report commissioned by the department of the...

of 56

Review of Disinfection and Associated Studies

on Cryptosporidium

Report commissioned by the Department of theEnvironment, Transport and the Regions (DETR),managed by the Drinking Water Inspectorate (DWI), fromYorkshire Environmental Alcontrol, UK.

Compiled by DP Casemore, Public Health Laboratory Service CryptosporidiumReference Unit (PHLS CRU), Rhyl, LL18 5UJ;

With John Watkins*, Alcontrol, Rotherham

(Alcontrol contract Ref. ALC 1796).

*Now of CREH Analytical Limited, Leeds.

of 56

Contents Page No

Executive Summary 3

1. Introduction 4-6

2. Disinfection - basic principles7-9

3. Studying the efficacy of disinfection10-11

4. Characteristics of different “strains” of oocysts 12

5. Methods for the collection, concentration, and storage of Cryptosporidium oocysts 13-15

6. Viability assessment methods16-20

7. General discussion of models used 21-23

8. Published studies 24-30

9. Conclusions 31

10. Tabulation of published study features 32-37

11. Bibliography 38-55

Acknowledgements 56

of 56

Review of Disinfection and Associated Studieson Cryptosporidium

Executive Summary

The project here reported was undertaken to review the published literature ondisinfection studies on Cryptosporidium. The purpose was to assess the variations inexperimental models and the effect of such variables on the results. A wide variety ofdisinfectants have been used to study the inactivation of Cryptosporidium parvum forclinical, veterinary and water treatment purposes. Test parameter variables include, pH,time of exposure, temperature, presence or absence of organic material, use of aneutralizing step, and measurement of disinfectant exposure and of post exposureresidual.

Of particular note is that such studies have employed oocysts of often ill definedcharacteristics, from different sources, which have been cleaned using differentprotocols and stored, with or without preservatives, under different conditions fordifferent lengths of time and temperature. All studies have been conducted usinganimal-derived oocysts, which are thus probably all genotype 2. No studies have usedthe apparently human-specific genotype 1, which has been responsible for a number ofmajor waterborne outbreaks. Oocysts have proved inherently difficult or impossible topreserve in the way that other inocula for conventional disinfection studies can bepreserved.

The assessment of disinfection efficacy for water treatment purposes presents specialdifficulties, and has been done using various methods. These include, excystation byvarying protocols, with and without measurement of sporozoite ratios, by dyeinclusion/exclusion, and by infectivity assessment using tissue culture or animalinoculation methods; in some cases additional disinfectant treatment has been a part ofthe excystation protocol. In disinfection studies for other potential pathogens it has notbeen the practice to demonstrate infectivity as opposed to viability.

These factors have led to poor standardisation of disinfection study protocols andrender the comparison of different research projects and their findings almostimpossible. In order to provide consistent and comparable test data, these variablesneed to be standardised. Whilst many research papers describe the test parameters insome detail, there is little critical appreciation of the fundamental importance of theneed particularly for standardisation of oocyst preparations for use in disinfectionstudies.

of 56

1. Introduction

The concept of the transmissibility of disease by means of water has existed sinceclassical times, and in the nineteenth century led to the recognition of the need forprotection or disinfection of potable water supplies, historically for bacterial pathogenscausing such diseases as typhoid and cholera. Subsequently, the use of faecal indicatororganisms such as Escherichia coli was introduced, including as a means of assessingdisinfection. Methods traditionally used to disinfect water for drinking purposesinclude chlorine and chlorine dioxide, ozone, and ultraviolet (Anon, 1990; Casemore,1995; Gates et al., 1989; Mc Feters, 1990; Russell et al., 1992). Generally speaking,water treatment has served public health well; the last outbreak of waterborne typhoidin the UK occurred in 1937 (Galbraith et al., 1987, Stanwell-Smith, 1994). However,recognition has grown of the importance of preventing the waterborne transmission ofthe enteric protozoan parasites, Giardia and Cryptosporidium, which are generallymore resistant to normal levels of water disinfection Anon, 1990; Casemore, 1990;Rose, 1990; West and Smith, 1995; Lisle and Rose, 1995; Meinhardt et al., 1996.

Disinfection can be defined as the removal, destruction or inactivation of infectiousagents. In its broadest sense, it includes both chemical and physical methods. A widevariety of disinfection procedures have been evaluated for their efficacy against C.parvum. Early disinfection studies on Cryptosporidium pre-dated awareness of theproblems of transmission by the water route. This applies particularly to studies donefor veterinary and hospital infection control purposes. The recognition of the problemof enteric protozoa in water led to the testing, using traditional methods and also novelmethods of treatment and disinfection, for efficacy against such parasites (Anon, 1990;Campbell et al., 1995; Casemore, 1995; Clancy et al., 1997 b and c; Finch et al., 1995;Korrich et al., 1990; Presdee et al., 1995; Robertson et al., 1994; Rose, 1990; Rose etal., 1997; Smith and Rose, 1998). Because of their innate resistance, however, reportsindicated that chemical disinfection was required at a level or concentration that cancause problems for other water treatment needs, such as the production of higher levelof disinfection by-products (DBPs) (Craun, 1988; Meyerhofer, 1988; LeChevallier andNorton, 1995; Pontius, 1997). Because of this, assays for efficacy are critical and yetsuch studies have used variable and often poorly defined parasite inocula and other testparameters, and reports often lack sufficient detail on this aspect (Casemore, 1995;Kloniki et al., 1997).

Physical methods for killing or removing Cryptosporidium have also been evaluated,many based on methods currently employed in water treatment works such as filtration,coagulation and sedimentation. Some have been laboratory or pilot plant scale studies(Hall et al., 1995; Ongerth and Pecoraro, 1995; Robertson and Smith, 1992) while otherstudies have attempted to measure the efficiency of oocyst removal during differentphysical processes at full-scale treatment works (Bellamy et al., 1993; Robertson et al.,1994). The effects of known physical disinfection exposures such as heat (Barbee etal., 1997; Anderson, 1985; Carrington and Ransome, 1994; Harp et al., 1996;Whitmore and Robertson, 1995; Fayer, 1994), drying (Anderson, 1986; Robertson etal., 1992), and ultra violet light (Bolton et al., 1998; Campbell et al., 1995; Clancy etal., 1997c; Lorenzo et al., 1993;), have also been examined for their effect onCryptosporidium. Other approaches have involved novel and developing techniques

of 56

such as electrostatic (charged resin) retention (Upton et al., 1988), modifications ofultrafiltration, depth filtration and coagulation/separation techniques e.g. magnetitetreatment (Robertson et al., 1994), pulsed light treatment (Arrowood et al., 1997),ultrasound (Watkins, 1995), and even the effects of microbial antagonism in sourcewater (Chauret et al., 1998; Medema et al 1997).

Disinfection studies are known to be affected by a number of variables, including testparameters such as pH, time of exposure, temperature of test exposure, presence orotherwise of organic (background demand) load, use of a specific test agent neutralisingstep, etc. A significant but under-recognised variable is the test organism used, in thiscase the oocysts of Cryptosporidium, their origin, characteristics, quality and quantityand the effect of different cleaning and concentration methods.

1.1 Early disinfection studies on Cryptosporidium oocysts

Early studies, which concentrated on the testing of compounds of veterinary and ofmedical interest (Angus et al., 1982; Campbell et al., 1982; Pavlasek, 1984; Blewett,1989; Casemore et al., 1989), were conducted in various (non-water) laboratories usingdifferent strains of the parasite, often of undefined characteristics in relation to:

• source host,• stage of infection cycle,• period elapsed since collection,• pre-test methods of preparation,• conditions of storage, etc• percentage viability,• characterisation.

They were assessed for viability by in vitro excystation or in vivo, usually byintragastric inoculation of suckling mice, a technically skilled procedure.

Such early studies showed that oocysts were largely unaffected by normal workingstrength dilutions of many common proprietary disinfectants and other recogniseddisinfecting agents, including UV, tested under appropriate conditions. Disinfectanttypes tested included aldehydes, cresols, halogens, quaternary ammonium compounds,alcohols, etc. Strong hydrogen peroxide (3%, 10vol) was found to be rapidly effectiveand has a place in dealing with spillages of oocyst suspensions in laboratory work(Blewett, 1989). Compounds also found to be effective included strong ammonia andsodium hydroxide solutions, which were generally impracticable for use but havesometimes been used for cleaning of infected animal accommodation (Angus et al.,1982; Pavlasek, 1984; Sunderman et al., 1987). It has subsequently been found thatthe ammonia present in slurry stores and animal muck heaps have a significantdisinfecting effect, in addition to the effects of pH and increased temperature throughfermentation (Bukhari and Smith, 1997; Bukhari et al., 1995; Jenkins et al., 1998;Ruxton, 1995). This is of significance for the water industry in relation to theproblems of leakage of agricultural wastes.

of 56

The treatment of human associated effluents may also impact on the water industry.For example, the inappropriate use of an ammonia based veterinary compound indealing with a faecally contaminated bathing pool (Watkins, J, pers comm) led toserious damage to an activated sludge treatment works when the contents of the poolwere disposed of to the mains sewers serving the urban area in which the leisurecomplex was sited. It is thus clear that suitable means for disinfection in suchsituations needs to be predicated on the needs of the water industry and its widercontext.

of 56

2. Disinfection - Basic Principles

2.1 The action of disinfectants

An understanding of the underlying processes involved in disinfection is essential tothe understanding of the testing of disinfection for Cryptosporidium. In 1908, Watsondescribed, and subsequently Chick modified, a general formula for the action ofdisinfectants (see Russell et al., 1992) according to concentration (C) and time (T). Inpractice, the assumption is made that η (the coefficient of dilution) = 1, for whichcondition both parameters are equal in importance. However, the effect of variations intemperature, and of pH, may be significantly unpredictable influences, particularly onthe activity of some compounds such as chlorine and ozone. Nonetheless, CT hastraditionally provided a useful rule of thumb for the indication of requirements forsatisfactory disinfection against bacteria, for operational use.

These parameters for the estimation of the efficacy of disinfection are predicated on theassumption that,

• the disinfectant reacts chemically as a first order reaction (time and concentrationbeing directly, inversely proportional), and

• that the contribution of the organism to the reaction is of zero order.

This, again, conveniently permits or supports the measure of the efficacy of adisinfectant solely in terms of time and concentration (CT). Although the assumptionmay hold true for most practical purposes this may not be adequate for a properscientific assessment of activity and the optimisation of disinfection (Armstrong et al.,1995a and b; Casemore, 1995). Given that many tests suggest disinfection efficacy atCT values that are at the limit of what is acceptable for other purposes (e.g. productionof DBPs), or is operationally practicable, these considerations may be crucial.

Microbial disinfection studies have traditionally been based primarily on models forbacterial inactivation, usually using standard "strains", which can be supplied fromrecognized sources (e.g. NCTC, ACTC). Stocks of well-characterised organisms can becultured under defined and controlled conditions, and stored under stable conditions(lyophilised or frozen at ultra-low temperature). For use, they can then be reconstitutedand grown in a controlled way, and their post-exposure viability determined by culturalmethods, for both inhibitory and cidal effects (Russell et al., 1992) having removed orneutralised all traces of the test compound prior to these tests to prevent effects fromcontinuing residual activity. Although not generally appropriate for water treatmentpurposes, potentially interfering substances such as protein may be incorporated intothe test model to reflect "in-use" conditions for general domestic and clinical use(Blewett, 1989; Casemore et al., 1989; Russell et al., 1992).

For a number of water treatment options, especially chemical disinfection, infectiveagents which need to be killed or inactivated form a gradient of responsiveness inwhich enteric parasites are the most resistant to conventional compounds and methodsor in-use conditions of exposure. The relative resistance, and the difficulties of testing,of parasitic protozoal transmissible forms (cysts and oocysts) began to be recognised,

of 56

primarily from work on Giardia (Dowd and Pillai, 1997; Hibler and Hancock, 1990;Jarroll et al., 1984; Labatiuk et al., 1991; Russel et al., 1992). Because of the obligateparasitism of the organism, means have had to be developed for assessing viability oforganisms exposed to test substances, which do not depend on conventional in vitrocultivation. Nonetheless, these tests clearly showed not only the relative resistance ofGiardia cysts, compared with vegetative bacteria, but also the significant contributionto the result of variations of factors such as pH and temperature (Hibler and Hancock,1990).

2.2 The problem posed by Cryptosporidium

In recent years, cryptosporidiosis has emerged as an important waterborne infectiousdisease of both the immuno-compromised and the otherwise healthy subjects(Casemore, 1990; Fayer, 1997). The recognition of waterborne cryptosporidiosis,primarily in the USA and the UK, has led to considerable public health concerns anddoubts about the safety of water supplies (Anon, 1990, 1995; Dawson and Lloyd 1994;Meinhardt et al., 1996). The direct cost to the community at large of significantcommunity-wide outbreaks of waterborne cryptosporidiosis is considerable, includinghealth, financial, legal and political impacts. Indirect but significant impact alsoincludes factors such as loss of public confidence in water quality and safety.

In the United States, Giardia accounts for the largest number of waterborne outbreakswhere a specific cause can be identified, while Cryptosporidium accounts for thelargest number of cases (Mc Feters, 1990). In recent years there have been a number ofoutbreaks of waterborne cryptosporidiosis in the UK (Anon, 1990; Casemore, 1990;Meinhardt et al., 1996; Furtado et al., 1997). These have encouraged large-scaleinvestment in research and development to determine inter alia laboratory evidence ofthe effect of disinfectants on Cryptosporidium and to investigate optimum watertreatment regimens (Anon, 1990, 1995, 1998; Casemore, 1995; Robertson et al., 1994;Presdee et al., 1995; West and Smith, 1995).

In the case of C. parvum, the transmissible form, the oocyst, consists of an outer,relatively robust shell, enclosing four sporozoites, the infective form. While exposedsporozoites are readily killed, the outer shell confers protection and thus relativeresistance to a wide range of disinfectants, etc. The outer shell is not, however, inertundergoing natural changes to its structure and may be affected by a variety ofenvironmental influences, both physical and chemical, which can affect permeability todisinfectants (Johnson et al., 1997; Parker and Smith, 1993; Robertson et al., 1992).

There is evidence that exposure to environmental stresses affect oocysts and mayenhance the action of disinfectants (Bukhari and Smith, 1995; Carrington & Ransome,1994; Drozd & Schwartzbrod, 1996; Johnson et al., 1997; Robertson et al., 1992). Theaddition of chlorine has been found to be more effective in bringing about the loss ofviability after sand mixing (Battigelli et al., 1996; Parker and Smith, 1993), but to alesser extent after freezing of oocysts at -20°C (Battigelli et al., 1996). The viability ofCryptosporidium oocysts stored in reservoir water at 4°C for 56-70 days and subjectedto a 500 PSI pressure drop was unaffected by exposure to 2.5 mgl-1 chlorine for 300min (Straub et al., 1994).

of 56

Johnson et al., (1997) and Medema et al 91997) showed that temperature and thepresence of light, salinity and water quality can all affect oocyst survival. The effect ofthese factors on disinfectant assays is unknown but it clearly emphasises the importanceof standardisation in test models particularly with reference to the source and age of theoocysts.

of 56

3. Studying the efficacy of disinfection

It is essential that disinfection studies should identify methods or forms of treatment,which provide optimum disinfection of Cryptosporidium with the minimum productionof DBPs or other unwanted side effects, at an acceptable cost. To ensure that the dataused to inform such decisions are valid, laboratory and pilot plant level studies need tobe based on defensible and demonstrably valid scientific methods and systems ofanalysis. Doubts have existed for some years now on the validity of some studymethodologies, the underlying assumptions about the nature of Cryptosporidium, theway in which the efficacy of disinfection is assessed in the laboratory and how that isextrapolated to full-scale water treatment.

