An adaptable organismUntil the late 1970s, the nOlmal habitatof V. cho/erae was thought by most tobe the human intestine, and it was notthought to be capable of surviving formore than a few days outside thishabitat. Other vibrios, both pathogenic(disease causing) and non-pathogenic,nonnally inhabit estuarine and marineenvironments, and so do some typesof V. cho/erae.

The disease cholera is caused by therelease of a toxin known as CT(choleragenic toxin) which not all V.cllO/erae can produce. V. cho/erae fromany subgroup mayor may not be ableto produce toxin but, unfortunately,testing for either the production or

as typhoid, hepatitis A, and most otherdian'hoeal diseases. The evidence forthis was a general failure to detect thecholera organism in water, except whencholera cases were in the immediatevicinity. While there is no doubt thatthe faecal-oral route is of major impor-tance in the development of secondarycases and in the spread of the diseasein most areas, it does not explainoutbreaks which occur where this routeis unlikely, such as in Louisiana in the

Table 2. Characterization of Vibrio cholerae.

The environmental transmission of choleraby Ann StoreyCholera transmission has been linked to waterfor over a hundred years, but faecalcontamination is not the only route to disease;new research points to other danger areas.

SINCE THE WORK done in Londonin 1854 by John Snow, and later byRobert Koch, it has been known boththat water is an important factor in thetransmission of cholera, and that waterfrom public supplies had been impli-cated in earlier pandemics (see Tablet). While water from treated publicsupplies does not appear to be a riskfactor in disease transmission in thepresent scventh pandemic, untreatedsurface water from lakes, ponds, ca-nals, and evcn wells continues to bean important source of infection.

presence of the toxin requires specialisttechniques and equipment. Finally,some V. cholerae can produce toxinsother than CT and may cause other,milder, illnesses.

The species V. cholerae is made upof many types or subgroups. Thesubgroup responsible for clinical chol-era is known as V. cho/erae 01, whichincludes two biotypes: 'Classical' and'EI Tor'. Both of these can also bebroken down into three serotypes,Ogawa, lnaba, and Hikojima (secTable 2). It is easy in a laboratory totest whether a V cholerae is 0 I or not:those found positive are designated01 s, those showing negative are desig-nated V. cho/erae non-O I (fOnllerly. non-agglutinating \'ihrios', althoughthey may agglutinate other antisera ifthese are available). Most V cllO/eraeisolated from the environment arenon-O Is. These were not usuallythought to cause cholera, althoughsome were found to produce all theknown toxins, including choleragenictoxin. and had caused small numbersof cases of diarrhoeal and cholera-likediseases.

In 1992, a major outbreak of choleracaused by V. cho/erae 0139, a non-Oltype, occurred in India, followed byanother outbreak of the same strain in1993 in Bangladesh, sparking fears ofan eighth pandemic before the seventhwas even over. At least 150000 people

Includes

EI Tor and Classical biotypes; Inaba, Ogawa, and Hikojimaserotypes. Both cholera toxin positive and negative strains.

013 serotypes including 0139. Other non-agglutinating V.cholerae. Both cholera toxin positive and negative strains.

V. cholerae non-01

Serotype

V. cholerae 01

United States, Using improved labora-tory techniques, Vihrio cho/erae hasnow been isolated in water even whenother faecal bacteria, in pal1icular Esch-erichia coli - the most widely ac-cepted indicator of pollution by faeces- were not present. This indicateseither than V. cho/erae can survivelonger in the environment than otherfaecal organisms, or that it can exist asan environmental organism in its ownright.

Oates

1817-231829-511852-591863-791881-961899-19231961-

Table 1. Cholera pandemics up tothe present day.

Pandemic number

1234567

It had generally been thought thatwater sources were contaminated byinfeCled human waste, and Ihal thegeneral spread of an outbreak was viathe faecal-oral route, as is the case withmany other important infections, such

a2;-'iiiill>C

::::lai

.~U5..s:UlcQJ.0oa:

While water Fom treated puhlic supplies does not appear to he a risk factor inthis pandemic, untreated slllface water cOllfinues to he a source of infection.

4 WATERLINES VOL. 12 NO.4 APRIL 1994

The existing methods for quantifying cholera are slow and complex.

toxin in laboratory tests. In fact toxin-producing strains do not lose the abilityto produce toxin after their introduc-tion to an aquatic environment.

