Cholera and its public health significance: a reviewBy Jemberu Alemu
Introduction
Foodborne illnesses are a continuous threat to public health with
increasing costs to society. One recent estimate is that the 31
major foodborne pathogens account for nearly 9.4 million people
becoming sick, more than 55,961 hospitalizations, and 1351 deaths
each year in the United States (Scallan et al., 2011) with an
estimated economic cost of $77.7 billion annually (Scharff, 2012).
Recent pathogenic outbreaks via food supplies and post-September 11
threats of bioterrorism have prompted the need for better detection
methodologies for foodborne pathogens and toxins that go beyond the
normal USDA, FDA and EPA concerns and into the realm of defense and
homeland security. Further, food safety concerns are not confined
to only the US, but are a global priority.
Cholera remains a major public health problem especially in
developing countries. The seventh pandemic of cholera which began
in1961 is still ongoing. In recent years, cholera cases have
steadily increased. In 2011, the cholera cases reported to WHO were
from 58 countries and accounted for 589,854 cases including 7,816
deaths (cholera, 2012). The most recent epidemic is striking in
Sierra Leone where over 20,000 cases including 280 deaths had been
reported before October 2012. Furthermore, the actual number of
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cholera cases is assumed to be much higher than those reported.
This discrepancy is attributed to the lack of dissemination of
effective surveillance system. Because under reporting or under
estimation impedes implementation of sufficient control measures,
further improvement in surveillance system, which contributes
largely to determine the true number of incidences, is still
required. Cholera is a severe diarrheal disease caused by the
bacterium Vibrio Cholerae, produced cholera toxin. Cholera toxin
transmits to humans frequently by water and food. This toxin is an
AB5 hexameric protein, composed of five identical B subunits and a
single A subunit. The A subunit is surrounded by five B subunits,
which is the potent activator of adenylate cyclase and the
pathogenic agent responsible for the symptoms of cholera (Dick et al,
2012 and Yamazaki, 2008). The B subunits are the nontoxic binding
components of the CT holotoxin and function in pentameric form to
specifically recognize the GM1 ganglioside receptor present on the
surface of mucous cells ( Palchetti and Mascini, 2008).
Currently, several diagnostic procedures including the “gold
standard” of culture test and rapid diagnostic tests are available
for V. cholerae detection (Dick et al, 2012). In culture test,
alkaline Peptone water (APW) and TCBS are commonly used as
enrichment and selective media, respectively. As many have noted,
the cultivation test is time consuming, but it has the advantage of
being able to isolate the causative bacterium which can be used for
further characterization. On the other hand, utility of various
rapid diagnostic tests such as polymerase chain reaction (PCR),
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quantitative PCR (qPCR), Loop mediated isothermal amplification
(LAMP), enzyme-linked immune sorbent assay (ELISA), reverse passive
latex agglutination test (RPLA), and immune chromatographic test
(IC) has been demonstrated. These rapid methods facilitate timely,
in some cases, on site responses. And, the rapid detections in
early stage of epidemic allow quick triggering of control measures.
In the case of diagnosis of cholera, after or along with the
detection of bacterium, verification of cholera toxin (CT)
production is required because only the V. cholerae which can
produce CT is responsible for cholera symptoms such as acute “rice
water” diarrhea. Some detection methods for toxigenic V. cholerae
have been described previously. The approaches to assay for CT can
be divided in terms of features to be detected: (1) bioassay
including rabbit ileal loop test, rabbit skin test, and cultured
CHO cell assay, (2) immune assay including ELISA and IC, and
(LMDVC,1999) DNA based assay including PCR, qPCR, DNA
hybridization, and LAMP ( Palchetti and Mascini, 2008). Combined
use of more than one detection method would be required to increase
the accuracy of a diagnosis. At that time, combination of different
target analytes; for example, immunoassay which detects the
existence of toxin and DNA based assay which detects the existence
of toxin coding DNA must be chosen. While DNA based assays may be
more sensitive than immunoassays, the latter has an important
advantage in the detection of extracellular bacterial toxin.
Recently, some new methodology of immunoassay with extremely high
sensitivity has been reported (Palchetti and Mascni, 2008:
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Shlyapnikove et al, 2012). However, RPLA is still one of the most
commonly utilized immunoassays because it is rapid and very easy to
conduct.