Laboratory based disinfection studies are known to be affected by a variety of variables,including test parameters such as pH, time of exposure, temperature of test exposure,presence or otherwise of organic (background demand) load, use of a specific test agentneutralising step, etc. (Table 1). A significant but under-recognised variable is the testorganism used, in this case oocysts of Cryptosporidium (Section 10, Table 3). Unlikebacteria, these have to be produced in vivo by animal inoculation and recovered fromthe faeces of the infected animal. They are then cleaned, further purified andconcentrated by various methods, and enumerated. The oocysts produced cannot bestored in, for example, liquid nitrogen, as can other test organisms, and thus continue todeteriorate by ageing with subsequent storage. It is also now known that the wall ofoocysts may be radically affected by different cleaning and concentration procedures(McLauchlin, J Watkins unpublished). Other study parameters that need to be takeninto account include various physical and chemical factors and appropriate studydesign.

of 56

Table 1. Experimental variables that may affect apparent disinfectant efficacy

Physical Chemical Study DesignTemperature during testexposure

Disinfectant type and testconcentration; method ofmeasuring concentration(e.g. start, end, integrated)

Oocyst source and straincharacterisation

Test system pH Disinfectant neutralisationprior to viability/infectivitytesting

Oocyst clean up method, ageand storage conditions.

Contact time Composition of disinfectant(e.g. presence of surfactantsor other agents in testproduct)

Oocyst test concentrationused; accuracy of counts

Effect of physicaldegradation andenvironmental stress onoocysts.

Denaturing/dissociation ofdisinfectant during contactperiod; method of measuring

Method of assessing efficacy- in vitro (excystation,sporozoite ratio, cellculture), infectivity in vivo.

Nature of test medium andorganic load*.

Method and timing ofmeasuring disinfectantconcentration (batch vscontinuous, etc).

* Size of the oocyst inoculum: the CT value may be greatly affected by the number ofoocysts present at the beginning of exposure to a disinfectant. This appears to havebeen orders of magnitude higher in some studies, e.g. Quinn et al., (1996) whenozone was reported to be the least effective of all the studies. Numbers of oocystspresent may markedly affect the dynamics of disinfection (Armstrong et al., 1995aand b; Peeters et al., 1989).

of 56

4. Characteristics of Different ‘Strains’ of Oocysts used in Disinfection Studies

A potentially major source of variation in tests is the failure to base tests of disinfectionon a standardised and well characterised test "strain" or isolate of Cryptosporidium.Various species of Cryptosporidium have been identified but C. parvum is the onlyspecies infectious to humans and to a wide variety of mammals, particularly to the new-born. There have been many reports of zoonotic infections with C. parvum and cattle andsheep have been strongly implicated in most of these (Casemore, 1990; Casemore et al.,1997). Differences in clinical responses to infection and in the antigenic properties ofsporozoite and oocyst wall proteins (Casemore 1998; Mc Lauchlin et al., 1998; Nichols etal.. 1991; Nina et al., 1992; Patel et al., 1998; Pozio et al., 1992) of C. parvum have beenobserved, indicating that distinct “strains” do exist. Recent molecular and other studiessupport the view that there are two groups of C. parvum, i.e. one which infects humansonly and one which infects humans and other animals (Pozio, 1992; Spano et al., 1997;Bonnin et al., 1996; Carraway et al., 1996; McLauchlin et al., 1998; Morgan et al., 1998;Patel et al., 1998; Awad El Kariem et al., 1998). At present however, these differencesare not well enough characterised for the allocation of isolates into discrete sub-speciesand are insufficient for division into separate species (Meinhardt et al., 1996). However,recent studies have shown that one genotype, referred to as Genotype 1, does not appearto be transmissible to animals but has been responsible for some major waterborneoutbreaks (Casemore, 1998; Patel et al., 1998). The inability to propagate this type meansthat all disinfection studies reported so far must have been conducted using the animaltype (Genotype 2). Oocyst viability also varies over time during the course of infection(Bukhari et al 1995; Casemore, unpublished observation).

An individual may harbour more than one strain during an infection episode (Casemore,1998; McLauchlin et al., 1998; Patel et al., 1998). The difficulties of Cryptosporidiumcharacterisation may then be exacerbated by the current lack of understanding of geneticrecombination within or between isolates. If full sexual recombination does occur,oocysts will contain genetically heterogeneous sporozoites. However, there is evidencefor a clonal mechanism with more restricted heterogeneity. Simple, routine techniques forthe in vitro purifying of clones of Cryptosporidium are not currently available and, as aconsequence, studies of disinfection efficacy have usually been carried out on oocystpopulations, which cannot be regarded as a single strain. An understanding of variationwithin C. parvum is necessary as it may lead to differences in the infectivity and severityof disease and also to the resistance of the oocyst stage to disinfection.

of 56

5. Methods for Collection, Concentration and Storage ofCryptosporidium Oocysts

5.1 Source of oocysts

The only source of oocysts used thus far in disinfection studies is the faeces of infectedanimals, which must be cleaned and concentrated. The main sources used (see Section10 Table 3) include:

• Random isolates of C. parvum from sporadic infections in humans or livestockanimals, usually calves or lambs, but also an avian species, C. baileyi;

• Specified and partially characterised isolates:

(i) The University of Idaho (Iowa) bovine isolate of C. parvum.

(ii) The Moredun (Scotland) Research Institute strain of C. parvum,derived originally from deer (i.e. Cervine), subsequently passagedthrough calves or lambs.

(iii) The RN66 isolate of C. muris.

Because of the ubiquity of the infection it is difficult to avoid the possibility of the"strain" being mixed with adventitious isolates. However, McLauchlin et al., (1998)using a provisional typing scheme based on phenotypic markers showed remarkablereproducibility in Western blot banding patterns with the Moredun isolate. It has beensuggested that one study on molecular aspects of the RN66 C. muris strain wascompromised because the isolate, derived from rats, was subsequently shown to bemixed with C. parvum (MacDonald, pers comm); mixed infections are found in rodents(Chalmers et al., 1997). A bovine isolate of C. muris has been described and has beenrecommended for disinfection studies as it is believed to be non-infectious for humans(Owens et al., 1994). The use of pheno- and genotyping may permit the bettercharacterisation of isolates in future.

5.2 Oocyst production and collection

The under standardised methods of stock oocyst production are thought to influenceoocyst quality. A number of different methods have been described for providing cleanoocyst suspensions for disinfection studies (Blewett, 1989; Bukhari and Smith, 1995;Campbell et al., 1993; Fayer 1997; Kilani and Sekla, 1987; Reduker and Speer, 1985;Suresh and Rehg, 1996; Whitmore and Sidorowicz, 1995). Oocysts fromexperimentally infected animals are usually obtained by total faecal collection over thecourse of the infection. Suitable animals, usually calves or lambs but in some casesmice, are infected with the designated isolate, usually within one week of birth.Animals are usually kept in isolators during the course of the procedure to preventintercurrent infection or cross-infection of or from other animals, and to facilitatefaecal collection. The feed used for animals may affect the purification process andsome workers avoid the use of normal milk-based feeds to improve subsequentpurification (J Watkins, SE Wright, unpublished). In the case of the livestock animals,

of 56

a harness is attached to the animal to collect the total faecal output. Daily faecalsamples may be screened, for example by using a haemocytometer or by auramine-phenol staining (Blewett, 1989a and b; Casemore, 1991), to determine when oocystexcretion commences. The pooled oocyst-positive faecal material is usually processedin bulk at the end of the collection period although prolonged storage may well affectoocyst quality.

5.3 Oocyst purification

A variety of methods is in use and may affect the quality of the oocysts and theircondition over time (Section 10 Table 3). Extraction methods generally involvemultiple steps, including combinations of:

Separation of gross debris by;

• settlement in a diluent,• sieving,

Wash/centrifugation steps with;

• water or buffered saline (e.g. PBS),• acid flocculation,• surfactants (SDS, Tween, Lauryl, etc),

Concentration methods include;

• formol-ether extraction,• flotation on sucrose, salt, zinc-sulphate,• sulphuric acid flocculation,• density gradient media such as caesium chloride, Percoll®.• chromatography (affinity, ion-exchange, HIC, etc),• newer methods such as IMS, flow-cytometry.

5.4 Oocyst preservation

Oocysts may subsequently be stored (usually at +4oC) in:

• water,• buffered saline (PBS, HBSS),• 2.5% potassium dichromate,• antibiotics solutions.

Recent studies (McLauchlin et al., unpublished) indicate that certain of these methods,especially those using chloride compounds, may radically alter surface characteristicsof oocysts detectable by variation in Western blot banding patterns.

Another source of experimental variation that may influence the susceptibility ofCryptosporidium to disinfection is the physiological condition of the stored oocysts

of 56

prior to disinfectant challenge exposure. This will in turn be greatly dependent uponthe storage time and conditions experienced by the oocysts. Contact with faeces hasbeen linked to an enhanced robustness due to a greater impermeability to smallmolecules (Robertson et al., 1992) while others have shown decreased viability (SEWright, pers comm). Potassium dichromate and formalin, commonly used for oocyststorage and faeces preservation prior to oocyst isolation, were both found to have adeleterious affect on oocyst viability (Campbell et al., 1993). Hence their use mayinterfere with disinfectant efficacy studies. Alcohol and acetone, used in the membranefilter dissolution process during purification of oocysts, however, did not appear toaffect infectivity (Graczyk et al., 1997).

of 56

6. Cryptosporidium - Viability Assessment in Disinfection Studies

6.1 Excystation of Oocysts as a Measure of Viability

Cryptosporidium is transmitted by means of oocysts, produced during the sexual stageof the developmental life cycle. These contain, by definition, four motile sporozoites,each capable of initiating infection of a suitable host cell. Structurally, cryptosporidialoocysts differ from those of other coccidia in some respects. However, viability of theoocyst is taken to be demonstrated if the oocyst wall opens, by means of a suture in theoocyst wall, and intact sporozoites are released. This is also taken to be an indicationof potential (but not confirmed) infectivity for an appropriate host cell (Fayer and Leek,1984; Reduker and Speer, 1985; Woodmansee, 1987; Blewett, 1989). Although thesporozoites are defined as motile, this is often not easy to see as, beyond the initialspurt of activity on release, they have only a slow, limax-like gliding motility (Sibley etal., 1997).

Early attempts to demonstrate excystation were by veterinary workers (Fayer and Leek,1984; Reduker and Speer, 1985; Woodmansee, 1987), often as part of studies on thebasic biology of the parasite. Their studies were usually informed by experience withother coccidia. Some of the first studies on excystation in the UK (Blewett, 1989;Blewett, Wright and Casemore, unpublished) were also based in a veterinary laboratorybut continued earlier studies on disinfection (see above) the efficacy of which had beenassessed by animal inoculation.

The conditions required for maximal excystation have been shown to include correcttemperature, pH, and the presence of trypsin and bile salt, for an appropriate length ofincubation time. Optimum conditions include a temperature of 37oC, pre-excystationincubation in acid trypsin (pH 3.0-3.5), followed by incubation in bile salt at pH 7.6,for a total incubation period of approximately 1hr. Sporozoite survival may fall off ifexposure to these conditions are extended to maximise their release (i.e. maximise thenumber of empty oocyst shells). Some disinfection studies have used maximizedexcystation, thus precluding the measurement of sporozoite ratios (Section 10 Table 3).For some coccidia, there is a requirement for a pre-excystation period under reducingconditions and the species of bile salt is critical, but this is not the case forCryptosporidium. Some excystation will also take place in the absence of the bile saltand trypsin, even in water. Armstrong et al., (1995a and b), like Finch et al., andOngerth (cited in Benton et al., 1991) emphasised the importance of countingsporozoites as well as intact oocysts and empty oocyst shells (the Korich model). It isalso important to recognise the delicate nature of excysted sporozoites and to minimisethe lysis of free sporozoites by, for example, removal of the sporozoites from theexcystment fluid, cooling of the excysted suspension to +4oC, glutaraldehyde fixation.

It has been reported that pre-treatment with chlorine and acidification may enhance theexcystation of oocysts (Black et al., 1996; Reduker and Speer, 1985; Sterling, 1990).Some disinfection studies have used this excystation protocol, which will serve tocomplicate interpretation and may invalidate comparisons with other disinfectionstudies where chlorine has not been used during excystation.

of 56

Armstrong et al., (1995) found that a 3-log reduction in viability could be achievedwith 2% wt/wt ozone for 120 seconds and that the rate of ozone aspiration controlledthe rate of inactivation. Thus, if the ozone had been fed faster then a higher rate ofinactivation would have been achieved. They described the probable stepwise effecton oocysts exposed to ozone which were consistent with the apparently anomalousfindings of Godfree and Casemore (1990) using a column diffusion plant with batchozonated water. Some other authors also referred to features of disinfection studiessuch as the anomalous apparent increase in excystation (pseudo-excystation) atincreasing exposures and the concurrent loss of sporozoites, neither of which is takeninto account by the Woodmansee model. The later (Armstrong et al.,) model permitteda more rapid mass transfer of ozone, which is important, because the reaction is rate-limited by the ability of ozone to be delivered to the water in the reaction chamber inclose proximity to the oocysts. The authors noted the probability of parallel events andthe importance of delayed secondary effects of ozone on the oocyst wall and thesporozoites within. This reaction is likely also to be affected by the number (i.e.biological mass) of oocysts used, another variable in reported studies.

The kinetic aspects of ozonization are particularly important and these authorsemphasised that the conventional linear CT value is not an appropriate model. Thefindings of the study by Armstrong et al., (1995a and b) concluded that with anappropriate delivery system, a 3 log (99.9%) reduction in viability could be achievedwith 2% (w/w) ozone for 120 seconds, without utilisation of all the available 03. Theuse of the higher concentrations of 03 which could be achieved (=>20%) wererecognised to be uneconomic, and to risk increased by-product production, but mightbe considered when dealing with backwash waters or major contamination events. Insome cases, the disinfection process may result in damage (reduction in viability) to, oreven destruction of sporozoites, while still permitting the opening of the apparentlyundamaged oocyst wall. Maximized excystation counts, which do not includesporozoite ratios, would therefore significantly underestimate the effect of a testcompound or disinfection system.

For this reason, in assessing disinfection efficacy by excystation, account needs to betaken of:

• total numbers of oocysts compared with the control suspension (to allow forcomplete destruction of some oocysts),

• ratio of full, partially emptied, and empty oocyst shells, and• sporozoite numbers (maximum ratio of sporozoites to empty or partially

empty oocyst 4:1).