Cholera in freshwaterSurface water that is generally used fordrinking has a salinity of less than 0.1per cent and organics below the levels

organism is shown to be seasonal in itsappearance, generally occurring duringthe wam1er months of the year orfollowing the monsoon, depending onthe part of the world. While in contactwith low salinity and relatively warmwater the organisms remain culturable.Once in contact with full-strength coldseawater «IO°C)they rapidly becomenon-culturable, but can still produce

Strict hygiene rules will prevent the contamination of water source~-

are believed to have been affected bythe end of 1993.

Whi Ie non-O I vihrios may be iso-lated in quite large numbers fromestuarine and marine waters, a Is areusually present only in low numbers,and most of these appear not to beproducing toxin. Much research hasbeen done on the survival of V.cholerue in different water types, in-cluding variations in salinity, organics,and temperatures. This research hasfound that when V. cholerae is addedto water, it adapts to its new environ-ment by becoming smal]er and slowingits metabolism to take into account itsnew nutrient-poor surroundings. It alsobecomes harder to grow and detect byconventional laboratory methods. Sur-veys have indicated that numbers of V.cholerae are at their highest whenwater temperatures are between 20°Cand 35°C, and at salinities between 1.0and 2.5 per cent, although this is veryvariable. Levels of organics above 1.0milligram per litre, which can oftenbe found in runoff from agriculturaland urban areas, decreases the organ-ism's need for salt. In addition V.cholerae is often found in highernumbers in aquatic plant life and insome fauna, especially crustacea andfilter feeders such as oysters.

When estuaries known to be con-taminated with V. cholerae are sam-pled over a period of a year, the

WATERLINES VOL.12 NO.4 APRIL 1994 5

Cholera has a high incidence in fishing coml1l11ni(ies in Bang/adesh. and many secondary cases arise (here.

be isolated from freshwater sourcesand, as in estuaries, it appears tosurvive there by adhering to waterplants, sediment, and crustacea. Ithas been found in algae, water hya-cinths, and in many other aquaticplants. In crustacea such as crabs andshrimp, V. cho/e/'(/e attaches to thechitin in their shells. It is not surprisingtherefore that another of the majorways of contracting cholera is byeating shellfish. This is the main waythat cholera is contracted in the south-ern states of the USA, where \'.cho/e/'(/e 0 I is known to be present inthe estuaries.

V. cho/e/'(/e does not tolerate dryconditions, and while it will survivein wet soil, it rapidly dies when it driesout. Likewise, it will survive in wethuman waste, but disappears when thatdrys. It is also killed off during somesewage treatment processes. especiallyanaerobic digestion.

Transmission by higher animalsdoes not appear to be important,although V. cllOle/'(/e non-O I has beenisolated from some water fowl andfish. It may be that waterfowl playarole in the transmission.

As previously discussed, V ('/101-

erae can maintain itself at low levelsin the aquatic environment, normallynot higher than 50 organisms per litre .The infecting dose for humans,however, is between 102 and lOS,depending on the individual. So forthe organisms in the water to cause

World Health OrganizationCollaborating Centre for theProtection of Drinking Water

Duality and Human Health

would seem to rule out colonizationby V. chole/'(/e, but V. cllO/e/'(/e 0 I can

Ins tit ute

Jennie LynchEnvironment Division

Robens InstituteUniversity of Surrey

GuildfordSurrey GU2 5XH

UK

Tel: 0483 259209; Fax: 0483 503517; Telex: 859331 UNIVSY G,

Environmental Monitoring and Managementin Developing Countries

An intensive ten week post-experience course for professionalsworking, or intending to work in developing countries

This annual course is to be held at the Robens Institute, University ofSurrey, from October to December 1994. The course will focus on thedevelopment of appropriate, low-cost monitoring activities, the linkage ofthese to preventative and remedial actions and monitoring as amanagement tool.