Taxonomy and serological classification
Vibrio cholerae, a member of the family Vibrionaceae, is a
facultatively anaerobic, Gram negative, non-spore forming curved
rod, about 1.4–2.6μm long, capable of respiratory and fermentative
metabolism; it is well defined on the basis of biochemical tests
and DNA homology studies (Bauman et al, 1984). The bacterium is
oxidase positive, reduces nitrate, and is motile by means of a
single, sheathed, polar flagellum. Growth of V. cholera is
stimulated by addition of 1% sodium chloride (NaCl). However, an
important distinction from other Vibrio spp. is the ability of V.
Cholerae to grow in nutrient broth without added NaCl.
Differences in the sugar composition of the heat stable surface
somatic “O” antigen are the basis of the serological classification
of V. cholerae first described by Gardner and Venkatraman (1935);
currently the organism is classified into 206 “O” serogroups
(Shimada et al., 1994; Yamai et al., 1997). Until recently, epidemic
cholera was exclusively associated with V. cholerae strains of the
O1serogroup. All strains that were identified as V. cholerae on the
basis of biochemical tests but that did not agglutinate with “O”
antiserum were collectively referred to as non-O1 V. cholerae. The
non-O1 strains are occasionally isolated from cases of diarrhoea
(Ramamurthy et al., 1993a) and from a variety of extra intestinal4
infections, from wounds, and from the ear, sputum, urine, and
cerebrospinal fluid (Morris & Black, 1985). They are ubiquitous in
estuarine environments, and infections due to these strains are
commonly of environmental origin (Morris, 1990). The O1 serogroup
exists as two biotypes, classical and El Tor; antigenic factors
allow further differentiation into two major serotypes—Ogawa and
Inaba. Strains of the Ogawa serotype are said to express the A and
B antigens and a small amount of C antigen, whereas Inaba strains
express only the A and C antigens. A third serotype (Hikojima)
expresses all three antigens but is rare and unstable. The
classical biotype was responsible for the fifth and sixth pandemics
and is believed to have been associated with the earlier pandemics
as well, although there is no hard evidence. The causative agent of
the seventh and current cholera pandemic, which began in 1961, is
the El Tor biotype. The classical biotype has been completely
displaced worldwide, except in Bangladesh where it reappeared in
epidemic proportions in 1982 (Samadi et al., 1983), remained
prominent there for a few years, and now seems to have become
extinct again (Siddique et al., 1991). The simple distinction between
V. cholerae O1 and V. cholerae non-O1 became obsolete in early 1993
with the first reports of a new epidemic of severe, cholera like
disease in Bangladesh (Albert et al., 1993) and India (Ramamurthy et
al., 1993b). At first, the responsible organism was referred to as
non-O1 V. cholerae because it did not agglutinate with O1
antiserum. However, further investigations revealed that the
organism did not belong to any of the O serogroups previously
5
described for V. cholerae but to a new serogroup, which was given
the designation O139 Bengal after the area where the strains were
first isolated (Shimada et al., 1993). Since recognition of the O139
serogroup, the designation non-O1 non-O139 V. cholerae has been
used to include all the other recognized serogroups of V. cholerae
except O1 and O139 (Nair et al., 1994a).
The emergence of V. cholerae O139 as the new serogroup associated
with cholera, and its probable evolution as a result of horizontal
gene transfer between O1 and non-O1 strains (Bik et al., 1995), has
led to a heightened interest in the V. cholerae non-O1 non-O139
serogroups. There is evidence for horizontal transfer of O antigen
among V. cholerae serogroups; Karaolis, Lan and Reeves (1995)
reported that isolates of nearly identical as gene (chromosomal
housekeeping gene, which encodes aspartate semi aldehyde
dehydrogenase) sequences had different O antigens and that isolates
with the O1 antigen did not cluster together but were found in
different lineages. There has been elevated activity of the non-O1
non-O139 serogroups in the recent past, and localized outbreaks of
acute diarrhoea caused by V. cholerae serogroups such as O10 and
O12 have been reported (Dalsgaard et al., 1995; Rudra et al., 1996).
General Characteristics of Pathogenic Vibrio species
The genus Vibrio contains at least twelve species pathogenic to
humans, ten of which can cause food borne illness. The majority of
food borne illness is caused by V. parahaemolyticus, cholera genic
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Vibrio cholerae, or Vibrio vulnificus. V. parahaemolyticus and V.
cholerae are solely or mainly isolated from gastroenteritis cases
that are attributable to consumption of contaminated food (both
species) or intake of contaminated water (V. cholerae). In
contrast, V. vulnificus is primarily reported from extra intestinal
infections (septicaemia, wounds, etc.) and primary septicaemia due
to V. vulnificus infection is often associated with consumption of
seafood
In tropical and temperate regions, these species of Vibrio occur
naturally in marine, coastal and estuarine (brackish) environments
and are most abundant in estuaries. Pathogenic Vibrio spp., in
particular V. cholerae, can also be recovered from freshwater
reaches of estuaries, where it can also be introduced by faecal
contamination. V. cholerae, unlike most other Vibrio species, can
survive in fresh water environments.