Failure to do so will invalidate the interpretation of disinfection data (Finch et al.,1994; Armstrong et al., 1995; Casemore, 1995). Models, which fail to take account ofsuch factors, cannot be considered sufficiently robust in scientific terms or fortranslation to operational needs.

6.2 Assessment of viability by the use of fluorogenic stains

A variety of fluorogenic stains have been used for assessment of viability, and as an aidto definitive identification, for a variety of parasites, including Cryptosporidium

of 56

(Belesovic and Finch, 1996; Belesovic et al., 1996, 1997; Campbell et al., 1992; Dowdand Pillai, 1997; Grimason et al., 1994; Jenkins et al., 1997; Labatiuk et al., 1991;Sauch et al., 1991). Their use may demonstrate integrity or permeability of the oocystwall, for example by resistance of the intact oocyst wall to penetration by propidiumiodide (PI), such integrity being lost during effective disinfection. Sporozoite nuclei ofboth viable and non-viable oocysts may be stained by intercalation of the fluorogenicdye 4',6-diamidino-2-phenylindole (DAPI) into the DNA within the nucleus, showingfour blue fluorescent dots within the oocyst by examination with appropriatewavelength UV illumination. Other stains used include fluorescein diacetate (FDA),hexidium, and the commercial nucleic acid stains SYTO-9® and SYTO-59®(Belesovic et al., 1997). It has been claimed that the viability of sporozoites within theintact oocyst may be inferred from the DAPI reaction. However, this is incorrect asnuclei in fixed (i.e. killed) oocysts also take up the stain. The application of DAPI is amethod used as an aid to identification. In addition, the DAPI/PI method on unfixedoocysts in suspension is poorly reproducible and may leave a variable number ofoocysts unstained by either dye (i.e. DAPI -, PI-). Although it is said that the latter areviable, much more needs to be known about these stains and the reasons for theseanomalies, probably related to changes in the oocyst wall.

The biological basis for these different tests and their function for evaluation oftreatment need to be properly understood. Viability as assessed by these methods is notthe same as that indicated by excystation and neither is it a measure of infectivityalthough some studies have shown good correlation (Robertson et al., 1995; Fricker etal., 1997; Korich et al., 1993). While figures have been derived for correlation ofresults between this technique and excystation there are two factors which need to beconsidered. Using the two methods in combination, the ratio of red (i.e. dead) oocyststo those with four blue dots (viable sporozoites) should indicate survival rates.However, a proportion of oocysts may:

• be destroyed totally;• take up neither stain .

It is thus important that the total number of oocysts post-treatment be compared withthe control. This is usually achieved by incorporating direct or indirectimmunofluorescence using a specific (usually genus-specific) anti-cryptosporidialmonoclonal antibody (MAb). It may still, however, be important to check total oocystnumbers by light microscopy, for example in a haemacytometer as treatment withchlorine may reduce the intensity of staining in immunofluorescence (D Casemore,unpublished observation).

6.3 In-vivo laboratory estimation of viability

For organisms that cannot readily be cultured, other means have to be used to estimatedisinfection efficacy in terms of infectivity. This has usually consisted of using anestablished laboratory animal model such as mice or even fertile hens' eggs.Cryptosporidium is an obligate parasite and the life cycle can therefore only occur in aliving host cell, either a suitable animal, or animal cells in culture (tissue culture), asused for disinfection studies on viruses. C. baileyi, an avian species, can be grown in

of 56

fertile hens eggs but this potential model does not seem to have been used fordisinfection study purposes. Several workers have proposed using animal models as ameasure of disinfection efficacy, even as a "gold standard" (Finch et al., 1993; Korichet al., 1990, 1993, 1998). This has a number of disadvantages (Casemore, 1995).Infectivity or otherwise in any animal model cannot safely be extrapolated to otherpotential host species, including from mice to humans. Indeed, there is increasingevidence that isolates differ in infectivity and that homologous host-to-hosttransmission might involve a lower level of infectivity than that between different hostspecies (Casemore, 1998). It should be noted that for the disinfection of no otherorganism is it a requirement to demonstrate post-treatment infectivity, as opposed toviability, as a measure of efficacy.

Although not strictly speaking "in-vivo", the use of living cells in laboratory culture(tissue culture) systems for detection of viable sporozoites excysted from oocysts, canconveniently be considered under this heading and have been used in viability assays,for example in drug susceptibility studies (Fayer 1997; Slifco et al., 1997; Woods andUpton, 1998), and including in testing oocysts recovered from water samples (Brasseuret al., 1998; Rochelle et al., 1997a and b; Slifco et al., 1997), including in disinfectionstudies (LeChevallier et al., 1997; Wilson and Margolin, 1999).

Whether or not viable oocysts, or more accurately excysted sporozoites, are infectivefor man is a separate issue - conventional disinfection studies on bacteria are notpredicated on demonstrating infectivity of any surviving bacteria but on survival (post-treatment viability) per-se. This is a crucial argument in the context of assessment ofefficacy of disinfection of Cryptosporidium.

6.4 Molecular methods for indicating viability

Although not so far reported specifically for use in disinfection studies, severalmolecular markers have been described for use in the detection of oocysts in water,which in some cases give an indication of viability (Battigelli et al., 1997; Deere et al.,1997; Mayer and Palmer, 1996; Rochelle et al., 1997a and b; Stinear et al., 1996;Vesey et al., 1995; Wick et al., 1997; Wagner-Wiening and Kimmig, 1995; Weidermanet al., 1997).

of 56

Table 2. Techniques for assessing oocyst viability after contact with disinfectant.

Method Advantages DisadvantagesIn vivo infectivity (usuallymice)

Only true measure ofinfectivity

Slow, expensive, difficult tointerpret, insensitive andethical problems.

Tissue culture Measures cell infectivitywithout using animals:quantifiable

Interpretationdifficult/uncertain

Fluorogenic dyeinclusion/exclusion (e.g.DAPI/PI)

Inexpensive, rapid andsensitive

Does not measureinfectivity. Results do notalways correlate withexcystation or infectivitystudies. Poor reproducibility.

In vitro excystation/sporozoite count

Inexpensive, rapid andsensitive

May over-estimateinfectivity; resultsinfluenced by assayconditions

Dielectrophoresis Rapid, automated and can beinstalled in situ at watertreatment works.

Rarely used, does notmeasure infectivity, lowsensitivity

Molecular methods May be possible to inferinfectivity

Still developmental:Interpretation uncertain

of 56

7. General Discussion of Test Models Used

Korich et al., (1990) estimated that Cryptosporidium oocysts were 14 times moreresistant to chlorine dioxide and 30 times more resistant to ozone than Giardia cysts. Aminimum of three logs reduction (e.g., in viable oocysts) is generally considered to berequired, at practicable CT values, with acceptable concentrations of by-productsformation, and which in many areas would have to operate at low ambienttemperatures. A direct relationship between concentration (C) and time (T) cannot,however, be assumed for oocysts, especially for ozone and the effect of likelyoperational temperatures needs also to be considered in laboratory models if the resultsof experiments are to be meaningful. The studies reported here used varying methodsof assessment in different experimental protocols; temperatures of test runs were oftenunrealistically high. In addition, the high concentrations of some compounds testedwould lead to unacceptably high concentrations of by-products, either directly or, forexample, by reaction with pipework materials in distribution (Anon, 1989). In the caseof ozone, concern has been expressed about the high concentrations of bromate inparticular, which might be produced and for which there is a stringent MaximumAdmissible Concentration (MAC) in drinking water. Reported studies have useddifferent means of assessing disinfection efficiency, some more rigorous than others,with different test protocols with, for example:

• different sources and pre-test conditions for oocysts;• either continuous flow loading, stabilised loads, or batch testing with pre-

disinfected water;• the disinfectant measured at different stages (input, average, integrated, and

residual);• different test conditions (temperatures, pH, water type, etc);• different sources and methods of preparation of oocysts used in testing;• different systems for oocyst viability assessment.

Conventionally, disinfection tests on vegetative bacteria have been conducted on“standard” strains of organism under reasonably well specified conditions. Thedisinfection process was often assumed to be a simple monomolecular “one hit”reaction. The reaction was therefore assumed to fit the Chick Watson formula as firstorder for the disinfectant and zero order for the organism. This assumption is notnecessarily correct, although for the action of chlorine on bacteria this has proved to bea reasonable model in practice. This leads to the expression of disinfection exposure asa direct linear relationship of concentration and time, CT.

Ozonation would appear to offer the most promise for an effective disinfectant, despitevarious possible disadvantages such as cost and DBPs. Ozone disinfection of oocysts isnot accurately modelled by the first order “one hit” reaction scheme. Variousmathematical models have been proposed to explain the kinetics involved, from thenon-first order “one hit” scheme of Hom (cited in Finch et al., 1994), to the first order“multiple hit” of Armstrong et al., 1995 or pseudo-first order. The concentration ofozone, and of any active daughter species, is definitely variable at the oocyst/solutioninterface. Background demand can be very variable in terms of quantity and“hardness” (defined as the susceptibility of ozone demand, the reaction rate constant),

of 56

both between water sources and seasonally within a single source. Oocysts arecomplex structures unlike vegetative organisms in that the disinfectant sensitiveinfective stage, the sporozoite, is within a protective case, the oocyst wall. The detailednature of the oocyst wall and its surface is poorly understood. When the multistagenature of the reaction, which this structure produces, is taken into account, some of theanomalies reported can be explained (Armstrong et al., 1995; Casemore, 1995).In particular, such sequential damage potentially from various disinfection processesmay result in:

• Unaffected oocysts which excyst normally.• Oocysts are destroyed by the disinfection process. The decrease in numbers is not

reflected in a viability assessment.• Release of intact sporozoites during excystment which are dead and which may then

lyse (break up) on exposure to the excystation fluid.• Dead oocysts which do not excyst.• Oocyst suture ruptures spontaneously with release of dead sporozoite remains from

the oocyst (lysis within). The oocyst then resembles a viable excysted or partiallyexcysted oocyst (pseudo-excystation).

• Oocysts disrupt.

Each of these stages may proceed in parallel with any of the others but an individualoocyst would experience the stages in sequence. These are not the characteristics of arobust model. Individual stages may be first order with respect to ozone and may alsobe very rapid, to the extent that they become rate limited by the supply of ozone, and itsdissolution, to the locality of the oocyst. The products of all stages of “disinfection”will be present almost immediately after first exposure to the ozone though theconcentrations from the slower reactions may only become measurable after a moreprolonged period of time has elapsed. For a stage-wise inactivation model it will benecessary to determine a 'k' value for each of the stages. These k values will then beindependent of the ozone delivery method and concentration, the concentration ofoocysts, and the degree of inactivation required. For the non-linear models it isreported that individual values of the rate constant (k), n (Cn), and m (Tm), need to bederived for each initial ozone concentration, each initial oocyst concentration, and foreach degree of inactivation required.

It is clear that tests must be able to show the ability to kill sporozoites, either by directobservation or, as suggested by some work , by infectivity studies. Finch et al., 1994have said that in vitro methods for measuring viability should not be used for assessingdisinfection as, in their view, this leads to an underestimation of the proportion ofoocysts killed.

A possible explanation for their findings is that the apparent differences in effectivenessof disinfection when assessed by animal inoculation as compared with excystation ordye exclusion may reflect the difference between:

• the difference between true and pseudo-excystation values;• the real significance of the biological state of sporozoite viability (as measured in

vitro);

of 56

• variable minimum infective dose (MID) according to oocyst strain• variable MID for the specific animal model used;• The dose needed to achieve 50% (ID50), 99%, 99.9%, or 100% (ID100) infection, or

any other defined level, which is not linear.

If mice are inoculated with too few viable oocysts they might show no signs ofinfection. However, some oocysts might still be viable and potentially infective. Thus,what is important is what is left (i.e. oocyst numbers) after water treatment, not what isremoved, given that the absolute number, which might be in water at any given time, isso variable. Any move to establish animal infectivity as the “gold standard” wouldhave to overcome other problems such as the technical difficulties of reliablyinoculating suckling mice, variable susceptibility of mice strains, the variableinfectivity for mice of oocysts from different sources, and the effect of varying ages andstorage conditions of the oocysts used. In addition, there are the difficulties implicit ina test, which relies on animal studies and the extrapolation of findings to other potentialhosts including humans. Animal models should thus not be regarded as a sine qua nonfor estimating efficacy of disinfection.

of 56

8. Published Studies on the Disinfection of Cryptosporidium inDrinking Water.

The increasing relevance and significance of disinfection studies for the water industryled to a number of reports of studies which have been conducted using widely varyingconditions, especially with regard to the temperature at which the test was performed,the way in which disinfectant concentration was measured, sources of oocysts,differing methods for assessing viability, viability vs infectivity etc; some studies havelooked at the response (% oocyst inactivation) to defined CT values, and others on theCT value required to achieve any particular level (e.g. 99%) inactivation.

8.1 Chlorine and related compounds

It has been reported that the total loss of excystation ability of a human derived strainof Cryptosporidium required exposure times of 24 hours to 8,000-16,000 mgl-1 freechlorine (Smith et al., 1989). Studies on chlorine and chloramine were reported bySterling et al., (1989), also using in vitro excystation, which also showed impracticablyhigh CT values. When oocysts were tested at 25oC, at pH7, a 100% kill CT of 9,600mg.min/l was found for chlorine as assessed by in vitro excystation. Similarexperiments assessed in vivo in mice gave a two-log reduction (99%) at a CT of 7,200mg.min/l. Chloramine tested by the same authors gave a CT for 100% kill of 9,600mg.min/l by in vitro excystation; a CT of 7,200 mg.min/l produced only 90% (1 log)reduction when assessed in vivo in mice. Studies on chlorine dioxide showed a CT of78 mg.min/l by in vitro excystation; assessed in vivo in mice the same CT yielded 90%inactivation. CT values required would generally be considerably higher at the loweroperating temperatures for many water treatment works, especially in winter.

Korich et al., (1990) found that oocysts of bovine origin were relatively more sensitiveto chlorine, achieving 90% inactivation within 90 min at 80 mgl-1. Similarly, 99.9%inactivation was achieved with 80 mgl-1 free chlorine after 2h, although the source ofthe strain was not specified (Anon, 1989). Ovine derived oocysts were relativelyunaffected by incubation in tap water with 5 mgl-1 chlorine for 7 days (Quinn andBetts, 1993). Similarly, Venczel et al., (1997) found the same concentration producedno measurable inactivation after 24 hours. The source of the C. parvum oocysts in thestudies where it was specified (Korich et al., 1990 and Peeters et al., 1989), was bovineand the CT values for these studies were 5-10 and 4 and 4.6 mg min l-1, respectively.