The principle areas of study include:• Environmental and public health• Environmental monitoring• Environmental quality and analysis• Environmental and health education• Technical and engineering interventions• Programme and project management• Sector planning and requirements

For further details on this and other courses please contact:

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which would negate the choleraorganisms salt requirement. This

6 WATERLINES VOL. 12 NO.4 APRIL 1994

Because of their working conditions fishermen have no option hut to he exposedto risk ji'om cholera in the aquatic environment.

disease, they must first multiplysomewhere. This could happen onfood or in stored water, or on infectedshellfish. Vihrios can stick to theshells of crabs in very high numbers,as well as colonizing the crab's hindgut, which also forms part of itsshell. This could then lead to a fewprimary cases caused by people eatinginfected crabs. In countries with goodhygiene and support services, such asin the USA, the number of secondarycases (caused by people infected bythe person infected by the crabs)would be low, but in countries whereservices were poor or non-existent,then the number of secondary casesresulting from the faecal-oral routecould be very high. Cholera ratesremain high in endemic areas evenwhen the population gets its drinking-water from supposedly uncontami-nated tubewells, because often thepeople are still bathing in contaminatedsurface water and eating shellfishfrom contaminated estuaries. InBangladesh, where cholera is en-demic and where it may haveoriginated, the peak cholera seasonsin rural areas are between Marchand April, and after the summermonsoon. This is not the season ofpeak salinity (which is pre-monsoon),but it is associated with a suddengrowth in algae and animal plankton,and a rise in both water recreation andthe consumption of shellfish. It has ahigh incidence in fishing communitiesand many secondary cases originatethere.

Testingv. cholerae can be isolated from theenvironment using modifications ofthe standard techniques used to culturefaecal bacteria from water. Thesemethods are described in referencebooks and involve concentrating agiven volume of water before testingfor the organism.4 The most suitableconcentration method is filtration us-ing membranes, borosilicate glass, ordiatomaceous earth. A filtration tech-nique using plastic containers packedwith gauze has been used successfullyby some workers. After concentration,the filter material is added to a givenvolume of an enrichment medium,usually alkaline peptone water. Fromhere the organisms can either becultured directly or a statistical MostProbable Number technique can beperformed. Alternatively, V. choleraecan be isolated directly from themembranes by laying the filtrationmembranes straight onto the selectivemedia. (This method grows lowernumbers of organisms than the other

methods.) Work being carried out atthe Robens Institute has shown that ifthe membranes are initially culturedonto an alkaline peptone water-soakedpad for four hours before transferringthe membrane onto selective agar, thenthe number of organisms isolated iscomparable to that obtained by themore laborious standard methods. Thismethod could be useful for workers inisolated areas because it does notrequire a full laboratory set-up, but canmake use of a field-test kit.

None of these methods will confirmV. cholerae's subgroups. For this it isnecessary to carry out more specializedtests in a laboratory. These includeconfirmation that the organism isindeed V. cholerae, subgroup determi-nation, and the detection of toxin-producing strains.

Other techniques are necessary todiscover non-culturable organisms;both fluorescent antibody tests andDNA probes have been used. Thesetechniques are both specialized and

expensive and are outside the scope ofmost workers in the field. They doprovide much higher counts than thoseobtained by culture alone, however,and so are of value in researchingfurther the environmental habitat of 1/.cholerae .•

ReferencesI. Cholera: Cllrre/ll IO{lics ill illli'clious di.l"-

ease. Barua and Greenough (cds). PlenumPress, London, IlJ92.

2. Colwell, Ed. Vihrios ill Ih(' /~'I1I'imllmelll,Wiley Inlerscience, Chichester, IlJ1l4.

3. 'New strains of Vihrio c/w{ef't/e () I.W in Indiaand Bangladesh: Lessons from the recentepidemics', Journal of Diarrhoeal Di.I·('aseReseardl, lin June IlJlJ3. pp.63-o.

4. Greenberg. Tussle and Clesceri (cds), SlulI-dun/ IIlt'lhod,,' jiil' Ihe ('.ramillalioll of wuleralld wuslewaler. APHA, AWA and WPCF.Washington DC, 19115.

AIIII Storey is a Sellior Micmhio{ogisl ill IheDiI'isioll of EII\'imll//l('/IIa{ lIeullh, RohelJs{IISlilll/e, Ulliversily (J{SIIIT('Y. Guile/I,ml, SIIITt',I',

GU2 5XII, UK.

WATERLINES VOL.12 NO.4 APRIL 1994 7