It is now possible to differentiate environmental strains of V.
cholerae and V. parahaemolyticus between virulent and avirulent
strains based on their ability or inability to produce their major
virulence factors. The pathogenic mechanisms of V. vulnificus have
not been clearly elucidated, and its virulence appears to be
multifaceted and is not well understood, and therefore all strains
are considered virulent.
The following are important characteristics common to all Vibrio
spp. Vibrio spp. are sensitive to low pH but grow well at high pH,
and thus infections caused by Vibrio spp. are frequently associated
7
with low-acid foods. In addition, the ingestion of a large number
of viable cells is needed for pathogenic Vibrio spp. to survive the
acidic environment of the stomach and establish an infection.
Cooking of food products readily inactivates Vibrio spp. even in
highly contaminated products. Hygienic practices used with all food
borne pathogens will in general control the growth of pathogenic
Vibrio spp.
There are, however, characteristics specific to each of the three
major pathogenic species of Vibrio that require attention as
described below.
1. Vibrio parahaemolyticus
Vibrio parahaemolyticus is considered to be part of the
autochthonous micro flora in the estuarine and coastal
environments in tropical to temperate zones. While V.
parahaemolyticus typically is undetectable in seawater at 10°C
or lower, it can be cultured from sediments throughout the
year at temperatures as low as 1°C. In temperate zones, the
life cycle consists of a phase of survival in winter in
sediments and a phase of release with the zooplankton when the
temperature of the water increases up to 14 - 19 °C.
V.parahaemolyticus is characterized by its rapid growth under
favourable conditions.
The vast majority of strains isolated from patient’s with
diarrhoea produce a thermostable direct hemolysin (TDH). It
has therefore been considered that pathogenic strains possess8
a tdh gene and produce TDH, and nonpathogenic strains lack the
gene and the trait. Additionally, strains that produce a TDH
related hemolysin (TRH) encoded by the trh gene should also be
regarded as pathogenic. Symptoms of V. parahaemolyticus
infections include explosive watery diarrhoea, nausea,
vomiting, abdominal cramps and, less frequently, headache,
fever and chills. Most cases are self-limiting; however,
severe cases of gastroenteritis requiring hospitalization have
been reported. Virulent strains are seldom detected in the
environment or in foods, including sea foods, while they are
detected as major strains from feaces of patients.
Vibrio parahaemolyticus was first identified as a foodborne
pathogen in Japan in the 1950s. By the late 1960s and early
1970s V. parahaemolyticus was recognized as a cause of
diarrhoeal disease worldwide. A new V. parahaemolyticus clone
of O3:K6 serotype emerged in Calcutta in1996. This clone,
including its serovariants, has spread throughout Asia and to
the USA, elevating the status of the spread of V.
parahaemolyticus infection to pandemic. In Asia, V.
parahaemolyticus is a common cause of foodborne disease. In
general, the outbreaks are small in scale, involving fewer
than 10 cases, but occur frequently. This pandemic V.
parahaemolyticus has now spread to at least 5 continents.
There is a suggestion that ballast discharge may be a major
mechanism for global spread of pandemic V. parahaemolyticus,9
but a possibility of export/import seafood-mediated
international spread cannot be ruled out.
From the point of controlling seafood borne V.
parahaemolyticus illnesses, harvest is probably the most
critical stage, since it is from this point onwards that
individuals can actually implement measures to control V.
parahaemolyticus.
Foods associated with illnesses due to consumption of V.
parahaemolyticus include for example crayfish, lobster,
shrimp, fish-balls, boiled surf clams, jack-knife clams, fried
mackerel, mussel, tuna, seafood salad, raw oysters, clams,
steamed/boiled crabmeat, scallops, squid, sea urchin, mysids,
and sardines. These products include both raw and partially
treated and thoroughly treated seafood products that have been
substantially re contaminated through contaminated utensils,
hands, etc.
