintained by fluids given orally if the
solutions contain a usable energy source such as glucose. Because of this discovery,
packets containing appropriate salts are distributed by such organizations as WHO and
UNICEF to cholera-afflicted areas, where they are dissolved in water as needed. One
such formulation, called ORS for oral rehydration salts, contains NaCl, 3.5 g; KCl,1.5 g;
NaHCO3, 2.5 g (or trisodium citrate, 2.9 g); and glucose, 20.0 g. This mixture is
dissolved in 1 L of water and taken orally in increments. Flavoring may be added.
Improved versions of ORS, including rice-based formulations that reduce stool output
and can be made at home, have been recommended. Unfortunately, this technique,
which will save countless millions of lives in developing countries, has not yet been
widely accepted by practicing physicians in developed countries.

The possibility of pharmacologic intervention (e.g., a pill that will stop choleraic
diarrhea after it has started), has been considered. Two drugs, chlorpromazine and
nicotinic acid, have been effective in experimental animals, although the precise
mechanism of action has yet to be defined.

Like smallpox and typhoid, choleraunder natural circumstancesappears to affect only

humans; therefore, V cholerae as an etiologic entity could conceivably disappear with
the last human infection. Nevertheless, the spectrum of cholera-like diarrheal diseases
probably will persist for some time.

Cholera is essentially a disease associated with poor sanitation. The simple application
of sanitary principlesprotecting drinking water and food from contamination with
human feceswould go a long way toward controlling the disease. However, at present,
this is not feasible in the underdeveloped areas that are afflicted with epidemic cholera
or are considered to be cholera receptive. Meanwhile, development of a vaccine that
would effectively prevent colonization and manifestations of cholera would be
extremely helpful. As indicated above, such vaccines are presently being tested.
Antibiotic or chemotherapeutic prophylaxis is feasible and may be indicated under
certain circumstances. It also should be mentioned that the incidence of cholera is
significantly higher in formula-fed than in breast-fed babies.

Present information indicates that V parahaemolyticus enteritis could be almost

completely prevented by applying appropriate procedures to prevent multiplication of
the organisms in contaminated seafood, such as keeping it refrigerated continually.

Other Vibrio Infections

Other vibrios may be clinically significant also. These include non-O group 1 V
cholerae. Vibrio parahaemolyticus, a halophilic (salt-loving) vibrio associated with
enteritis is acquired by ingestion of raw or improperly cooked seafoods. Another
halophilic vibrio, which ferments lactose and for this reason was called the L + vibrio,
has recently been identified as V vulnificus. It has been associated with wound
infections as well as fatal septicemias. Other groups of vibrios, previously referred to as
group F and EF-6, have recently been classified into species: V fluvialis, V hollisae, V
furnissia, and V damsela. Vibrio mimicus is a recently described sucrose-negative
species. Vibrio fetus, a group of anaerobic to microaerophilic spirally curved rods
associated with venereally transmitted infertility and abortion in domestic animals, is
now called Campylobacter jejuni and is considered to belong in the family Spirillaceae
rather than in the family Vibrionaceae. Campylobacter jejuni has been associated with
dysentery-like gastroenteritis, duodenal and gastric ulcers, as well as with other types of
infection, including bacteremic and central nervous system infections in humans (see
Ch. 23). Another vibrio-like organism, Helicobacter pylori (formerly known as C
pylori) causes gastritis and predisposes to duodenal ulcers and gastric cancer. Although
some similarities in habitat and other properties occur, members of the family
Vibrionaceae are separated taxonomically from members of the family
Enterobacteriaceae. The oxidase test (vibrios are usually oxidase positive) is particularly
useful. Other vibrios exist, and some of these may be responsible for diseases in fish
and other lower animals. As vibrios are widely distributed in the environment,
particularly in estuarine waters and in seafoods, reports of their isolation from patients
with diarrheal disease do not necessarily always imply an etiologic relationship.

Cholera-like vibrios have been reported in Maryland's Chesapeake Bay but have not
been associated with any human cases despite more than 15 years of extensive
surveillance. These vibrios are probably nonpathogenic nonagglutinable (non-O group
1) vibrios, or the atypical O group 1 vibrios mentioned above, which do not contain the
genes for toxin production, do not colonize, and are avirulent.

Relatively little is known about the epidemiology of nonagglutinable vibrios. When

sought, these vibrios have been found widely in brackish surface waters (sewers,
marshes, bogs, and coastal areas), and are generally more numerous in warmer months.
They appear to be free-living aquatic organisms; whether particular subsets are potential
pathogens is not yet clear. Strains isolated from humans with diarrheal disease more
frequently give positive responses in assays for enterotoxins or enteropathogenicity, but
the pathogenic mechanism of other isolates associated with shellfish remains
undefined.An epidemiologic pattern is more evident with V parahaemolyticus, which is
clearly part of the normal flora of coastal and estuarine waters throughout the world.
Although originally recognized in Japan, V parahaemolyticus enteritis has been reported
virtually worldwide within the last decade. Its reported frequency varies widely, partly
because of inherent differences in distribution and partly because many laboratories do
not use the appropriate culture medium (TCBS) to isolate these organisms. Two types
of clinical syndromes, both usually self-limited, have been observed. The most common
is a watery diarrhea, perhaps with associated abdominal cramps, nausea, vomiting, and
fever, with a modal incubation period of 15 hours. A dysenteric syndrome with a short
incubation period of 2 1/2 hours also has been described. In Japan, about 24 percent of
reported cases of food poisoning are attributed to V parahaemolyticus. The disease
occurs primarily during summer, possibly reflecting the increased presence of the
organism in the marine environment during those months, as well as the enhanced
opportunity for it to multiply in unrefrigerated foods. It appears to be transmitted
exclusively by food, primarily raw or improperly prepared seafood. As growth of this
organism is inhibited at temperatures below 15° C, rapid cooling and refrigeration of
seafoods that are eaten raw would vastly reduce the incidence of disease. The organisms
are killed by heating to 65° C for 10 minutes; therefore, properly handled cooked
seafood should present no problem. The role played in virulence and pathogenesis by
the thermostable direct hemolysin, which is responsible for the positive Kanagawa
phenomenon (a hemolytic reaction around colonies growing on a particular blood agar
medium), is not yet fully defined. This hemolysin is clearly associated with
pathogenicity, but whether it is merely an associated marker or intimately involved in
the disease process awaits further research. Be this as it may, only strains that possess
the Kanagawa hemolysin are considered pathogenic. In laboratory studies, the isolated
hemolysin has been reported to be cytotoxic, cardiotoxic, and lethal.
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