Peeters et al., (1989) reported on studies using chlorine dioxide and mouse infectivityas a measure of efficacy showing somewhat lower CTs of 5-12.9 mg.min/l gave 1.5logs (93.5-97%) inactivation. Korich et al., (1990), using both methods of assessment,showed chlorine dioxide to be more effective than either chlorine or monochloramine.Exposure to 1.3 mg/l chlorine dioxide yielded 90% reduction after 1 hour while 80mg/l of the other two compounds were required for 90% inactivation in 90 minutes.Ransome et al., (1992), using varying times and concentrations, found high levelresistance to chlorine, but a reduction of >90% in excystation after 15 mins exposure toan initial 5 mg/l of chlorine dioxide.

of 56

Other investigators have assessed the efficacy of chlorine based compounds such aschlorine dioxide (LeChevallier et al., 1997), chloramines (Korich et al., 1990) and freechlorine (Smith et al., 1989; Smith and Rose, 1990; Venczel and Sobsey, 1995; Korichet al., 1990; Robertson et al., 1994; Anon, 1989;) and a mixture of free chlorine andchloramine (LeChevallier and Norton, 1995; Gyurek, et al., 1997).

Chlorine dioxide is sometimes generated by the reaction of chlorine and sodiumchlorite and may hydrolyse into chlorate, chlorite and chloride ions. It is possibletherefore, that all these components would be present simultaneously in waterdisinfected with chlorine dioxide and the proportions of each determined by thephysico-chemical conditions of the water. Liyanage et al., (1997) have recentlydetermined that it is the un-dissociated chlorine dioxide that is the main anti-cryptosporidial component.

8.2 Ozone

Peeters et al., (1989) found that an initial concentration (Co) of 1.11 mg/l of ozone for6 mins (CT 6.7 mg.min/l) completely inactivated 104 oocysts per ml when infectivitywas tested in mice (for which 1000 oocysts were needed to establish infection); 2.27 Co

for 8 mins (CT 18.2 mg.min/l) were needed for 5x105 oocysts per ml. The temperaturefor the test exposures was not specified. Similar values were obtained in preliminarytests at 20oC (range used 16-30oC) by Godfree and Casemore (unpublished report), intests designed for investigation of the potential for use in swimming pools (1990).These workers, using in vitro excystation, noted some anomalous effects duringexcystation including pseudo-excystation (see above) also noted by some otherworkers. Sterling et al., (1989) found variable results depending upon the method andtiming of assessment (see below) but results suggested a CT of 5-10 mg.min/l gave99% inactivation. Korich et al., (1990) found greater than 90% inactivation as measureby infectivity with 1mg/l for 5 mins; temperature of test exposure was not specified.

Parker et al., (1993), using oocysts of bovine origin, found that at a startingconcentration of 5 mgl-1 ozone, total viability was lost after 2 min. The oocysts used inthe study of Perrine et al., (1990) and which yielded the lowest CT value were derivedfrom horse faeces. It is possible that C. parvum oocysts of ovine origin may be mostresistant to ozone, as at CT of 14.88 mg min l-1 only succeeded in approximately 95%inactivation of oocysts (Quinn et al., 1996). There is also some evidence that C. muris(Owens et al., 1994) and C. baileyi (Langlais et al., 1990, 1991) may be moresusceptible to ozone than C. parvum. These observations however, are only tentativeat present and may be a result of the different experimental protocols used in differentstudies. The different experimental protocols and apparatus design used in the abovestudies (Section 10 Table 3) makes any conclusions drawn about the effect of straindifferences very tentative.

Parker et al., (1993) studied the effects of using different test systems and protocols forozone exposure, using fluorogen (DAPI/PI) inclusion/exclusion to assess viability.They showed the critical effect of temperature and of maintaining a continuousconcentration (C) rather than starting with a particular residual value (batch test) whichwould further decay to lower levels. 100% reduction in viability was found after 2

of 56

mins with 5 mg/l, and 6 mins at 3 mg/l when oocysts were exposed to continuouslevels of ozone at 20oC. This temperature would, however, not normally be reachedunder normal operational conditions in water treatment. Ransome et al., (1990) usingtimes varying from 10-30 mins, and mean concentrations of ozone of 0.1-1.8 mg/lfound >90% (92.3-98.6%) inactivation.

Owens et al., (1994) in pilot-scale studies used C. muris as a surrogate for C. parvumand a counter-current ozone delivery system with river water to evaluate bench-scalestudies. Their results suggested CT values midway between those for C. parvumexcystation and mouse infectivity assays. Further pilot-scale studies on ozone treatmentwere reported by Milner et al., (1997), again using both C. muris and C. parvum, aswell as other organisms including spores as a potential surrogate model. Other workershave also suggested the use of surrogate markers (Chiou et al., 1997; Jakubowski etal., 1996; Nieminski and Ongerth, 1995; Payment and Franco, 1993).

Quinn et al., (1993) used dielectrophoresis to detect differences in response in ozonetreated oocysts, compared with untreated oocysts. They found that there was arelationship between the ratio obtained and that found with excystation and that therelationship was non-linear with increasing ozone dose, and that these findings wereconsistent with the results of a mathematical model for the system.

Ransome et al., (1992), using simple excystation ratios (i.e. without noting sporozoitenumbers) reported the effect of a range of ozone exposures and reported that nominalozone residuals of 1 mgl-1 caused over 95% average reduction in viability in 10mins.

Finch et al., (1993; 1994a and b) have carried out the most comprehensive studies todate and have extensively explored the inactivation kinetics of ozone and otherdisinfectants such as chlorine, against both Giardia and Cryptosporidium. Using bothin vitro excystation and animal infectivity as methods of assessment, they tested ozonedisinfection with protocols using temperatures of 7oC and 22oC, and ozoneconcentrations which conventionally expressed CT values of 3.5-7 mg.min/l (Finch etal., 1993). The ozone value was based on an integrated residual. Further studies(Finch et al., 1994) gave conventionally expressed CT values of 7.9-16.0 mg.min/l at7oC, and 3.1-15.3 mg.min/l at 22oC. However, using a non-linear kinetic model basedon that described by Hom (1972) yielded Cn Tm values of 10.3 mg.min/l at 7oC and 3.7mg.min/l at 22oC. The authors emphasised the importance of counting sporozoites, theKorich model, as opposed to the simple ratio of full to empty oocysts shells, describedas the Woodmansee model. However, they also expressed the view that animalinfectivity studies were a better measure of activity (see above).

In some preliminary studies, Armstrong et al., (1995a and b) also explored theinactivation kinetics for ozone using a novel electrolytic system capable of producinghigh concentrations (2-=>20%) of 03 with continuous charging of the exposure vesseland intense exposure of oocysts to 03. These test parameters were designed to look attreatment systems, which would maximise exposure and minimise by-productproduction, particularly for the disinfection of water treatment effluents. Smallnumbers of oocysts were exposed to low concentrations of ozone by Cambell et al.,(1997) and the results assessed by the DAPI/PI method and excystation. It was found

of 56

that the former method significantly over-estimated viability if the test was applied toosoon after exposure. Such delayed effects are implicit in the cascade model describedby Armstrong et al., (1995).

8.3 Mixed oxidants

There has been some interest in the use of mixed oxidants. Although Robertson et al.,(1994) reported that ozone appeared to be more effective alone than in combinationwith either chlorine or hydrogen peroxide, synergy was found between ozone andchlorine by Gyurek et al., (1997). The electrochemical generation of mixed oxidantscan be carried out on-site, relatively inexpensively. Mixed oxidants have been foundto be very effective at almost totally eliminating C. parvum within 4 hours at a totaloxidant concentration of 2 mgl-1 (Venczel and Sobsey, 1995). In comparison, the sameconcentration of chlorine alone had no effect. Similar results were achieved byVenczel et al., (1997), with concentrations of 5 mgl-1 total mixed oxidants or chlorine.Concentrations of mixed oxidants less than 1 mgl-1, however, were of little value in theinactivation of oocysts (Casteel and Sobsey, 1997). Yozwiak et al., (1994) used MIOXsystem that generates mixed oxidant solution electrolytically from sodium chloridebrine. Effect was determined by excystation following exposure to variousconcentration, time and temperature combinations and suggested that this provided apotential means for disinfection of potable water.

8.4 Other methods

8.4.1 Iodination, etc

There have been a few studies carried out on iodine and its salts asdisinfectants, primarily for treating small volumes of water. Ransome et al.,(1992) showed elemental iodine and hypo-iodous solution to be relativelyineffective at practicable concentrations. They also evaluated the effect ofbromine and iodine-bromine with similar results. Iodinated water treatmenttablets for travellers were evaluated by Gerba et al., (1997), who found them tobe of poor efficacy.

8.4.2 Combined methods

Another approach to the inactivation of Cryptosporidium in water has been tocombine physical with chemical methods. Various combinations have beenevaluated. These have included synergy between different disinfectants (Finch etal., 1997), freezing and thawing with chlorine (Battigelli et al., 1996); sandmixing with chlorine (Battigelli et al., 1996; Haas, 1995; Parker and Smith,1993), the combined effect of other treatment procedures at water treatmentplants (Rose et al., 1996, Bellamy et al., 1993, Payment and Franco, 1993).Watkins, (1995), using DAPI/PI and excystation, evaluated the use of ultrasoundto enhance disinfection. The effect of iodinated resin treatment, for use bytravellers, in conjunction with reverse osmosis or other cartridge filter, provedmore effective in killing or removing Cryptosporidium and other potentialpathogens from raw surface water (Naranjo and Gerba, 1997; Naranjo et al.,

of 56

1997). Iodide resin, an effective disinfectant against bacteria and viruses, wasreported as having little effect on the viability of Cryptosporidium oocysts. Itdid, however, provide an effective matrix for the removal of oocysts from waterby electrostatic retention (Upton et al., 1988).

8.5 Non-chemical methods of disinfection

8.5.1 Effects of Raised Temperature

Several workers have commented on the effects of moderately increasedtemperatures on oocysts (Anderson, 1985; Blewett, 1989; Casemore et al.,1989; Casemore, 1995; Fayer, 1994; Fayer and Leek, 1984; Fayer and Nerad,1996; 1997; Harp et al., 1996), which seem to reflect:

• Changes in oocyst wall permeability leading to increased penetration ofdisinfectants;

• Premature activation of sporozoites leading to exhaustion of energyreserves and thus reducing subsequent viability independently of anydisinfection effect.

Generally, the activity of all disinfectants examined in the study by Blewett andcolleagues (1989) was higher at 37°C than at 22°C. This was attributed to theoocysts becoming more permeable at host body temperature in order to aidexcystation in vivo. Ozone has been shown to be more effective at 20°C than at5°C in one study (Parker et al., 1993), and to have a higher CT value at 7°Cthan at 22°C in another (Finch et al., 1993).

Inactivation values at such increased temperatures are not appropriate forpotable water treatment but may be of value for special applications such asswimming pool water disinfection. Here, the increased water temperatures usedmay, particularly with the prolonged disinfectant exposures involved, and thephysical effects on oocysts walls of continuous filtration, enhance the effect ofotherwise ineffective disinfectant levels.

8.5.2 U.V. Irradiation

Studies have indicated that UV irradiation is ineffective in the normal formatand levels used for water treatment. It required 80 mW s cm-2 to achieve a 90%reduction in viability and to achieve a 99% reduction would require 120 mW scm-2 which is considerably greater then the levels used in practice (Ransome etal., 1992). Preliminary studies by the author also showed the normal operatinglevels to be ineffective. Approaches to improving the efficiency of UVtreatment and increasing the exposure achievable promises to be capable ofeffective disinfection (Abbaszadegan et al., 1997; Bolton et al., 1998; Campbellet al., 1995; Clancy et al., 1997c; Lorenzo et al., 1993).

of 56

8.6 Other (non-water related) disinfection studies

8.6.1 Veterinary/Hospital use

Disinfection of Cryptosporidium was studied initially, mainly for veterinary,and hospital and domestic purposes. Studies on disinfectants routinely used inveterinary laboratories described by Campbell et al., 1982; Blewett, 1989;Casemore et al., 1989; Fayer et al., 1996; Vassal et al., 1998. Some commonlyused agents for disinfecting surfaces and instruments in hospitals have beenevaluated, include peroxygens (Ares-Mazas et al., 1997; Holton et al., 1994),aldehydes (Holton et al., 1994; 1995; Angus et al., 1982), iodine-basedcompounds (Upton et al., 1988; Holton et al., 1994), peracetic acids (Holton etal., 1994),and various domestic and clinical compounds (Angus et al., 1982;phenol (Blewett, 1988; Casemore et al., 1989; Holton et al., 1995; Vassal et al.,1998; Wilson and Margolin, 1999). In the latter study, the authors used chlorinetreatment excystation protocol of Finch et al., (1993), thus complicating theinterpretation of their findings.

Although the findings of some of these studies may be of value in evaluatingtest models and in assessing potential compounds for water treatment thetoxicity or other characteristics of some of these agents makes them generallyunsuitable for use in disinfection of potable water. It has been stated that manycommon disinfectants, including pine oil, phenol, lysol, ethanol, aldehydes andquaternary ammonium compounds, have little effect on oocyst viability (Cordelland Addiss, 1994). Of the various disinfectants tested, hydrogen peroxide andammonia have both been observed to bring about reduction in the excystationability of Cryptosporidium sp. after relatively short contact times with oocysts(Blewett, 1989; Casemore et al., 1989).

Disinfectants routinely used in veterinary investigation laboratories had anegligible effect on calf-derived oocysts after a 2 h exposure period, althoughammonia and formalin did completely destroy infectivity after 18 h exposure(Campbell et al., 1982). As intestinal homogenates were used in place ofpurified oocysts, the efficacy of disinfection was probably reduced due to theadditional organic load.

A 1% solution of 'Virkon', a mixture of oxidising agents, was appraised as apossible anti-cryptosporidial agent, and found to reduce the excystationpotential by approximately 50% after 30 min contact at 22°C (Blewett, 1988).In another study using calf-derived C. parvum oocysts (Ares-Mazas et al.,1997), the same exposure conditions of time, concentration and temperature hadno effect on infectivity. Exposure times of greater than 2 hours did show somereduction, although infectivity was never totally abolished even after exposureto 8- 10% 'Virkon' for time periods up to 6 h. The lack of any anti-cryptosporidial activity of 1% 'Virkon' and an iodine compound against bovineoocysts was confirmed by Holton et al., (1994), at 37°C for 60 min. However,this study did reveal that 2 % glutaraldehyde, a quaternary ammoniumcompound and a beta-ene compound ('Pentapon DC1') were highly effective in

of 56

reducing excystation ability under the same conditions. Infectivity of oocystsobtained from bovine faeces and passaged 15 times in mice was found to beunaffected by high concentrations of aldehydes-based disinfectants at roomtemperature (Angus et al., 1982). Again these results may have been influencedby the fact that intestinal homogenates were used in this study. Holton et al.,(1995) found that glutaraldehyde did not completely destroy the excystation ofoocysts of human origin. In contrast, 'Nu-cidex', a mixture of acetic acid,peracetic acid and hydrogen peroxide, was considerably more effective with fiveminutes exposure being sufficient for total eradication of viable oocysts.Studies conducted on commercial disinfectants are generally difficult toevaluate as they often containing a mixture of active agents.