2. Vibrio cholerae
Vibrio cholerae is indigenous to fresh and brackish water
environments in tropical, subtropical and temperate areas
worldwide. Over 200 O serogroups have been established for V.
cholerae. Strains belonging to O1 and O139 serotypes generally
possess the ctx gene and produce cholera toxin (CT) and are
responsible for epidemic cholera. Epidemic cholera is confined
mainly to developing countries with warm climates. Cholera is
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exclusively a human disease and human feaces from infected
individuals are the primary source of infection in cholera
epidemics. Contamination of food production environments
(including aquaculture ponds) by feaces can indirectly
introduce cholera genic V.cholerae into foods. The
concentration of free-living cholera genic V. cholerae in the
natural aquatic environment is low, but V. cholerae is known
to attach and multiply on zooplankton such as copepods.
Seven pandemics of cholera have been recorded since 1823.The
first six pandemics were caused by the classical biotype
strains, whereas the seventh pandemic that started in 1961 and
has lasted until now, is due to V. cholerae O1 biotype El Tor
strains. Epidemic cholera can be introduced from abroad by
infected travellers, imported foods and through the ballast
water of cargo ships. Detection frequencies of cholera genic
strains of V. cholerae from legally imported foods were very
low and they have seldom been implicated in cholera outbreaks.
V. cholerae O139 has been responsible for the outbreaks of
cholera in the Bengal area since 1992, and this bacterium has
spread to other parts of the world through travellers. The
cholera genic strains of V. cholerae that spread to different
parts of the world may persist, and some factors may trigger
an epidemic in the newly established environment.
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Some strains belonging to the O serogroups other than O1 and
O139 (referred as non-O1/non-O139) can cause food borne
diarrhoea that is milder than cholera.
Outbreaks of food borne cholera have been noted quite often in
the past 30 years; seafood, including bivalve molluscs,
crustaceans, and finfish, are most often incriminated in food-
borne cholera cases in many countries. While shrimp has
historically been a concern for transmission of cholera genic
V. cholerae in international trade, it has not been linked to
outbreaks and it is rarely found in shrimp in international
trade.
3. Vibrio vulnificus
Vibrio vulnificus can occasionally cause mild gastroenteritis
in healthy individuals, but it can cause primary septicaemia
in individuals with chronic pre-existing conditions,
especially liver disease or alcoholism, diabetes,
haemochromatosis and HIV/AIDS, following consumption of raw
bivalve molluscs. This is a serious, often fatal, disease with
one of the highest fatality rates of any known foodborne
bacterial pathogen. The ability to acquire iron is considered
essential for virulence expression of V. vulnificus, but a
virulence determinant has not been established and, therefore,
it is not clear whether only a particular group of the strains
are virulent. The host factor (underlying chronic diseases)
appears to be the primary determinant for V. vulnificus infection.
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Incubation period ranges from 7 hours to several days, with
the average being 26 hours. The dose response for humans is
not known.
Of the three biotypes of V. vulnificus, biotype 1 is generally
considered to be responsible for most seafood-associated human
infection and thus the term V. vulnificus refers to biotype 1 in
this Code.
Foodborne illness from V. vulnificus is characterized by sporadic
cases and an outbreak has never been reported. V. vulnificus has
been isolated from oysters, other bivalve molluscs, and other
seafood worldwide.
