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

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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

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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

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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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