Some success has been reported for gaseous disinfectants. Cryptosporidiumoocysts derived from infected calves and subjected to an ammonia, ethyleneoxide or methyl bromide atmosphere for 24 h, failed to initiate infections inmice (Fayer et al., 1996). Formaldehyde, on the other hand, only brought abouta partial reduction in infectivity. The volatilisation of ammonia in slurry stores(Ruxton, 1995) and during silage production (Merry et al., 1997) has beenlinked with a decrease in the viability of oocysts, although any quantitativeevaluation is difficult due to the undefined conditions associated with theseenvironments. Sundeman et al (1987) reported trials on a number of "domestic"and other disinfectants for cleaning avian accommodation to treat contaminationwith C. baileyi.

of 56

9. Conclusions.

Disinfection studies have been conducted under very varying conditions, partlyreflecting the difficulties and constraints of handling this obligate parasite underspecified controlled conditions. The conditions used, however, do not alwaysrealistically reflect the physical and chemical conditions that would apply inoperational use. Such studies may suffer because some workers with expertise inparasitology have limited understanding of disinfection and water treatment, whilewater experts may have limited understanding of the nature of this parasite.However, studies have increasingly used appropriate multidisciplinary groups. Manystudies have indicated the unusual degree of resistance of Cryptosporidium oocyststo disinfection, especially with fresh oocysts tested in laboratory conditions. There issome evidence that, in practice, many oocysts found in the environment are non-viable. In addition, the combined effects of current water treatment processes andother environmental stresses may make oocysts more susceptible to the effects ofconventional or achievable levels of disinfectants, including chlorine (LeChevallier,cited in Benton, 1991; LeChevallier and Moser, 1993; Parker et al., 1993; Robertsonet al., 1992). The possibility also exists for synergistic effects from mixtures ofdisinfectants such as moderate levels of ozone followed, for example, by chlorine-based disinfectants required for maintaining residuals in distribution (Finch et al.,1994). Ozone is expensive but may be required in some treatment systems forremoval of pesticides or the control of colour, taste and odour problems. In that casethe additional benefit of an effect on any oocysts present would be a bonus. Whetherit could be justified on disinfection grounds alone is questionable. It may however,be considered in newer, more efficient delivery formats for the treatment ofbackwash waters, or dealing with contamination episodes, which would carrybenefits of both cost and resource management.

The various methods described above give differing measures of the biological stateof oocysts and the sporozoites within them, following exposure to disinfection andother treatment processes. In addition, conditions of oocyst production and pre-teststorage, the inability to store standardised inocula in a suspended state of animation,etc, all militate against the production of reliable and comparable, robust data fromdifferent studies. No studies have been published to date which compare efficacyagainst animal-derived oocysts (all studies to date), with those human-derivedisolates characterised as apparently confined to humans (Genotype 1) and whichappear to have been responsible for some major waterborne outbreaks (Casemore,1998; Patel et al., 1997). There are thus a variety of caveats that need to beconsidered in using data derived from currently published disinfection tests, basedon the existing methods, to provide information for practical water disinfectionpurposes, and further work is clearly required. In addition, relatively little is knownabout the surface characteristics of oocysts (Ives, 1995). The effect that this mighthave on disinfection studies has not generally been recognised in published work.The cascade mechanism of action proposed by Armstrong et al (1995a,b) wouldsuggest that a more detailed understanding of the oocyst wall is a critical forunderstanding the process of disinfection of Cryptosporidium.

of 56

10. TABLE 3 Experimental disinfection models - oocyst sources, preparation and viability assessment methods

REFERENCE TYPE OFDISINFECTION

SOURCE ANDSTRAIN OFOOCYSTS

CLEAN UPMETHOD

STORAGEDETAILS (time, temperatureand method)

METHOD OFTESTINGOOCYSTVIABILITY

ANY OTHERCOMMENT

Abbaszadegan etal.,1997

POU + UV University of Idaho Sheather's sugarflotation.

Not specified Not applicable: countonly if passedthrough system.

No oocysts ineffluent to testviability. ProbablyIowa bovine isolate

Angus et al., 1982 Tegodor, Formula-H(aldehydes)

Bovine sporadicisolate passaged inPorton SPF mice.

None - suspension offaecal + mouse gutmaterial in PBS.

Not specified Infectivity for mice. For veterinary use.

Archer et al., 1993 Ozone N/S (as supplied byYorkshire Water Plc- probably Moredun)

Stock suspensionfurther washed in 0.5mM SDS

Stored in distilledwater.

Experimentaldiaelctrophoresiscompared withcontrols

Large numbers ofoocysts used

Archer et al., 1995 Chlorine and ozone Moredun isolate Ready preparedsuspension.

Not specified. Dialectrophoresis NB Moredun strain isa cervine isolatemaintained in calvesor lambs

Ares-Mazas etal.,1997

Virkon (mixture withpotassiummonopersulphate)

Bovine isolate Ether extraction+ Percoll gradient.

Not specified Mouse infectivitywith (CD-1 Swiss).

Hospital infectioncontrol study

of 56



CLEAN UPMETHOD

STORAGEDETAILS

(time, temperatureand method)

METHOD OFTESTINGOOCYST

VIABILITY

ANY OTHERCOMMENT

Armstrong et al.,1994 and 1995

Ozone Moredun isolate Ready preparedsuspension in PBS.

Not specified(Known to be in PBS

at +4oC <=3/12th).

Excystation includingsporozoite counts.

Precise details ofoocysts preparation

not given.

Arrowood et al.,1996

Pulsed light Iowa bovine isolate Sucrose followed bycaesium chloride

gradient thensuspended in PBS.

Not specified Mouse infectivity(BALB-c) checked

by flow cytometry ofgut contents.

Large numbers ofoocysts used.

Black et al., FEMSLetters 1996

Chlorine, ozone As described forFinch et al.

As described forFinch et al.

Not specified DAPI/PICD-1 Mouse

infectivity

Comparison of assaymodels.

Blewett ,1989 Various hospital,domestic and

veterinary.

Not specified(Moredun).

Not specified. Not specified. Excystation andmouse infectivity.

Early developmentalwork.

Bolton et al., 1998 Modified (mediumpressure) UV

University of Arizona(Harley Moon Iowa

bovine strain).

Caesium chloride PBS with antibiotics DAPI/PI, maximisedexcystation and

mouse infectivity.Campbell et al., 1995 Modified UV Sporadic bovine

isolate.Sucrose and Percoll +4oC in water. DAPI/PI and

excystation withsporozoite ratios.

of 56



CLEAN UPMETHOD

STORAGEDETAILS

(time, temperatureand method)


VIABILITY

ANY OTHERCOMMENT

Campbell et al., 1982 Various "domestic"chemical

disinfectants

Sporadic bovineisolate passaged in

rats and mice.

20% intestinalhomogenate in PBS

Not specified Mouse infectivity Veterinary study

Carrington andRansome, 1994

Chlorine and ozonewith various other

stress factors

Moredun (severalbatches)

As supplied PBS DAPI/PI Study primarily ofeffect of stressors on

oocyst survival.Casteel and Sobsey,

1997Mixed oxidants

(MIOX)Not specified Not specified Not specified Tissue culture assay. Abstract only

Clancy et al., 1997 Modified UV Bovine Not specified Not specified Mouse infect; excyst;DAPI/PI; SYTO9/59;

Fayer, 1995 Sodium hypochlorite Bovine AUCP-1strain

Pot. Dichromate,used <1/12; sieved;caesium chloride,

water.

DDW, used at <1/12 Mouse infectivity Oocyst productiongiven in detail in

earlier paper.

Fayer et al., 1996 Ammonia, carbonmonoxide, ethylene

oxide, formaldehyde,methyl bromide

As above As above As above As above As above

Finch et al., 1993 Ozone Not specified Not specified DDW Mouse infectivity;simple excystation

(no sporozoite count)Finch et al., 1994 Ozone Bovine Iowa (Harley

Moon strain) viaUniv Arizona

Sieved/Tween 20;Sucrose/tween 80 x3;

0.01% tween 20 +antibiotics, +40C

Mouse infectivityCD-1 mice; simple

excystation (nosporozoite count).

Very comprehensivestudy.

of 56



CLEAN UPMETHOD

STORAGEDETAILS

(time, Temperaturemethod)


VIABILITY

ANY OTHERCOMMENT

Finch et al., 1995 Ozone and Chlorine Not specified Not specified Not specified Mouse infectivityCD-1 mice; simple

excystation (nosporozoite count) and

DAPIGerba et al., 1997 Iodine Bovine (Waterborne

Inc).Sucrose Not specified Simple excystation

(no sporozoitecount).

Gyurek et al., 1997 Chlorine,monochloramine

Not specified Not specified Not specified Neonatal CD-1 mice

Holton et al.,1994;95

Glutaraldehyde;Peracetic acid, etc

Unspecified bovine Previous referencecited

2.5% Pot dichromate Excystation withsporozoite ratio.

Hospital infectioncontrol study

Jenkins et al., 1998 Ammonia Unspecified bovine Previous referencecited (continuousflow differential

density)

DDW + antibiotics DAPI/PI

Korich et al.,1989;90

Ozone, free chlorine,Chlorine dioxide,Monochloramine

Unspecified bovine Sucrose, Percoll 2.5% Pot dichromate+40C

Excystation withsporozoite count;

Balb/c miceFDA/PI

LeChevallier et al.,97th Gen Mtg 1997

Chlorine dioxide Not specified Not specified Not specified ExcystationTissue culture

Conference abst

Liyanage et al.,1997a,b

Chlorine compounds See Finch et al See Finch et al See Finch et al See Finch et al

Lorenzo et al., 1993 UV Unspecified bovine Collected in Potdichromate; within48hrs sieved, ether;

Percoll; DDW

Not specified Mouse infectivity

of 56



CLEAN UPMETHOD

STORAGEDETAILS

(time, Temperaturemethod)


VIABILITY

ANY OTHERCOMMENT

Miltner et al., 1997 Ozone (pilot plant) Not specified Not specified Not specified Animal infectivity Conference abstrOwens et al., 1994 Ozone (pilot plant) C. muris strain RN66

from Dr Iseki, Japan,propagated in mice

Tween 20; sieved;sucrose.

Stored in unspecifieddiluent withantibiotics.

Excystation only Large numbersoocysts used.

Parker and Smith, 1993 Chlorine + sand Moredun isolate Stock suspension assupplied.

Not specified DAPI/PI

Pavlasek, 1999 Various domesticdisinfectants

Not specified Not specified Not specified Mouse infectivity Veterinary study -Czech language

paperPeeters et al., 1989 Ozone, chlorine

dioxideUnspecified bovine Pot Dichromate at

+4oC <24hrs; sieved;ether; Percoll; DDW;then to mice and guttissue homogenized

in DDW + antibiotics<24hrs.


Quinn et al., 1996 Ozone Moredun isolate vialambs

Stock suspension assupplied in PBS.

Stock suspension assupplied.

Diaelectrophoresis Oocyst purificationreference to Hill et al

Quinn and Betts, 1993 Chlorine Moredun isolate vialambs

Stock suspension assupplied in PBS

Stock suspension assupplied.

Maximisedexcystation, no

sporozoite countRansome et al., 1992 Various including

chlorine, ozone,peroxone, UV

Not specified Not specified Not specified Excystation withoutsporozoite counts

Oocysts believed tobe Moredun

of 56



CLEAN UPMETHOD

STORAGEDETAILS(time, Temperaturemethod)

METHOD OFTESTINGOOCYSTVIABILITY

ANY OTHERCOMMENT

Rasmussen et al.,1995

Chlorine/chloramine Not specified Not specified Not specified Excystation withoutsporozoite counts

Chlorine exposuredetermined by redox

Smith et al., 1989 Chlorine Pooled humanisolates

Formol-ether; sieved;sucrose; DDW

DDW +4oC Excystation withoutsporozoite counts;

DAPI/PI

See also Anon 1990

Sundermann et al.,1987

Various "domestic" C. Baileyi fromchicks

Refers to earlierreference

Not specified Excystation Veterinary

Upton et al., 1988 Pentaiodide resin Bovine sporadicisolate via goat

Sieved; potdichromate; nonidetP40; sucrose;caesium chloride.

Not specified butused at 6/12th,washed in DDWprior to use

Excystation; mouseinfectivity.

Venczel et al., 1997 Mixed oxidents Iowa bovine Filtration; sucrose;Percoll; PBS + BSA


Watkins, 1995 Ultrasound Moredun isolate Not specified (assupplied)

Not specified (assupplied)

Excystation; DAPI/PI

Yozwiak et al., 1997 Mixed oxidants Not specified Not specified Not specified Excystation

of 56

11. Bibliography

Abbaszadegan M, Hasan MN, Gerba CP, Roessler PF, Wilson BR, Kuennen R, VanDellen E. The disinfection efficacy of a point-of-use water treatment system againstbacterial, viral and protozoan waterborne pathogens. Water Research 1997; 31 (3):574-582.

Anderson BC. Effect of drying on the infectivity of cryptosporidia-laden calf faeces for3- to 7-day-old mice. American Journal of Veterinary Research 1986; 47 (10): 2272-2273.

Anderson BC. Moist heat inactivation of Cryptosporidium sp. American Journal ofPublic Health 1985; 75 (12): 1433-4.

Angulo FJ, Tippen S, Sharp DJ, Payne BJ, Collier C, Hill JE, Barrett TJ, Clark RM,Geldreich EE, Donnell HD et al. A community waterborne outbreak of salmonellosisand the effectiveness of a boil water order. American Journal of Public Health 1997; 87(4): 580-584.

Angus KW, Sherwood D, Hutchison G, Campbell I. Evaluation of the effect of twoaldehyde-based disinfectants on the infectivity of faecal cryptosporidia for mice.Research in Veterinary Science 1982; 33 (3): 379-381.

Anon. Chlorine and ozone inactivation of Cryptosporidium oocysts. Journal of theAmerican Water Works Association 1989; 81:No. 9: 46.

Anon (1990). Water treatment processes and removal of Cryptosporidial Oocysts.In: DoE/DoH. Cryptosporidium in Water Supplies. First Report of the Group ofExperts. Chairman Sir John Badenoch. London HMSO, Pp 23-30.

Anon (1990). Resistance of Oocysts. In: ibid, Pp 90-95.

Anon (1990). Disinfection. In ibid, Pp 173-177.

Anon (1995). DoE/DoH. Cryptosporidium in Water Supplies. Second Report of theGroup of Experts. Chairman Sir John Badenoch. London HMSO.

Anon (1998). Cryptosporidium in Water Supplies. Third Report of the Group ofExperts, Chairman Prof I Bouchier. DETR/DoH, London, 171.

Anon (1997). Cryptosporidium and water: A Public Health Handbook.

Anon 1997. Draft guidance manual on implementation of Badenoch water treatmentrecommendations. Contract DW06 Cryptosporidium, Giardia and other encystingparasites: origins, analytical methodology, the significance of water vs other vectors.UK Water Industry Research Limited. 77.

of 56

Anon 1997. Report on Evaluation of CID-2/CID-4 [previously WR5000]Cryptosporidium parvum inactivation device. Water Recovery PLC Monmouth Gwent.Archer GP, Betts WB, Haigh T. Rapid differentiation of untreated, autoclaved andozone-treated Cryptosporidium parvum oocysts using dielectrophoresis. Microbios1993; 73: 165-72.

Archer GP, Quinn CM, Betts WB, Allsopp DWE, and O’Neill JG. Physical separationof untreated and ozone-treated Cryptosporidium parvum oocysts using non-uniformelectric fields. In: Protozoan Parasites and Water Eds Betts WB, Casemore DP,Fricker C, Smith H, Watkins J; The Royal Society of Chemistry Cambridge, 1995 143-145.