The densities of V. vulnificus are high in oysters at harvest when
water temperatures exceed 20°C in areas where V. vulnificus is
endemic; V. vulnificus multiplies in oysters at a temperature
higher than 13°C. The salinity optimum for V. vulnificus appears
to vary considerably from area to area, but highest numbers
are usually found at intermediate salinities of 5 to 25 g/l
(ppt: parts per thousand). Relaying oysters to high salinity
waters (>32 g/l (ppt: parts per thousand) was shown to reduce
V. vulnificus numbers by 3–4 logs (<10 per g) within 2 weeks.
Pathogenicity for humans, and virulence factors
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The major features of the pathogenesis of cholera are well
established. Infection due to V. cholera begins with the ingestion
of contaminated water or food. After passage through the acid
barrier of the stomach, the organism colonizes the epithelium of
the small intestine by means of the toxin-coregulated pili (Taylor
et al., 1987) and possibly other colonization factors such as the
different haemagglutinins, accessory colonization factor, and core-
encoded pilus, all of which are thought to play a role. Cholera
enterotoxin produced by the adherent vibrios is secreted across the
bacterial outer membrane into the extracellular environment and
disrupts ion transport by intestinal epithelial cells. The
subsequent loss of water and electrolytes leads to the severe
diarrhoea characteristic of cholera. The existence of cholera
enterotoxin (CT) was first suggested by Robert Koch in 1884 and
demonstrated 75 years later by De (1959) and Dutta, Pause and
Kulkarni (1959) working independently. Subsequent purification and
structural analysis of the toxin showed it to consist of an A
subunit and 5 smaller identical B subunits (Finkelstein and
LoSpalluto, 1969). The A subunit possesses a specific enzymatic
function and acts intracellularly, raising the cellular level of
cAMP and thereby changing the net absorptive tendency of the small
intestine to one of net secretion. The B subunit serves to bind the
toxin to the eukaryotic cell receptor, ganglioside GM1. The binding
of CT to epithelial cells is enhanced by neuraminidase. Apart from
the obvious significance of CT in the disease process, it is now
clear that the production of CT by V. cholerae is important from
14
the perspective of a serogroup acquiring the potential to cause
epidemics. This has become particularly evident since the emergence
of V. cholerae O139. A dynamic 4.5-kb core region, termed the
virulence cassette (Trucksis et al., 1993), has been identified in
toxigenic V. cholerae O1 and O139 but is not found in non-toxigenic
strains. It is known to carry at least six genes, including
ctxAB(encoding the A and B subunits of CT), zot(encoding zonula
occludens toxin (Fasano et al., 1991)), cep(encoding core-encoded
pilin (Pearson et al., 1993)), ace(encoding accessory cholera
enterotoxin (Trucksis et al., 1993)), and orfU(encoding a product
of unknown function (Trucksis et al., 1993)). In the El Tor biotype
of V. cholerae, many strains have repetitive sequence (RS)
insertion elements on both sides of the core region; these are
thought to direct site-specific integration of the virulence
cassette DNA into the V. cholerae chromosome (Mekalanos, 1985;
Goldberg and Mekalanos, 1986; Pearson et al., 1993). The core
region, together with the flanking RS sequences, makes up the
cholera toxin genetic element CTX (Mekalanos, 1983). Recent
studies have shown that the entire CTX element constitutes the
genome of a filamentous bacteriophage (CTXf). The phage could be
propagated in recipient V. cholerae strains in which the CTXf
genome either integrated chromosomally at a specific site, forming
stable lysogens, or was maintained extra-chromosomally as a
replicative form of the phage DNA (Waldor & Mekalanos, 1996).
Extensive characterization of the CTXf genome has revealed a
modular structure composed of two functionally distinct genomes,
15
the core and RS2 regions. The core region encodes CT and the genes
involved in phage morpho-genesis, while the RS2 region encodes
genes required for replication, integration, and regulation of CTXf
(Waldor et al., 1997). Generally, CTXf DNA is integrated site-
specifically at either one (El Tor) or two (classical) loci within
the V. cholerae genome (Mekalanos, 1985). In El Tor strains, the
prophage DNA is usually found in tandem arrays that also include a
related genetic element known as RS1. The RS1 element contains the
genes that enable phage DNA replication and integration, plus an
additional gene (rstC) whose function is unknown but that does not
contain ctxAB or the other genes of the phage core region that are
thought to produce proteins needed for virion assembly and
secretion (Davis et al., 2000). CTXf gains entry to the V. cholera
cell by way of the toxin regulated pili the surface organelles
required for intestinal colonization. Its genes are then
incorporated into host chromosome, inducing the cell to secrete CT.
The zot gene increases the permeability of the small intestinal
mucosa by an effect on the structure of the intestinal tight
junctions (Fasano et al., 1991), while ace affects ion transport in
the intestinal epithelium. Another factor whose gene resides
outside the CTX genetic element and which is thought to contribute
to the disease process is haemolysin/cytolysin (Honda and
Finkelstein, 1979). In contrast to the watery fluid produced by CT,
the haemolysin can cause accumulation in ligated rabbit ileal loops
of fluid that is bloody with mucous (Ichinose et al., 1987). Although
not fully characterized, other toxins produced by V. cholera
16
include the shiga like toxin (O’Brien et al., 1984), a heat-stable
enterotoxin (Takeda et al.,1991), new cholera toxin (Sanyal et al.,
1983), sodium channel inhibitor (Tamplin et al., 1987),
thermostable direct haemolysin like toxin (Nishibuchi et al.,
1992), and a cell-rounding cytotoxic enterotoxin known as the non-
membrane-damaging cytotoxin (Saha, Koley and Nair, 1996; Saha and
Nair, 1997).
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