Ares-Mazas E, Lorenzo MJ, Casal JA. Effect of a commercial disinfectant ('Virkon') onmouse experimental infection by Cryptosporidium parvum. Journal of HospitalInfection 1997; 36: No.2: 141-145.

Armstrong DC, Casemore DP, Couper AM, Martin AD, and Naylor PJ. Disinfection ofCryptosporidium parvum with ozone at high concentrations. In: Protozoan Parasitesand Water Eds Betts WB, Casemore DP, Fricker C, Smith H, Watkins J; The RoyalSociety of Chemistry; Cambridge 1995a; 226-229.

Armstrong DC, Casemore, DP, Couper AM, Martin AD, and Naylor PJ. Disinfectionof Cryptosporidium parvum with ozone at high concentrations “Crypto busting withconcentrated ozone”. 1995b, Report for ICI Chemicals & Polymers Ltd.

Arrowood MJ, Xie LT, Rieger K, Dunn J. Disinfection of Cryptosporidium parvumoocysts by pulsed light treatment evaluated in an in vitro cultivation model. Journal ofEukaryotic Microbiology 1997; 43 (5):88S-Oct.

Awad-El-Kariem FM, Robinson HA, Petry F, McDonald V, Evans D, Casemore DP.Differentiation between human and animal isolates of Cryptosporidium parvum usingmolecular and biological markers. Parasitological Research 1998; 84: 297-301.

Bailey SW. Management of livestock manure. In: DoE/DoH Cryptosporidium inWater Supplies. First Report of Group of Experts London HMSO 1990; Pp 100-107.

Barbee SL, Sobsey MD, Arrowood M. Effect of elevated temperatures and thermo-philic anaerobic digestion on C. parvum oocyst viability as determined by cell cultureinfectivity assay. Proceedings: 97th General Meeting of the American Society forMicrobiology 1997; 8.

Battigelli DA, LeChevallier MW, Abbaszadegan M. Environmental resistance ofCryptosporidium parvum and Giardia muris. Proceedings: 96th General Meeting of theAmerican Society for Microbiology 1996; 7: 23, 1996.

Battigelli DA, LeChevallier MW, Abbaszadegan M, and Upton S. Viability assessmentof Cryptosporidium parvum by an integrated cell culture/RT-PCR method.

of 56

Proceedings: International Symposium on Waterborne Cryptosporidium. NewportBeach Ca, USA, 1997.

Bellamy WD, Cleasby JL, Logsdon GS, Allen MJ. Assessing treatment plantperformance. Journal of the American Water Works Association 1993; 85: 34-38.

Belosevic M, Finch GR. Development of a vital stain methodology for Giardia andCryptosporidium. 1996 AWWARF Report.

Belosevic M, Guy RA, Neumann NF, and Finch GR. Viability Assessment ofCryptosporidium parvum Oocysts using Nucleic Acid Stains. Proceedings:International Symposium on Waterborne Cryptosporidium. Newport Ca, USA, 1997.

Belesovic M, Guy RA, Taghi-Kilani R, et al. Nucleic acid stains as indicators ofCryptosporidium parvum oocyst viability. International Journal of Parasitology 1997;27: 787-798.

Benton C, Ives KJ, Miller DG, West PA. (1991). Cryptosporidium in water - progressin the United States: Report of an OSTEMS mission. WRc Medmenham.

Black EK, Finch GR, Taghi Kilani R, Belosevic M. Comparison of assays forCryptosporidium parvum oocyst viability after chemical disinfection. FemsMicrobiology Letters 1996; 135 (23): 187-189.

Blewett, DA. Quantitative techniques in Cryptosporidium research. In: Eds Angus,KW., Blewett, DA. Cryptosporidiosis. Proceedings of International Workshop onCryptosporidiosis. Animal Diseases Research Association, Moredun ResearchInstitute, Edinburgh. 1989; Pp 85-95.

Blewett DA. Disinfection and oocysts. In: Eds Angus, KW., Blewett, DA.Cryptosporidiosis. Proceedings of International Workshop on cryptosporidiosis.Animal Diseases Research Association, Moredun Research Institute, Edinburgh. 1989;Pp 107-115.

Bodley-Tickell A, Kitchen S, Sturdee A. Cryptosporidium parvum in rural surfacewaters. In proceedings: International Symposium on Waterborne CryptosporidiumWarwick UK Eds: Morris, R., Gammie, A 1997; Pp 138-143.

Bolton JR, Dusseri B, Bukhari Z, Hargy T, Clancy JL. 1998. Inactivation ofCryptosporidium parvum by medium pressure ultraviolet light in finished drinkingwater. AWWA Proceedings AGM 1998, 15 Pp.

Bonnin A, Fourmaux MN, Dubremetz JF, et al. Genotyping human and bovine isolatesof Cryptosporidium parvum by polymerase chain reaction-restriction fragment lengthpolymorphism analysis of a repetitive DNA sequence. FEMS Microbiology Letters1996; 137: 207-11.

of 56

Brasseur P, Uguen C, Moreno-Sabater A, et al. Viability of Cryptosporidium parvumoocysts in natural water. Folia Parasitologica. 1998; 45: 113-6.

Brush CF, Walter MF, Anguish LJ, Ghiorse WC. Influence of pre-treatment andexperimental conditions on electrophoretic mobility and hydrophobicity of Crypto-sporidium parvum oocysts. Journal of Applied and Environmental Microbiology 1998;64: 4439-45.

Bukhari Z, and Smith HV. Cryptosporidium parvum: oocyst excretion and viabilitypatterns in experimentally infected lambs. Epidemiology and Infection 1997; 119: 105-108.

Bukhari Z, and Smith HV. Effect of three concentration techniques on viability ofCryptosporidium parvum oocysts recovered from bovine faeces. Journal of ClinicalMicrobiology 1995; 33;10:2592-2595.

Bukhari Z, Smith HV, Humphreys SW, Kemp J, Wright SE. Comparison of excretionand viability patterns in experimentally infected animals: potential for release of viableC. parvum oocysts into the environment. In: Protozoan Parasites and Water Eds. BettsWB, Casemore DP, Fricker C, Smith H, Watkins J; The Royal Society of ChemistryCambridge 1995;188-191.

Campbell AT, Robertson LJ, Snowball MR, Smith HV. Inactivation of oocysts ofCryptosporidium parvum by ultraviolet UV irradiation. Water Research 1995; 29 (11):2583-2586.

Campbell AT, Robertson LJ, Smith HV. Viability of Cryptosporidium parvum oocysts:Correlation of in vitro excystation with inclusion or exclusion of fluorogenic vital dyes.Applied and Environmental Microbiology 1992; 58; No 11: 3488-3493.

Campbell AT, Robertson LJ, and Smith HV. Effects of preservatives on viability ofCryptosporidium parvum oocysts. Applied and Environmental Microbiology 1993;59:12: 4361-4362.

Campbell AT, Robertson L, Parker J, Anderson R, and Smith HV. Viability ofCryptosporidium oocysts: assessment following disinfection with low concentrations ofozone. Proceedings: International Symposium on Waterborne Cryptosporidium.Newport Beach Ca USA, 1997.

Campbell I, Tzipori S, Hutchison G, Angus KW. Effect of disinfectants on survival ofCryptosporidium oocysts. Veterinary Record 1982; 111, No 18: 414-415.

Carrington E.G. and Ransome ME. Factors influencing the survival ofCryptosporidium oocysts in the environment. Report to Foundation for WaterResearch 1994.

of 56

Casemore DP. Epidemiological aspects of human cryptosporidiosis. Epidemiology andInfection 1990; 104: 1-28.

Casemore DP. Disinfection options. In: West PA & Smith MS, Eds, DoE/WelshOffice/UK Water Industry Research Ltd. Proceedings of Workshop on TreatmentOptimisation for Cryptosporidium Removal from Water Supplies. London HMSO1995; 19-24.Casemore, DP. Laboratory methods for diagnosing cryptosporidiosis. Journal ofClinical Pathology 1991; 44: 445-451.

Casemore DP. Molecular and antigenic aspects of Cryptosporidium andcryptosporidiosis (a brief review). In: Anon (1998). Cryptosporidium in WaterSupplies. Third Report of the Group of Experts, Chairman Prof I Bouchier.DETR/DoH, London, 137-142.

Casemore DP, Blewett DA, Wright SE. Cleaning and disinfection of equipment for gastro-intestinal flexible endoscopy. Gut 1989; 30: 1156.

Casemore DP, Wright SE, and Coop RL. Cryptosporidiosis – human and animalepidemiology. In: Ed R Fayer Cryptosporidium and cryptosporidiosis CRC Press Inc 1997;65-92.

Casteel MJ, Sobsey MD. Inactivation of Cryptosporidium parvum oocysts andClostridium perfringens spores in water by electrochemically generated mixedoxidants. Proceedings: 97th General Meeting of the American Society forMicrobiology, Miami Beach, Florida, USA, May 4-8, 1997 Abstracts of the GeneralMeeting of the American Society for Microbiology Q-11, 456.

Chalmers RM, Sturdee A, Casemore DP, et al. Cryptosporidium muris and C. parvumin wild house mice (Mus musculus): first report in the UK. European Journal ofProtistology 1994; 30:151-5.

Chauret C, Bagwell B, Meybodi S, et al. 1998. Effect of microbial antagonism on thesurvival of Cryptosporidium parvum oocysts in fresh natural waters. 7 Pp.

Chiou C, Torees-Lugo M, Marinas BJ, Adams JQ. Non-biological surrogate indicatorsfor assessing ozone disinfection. Journal of the American Water Works Association1997; 89: 54-66.

Clancy JL, Fricker CR, Telliard W. New US EPA standard method forCryptosporidium analysis in water (Draft unpublished). Proceedings: InternationalSymposium on Waterborne Cryptosporidium Warwick UK Eds: Morris, R., Gammie, A1997a; 134-125.

Clancy JL, Marshall MM, and Dyksen, JE. Innovative electro-technologies forinactivation of Cryptosporidium. Proceedings: International Symposium onWaterborne Cryptosporidium Newport Beach Ca USA, 1997b; 192-199.

of 56

Clancy JL, Marshall MM, and Dyksen JE. Inactivation of Cryptosporidium parvumoocysts in water using advanced ultraviolet irradiation. Proceedings: InternationalSymposium on Waterborne Cryptosporidium Warwick UK Eds: Morris R, Gammie A.1997c; 219-227.

Cordell RL, Addiss DG. Cryptosporidiosis in a childcare setting: a review andrecommendations for prevention and control. Pediatric Infectious Disease Journal1994; 13: 310-317.

Craun GF, Bull RJ, Clark RM, et al. Balancing chemical and microbial risks ofdrinking water disinfection: benefits and potential risks. Aqua 1994; 43: 192-199.

Dawson D, Lloyd A. Eds, Proc of Workshop on Cryptosporidium in water supplies.HMSO London, 1994.

Deere D, Vesey G, Dorsch M, Ashbolt N, Williams K, Veal D. Optimisation offluorescent in-situ ribosomal RNA labelling of Cryptosporidium parvum in suspension.Proceedings: International Symposium on Waterborne Cryptosporidium Warwick UKEds: Morris R, Gammie A. 1997; 144-156.

Drozd C, Schwartzbrod J. Hydrophobic and electrostatic cell surface properties ofCryptosporidium parvum. Applied and Environmental Microbiology 1996; 62: 1227-1232.

Dowd SE, Pillai SD. A rapid viability assay for Cryptosporidium oocysts and Giardiacysts for use in conjunction with indirect fluorescent antibody detection. CanadianJournal of Microbiology 1997; 43: 658-62.

Fayer R. Effect of high temperature on infectivity of Cryptosporidium parvum oocystsin water. Applied and Environmental Microbiology 1994; 60: 2732-5.

Fayer R. Effect of sodium hypochlorite exposure on infectivity of Cryptosporidiumparvum oocysts for neonatal balb-c mice. Applied and Environmental Microbiology1995; 61: 844-846.

Fayer R. Ed: Cryptosporidium and cryptosporidiosis. CRC Press Inc, Boca Raton. 1997.

Fayer R, Graczyk TK, Cranfield MR, Trout JM. Gaseous disinfection ofCryptosporidium parvum oocysts. Applied and Environmental Microbiology 1996; 62:3908-3909

Fayer R and Leek RG. The effects of reducing conditions, medium, pH, temperatureand time on in vitro excystation of Cryptosporidium. Journal of Protozoology 1984;31: 567-569.

Fayer R, and Nerad T. Effects of low temperatures on viability of Cryptosporidiumparvum oocysts. Applied and Environmental Microbiology 1996; 4: 1431-1433.

of 56

Fayer R, Trout J, Nerad T. Effects of a wide range of temperatures on infectivity ofCryptosporidium parvum oocysts. Journal of Eukaryotic Microbiology; 43: 64S.

Finch GR, Black KE, Gyurek L, Belosevic M. Ozone inactivation of Cryptosporidiumparvum in demand-free phosphate buffer determined by in vitro excystation and animalinfectivity. Applied and Environmental Microbiology 1993a; 59 (12): 4203-4210.

Finch GR, Daniels CW, Black EK, Schaefer FW, 3d, Belosevic M. Dose response ofCryptosporidium parvum in outbred neonatal CD-1 mice. Applied and EnvironmentalMicrobiology 1993b; 59 (11): 3661-3665.Finch GR, Black KE, Gyurek LL. 1994. Ozone disinfection of Giardia andCryptosporidium. Report to AWWA Research Foundation. i-xxvi + 56 Pp.

Finch GR, Black KE, and Gyurek LL. Ozone and chlorine inactivation ofCryptosporidium. Proceedings JAWWA Water Quality Technology Conference, SanFrancisco CA, USA, 1995; 1303-18.

Finch GR, et al. Synergistic effects of sequential exposure of Cryptosporidium oocyststo chemical disinfectants. Proceedings: International Symposium on WaterborneCryptosporidium Newport Beach Ca USA, 1997.

Fricker CR, Clancy J, Smith HV. Factors involved in the variation of performance datacollected during trials of new methods for protozoan parasites. Proceedings:International Symposium on Waterborne Cryptosporidium Warwick UK Eds: Morris R,Gammie A. 1997; 238.

Fricker CR, Smith HV, Marshall MM, Clancy JL. Comparison of viability/infectivityof Cryptosporidium in disinfection studies. Proceedings: International Symposium onWaterborne Cryptosporidium Warwick UK Eds: Morris R, Gammie A. 1997 ; 240.

Furtado C, Adak GK, Stuart JM, Wall PG, Evans HS, Casemore DP. Outbreaks ofwaterborne infectious intestinal disease in England and Wales, 1992-5. Epidemiologyand Infection 1998; 121: 109-119.

Galbraith NS, Barrett NJ, Stanwell-Smith R. Water and disease after Croydon: a reviewof the water-borne and water-associated disease in the UK 1937-86. Journal of theInstitute of Water and Environment 1987; 1: 7-21.

Gates D, Harrington RM, Romano RR. 1989 A review of the uses, chemistry and healtheffects of chlorine dioxide and the chlorite ion. Prepared by: The chlorine dioxidepanel of the chemical manufacturers association Washington DC.

Gerba CP, Johnson DC, Hasan MN. Efficacy of iodine water purification tabletsagainst Cryptosporidium oocysts and Giardia cysts. Wilderness and EnvironmentalMedicine 1997; 8: 96-100.

of 56

Graczyk TK, Fayer R, Cranfield MR, and Owens R. Cryptosporidium parvum oocystsrecovered from water by the membrane filter dissolution method retain their infectivity.Journal of Parasitology 1997; 83 (1): 111-4.

Godfree AF, Casemore DP. Report of studies to assess the effectiveness of ozoneagainst Cryptosporidium. Contract Report to Electricity Council Research Centre,Capenhurst.

Grimason AM, Smith HV, Parker JFW, Bukhari Z, Campbell AT, Robertson LJ.Application of DAPI and immunofluorescence for enhanced identification ofCryptosporidium Spp oocysts in water samples. Water Research 1994; 28: 733-36.

Gyurek LL, Finch GR, Belosevic M. Modelling chlorine inactivation requirements ofCryptosporidium parvum oocysts. Journal of Environmental Engineering 1997;123:865-75.

Gyurek LL, Finch GR, Belosevic M. Inactivation of Cryptosporidium using single andsequential application of ozone and chlorine species. Proceedings: InternationalSymposium on Waterborne Cryptosporidium Newport Beach Ca USA, 1997.

Haas CN, Parker JFW, Ongerth JE, Smith HV. Destruction of oocysts ofCryptosporidium parvum by sand and chlorine. Water Research 1995; 29: 1615-1618.

Hall T, Pressdee J. 1995. Removal of Cryptosporidium during water treatment. In:Anon Second report of the Group of Experts, Chairman Sir John Badenoch. DoE/DoH.HMSO London.

Hall T, Pressdee J, and Carrington E. Removal of Cryptosporidium during watertreatment. In: Protozoan Parasites and Water Eds. Betts WB, Casemore DP, Fricker C,Smith H, Watkins J; The Royal Society of Chemistry, Cambridge. 1995; 192-5.

Harp JA, Fayer R, Pesch BA. Effect of pasteurisation on infectivity of Cryptosporidiumparvum oocysts in water and milk. Applied and Environmental Microbiology 1996; 62(8): 2866-8.

Hibler CP, Hancock CM. Waterborne giardiasis. In: Ed GA McFeters Drinking WaterMicrobiology. Springer-Verlag, New York, 1990. 271-293.

Holton J, Shetty N, McDonald V. Efficacy of 'Nu-cidex' (0.35 percent peracetic acid)against mycobacteria and cryptosporidia. Journal of Hospital Infection 1995 31; 3:235-237.

Holton J, Nye P, McDonald V. Efficacy of selected disinfectants against mycobacteriaand cryptosporidia. Journal of Hospital Infection 1994 27; 2: 105-115.

Hom LW. Kinetics of chlorine disinfection in an ecosystem. Journal of the SanitaryEngineering Division, American Society of Civil Engineering. 1972; 98: 183-193.

of 56

Ives KJ. Coagulation, Clarification and Filtration. DoE/Welsh Office UK WaterIndustry Research Ltd. Proceedings: Workshop on Treatment Optimisation forCryptosporidium Removal from Water Supplies. Eds West PA, Smith MS. 1995London HMSO.

Jacangelo JG, Adham SS, Laine JM. Mechanism of Cryptosporidium, Giardia andMS2 virus removal by MF and UF. Journal of the American Water Works Association1995; 87: 107-21.

Jacobowski W, Boutros S, Faber W, et al. Environmental methods for Crypto-sporidium. JAWWA 1996; 88: 107-21.

Jarroll, EL. 1992. Sensitivity of protozoa to disinfectants. In: Principles and Practiceof Disinfection, Preservation and Sterilization. Eds Russell AD, Hugo WB, AyliffeGAJ. Blackwell Scientific Publications Oxford. Pp 180-186.

Jenkins MB, Anguish LJ, Bowman DD, et al. Assessment of a dye permeability assayfor determination of inactivation rates of Cryptosporidium parvum oocysts. Appliedand Environmental Microbiology 1997; 63: 3844-3850.

Jenkins MB, Bowman DD, Ghiorse WC. Inactivation of Cryptosporidium parvumoocysts by ammonia. Applied and Environmental Microbiology 1998; 64 (2): 784-788.

Johnson DC, Enriquez CE, Pepper IL et al. Survival of Giardia, Cryptosporidium,poliovirus and salmonella in marine waters. Water Science and Technology 1997; 35:261-268.

Juranek DD, Addiss DG, Bartlett ME, Arrowood MJ, Colley DG, Kaplan JE,Perciasepe R, Elder JR, Regli SE, Berger PS. Cryptosporidiosis and public health:workshop report. Journal of the American Water Works Association 1995; 87: 69-80.

Kilani RT, Sekla SL. Purification of Cryptosporidium oocysts and sporozoites bycaesium chloride and Percoll gradients. American Journal of Tropical Medicine andHygiene 1987; 36: 505-508.

Klonicki PT, Hancock CM, Straub TM, Harris SI, Hancock KW, Alyaseri AN, MeyerCJ, Sturbaum GD. Crypto research: are fundamental data missing? Journal of theAmerican Water Works Association 1997; 89 (9): 97-103.

Korich DG, Mead JR, Madore MS, Sinclair NA, and Sterling CR. Chlorine and ozoneinactivation of Cryptosporidium oocysts. Water Quality Technical Conference 1989;681-693.

Korich DG, Mead JR, Madore MS, Sinclair NA, Sterling CR. Effects of ozone, chlorinedioxide, chlorine, and monochloramine on Cryptosporidium parvum oocyst viability.Applied and Environmental Microbiology 1990; 56 (5): 1423-1428.

of 56

Korich DG, Yozwiak ML, Marshall MM, Sinclair NA, Sterling C.1993. Developmentof a test to assess Cryptosporidium parvum oocyst viability: correlation with infectivitypotential. Report published by the American Water Works Research Foundation.

Korich DG, Clancy JL, Fricker C, Marshall MM, Smith HV. Comparison ofCryptosporidium viability/infectivity methods. Report (draft) to American WaterWorks Association Research Fund, 1997.

Labatiuk, CW., Belosevic, M., and Finch, GR. Factors influencing the infectivity ofGiardia muris cysts following ozone inactivation in laboratory and natural waters.Water Research 1992; 26; 6: 733-743.

Labatiuk CW, Schaefer III FW, Finch GR, Belosevic M. Comparison of animalinfectivity, excystation and fluorogenic dye as measures of Giardia muris cystinactivation by ozone. Applied and Environmental Microbiology 1991; 57: 3187-3192.

Langlais BD, Perrine JC, Joret JC, Chenu JP. The CT value concept for evaluation ofdisinfection process efficiency. Proc Internat Ozone Assoc Spring Conference,Shreveport, LO, USA. 1990

Langlais BD, Joret JC, Perrine JC, Chenu JP, LeGardinier JC. Inactivation ofCryptosporidium, a waterborne protozoan parasite, using ozone. TSM-L'Eau 1991; 86:197-200.

LeChevallier MW, Battigelli DA, Arora H, Abbaszadegan M. Tissue culture method forCryptosporidium parvum inactivation by chlorine dioxide. Proceedings: 97th GeneralMeeting of the American Society for Microbiology 1997; Abstr Q-12, Pp 457.

LeChevallier MW, Moser RH. Monitoring of Giardia and Cryptosporidium in theAmerican water system. Report to the American Water Works Service Co, Belleville,Ill. 1993.

LeChevallier MW, Norton WD. Giardia and Cryptosporidium in raw and finishedwater. Journal of the American Water Works Association 1995; 87 (9): 54-68.

LeChevallier MW, Arora H, Battigelli DA. Inactivation of Cryptosporidium bychlorine dioxide: balancing risks. Proceedings: International Symposium onWaterborne Cryptosporidium. Newport Beach Ca USA, 1997.

Lisle JT and Rose JB. Cryptosporidium contamination of water in the USA and UK: amini-review. Aqua (Oxford) 1995; 44 (3): 103-117.

Liyanage LRJ, Finch GR, Belosevic M. Effect of aqueous chlorine and oxy-chlorinecompounds on Cryptosporidium parvum oocysts. Environmental Science andTechnology 1997; 31: 1992-1994.

of 56

Liyanage LRJ, Finch GR, Belosevic M. Sequential disinfection of Cryptosporidiumparvum by ozone and chlorine dioxide. Ozone Science Engineering 1997; 19 :409-423.

Liyanage LRJ, Finch GR, Belosevic M. Synergistic effects of sequential exposure ofCryptosporidium oocysts to chemical disinfectants. Proceedings: InternationalSymposium on Waterborne Cryptosporidium California Eds. Fricker CR, Clancy JL,Rochelle PA. American Water Works Association Denver 1997 Pp 41-51.

Lorenzo-Lorenzo MJ, Ares-Mazas ME, Villacorta-Martinez de Maturana I, Duran-Oreiro D. Effect of ultraviolet disinfection of drinking water on the viability ofCryptosporidium parvum oocysts. Journal of Parasitology 1993; 79 (1): 67-70.

Marshall MM, Naumovitz D, Ortega Y, Sterling CR. Waterborne protozoan pathogens.[Review]. Clinical and Microbiological Reviews 1997; 10 (1): 67-85.Mayer C, Palmer CJ. Evaluation of PCR, nested PCR, and fluorescent antibodies fordetection of Giardia and Cryptosporidium species in wastewater. Applied andEnvironmental Microbiology. 1996; 62: 2081-2085.

Mayerhofer P. Control of disinfection by-products and the inactivation of parasites.Proc Annual Conf and Exposition of AWWA. Orlando Fla USA 1988. Journal of theAmerican Water Works Association 1988; 80 (4): 79.

Mc Feters GA. Ed: Drinking Water Microbiology: Progress and recent developments.Springer-Verlag. 1990; New York.

McLauchlin J, Casemore DP, Moran S, Patel S. The epidemiology of cryptosporid-iosis: application of experimental sub-typing and antibody detection systems to theinvestigation of waterborne outbreaks. Folia Parasitologica 1998; 45: 83-92.

Medema GJ, Bahar M, Schets FM. Survival of Cryptosporidium parvum, Escherichiacoli, faecal enterococci and Clostridium perfringens in river water: influence oftemperature and autochthonous microorganisms. Wat Sci Tech 1997; 35: 11-2.

Meinhardt PL, Casemore DP, Miller KB. Epidemiologic aspects of humancryptosporidiosis and the role of waterborne transmission. Epidemiology Reviews1996; 18 (2): 118-136.

Merry RJ, Mawdsley JL, Brooks AE, and Davies DR. Viability of Cryptosporidiumparvum during ensilage of perennial ryegrass. Journal of Applied Microbiology 1997;82 (1): 115-20.

Miltner D, Rice, Owen, Schaefer, and Shukairy. Comparative ozone inactivation ofCryptosporidium and other micro-organisms. Proceedings: International Symposiumon Waterborne Cryptosporidium California Eds. Fricker CR, Clancy JL, Rochelle PA.American Water Works Association Denver 1997 Pp 229-241.

of 56

Morgan UM, Sargent KD, Deplazes P, et al. Molecular characteristics ofCryptosporidium from various hosts. Parasitology 1998; 117: 31-7.

Naranjo JE, Gerba CP. Evaluation of portable reverse osmosis and iodine watertreatment system for removal of enteric pathogens. Proceedings: 97th General Meetingof the American Society for Microbiology, Miami Beach, Florida, USA, May 4-8, 1997Abstracts of the General Meeting of the American Society for Microbiology 97 (0)1997; 455.

Naranjo JE, Chaidez CI, Quinonez M, Gerba CP, Olson J, Dekko J. Evaluation of aportable water purification system for the removal of enteric pathogens. Water Scienceand Technology 1997; 35: 55-58.

Nichols GL, McLauchlin J, Samuel D. A technique for typing Cryptosporidiumisolates. Journal of Protozoology 1991; 38: 237S-240S.

Nieminski EC and Bradford SM. Impact of ozone treatment on selected microbiologicalparameters. Ozone Science and Engineering 1991; 13 (2): 127-146.

Nieminski EC, Ongerth, JE. Removing Giardia and Cryptosporidium by conventionaltreatment and direct filtration. Journal of the American Water Works Association 1995;97: 96-106.

Nina JMS, McDonald V, Deer RMA, et al. Comparative study of the antigeniccomposition of oocyst isolates of Cryptosporidium parvum from different hosts.Parasitology and Immunology 1992; 14: 227-232.

Nina JMS, McDonald V, Dyson DA, et al. Analysis of oocyst wall and sporozoiteantigens from three Cryptosporidium species. Infection and Immunity 1992; 60: 1509-1513.

Ongerth JE, Pecoraro J. Electrophoretic mobility of Cryptosporidium oocysts andGiardia cysts. Journal of Environmental Engineering 1986; 122: 228.

Ongerth JE, Pecoraro J. Removal of Cryptosporidium in rapid sand filtration. Journalof the American Water Works Association 1996; 87: 83.

Oppenheimer JA, Aieta EM, Jacanqelo J, Najm I, Selby DA, and Rexing D.Disinfection of Cryptosporidium: design criteria for North American water agencies.Proceedings: International Symposium on Waterborne Cryptosporidium California Eds.Fricker CR, Clancy JL, Rochelle, PA. American Water Works Association Denver1997 Pp 275-280.

Otterstetter H, Craun G. Disinfection in the Americans: A necessity. Journal of theAmerican Water Works Association 1997; 89 (9): 8-10.

Owens JH, Miltner RJ, Schaefer FW, 3rd, Rice EW. Pilot-scale ozone inactivation ofCryptosporidium. Journal of Eukaryotic Microbiology 1994; 41 (5): 56S-57S.

of 56

Parker JFW, Smith H.V. Destruction of oocysts of Cryptosporidium parvum by sandand chlorine. Water Research 1993: 27 (4): 729-731.

Parker JFW, Smith HV. Comment on “Destruction of oocysts of Cryptosporidiumparvum by sand and chlorine”. Water Research 1995; 29 (6): 1615-1618.

Parker JFW, Greaves GF, Smith HV. The effect of ozone on the viability ofCryptosporidium parvum oocysts and comparison of experimental methods. WaterScience and Technology 1993; 27 (3-4): 93-96.

Patel S, McLauchlin J, Casemore DP. A simple SDS-PAGE immunoblotting techniqueusing an enhanced chemiluminescence detection system to identify polyclonal antibodyresponses to complex cryptosporidial antigen preparations following a monoclonalantibody retest and image overlay technique. Journal of Immunological Methods 1997;205: 157-161.

Patel S, Pedraza-Diaz S, McLauchlin J, Casemore DP. Molecular characterisation ofCryptosporidium parvum from two large suspected waterborne outbreaks.Communicable Disease and Public Health 1998; 4: 231-3.

Pavlasek I. [The effect of disinfectants on the infectivity of Cryptosporidium sp.oocysts]. [Czech]. Ceskoslovenska Epidemiologie, Mikrobiologie, Imunologie 1984; 33(2): 97-101. (English summary)

Payment P, Franco E. Clostridium perfringens and somatic coliphages as indicators ofthe efficiency of drinking water treatment for viruses and protozoan cysts. Applied andEnvironmental Microbiology 1993; 59: 2418-24.

Peeters JE, Mazas EA, Masschelein WJ, Villacorta Martiez de Maturana I, Debacker E.Effect of disinfection of drinking water with ozone or chlorine dioxide on survival ofCryptosporidium parvum oocysts. Applied and Environmental Microbiology 1989; 55(6): 1519-1522.

Perrine D, Georges P, Langlais B. The efficacy of water ozonation on the inactivationof oocysts of Cryptosporidium. [French]. Bulletin de l Academie Nationale deMedecine 1990; 174 (6): 845-850; discussion 850-851.

Pontius FW. Expedited microbial/disinfection by-product rules defined. Journal of theAmerican Water Works Association 1997; 89 (9): 20-24.

Pozio E, Morales MAG, Barbieri FM, LaRosa G. Cryptosporidium: different behaviourin calves of isolates of human origin. Transcriptions of the Royal Society of TropicalMedicine and Hygiene 1992; 86: 636-638.

Pressdee JR, Hall T, Carrington E. Practicalities of disinfection for control ofCryptosporidium and Giardia. In: Protozoan Parasites and Water Eds Betts WB,

of 56

Casemore DP, Fricker C, Smith H, Watkins J; The Royal Society of ChemistryCambridge 1995; Pp 206-208.

Quinn CM, Archer GP, Betts WB, and O’Neill JG. Dose-dependent dielectrophoreticresponse of Cryptosporidium oocysts treated with ozone. Letters in AppliedMicrobiology 1996; 22: 224-228.

Quinn CM, Betts W.B. Longer term viability status of chlorine-treatedCryptosporidium oocysts in tap water. Biomedical-Letters 1993; 48: 315-318.

Ransome ME, Carrington E.G., Whitmore TN, and Harman SA. Evaluation of theefficiency of alternative disinfectants to chlorine. (WRC Report) 1992.

Rasmussen V, Nissen B, Lund E, Strand RL. Using redox potential to assessCryptosporidium inactivation by chlorine. In: Protozoan Parasites and Water Eds.Betts WB, Casemore DP, Fricker C, Smith H, Watkins J; The Royal Society ofChemistry Cambridge 1995; Pp 214-222.

Reduker DW, Speer CA. Factors influencing excystation in Cryptosporidium oocystsfrom cattle. Journal of Parasitology 1985;71 (1): 112-115.

Restaino L, Frampton EW, Hemphill JB, Palnikar P. Efficacy of ozonated wateragainst various food-related micro-organisms. Applied and EnvironmentalMicrobiology 1995; 61 (9) : 3471-3475.

Robertson LJ. Viability of Giardia cysts and Cryptosporidium oocysts. In: ProtozoanParasites and Water Eds. Betts WB, Casemore DP, Fricker C, Smith H, Watkins J; TheRoyal Society of Chemistry Cambridge 1995; Pp 97-103.

Robertson LJ, Campbell AT, and Smith HV. Survival of Cryptosporidium parvumoocysts under various environmental pressures. Applied and EnvironmentalMicrobiology 1992; 58 (11): 3494-3500.

Robertson LJ, Smith HV. Cryptosporidium and cryptosporidiosis. Part I: currentperspectives and present technologies. European Microbiology Nov/December 1992;20-29.

Robertson LJ, Smith HV, Ongerth JE. Cryptosporidium and cryptosporidiosis. Part III:development of water treatment technologies to remove and inactivate oocysts.European Microbiology 1994; 2 (1): 18-26.

Rochelle PA, Ferguson DM, Handojo TJ, DeLeon R, Stewart MH, Wolfe RL.Development of a rapid detection procedure for Cryptosporidium, using in vitro cellculture combined with PCR. Journal of European Microbiology 1996; 43: 72S.

Rochelle PA, Ferguson DM, Handojo TJ, DeLeon R, Stewart MH, and Wolfe RL. Anassay combining cell culture with reverse transcriptase PCR to detect and determine the

of 56

infectivity of waterborne Cryptosporidium parvum. Applied and EnvironmentalMicrobiology 1997; 63 (5): 2029-37.

Rochelle, PA., De Leon, R., Ferguson, DM., Stewart, MH., and Wolfe, RL.Optimization of an infectivity assay, combining cell culture and PCR, for waterborneCryptosporidium parvum. Proceedings: International Symposium on WaterborneCryptosporidium California Eds. Fricker CR, Clancy JL, Rochelle PA. American WaterWorks Association Denver 1997 Pp 31-40.

Rose JB. Occurrence and control of Cryptosporidium in drinking water. In: DrinkingWater Microbiology. Ed McFeters, GA, Springer-Verlag New York Inc. 1990. Pp 294-317.

Rose JB, Dickson LJ, Farrah SR, Carnahan RP. Removal of pathogenic and indicatormicro-organisms by a full-scale water reclamation facility. Water Research 1996; 30(11): 2785-97.

Rose JB, Lisle JT, and LeChevallier M. Waterborne cryptosporidiosis: Incidence,outbreaks, and treatment strategies. In: Ed R Fayer Cryptosporidium andCryptosporidiosis CRC Press Inc Boca Raton USA 1997; Pp 93-109.Russell AD, Hugo WB, Ayliffe GAJ. Eds: Disinfection, preservation and sterilization.Blackwell Scientific Publications, London, 1992.

Ruxton, GD. Mathematical modelling of ammonia volatilization from slurry stores andits effect on Cryptosporidium oocyst viability. Journal of Agricultural Science 1995;124: 55-60.

Sauch JF, Flanigan D, Galvin MI, Berman D, Jakubowski W. Propidium iodide as anindicator of Giardia cyst viability. Applied and Environmental Microbiology 1991; 57:3243-3247.

Schaub S, Fox J, Regli S, Schaefer F, Vasconcellos J, Harris S, Jakubowski W, LustikM. EPA protozoa method criteria document for guidance in development of analyticalmethods US Environment Protection Agency (Draft unpublished) 1997.

Sibley LD, Hakansson S, Carruthers VB. Gliding motility: An efficient mechanism forcell penetration. Current Biology 1998; 8: R12-R14.

Slifko TR, Friedman D, Rose JB, Jakubowski W. An in vitro method for detectinginfectious Cryptosporidium oocysts with cell culture. Applied and EnvironmentalMicrobiology 1997; 63: 3669-75.

Smith HV, Marshall MM, O’Grady J, Korich DG, Olfers S, Millan D, Clancy JL,Fricker CR, Campbell B, Bukhari Z, Rosen J. Usefulness of the CD1 neonatal mousemodel for determining the infectivity of Cryptosporidium parvum oocysts. Proceedings:International Symposium on Waterborne Cryptosporidium Warwick UK Eds: Morris R,Gammie A. 1997; Pp 200-206.

of 56

Smith HV, Rose JB. Waterborne cryptosporidiosis. Parasitology Today 1990; 6: 8-12.

Smith HV, Rose JB. Waterborne cryptosporidiosis: current status. Parasitology Today1990; 14: 14-22.

Smith HV, Smith AL, Girdwood RWA, Carrington E.G. The effect of free chlorine onthe viability of Cryptosporidium sp. oocysts isolated from human faeces. WRcMedmenham 1989; Pp 185-204. Also see Anon 1990, DoE/DoH First Report of theGroup of Experts, Chairman Sir John Badenoch. HMSO London, Pp 185-204.

Spano F, Putagnani L, Mc Lauchlin J, Casemore DP, Crisanti A. PCR-RFLP analysis ofthe Cryptosporidium oocyst wall protein (COWP) gene discriminates between C. wrairiand C. parvum isolates of human and animal origin. FEMS Letters 1997; 150: 209-217.

Stanwell-Smith R. Water and public health in the United Kingdom. Recent trends in theepidemiology of water-borne disease. In: Eds AMB Golding, N Noah, R Stanwell-Smith, Water & Public Health. 1994; Smith-Gordon London, 39-57.

States S, Stadterman K, Ammon L, Vogel P, Baldizar J, Wright D, Conley L, Sykora J.Protozoa in river water: sources, occurrence, and treatment. Journal of the AmericanWater Works Association 1997; 89 (9): 74-83.Sterling CR. Waterborne cryptosporidiosis. In: Eds JP Dubey, CA Speer, R Fayer.Cryptosporidiosis of man and animals. CRC Press, Boca Raton. Pp

Sterling CR, Korich DG, Mead, Madore MS, Sinclair NA. Chlorine and ozoneinactivation of Cryptospordium oocysts. Proc AWWA Water Quality Technol Conf,Philadelphia, USA, 1989.

Stinear T, Matusan A, Hines K, Sandery M. Detection of a single viableCryptosporidium parvum oocyst in environmental water concentrates by reversetranscription-PCR. Applied and Environmental Microbiology 1996; 62 (9); 3385-3390.

Straub T, Mena H, Gerba C, Daniel P, West T. Viability of Giardia muris cysts andCryptosporidium parvum oocysts after ageing pressure, pH manipulations anddisinfection in mountain reservoir water. Proceedings: 94th General Meeting of theAmerican Society for Microbiology 1994; 473.

Sunderman CA, Lindsay DS, Blagburn BL. Evaluation of disinfectants for ability to killavian Cryptosporidium oocysts. Companion Animal Practice 1987; 1 (6): 36-9.

Suresh P, Rehg JE. Comparative evaluation of several techniques for purification ofCryptosporidium parvum oocysts from rat feces. Journal of Clinical Microbiology1996; 34 (1): 38-40.

Ungar BLP, Burris JA, Quinn CA, Finkelman FD. New mouse models for chronicCryptosporidium infection in immunodeficient hosts. Infection and Immunology 1990;58: 961-969.

of 56

Upton SJ, Tilley M, Marchin GL, and Fina LR. Efficacy of a pentaiodide resindisinfectant on Cryptosporidium parvum (Apicomplexa: Cryptosporidiidae) oocysts InVitro. Journal of Parasitology 1988; 74: 719-721.

Vassal S, Favennec L, Ballett JJ, Brasseur P. Lack of activity of an association ofdetergent and germicidal agents of the infectivity of Cryptosporidium parvum oocysts.Journal of Infection 1998; 36: 245-247.

Veal D, Vesey G, Fricker CR, Ongerth J, Moenic S.Le, Rossington ACG, Faulkner B.Routine cytometric detection of Cryptosporidium and Giardia: recovery rates andquality control (draft unpublished). Proceedings: International Symposium onWaterborne Cryptosporidium California Eds: Fricker CR, Clancy JL, Rochelle PA .American Water Works Association Denver 1997; Pp 9-19.

Venczel L & Sobsey M. Inactivation kinetics of waterborne pathogens using a mixedoxidant disinfectant. Abstracts of the 95th General Meeting of the American Society forMicrobiology, Washington DC, USA, 1995. 454.

Venczel LV, Arrowood M, Hurd M, and Sobsey MD. Inactivation of Cryptosporidiumparvum oocysts and Clostridium perfringens spores by a mixed-oxidant disinfectantand by free chlorine. Applied and Environmental Microbiology 1997; 63 (4): 1598-1601.

Vesey G, Ashbolt N, Wallner G, Dorsch M, Williams K, Veal D. AssessingCryptosporidium parvum oocyst viability with fluorescent in situ hybridisation usingribosomal RNA probes and flow cytometry. In: Protozoan Parasites and Water Eds.Betts WB, Casemore DP, Fricker C, Smith H, Watkins J. The Royal Society ofChemistry Cambridge 1995; Pp 133-138.

Vesey G, Deere D, Weir C, Ferrari B, Gauci M, Williams K, Veal D. Optimising theuse of monoclonal antibodies for the detection of Cryptosporidium in water samples.Proceedings: International Symposium on Waterborne Cryptosporidium Warwick UKEds: Morris R, Gammie A. 1997: Pp 214-219.

Wagner-Wiening C, and Kimmig P. Detection of viable Cryptosporidium parvumoocysts by PCR. Applied and Environmental Microbiology 1995; 61 (12): 4514-4516.

Watkins J. 1995. Report to Yorkshire Water. Determination of the effect of ultrasoundon the efficacy of disinfectants for inactivating Cryptosporidium oocysts.

West PA and Smith MS, Eds. 1995. DoE Welsh Office/UK Water Industry ResearchLtd. Proceedings of Workshop on Treatment Optimisation for CryptosporidiumRemoval from Water Supplies. London HMSO.

Whitmore TN, Sidorowicz S. Novel methods for the concentration and selectivepurification of micro-organisms and their potential application for water analysis.Microbiology Europe 1995; 3, No 5: 16-22.

of 56

Wick JF, Blassak M, Mueller R, Jolly J, Kosler E. Detection of Cryptosporidium usingthermophilic strand displacement amplification and a calorimetric probe capturesystem. Proceedings: International Symposium on Waterborne CryptosporidiumWarwick UK Eds: Morris R, Gammie A. 1997.

Wiederman A, Steuer S, Botzenhardt K. A simple procedure for an exact evaluation ofthe sensitivity of the selective detection of viable Cryptosporidium oocysts by PCR.Proceedings: International Symposium on Waterborne Cryptosporidium NewportBeach Ca USA, 1997.

Wilson JA & Margolin AB. The efficacy of three common hospital liquid germicides toinactivate Cryptosporidium parvum oocysts. Journal of Hospital Infection 1999, In-press.

Woodmansee DB. Studies of in vitro excystation of Cryptosporidium parvum fromcalves. Journal of Protozoology 1987; 34 (4): 398-402.

Woods KM, Upton SJ. Efficacy of select antivirals against Cryptosporidium parvum invitro. FEMS Letters 1997; 168: 59-63.

Xiangdong You, Arrowood MJ, Lejkowski M, Xie L, Schinazi RF, Mead JR. Achemiluminescence immunoassay for evaluation of Cryptosporidium parvum growth invitro. FEMS Microbiology Letters 1996; 136 : 251-256.

Yang S, Healey MC, Du C. Infectivity of preserved Cryptosporidium parvum oocystsfor immunosuppressed adult mice. FEMS Immunology and Medical Microbiology1996; 13 : 141-145.

Yozwiak ML, Bradford FA, Baker FA, Olfers SD, Marshall MM, Sterling CR. Effectof a mixed oxidant solution (MIOX) on Cryptosporidium parvum oocyst viability.Presented at the 47th Annual Meeting of the Society of Protozoologists, June 24-29,1994, Cleveland, Ohio.

Zuckerman V, Gold D, Shefet G, Yuditsky A, Armon R. Microbial degradation ofCryptosporidium parvum by Serratia, marcescens with high chitinolytic activity.Proceedings: International Symposium on Waterborne Cryptosporidium California Eds:Fricker CR, Clancy JL, Rochelle PA. American Water Works Association Denver 1997Pp 297-304.

of 56

Acknowledgements. Dr Mark Rutter and Dr Gordon Nichols, PHLS EnvironmentalSurveillance Unit, PHLS Library staff, and Paula Jones, are thanked for theircontribution to this study.

report commissioned by the department of the...

Documents